Dissertação de candidatura ao grau de Doutor apresentada à

Faculdade de Medicina do Porto

Role of Tlx3 in the development of the

somatosensory system

Cláudia Sofia Amorim Saraiva Lopes

Orientação do Professor Doutor Qiufu Ma

Co-orientação da Professora Doutora Deolinda Maria Alves de Lima

Teixeira

Artigo 48º, § 3º

A Faculdade não responde pelas doutrinas expendidas na dissertação.

(Regulamento da Faculdade de Medicina do Porto, Decreto-Lei Nº 19 337, de 29 de

Janeiro de 1931).

MEMBROS DO JURI:

Vogais:

Doutor Aziz Moqrich

Doutor Qiufu Ma

Doutora Isabel Maria Mestre Marques Palmeirim de Alfarra Esteves

Doutora Alexandra Matias da Cunha Coelho de Macedo

Doutor Carlos Manuel Gomes Reguenga

Doutor Paulo Jorge Sousa Nunes Pereira

CORPO CATEDRÁTICO DA FACULDADE DE MEDICINA DO

PORTO

Professores Efetivos

Alberto Manuel Barros da Silva

Altamiro Manuel Rodrigues Costa Pereira

Álvaro Jerónimo Leal Machado de Aguiar

António Carlos Freitas Ribeiro Saraiva

Daniel Filipe Lima Moura

Deolinda Maria Valente Alves Lima Teixeira

Francisco Fernando Rocha Gonçalves

Isabel Maria Amorim Pereira Ramos

João Francisco Montenegro Andrade Lima Bernardes

Jorge Manuel Mergulhão Castro Tavares

José Agostinho Marques Lopes

José Carlos Neves da Cunha Areias

José Eduardo Torres Eckenroth Guimarães

José Henrique Dias Pinto de Barros

José Manuel Lopes Teixeira Amarante

José Manuel Pereira Dias de Castro Lopes

Manuel Alberto Coimbra Sobrinho Simões

Manuel António Caldeira Pais Clemente

Manuel Jesus Falcão Pestana Vasconcelos

Maria Amélia Duarte Ferreira

Maria Dulce Cordeiro Madeira

Maria Fátima Machado Henriques Carneiro

Maria Leonor Martins Soares David

Patrício Manuel Vieira Araújo Soares Silva

Rui Manuel Almeida Mota Cardoso

Rui Manuel Lopes Nunes

Professores Jubilados ou Aposentados

Abel José Sampaio da Costa Tavares

Abel Vitorino Trigo Cabral

Alexandre Alberto Guerra Sousa Pinto

Amândio Gomes Sampaio Tavares

António Augusto Lopes Vaz

António Carvalho Almeida Coimbra

António Fernandes da Fonseca

António Fernandes Oliveira Barbosa Ribeiro Braga

António Germano Pina Silva Leal

António José Pacheco Palha

António Luís Tome da Rocha Ribeiro

António Manuel Sampaio de Araújo Teixeira

Belmiro dos Santos Patrício

Cândido Alves Hipólito Reis

Carlos Rodrigo Magalhães Ramalhão

Cassiano Pena de Abreu e Lima

Daniel Santos Pinto Serrão

Eduardo Jorge Cunha Rodrigues Pereira

Fernando de Carvalho Cerqueira Magro Ferreira

Fernando Tavarela Veloso

Francisco de Sousa Le

Henrique José Ferreira Gonçalves Lecour de Menezes

Joaquim Germano Pinto Machado Correia da Silva

José Augusto Fleming Torrinha

José Carvalho de Oliveira

José Fernando Barros Castro Correia

José Luís Medina Vieira

José Manuel Costa Mesquita Guimarães

Levi Eugénio Ribeiro Guerra

Luís Alberto Martisn Gomes de Almeida

Manuel Augusto Cardoso de Oliveira

Manuel Machado Rodrigues Gomes

Manuel Maria Paula Barbosa

Maria da Conceição Fernandes Marques Magalhães

Maria Isabel Amorim de Azevedo

Mário José Cerqueira Gomes Braga

Serafim Correia Pinto Guimarães

Valdemar Miguel Botelho dos Santos Cardoso

Walter Friedrich Alfred Osswald

Ao Professor Doutor Qiufu Ma

À Professora Doutora Deolinda Maria Alves de Lima Teixeira

Aos meus Pais e Irmã

À memória do meu avô

Prefácio

Na fase final deste longo percurso é com muita satisfação que escrevo estas

linhas de gratidão a todos aqueles que me ajudaram no meu doutoramento. Significa

também que estou a ultimar os preparativos da tese e não escondo a alegria que me traz.

Agradeço em primeiro lugar aos meus orientadores pelo apoio, paciência,

compreensão, imensa disponibilidade e acima de tudo a contagiante paixão que ambos

apresentam pela ciência e que com sucesso incutiram em mim. Sem eles a conclusão

desta tese seria sumariamente impossível e é sem dúvida difícil exprimir em palavras o

quanto lhes estou grata.

Pela mão da Professora Doutora Deolinda Lima fui iniciada na belíssima área de

neurociências na Faculdade de Medicina Do Porto como bolseira de investigação para

ajudar a desenvolver um dos projectos no seu grupo. Devido a ela e a incríveis e

talentosos colegas que me ajudaram a dar os primeiros passos no mundo da investigação

percebi que era nesta área que queria desenvolver o meu doutoramento. Assim e devido

a uma colaboração entre o nosso grupo na FMUP e o grupo do Professor Qiufu Ma em

iniciei o meu doutoramento em Harvard Medical School em Boston. Por isso tive a

imensa sorte e prazer de ter um próximo contacto não com um mas com dois

extraordinários grupos de investigação. E agradeço às duas instituições o calor, carinho

e respeito com os quais fui recebida.

Na FMUP, agradeço à Professora Deolinda Lima, cujo exemplar empenho rigor

e paixão pela ciência pautados na sua carreira fazem dela uma cientista impar. Ter tido a

oportunidade de trabalhar com ela foi sem dúvida uma honra, e espero continuar a

merecer a sua consideração.

No grupo de investigação da FUMP onde estive inserida, tenho que agradecer ao

Professor Carlos Reguenga que tive a sorte de ser o meu mentor. Marcada pelo seu

dinamismo e profundo conhecimento científico, dei os primeiros passos na ciência. Não

esquecerei a sua disponibilidade. Com a Professora Sandra Rebelo aprendi que é

possível fazer varias coisas bem ao mesmo tempo. Sandrinha o teu dinamismo é

impressionante e prezarei a tua amizade para sempre. Ao Professor Filipe Monteiro,

agradeço as discussões científicas e amizade.

Aos vários amigos que fiz aquando da minha estadia na FMUP, às Doutoras,

Raquel Carvalhosa, Catarina Potes, Joana Gomes. Minhas amigas estarão sempre no

meu pensamento e sei que posso contar com vocês para tudo. Doutoras Ana Charrua,

Sara Adães e Isabel Martins, minhas companheiras de gabinete, e ao Doutor Carlos

Pereira, nunca teria sido tão divertido sem vocês. À Ana Tavares quero agradecer o

profissionalismo e amizade.

Em Boston, iniciei o meu doutoramento pelas mãos do Professor Doutor Qiufu

Ma, no Dana Farber Cancer Institute. Os tempos passados no seu laboratório

permitiram-me desenvolver e crescer como cientista. O Professor Qiufu Ma é um

cientista com uma inteligência invulgar e criatividade única. Foi um privilégio trabalhar

com ele. Posso dizer, que em todos os meus anos de doutoramento, não me lembro de

uma única vez que não estivesse disponível para me receber no seu gabinete. Foi sem

dúvida um mentor exemplar e espero continuar a merecer a sua estima. Os anos

formativos no seu laboratório, permitiram-me aprender não só com ele, mas com um

grupo de cientistas altamente qualificados que em muito me moldaram. Entre eles,

membros passados e presentes, Fuchia-Yang, Bo Duan, Tianwen Huang, Shan Lou,

Longzhen Cheng, Omar Abdelsamad, Yang Liu, Yi Xu, Zijing Liu, Tari Tan,Wendy

Knowlton e Martin Tamte. Aos Doutores Zijing Liu e Yi Xu, agradeço em particular

pois tive a oportunidade de trabalhar em vários projectos e trabalhar mais proximamente

com ambos. Mas tive a oportunidade de usufruir de enriquecedoras discussões

cientificas com todos eles. Agradeço ao Professor Doutor Charles Stiles, chefe do

departamento no Dana Farber, todas as discussões científicas e disponibilidade com que

me brindou. Acima de tudo agradeço o fato de estar presente e de me encaminhar nas

etapas que se seguem. Aos meus queridos amigos no instituto, Dimphna Meijer,

Katharina Cosker, e a todo o pessoal administrativo que torna a nossa vida mais fácil.

A todos os meus amigos em Boston, sem dúvida a minha família longe de casa,

Susana Godinho, Rita Teodoro, Miguel Remondes, Cláudio Alves, Cátia Fonseca, Dinis

Calado, Douglas MacMillin, Joep Pijpers, Yoav Tadmor, Marieke Liem, Stephanie

Lewis, Tana Ruegamer e Helene Bacherman. Vocês tornam o insuportável suportável.

Inbal Israeli e Rui Costa, obrigada pelo carinho e estima, e ao Rui que sempre me

guiou na minha carreira.

Aos meus amigos em Portugal, Ilda Cruz, Liliana Silveira, Susana Sá Carneiro,

Pedro Mena, Marcelo Igreja, Mafalda Regado,Sara Mesquita, Raquel Carvalhosa, Susana

Aragão, vocês são a família que eu escolhi.

Aos meus pais. Não há palavras que possam explicar o meu enorme orgulho em

vocês. Tenho os melhores pais do mundo. Tudo o que eu sou, foram vocês que me

incutiram. Obrigada pelo apoio incondicional e por acreditarem sempre em mim,

mesmo quando eu não sou capaz.

Ao meu querido avô que sempre acreditou em mim e acima de tudo me incentivou

a seguir as minhas paixões.

À minha irmã. Mana, és a minha melhor amiga. Mesmo que pudesse escolher

não escolheria outra. A nossa amizade e cumplicidade, as tuas palavras de coragem e

ânimo, o teu sentido de humor, o teu apoio incondicional, fazem com que tudo seja mais

fácil. Para alem disso, deste-me as sobrinhas mais bonitas do mundo. E uma bênção ter-

te como irmã.

Ao meu cunhado, que sempre me ajudou em tudo e sempre se mostrou

disponível. Pela amizade e compreensão. É um privilégio ter-te na família.

Ao meu namorado, Jonathan. Todos os dias me inspiras a ir mais além e a dar o

meu melhor, mesmo quando parece impossível fazê-lo. Tu tiveste que lidar,

especialmente nos últimos tempos com o meu desgaste e cansaço e mesmo assim,

conseguiste fazer com que o escasso tempo que passássemos juntos fosse único e algo

pelo qual ansiar. Estou desejosa por um futuro contigo, e por partilhar tudo o que a vida

nos reserva. Sou a miúda mais sortuda do mundo.

Agradeço ainda o fato do meu doutoramento ter sido financiado por uma bolsa

da Fundação para a ciência e Tecnologia (SFRH/BD/36380/2007), e aminha estadia

inicial em Boston foi possível devido a uma bolsa da Fundação Calouste Gulbenkian e

Fundação FLAD/Fulbright.

Em obediência ao disposto no Decreto-Lei n 388/70, Artigo 8, paragrafo 2, declaro que

efetuei o planeamento e execução das experiencias, observação e análise de resultados e

participei ativamente na redação de todas as publicações que fazem parte integrante

desta dissertação:

Lopes C, Liu Z, Xu Y, Ma Q (2012) Tlx3 and Runx1 act in combination to

coordinate the development of a cohort of nociceptors, thermoceptors, and pruriceptors.

J Neurosci. 2012 Jul 11;32(28):9706-15.

Xu Y, Lopes C, Wende H, Guo Z, Cheng L, Birchmeier C, Ma Q.(2013)

Ontogeny of excitatory spinal neurons processing distinct somatic sensory modalities.J

Neurosci. 2013 Sep 11;33(37):14738-48.

Liu Y, Abdel Samad O, Zhang L, Duan B, Tong Q, Lopes C, Ji RR, Lowell

BB, Ma Q.( 2010) VGLUT2-dependent glutamate release from nociceptors is required

to sense pain and suppress itch. Neuron 2010 Nov 4;68(3):543-56.

A reprodução destas publicações foi feita com autorização das respetivas

editoras.

Índice

I. Introduction

A. Overview of somatosensary system 17

B. Development of primary sensory neurons In DRG 19

B1. Genesis of primary sensory neurons in DRG 19

B2. Segregation of TrkA lineage neurons into TrkA+ and Ret+ subpopulations 20

B3. Channel receptors and functional heterogeneity of TrkA lineage neurons 22

B4. Developmental regulation of sensory channels and receptors 24

C. Spinal cord circuitry and development 26

C1. Lamina organization of dorsal spinal cord 26

C2. Ontogeny of dorsal horn neurons 29

C3. From developmental ontogeny to physiological functions of spinal neurons 32

D. The encoding of pain versus itch 33

E. Questions addressed in this thesis 35

II. Publications 37

Publication I 38

Publication II 39

Publication III 40

III. Discussion 41

A. Generation of primary somatic sensory neuron diversity 42

A1. Runx1 and Tlx3 are selector-like factors that act in combination to control

the development of the Ret+ subset of TrkA lineage sensory neurons 42

A2. How do Runx1 and Tlx3 form a combinatorial code 44

A3. How are different submodalities further segregated 45

A4. A summary of sensory neuron subtype specification and their implication

on sensory coding 46

B. Developmental ontogeny of spinal neurons processing distinct sensory

modalities 48

C. Unsolved problems and future directions 52

References 54

IV. Summary and conclusions 71

V. Resumo e conclusões 74

I. Introduction

17

A. Introduction

The somatosensory system mediates fundamental physiological functions, such as

nociception, thermoception, pruriception, proprioception and mechanoception. Its ability to do so

is endowed by virtue of distinct somatic sensory neurons, sensory receptors and processing

centers that will allow for different percepts to form in higher brain centers. Primary somatic

sensory neurons receive information from the periphery and have their cell bodies in both cranial

and dorsal root ganglia. They transmit the information from skin, muscle, bones, joints and

visceral organs, constituting a highly heterogeneous population of neurons specialized to provide

modality specific information to the central nervous system. These neurons are classified into

several major subtypes including low threshold mechanoreceptors that sense touch, pressure and

vibration; proprioceptors that sense body position; thermoceptors that respond to innocuous cold

and warm temperatures; nociceptors that respond to noxious stimuli and tissue damage, and

pruriceptors that respond to itch inducing compounds (Marmigere et al, 2007).

Based on different degrees of myelination and distinct conduction velocities, somatic

sensory nerve fibers can also be distinguished into three types: Aβ, Aδ, and C nerve fibers. The

Aβ fibers are heavily myelinated, the Aδ fibers are thinly myelinated, and C fibers are

unmyelienated. The speed at which an individual nerve fiber carries action potentials varies

according to its diameter and the degree of myelination. Accordingly, large-diameter Aβ and

medium Aδ fibers show fast and medium conduction speeds, whereas the slowest ones are

unmyelinated small-diameter C fibers. This classification is particularly important in the realm of

pain conduction. For instance, nociceptors are normally divided into two classes, Aδ and C fiber

nociceptors (though rare Aβ nociceptors also exist) (Djouhri et al., 2004). The fast-conducting

Aδ nociceptors mediate the first/sharp pain, whereas C fiber nociceptors mediate the second dull

pain in humans (Lawson SN, 1992; McCarthy and Lawson 1990; Lawson et al., 1996).

The different subtypes of somatic neurons innervate different organs and also have

distinct patterns of termination peripherally. Proprioceptive neurons are myelinated neurons that

innervate deep structures such as Golgi tendon organs, which are sensors for detecting strain, and

muscle spindles that detect contraction and stretch (Patel et al., 2003; Marmigere and Ernfors.,

18

2007; Dalla Torre et al., 2008; Lee J. et al., 2012; Tripodi M. and Arber S., 2012; Arber S.

2003& 2012). Low threshold Aβ mechanosensitive neurons terminate in the skin, tendons,

muscles, joint capsules and viscera, and transduce touch, pressure and vibration. These

mechanoreceptors form specialized nerve endings, including Meissner corpuscles, Pacinian

corpuscles, Ruffini corpuscles and Merkel cells (Li et al., 2011; Luo et al., 2009; Abraira and

Ginty, 2013). Merkel cells are situated at the base of the epidermis and respond to gentle

localized pressure. Meissner corpuscles are situated immediately below the epidermis and are

particularly sensitive to light touch and the speed of the stimulus. Ruffini corpuscles are located

in the dermis and articulations and are sensitive to vibration and stretching of the skin and

tendons, and finally Pacinian corpuscles are present in the dermis and hypodermis and are

involved in the discrimination of fine surface textures or other moving stimuli that produce high-

frequency vibration of the skin. Aδ and C fiber neurons signal nociception, thermoception,

pruriception or sensory touch, and they terminate throughout the peripheral tissues, though

pruriceptors only innervate the skin (Delmas et al., 2011; Lallemond and Ernfors., 2012).

Different types of primary sensory fibers project to distinct laminae in the spinal cord

(Lallemond and Ernfors, 2012). Group Ia and some group II proprioceptors connect to motor

neurons in the ventral spinal cord, whereas group Ib proprioceptors connect to interneurons in

the intermediate zone (Patel et al., 2000; Tripodi et al., 2011; Arber S. 2012). Aβ, Aδ and C-low

threshold mechanoreceptor (C-LTMR) fibers project to the dorsal horn in a partially overlapping

manner (Li et al., 2011; Abraira and Ginty, 2013). C-LTMRs terminate in lamina IIi and partly

overlap with Aδ-LTMRs on the same lamina, although the latter mostly projects to lamina III.

The Aβ-LTMR fibers terminate in laminae III through V. Nociceptive, thermoceptive and

pruriceptive neurons are either unmyelinated C fibers that terminate in lamina I and II or lightly

myelinated Aδ sensory neurons that terminate in laminae I and V (Lallemond and Ernfors,

2012).

Sensory neurons, from a molecular point of view, encompass a remarkably

heterogeneous population of neurons, as indicated by the expression of neurotrophin receptors, a

large cohort of sensory receptors and ion channels that allow individual sensory fibers to respond

to specific stimuli, and others (see below).

19

The goal of my thesis work is to characterize the genetic programs that control the

development of somatic sensory circuitry. We focused on two areas. One is to understand how

different types of primary sensory neurons are specified, focusing on the study of nociceptors,

pruriceptors and thermoceptors. The other is to understand how spinal neurons processing

distinct sensory modalities are formed.

B. Development of primary sensory neurons in DRG

B1. Genesis of primary sensory neurons in DRG

Dorsal root ganglion neurons derive from neural crest cells that delaminate from the

dorsal neural tube. These cells are born in three successive waves of neurogenesis (Ibanez and

Ernfors, 2007; Lallemend and Ernfors, 2012; Marmigere and Ernfors, 2007). The first wave

starts between E9.5 and E11.5 and produces Aβ proprioceptive and Aβ/Aδ mechanoceptive

neurons. Proprioceptive neurons are marked by the expression of tyrosine kinase receptor C

(TrkC), the receptor for neurotrophin 3 (NT-3). Mechanoceptors are marked by the expression

of TrkB, the receptor for brain derived neurotrophic factor (BDNF) and Ret, the receptor for glial

derived neurotrophic factor (GDNF). The second wave occurs between E10.5 and E13.5, and

gives rise to a majority of Aδ and C fiber neurons that mediate nociception, thermoception and

pruriceptors. About 5% of DRG neurons that belong exclusively to small diameter c-fiber

neurons arise in a third wave of neurogenesis from cells of the boundary cap, a neural crest

derivative (Maro et al, 2004; Lallemend and Ernfors, 2012). All these nociceptors, thermoceptors

and pruriceptors produced during late waves of neurogenesis are initially marked by the

expression of TrkA, the receptor for nerve growth factor (NGF) (Marmigere and Ernfors, 2007;

Luo et al., 2009; Bourane et al., 2009; Honma, et al, 2010).

Two basic helix loop helix (bHLH) transcription factors have a determinant role in

committing the neural crest cells to its sensory fate. Neurogenin2 (ngn2) is expressed during the

first wave of migratory sensory precursors, whereas the second and third waves are marked by

the expression of Neurogenin1 (ngn1). Mice that lack both of these two transcription factors

20

show impairment in sensory neurogenesis altogether (Ma Q. et al, 1999). Analysis of Trk

expression in dorsal root ganglia in mice revealed that ngn2 is required for the differentiation of

most TrkB and TrkC positive neurons and a small subset of TrkA+ neurons, while ngn1 is

required later for most of TrkA positive neurons while contributing to a fraction of TrkC and

TrkB positive ones (Ma Q. et al, 1999; Bachy et al, 2011). The expression of Neurogenin1/2 in

embryonic sensory precursors is followed by the expression of two other bHLH transcription

factors, NeuroD1 and NeuroD4, whose expression is dependent on Neurogenin1/2 (Sun et al.,

2008; Eng et al., 2004; Ma et al., 2003; Lanier et al, 2009). The neurogenic phase is followed by

cell cycle exit, axon growth and expression of genes characteristic of neuronal function.

Concomitantly, around E9.5/E10.5, newly formed sensory neurons start to express the

panneuronal markers Islet1 and Brn3a (Sun et al, 2008; Dykes et al., 2011; Zou et, 2012 ; Ma et

al, 2003; Lei et al, 2006; Eng et al., 2004). Both transcription factors have an overlapping role in

ending the expression of neurogenic bHLH factors by direct repression and facilitating the

progression to the phase of sensory neuron specification (Lanier et al, 2009; Dykes et al, 2011).

Brn3a is required for proper specification and survival of proprioceptive, mechanoreceptive and

nociceptive neuronal populations, partly by controlling the expression of neurotrophin receptors

(Huang et al., 1996, McEvilly et al., 1996, Xiang et al., 1999, Dykes et al; 2010, Lei et al. 2006,

Eng et al, 2004). Both Brn3a and Islet1 are also required for terminal differentiation of

nociceptors, mechanoceptors and thermoceptors (Sun et al., 2008, Lanier et al 2009).

B2. Segregation of TrkA lineage neurons into TrkA+ and Ret+ sub-

populations

A main focus of my thesis is to study how TrkA lineage neurons produced during second

and third waves of sensory neurogenesis are further segregated into different subtypes.

TrkA lineage neurons can be broadly divided into two major sub-populations: peptidergic

and non-peptidergic (Molliver et al., 1997; Ibanez and Ernfors, 2007; Luo et al, 2007; Woolf and

Ma, 2007; Gascon et al, 2010; Liu and Ma., 2011). The former is marked by the expression of

TrkA, as well as two neuropeptides, the calcitonin gene related peptide (CGRP) and Substance P

21

(SP). The latter is mostly marked by the expression of Ret and can be labeled by the binding of

Isolectin B4 (IB4). These two separate classes of nociceptors have distinct innervation targets. In

peripheral tissues, TrkA positive neurons terminate in the stratum spinosum of the skin epidermis

as well as many deep tissues, and centrally, they terminate in lamina I and outer lamina II. Ret

positive neurons have peripheral projections terminating in the superficial stratum granulosum of

the epidermis while central projections end in inner lamina II of the spinal cord, implying that

these two classes of nociceptors are anatomically segregated (Basbaum, et al., 2009, Woolf and

Ma, 2007).

Studies in my lab show that the runt domain transcription factor Runx1 plays a critical

role for the segregation of these two sub-populations. At early embryonic stages, Runx1 is

expressed in about 93% of TrkA+ neurons, and its expression starts right after the onset of TrkA

expression (Chen et al., 2006, Kramer et al., 2006, Levanon et al., 2002, Theriault et al., 2005,

Marmigere et al., 2006). However, during perinatal and postnatal stages, Runx1 is no longer

expressed in those peptidergic neurons that retain TrkA and becomes restricted to non-

peptidergic Ret+ neurons that will have switched off TrkA (Figure 1). By genetically removing

Runx1 in neuronal precursors in mice, my colleagues showed that Runx1 has a dual role: 1)

activating Ret and 2) suppressing TrkA and CGRP, and is thereby critical for determining the

Ret+ nonpeptidergic sensory fate (Chen et al., 2006). Subsequent gain-of-function studies further

showed that Runx1 downregulation is critical for the establishment of peptidergic neurons.

(Kramer et al., 2006; Samad et al., 2010).

Figure 1: Runx1 is critical in the segregation between peptidergic and non-peptidergic populations. a)Molecular diagram showing he suppression of TrkA by Runx1 and Runx1-dependent Ret.

Runx1

Ret

TrkA

TrkA+Runx1+

TrkA

Runx1-Runx1+

Ret+

Non-peptidergic peptidergic

a) b)

E12

Adult

22

There are two follow-up questions: (1) what is the mechanism by which Runx1

suppresses TrkA expression in Ret+ nonpeptidergic neurons, and (2) how is Runx1 expression

extinguished in peptidergic neurons? Luo et al addressed the first question by proposing a

feedforward control mechanism, in which they envisioned that Runx1 is necessary for Ret

activation, and that Ret mediated signaling will in turn suppress TrkA expression (Luo et al.,

2007). For the second question, Gascon et al. showed that Runx1 downregulation in a subset of

peptidergic neurons requires hepatocyte growth factor-Met signaling working in synergy with

TrkA (Gascon et al., 2010).

B3. Channel receptors and functional heterogeneity of TrkA lineage neurons

The expression of ion channels and receptors lends neurons the unique ability to respond

to different somatosensory stimuli, including those that will lead to the perception of pain, itch,

touch, and temperature (Basbaum et al., 2009, Woolf and Ma, 2007).

A major breakthrough in the somatic sensory neuron field was the discovery of Transient

Receptor Potential (TRP) channels as the sensors of thermal and chemical stimuli, starting with

the finding that TRPV1 receptor is a receptor for heat and capsaicin (Caterina et al., 1997). In

mammals, there are over thirty different members of the TRP family grouped into six different

classes: TRPC, TRPV, TRPM, TRPA, TRPP, TRPL (Caterina et al, 1997, 1999). These Trp

channels are involved in an array of distinct functions, including olfaction, taste, vision,

osmoregulation, mechanosensation and temperature perception (Clapham, 2003; Caterina et al.,

2007; Venkatachalam and Montell, 2007).

TRPV1 was initially described as the endogenous heat transducer being expressed in the

majority of heat sensitive nociceptors (Caterina et al., 1997). It is activated by a range of other

stimuli including capsaicin, low pH, toxins, and endogenous lipids. Similarly to how capsaicin

activates TRPV1 sensitive heat channels, other compounds such as eucalyptol and menthol have

been used to search for cold sensitive fibers and cells. TRPM8 is required for multiple types of

cold signaling, including innocuous cool, noxious cold, cold allodynia and cooling analgesia

(Proudfoot et al., 2006; Colburn et al 2007; Knowlton et al., 2011 & 2013). TRPM8 positive

23

neurons are molecularly heterogeneous and approximately half of them are also capsaicin

sensitive and express TRPV1 (Mckemy et al., 2002 & 2013; Viana et al., 2002; Babes et al.,

2004; Takashima et al., 2010), meaning that subsets of neurons are both heat and cold sensitive.

TRPA1 was initially reported as an ion channel that would respond to noxious cold (Story

et al., 2003). However some controversy has been raised regarding the physiological stimuli that

activates TRPA1, and the generation of TRPA1 deficient mice by two independent groups did

not help to clarify it (Bautista et al., 2006; Kwan et al., 2006). On the contrary its functions as a

chemical sensor responding to a range of pungent or irritant chemicals, such as mustard oil,

cinnamon, gas exhaust, garlic and formalin are widely accepted (Patapoutian et al., 2009, Stucky

et al., 2009). Moreover, in a TRPA1 and TRPM8 double knockout, there are neurons that still

respond to noxious cold stimuli (Knowlton et al., 2010), indicating that noxious cold transducers

other than TRPA1 exist. TRPA1 is also activated by bradykinin, an inflammatory mediator, and

TRPA1 deficient mice show marked deficits in thermal and mechanical hyperalgesia induced by

bradikynin injection (Bautista et al., 2006; Kwan et al., 2006). Notably, TRPV1 and TRPA1 are

also expressed in partially overlapping neuronal subpopulations (Bautista et al., 2005; Kobayashi

et al., 2005).

The Mrgpr G-protein coupled receptor family has also been shown to have key roles in

the signaling of somatosensation (McNeil, 2012). For instance, MrgprA3 positive neurons are

pruriceptors that are required to sense itch evoked by choroquine, a compound used to treat

malaria (Liu et al., 2009). MrgprC11 is also a pruriceptor activated by BAM8-22 (Liu et al.,

2011). MrgprB4 positive neurons innervate exclusively the hairy skin and seem to function as C-

low threshold mechanoreceptors that are important for pleasant touch sensation, although further

studies are required to verify this assumption (Liu et al., 2007; Vrontou et al., 2013). MrgprD

positive neurons are polymodal nociceptors that respond to mechanical and heat stimuli (Zilka et

al., 2005; Rau et al., 2009; Cavanaugh et al., 2009).

The process through which cutaneous mechanoresponsive cells transform mechanical

stimuli into electric signals has been harder to decipher (Eijkelkamp et al., 2013). The acid

sensing ion channels ASIC1 and ASIC2, which are mammalian homologs to the C. elegans

degenerin/epithelial Na+ channel (DEG/ENaC), are localized in the Pacinian corpuscles,

24

potentially ascribing them to a role in mechanotransduction ( Delmas et al., 2011). Piezo1 and

Piezo2 have recently been discovered to be multipass membrane proteins required for rapidly

adapting mechanically activated currents in DRG neurons (Coste et al., 2010& 2012; Hao et al.,

2011; Kim et al., 2012). These afferents also express a myriad of ion channels that are involved

in pain transmission (Coste et al., 2012; Hao and Delmas, 2011; Kim et al., 2012). These include

Nav1.8 and Nav1.9 ion channels expressed in small nociceptive neurons (Aktopian et al., 1996,

Black et al., 1996), and potassium channels like TRAAK and TREK-1 that modulate excitability

and contribute to action potential propagation (Noel et al., 2011, Alloui et al., 2006).

B4. Developmental regulation of sensory channels and receptors

One key question in somatic sensory neuron development is to understand how individual

sensory neurons acquire the expression of specific sensory channels and receptors. In the past

years, two themes start to emerge. One is that most of these ion channels/receptors are under the

control of a combination of mostly three transcription factors: Runx1, Brn3a and Islet1. The

other is that target derived signals also play a role.

Runx1 in particular is required for the expression of many sensory channels and receptors

in those sensory neurons innervating the skin, including TRP channels and Mrgpr proteins (Chen

et al., 2006). Moreover, studies in our lab have further helped to understand how functionally

distinct sensory neuron subtypes are segregated. Take three non-overlapped populations of

neurons expressing different Mrgpr proteins as an example: one with MrgprD marking

polymodal nociceptors for mechanical pain, one with MrgprA3 marking itch-related

pruriceptors, and one with MrgprB4 marking neurons involved with pleasant touch (Figure 2)

(Liu et al., 2008). It turns out that genetically, Runx1 functions as both a transcriptional activator

and repressor in controlling the segregation of these neurons. Runx1 acts as a transcriptional

activator for MrgprD throughout development. However, for MrgprA3 and MrgprB4, it initially

acts as an activator, but switches to be a transcriptional repressor at postnatal stages. In

consequence, MrgprD expression requires Runx1 persistent expression, and MrgprA3/B4/C11

can only be sustained in neurons where Runx1 is transiently expressed. Hence, Runx1 dual

25

transcriptional activity associated with its dynamic expression can help to explain the segregation

of distinct sensory modalities (Liu et al., 2008).

But what controls the dynamic expression and activity of Runx1? Knowing that the

expression of sensory channels and receptors happens about the time when axons reach their

peripheral targets, target derived signals may have a role in their expression. Indeed, TrkA

signaling that is required for axons to reach their peripheral targets is also necessary to control

many Runx1-dependent genes (Luo et al., 2007). Ret signaling controls a subset of these genes,

TrpA1, MrgprA3 and MrgprB4 (Luo et al., 2007), and Smad4-mediated BMP signaling is

selectively required for the expression of MrgprB4 (Liu et al., 2008) (Figure 3). These studies

strongly suggest that target derived signals must somehow interface with intrinsic transcription

factors, such as Runx1, to control the expression of these sensory channels and receptors.

Figure 2: Runx1 controls the expression of functionally distinct sensory channels and receptors.

26

Despite this progress, it was still not fully understood how Runx1 could coordinate the

development of such a large cohort of cutaneous sensory neurons involved in nociception,

pruriception and thermoception. Addressing this question forms the basis of the first part of my

thesis.

C. Spinal cord circuitry and development

In order to appropriately distinguish between different somatosensory sensations, sensory

neurons in the DRG must establish precise connections with their targets in the CNS. The dorsal

spinal cord is the first relay center that receives processes and transmits somatic sensory

information. For my second part of the thesis, I will study how spinal neurons processing distinct

somatic sensory modalities are specified during development.

C1. Lamina organization of dorsal spinal cord

The dorsal horn of the spinal cord is the first integrating center for somatosensory

perception receiving sensory signals from peripheral neurons and conveying distinct sensory

inputs to higher brain centers (Willis and Coggeshall, 2004; Todd AJ., 2010). Neurons that

Figure 3: Broadly expressed transcription factors, like Runx1, interface with target derived signals in order to progressively segregate distinct sensory modalities.

27

process and integrate different somatosensory inputs are segregated in different laminae in the

spinal cord. The spinal cord can be divided into two organizing and integrative centers; the

dorsal spinal cord that integrates somatosensory information and the ventral spinal cord that

integrates motor related inputs (Fitzgerald M., 2005; Todd AJ., 2010). The dorsal horn of the

spinal cord receives and processes inputs from a wide variety of primary afferent fibers,

including nociceptors, thermoceptors, pruriceptors, and chemoreceptors that respond to stimuli

from the skin, muscles, joints and viscera (Takazawa and MacDermott , 2010).

The primary afferent terminations exhibit a specific laminar organization, such that

mechanosensitive Aβ fibers, that carry tactile information, terminate primarily in the deep

laminae (III–V), whereas the superficial laminae (I–II) receives projections primarily from the

smaller Aδ and C fibers, many of which are nociceptive (Light, 1992). These primary afferents

target mostly two different classes of neurons in the dorsal horn: interneurons and projection

neurons (Todd AJ., 2002). Interneurons consist of all neurons in lamina II and most neurons in

lamina I-III (Todd AJ., 2010), being the best characterized ones lamina II interneurons. They

receive sensory information and make local connections with other association neurons

projecting ipsilaterally to higher brain centers (Lu Y and Perl ER., 2003). There are no projection

neurons in lamina II and most of them are excitatory using glutamate as a main transmitter

(Yasaka et al., 2010). Up to one third of them are inhibitory GABAergic neurons and a subset of

these coexpress glycine (Takazawa and McDermott, 2010). Through a combination of

electrophysiological studies and morphological analysis, lamina II interneurons can be

categorized into four distinct groups according to somatodendritic morphology: islet, radial,

vertical and central cells (Maxwell et al., 2007; Lu Y and Perl ER, 2007; Grudt TJ and Perl ER,

2002). Neurotransmitter release, is not the only way to classify interneurons in lamina II, other

parameters such as somatodendritic morphology, innervation pattern from periphery, firing

pattern and neurochemical expression comprise other ways of classification. There seems to be a

consensus between morphology and neurotransmitter, as Islet cells are GABAergic, radial and

vertical cells are glutamatergic and central cells can be either glutamatergic or GABAergic

(Yasaka et al., 2010, Todd AJ, 2010). In terms of innervation Islet and most central cells receive

input from C-fibers while vertical and radial cells receive monosynaptic input from both Aδ and

C afferents (Light, 1992).

28

There is very limited knowledge on lamina I interneurons and they are harder to classify

probably due to confusion with lamina I projection neurons but nonetheless they can be

distinguished into pyramidal, flattened, fusiform and multipolar cells according to morphology

and firing pattern (Lima D and Coimbra A, 1986). Another way of classifying functional

populations in the dorsal horn is by utilizing neurochemical markers that are expressed either in

glutamatergic or GABAergic populations. For instance, somatostatin, neurokenin B, neurotensin

and substance P-expressing are glutamatergic neurons, as neuropeptide Y and galanin-expressing

are inhibitory ones. Enkephalin and dynorphin can be espressed by both types ( Proudlock et al.,

1993; Xu et al., 2008).

The other type of neuron that populates the dorsal horn of the spinal cord is projection

neurons. These are located in lamina I also referred to as marginal zone, lamina V and ventral

spinal cord. These projection neurons are mostly commissural neurons that project

contralaterally to the hindbrain, midbrain and thalamus along spinocerebellar, spinocervical,

spinotectal and spinothalamic tracts (Brown AG., 1981 and Tracey, 1985).

Lamina I projection neurons constitute a large fraction of the spinothalamic tract conveying

information related to pain, itch and temperature (Hodge and Apkarian, 1990). Even though the

preferred postsynaptic targets for primary afferents are interneurons they also synapse onto

projection neurons, particularly peptidergic ones. The main supraspinal targets of lamina I

projection neurons include the caudal ventrolateral medulla, the nucleus of solitary tract, the

lateral parabrachial area, the periacqueductal gray matter and some thalamic nuclei, imcluding

the ventral posterolateral nucleus, the posterior group and the posterior triangular nucleus (Spike

et al., 2003). Even though they send extensive projections to higher brain centers they sum up to

only 5% of lamina I neurons and almost all of them project to the lateral parabrachial area, with

the remainder projecting to the nucleus of solitary tract and less than 5% of these to the thalamus,

showing that there is collateralization of axons to these three targets (Spike et al., 1993).

Lamina I projection neurons constitute a very heterogeneous population including nociceptive

specific neurons that respond to pinch and/or noxious heat (Han et al., 1998). These neurons are

marked by the expression of the neurokinin1 receptor (NK1R), the main target for substance P.

NK1R expression is associated with noxious activated neurons and marks over 80% of lamina I

29

projection excitatory neurons (Todd et al., 2002). Besides nociceptive specific projection neurons

lamina I also contains: 1) COLD neurons that are responsive to innocuous cooling and inhibited

by warm and show no NK1R expression, 2) HPC neurons that respond to noxious heat, pinch

and innocuous and noxious cold and 3) itch selective neurons (Todd AJ, 2010).

In addition to receiving direct primary afferent input, dorsal horn neurons are modulated

by a high degree of local interneuronal connectivity that can also influence their sensory

response properties (Yoshimura and Nishi, 1992; Torsney and MacDermott, 2006; Yasaka et al.,

2007). For instance, a small percentage of superficial dorsal horn neurons normally receive

polysynaptic Aβ input, but this percentage is greatly increased under conditions of central

disinhibition (Torsney and MacDermott, 2006) and in animal models of inflammatory or

neuropathic pain hypersensitivity (Baba et al., 1999; Nakatsuka et al., 1999; Okamoto et al.,

2001; Kohno et al., 2003). It has been hypothesized that this polysynaptic Aβ input is mediated

by an unidentified interneuronal connection from the deep to the superficial laminae where in

conditions of nerve injury, a lamina I nociciceptive specific neuron is able to respond to

innocuous stimuli after the unmasking of polysynaptic pathways that carry low-threshold input to

superficial laminae (Torsney and MacDermott, 2006, Takazawa and MacDermott, 2010).

C2. Ontogeny of dorsal horn neurons

The spinal cord is built on a ventral to dorsal gradient along the neural tube. Spinal

neurons have two major functions. One class of spinal neurons relays cutaneous information to

higher brain centers and the other integrates proprioceptive input and motor output. These two

distinct systems are anatomically segregated, and are assigned to the dorsal and ventral spinal

cord, respectively. Motor neurons are the first to be generated and reside in the ventral (basal)

region, whereas neurons that process somatosensory information reside in the dorsal (alar) plate.

(Wilson and Madden, 2005).

Family members of the homeodomain family and the basic helix loop helix family of

transcription factors are expressed in restricted dorsoventral domains (Timmer et al., 2002; Lee

and Pfaff., 2001). As such the spinal cord will be divided into eleven progenitor domains and

each one is identified by a specific transcription factor code (Lee and Pfaff., 2001; Marquardt

30

and Pfaff., 2001; Shirasaki and Pfaff, 2002). There are five ventral domains (p3, pMN, p2, p1

and p0) and six dorsal domains (dP1-dP6, from dorsal to ventral). This combinatorial

transcription factor code assigned to distinct progenitor domains will dictate the neuronal

subtype progeny that they will produce (Anderson et al., 1997; Briscoe et al., 2000; Briscoe and

Ericson, 1999& 2001; Gowan et al., 2001; Lee and Jessell, 1999; Lee and Pfaff, 2001; Marquardt

and Pfaff, 2001; Mizuguchi et al., 2001).

Expression of these transcription factors is determined by patterning signals secreted from

two signaling centers, the ventral floor plate and the dorsal roof plate. The ventral floor plate

secretes sonic hedgehog (Shh) signaling while the roof plate secretes members of the Wingless-

type MMTV integrator site (Wnt) and bone morphogenetic protein (BMP) (Kuschel et al., 2003;

Wilson and Madden., 2005). Other patterning signals, also involved in dorsal ventral patterning,

although to a lesser extent, include transforming growth factor Beta (TGF-beta) and retinoic

acids (Pituello et al., 1995; Liem et al., 1997; Garcia-Campmany and Marti, 2007; Novitch et al.,

2003; Wilson and Maden., 2005). For the purpose of this thesis I will focus on the dorsal neural

tube development.

The Wnt canonical pathway and the BMP are the two patterning signals arising from the

roof plate, and ablation studies in which the roof plate signaling was removed led to a complete

loss of the three most dorsal populations of interneurons (dI1-dI3), which are derived from dP1-3

progenitors (Lee et al., 2000).

Wnts are related to the Drosophila wingless family. They are a highly conserved family

of secreted proteins with prominent roles in cell to cell interactions through embryogenesis

(Logan and Nusse, 2004). Overexpression studies have shown that Wnt signaling is able to

induce genes normally expressed in dorsal progenitor cells, such as Pax7 and Pax6, and

suppresses genes normally expressed in ventral progenitors, such as Nkx6.1, Olig2 and Nkx2.2

(Alvarez-Medina et al., 2008; Yu et al., 2008). Mechanistically, the Wnt canonical pathway is

able to antagonize the ventralizing activities of SHH signaling (Alvarez-Medina et al, 2008; Lei

et al, 2006; Yu et al., 2008). This is a somewhat conserved mechanism throughout the neuronal

axis given that a proper balance between Wnt and Shh is required for the determination of dorsal

31

and ventral telencephalic types (Jun Motoyama and Kazushi Aoto, 2000; Diana S Himmelstein et

al., 2010).

After neural tube closure, several members of the BMP family are expressed in the roof

plate, including BMP4, BMP5 and BMP7. These proteins were shown to have a seminal role in

neural development by promoting the generation of dorsal interneurons, particularly, dI1 and dI3

(Liem et al., 1995, 1997). Progenitor cells in the dorsal horn patterned by BMP and Wnt

signaling show differential expression of the bHLH factors, including Olig3, Math1, Ngn1/2 and

Mash1 (Timmer et al., 2002; Barth et al., 1999; Lee et al., 1999; Muroyama et al., 2002; Wodarz

et al., 1998). During the first neurogenic wave, that in mice starts around E10.5 through E11.5,

six different populations of neurons arise at stereotyped positions from non-overlapping domains

of progenitor populations, dI1-dI6. The second neurogenic wave that starts at E11 through E13

will give rise to two late born populations, dILA and dILB (Gross et al., 2002; Muller et al.,

2002; Helms and Johnson., 2003). These dorsal horn neurons are divided into two large classes,

“A” and “B”, based on the expression or a lack of expression of a Lim homeodomain factor,

Lbx1. Class A neurons, dI1-dI3, are Lbx1 negative and are derived from Olig3+ positive

precursors, require roof plate signaling for their specification, and settle in the deep dorsal horn.

Class B neurons, dI4-dI6, dILA and dILB, are Lbx1 positive and derived from Olig3 negative

precursors; they emerge independently from roof plate signaling; and settle mainly in the

superficial laminae of the dorsal horn, but also minorly in the deep dorsal horn and even in the

ventral spinal cord (Gross et al., 2002; Muller et al., 2002&2005).

Class B neurons are further divided into glutamatergic/excitatory neurons (dI5 and dILB)

and GABAergic/glycinergic inhibitory neurons (dI4, dI6 and dILA). The former are marked by

the expression of another homeodomain transcription factor Tlx3 and Lmx1b, whereas the latter

one is marked by the expression of another transcription Pax2 ( Gross et al., 2002; Muller et al.,

2002; Qian et al., 2002; Helms and Johnson, 2003; Cheng et al., 2004 & 2005; Glasgow et al.,

2005; Rebelo et al., 2010).

Studies from our lab show that Lbx1 specifies default GABAergic differentiation,

whereas Tlx3 antagonizes Lbx1 to allow a subset of Lbx1 positive neurons to become

glutamatergic neurons (Cheng et al., 2005) (figure 4). Other transcription factors, such as

32

Gsx1/2, Ptf1a, and Prdm13 also play a role in specifying glutamatergic versus GABAergic cell

fates in the dorsal horn (Mizugushi et al., 2006; Glasgow et al., 2005; Hoshino et al., 2005;

Huang et al., 2008). Furthermore, many of these transcription factors also control other features

in these neurons, such as the peptide transmitters and neurotransmitter receptors (Qian et al.,

2002, Cheng et al., 2004, Xu et al., 2008, Guo et al., 2012, Huang et al., 2008, Brohl et al.,

2008).

C3. From developmental ontogeny to physiological functions of spinal neurons

The spinal cord receives and integrates somatosensory information regarding pain, touch,

itch, cool and warm. Both electrophysiological and ablation studies in recent years have started

to reveal modality selective spinal neurons. For example, neurons expressing the gastrin-

releasing peptide receptor GRPR are critical for the sense of itch (Sun and Chen, 2007; Sun et

al., 2009).

Studies in recent years have also begun to bridge the gap of knowledge between

developmental classification of dorsal horn neurons (dI1-6, dILA, and dILB) and their

physiological functions. For instance, dI1 excitatory neurons are important for the formation of

spinocerebellar tracts and thereby for proprioception, dI3 excitatory neurons mediate grasping

responses (Bermingham et al., 2001; Bui et al., 2013), and dI6 inhibitory neurons are involved in

motor pattern generation (Anderson et al., 2012).

The goal of the second part of my thesis is to study the physiological function of two

groups of dorsal horn excitatory neurons (dI5 and dILB), (Figure 4). Previously, my lab had

shown that Tlx3 is required for the proper development of dI3, dI5 and dILB excitatory

glutamatergic neurons (Qian et al., 2002; Cheng et al., 2004; Xu et al., 2008; Guo et al., 2012).

In the second part of my thesis, I, together with a postdoctoral fellow in the lab, investigated the

role of dI5 and dILB neurons by creating mice with selective development impairment of these

neurons.

33

D. The encoding of pain versus itch

While my thesis work focuses mainly on the development of somatic sensory circuits, I

also contributed to a study on the neural basis of somatic sensory encoding, mainly on pain

versus itch. Pain and itch are distinct somatosensory sensations evoking distinct reflexes. While

pain can be elicited from anywhere in the body triggering a withdrawal reflex, itch can only be

elicited in the skin, evoking a scratching response (Ross S , 2011). The coding of pain versus itch

has been the subject of several different theories (Norrsell et al., 1999; Ma Q., 2010, Noordenbos

W., 1959 & 1987; Melzack and Wall., 1965; Wall PD., 1978). The specificity or the labeled line

theory argues that primary afferents that signal itch sensation are distinct from the ones that

signal pain. Several studies make a case for the labeled line theory. Studies in humans show the

Figure 4: a) Molecular diagram showing how Tlx3 antagonizes Lbx1 expression in order to promote glutamatergic differentiation . b) Segregation between glutamatergic and GABAergic populations and linkage between developmental ontogeny to physiological functions in the dorsal spinal cord.

Tlx3

Lbx1

Pax2

GABAergic

Lbx1+

Lbx1+Tlx3+

Lbx1+Pax2+

Vglut2+ Pax2+

dI3 dI5 dILB dI4 dI6 dILA

Grasping ? ?

Glutamatergic GABAergic

a) b)

VGlut2

Glutamatergic

34

existence of histamine-sensitive C fibers whose activation correlates with itch perception

(Schmelz et al., 1997; Namer et al., 2008). In mice, MrgprA3+ DRG neurons and GRPR+ spinal

neurons are required to sense itch, but not pain (Han et al., 2013; Sun et al., 2009). However,

itch-related neurons, such as MrgprA3+ DRG neurons, also respond to stimuli that normally

evoke pain, such as capsaicin and mustard oil that bind and activate TRPV1 and TRPA1,

respectively (Wilson et al., 2011; Shim et al., 2007; Sikand et al., 2009). Indeed, TRPV1 and

TRPA1 are essential for sensing both pain and itch. These findings appear to argue against the

strict definition of the labeled line encoding theory, and also raise the puzzling question as to

why in normal healthy human subjects intradermal injection of capsaicin or mustard oil only

evokes burning pain, but not itch, even though both pain and itch fibers will be concurrently

activated (Simone et al., 1989; LynnB., 1992, Shim and Oh., 2008; Imamachi et al., 2008).

To explain this puzzle, an alternative theory, called the selectivity hypothesis or the

population coding theory, was proposed by several investigators (Akiyama et al., 2009; Campero

et al., 2009; Ma Q., 2010). This theory on one hand supports the existence of specific neural

circuits/sensory labeled lines processing specific modalities, but also argues a cross interaction

among these sensory labeled lines, such as a dominant suppression of itch when pain fibers are

concurrently activated. Itch will be evoked only if itch-related sensory fibers are preferentially

activated.

Indeed, it has been long recognized that pain and itch counteract each other. The

sensation of itch can be relieved by a counter painful (or even non-painful) stimulus, such as

scratching, heat, cold, pinprick, capsaicin injection and electrical stimulation (Ikoma et al.,

2006). Clinically, the sensation of itch can be unmasked following pain reduction, such as

opioid-induced itch or pruritus (Davidson and Giesler 2010; Ikoma et al., 2006; Paus et al.,

2006). Indeed, pruritus is one of the most prevalent acute side effects of spinal or epidural usage

of opioids in patients who undergo pain treatment (Chaney et al., 1995; Hales et al., 1980). Itch

inhibition can be achieved even when the counter stimuli is applied to a few centimeters from the

affected area, which means that these counter stimuli do not need to act on the same primary

afferents responsible for signaling itch. This leads to the hypothesis that scratching and other

counter stimuli activate mechanically sensitive polymodal C and Aδ fibers that probably inhibit

itch though a central mechanism. This cross-inhibition between pain versus itch allows specific

35

sensation to be evoked even when two types of sensory fibers are concurrently activated, thereby

drawing a parallel to what happens in the visual system, in which inhibition between red and

green sensitive neurons improves color discrimination (Solomon and Lennie, 2007).

The nature of spinal inhibitory neurons involved with itch inhibition is beginning to be

understood. Electrophysiology studies showed that scratching inhibits histamine responsive

projection neurons in the dorsal horn of the spinal cord (Davidson et al., 2009), possibly via

GABAergic or glycinergic interneurons. Ross et al. then reported that a subpopulation of

inhibitory neurons in the dorsal horn of the spinal cord, whose development is dependent on the

basic helix loop helix transcription factor bHLHb5, have an essential role in blocking itch. Mice

lacking bHLHb5 exhibited excessive spontaneous scratching and self inflicted skin lesions (Ross

et al., 2010).

Despite these discoveries, the neural basis of pain-induced itch inhibition is still not fully

understood. In the last part of my thesis, I will describe my contribution to a study that reveals a

neural component in DRG neurons that is required for pain sensation and itch inhibition.

E. A summary of the questions addressed in my thesis

For somatic sensory neuron development, we want to address the following questions:

How are different types of primary and spinal sensory neurons specified and how are they

assembled into specific neural circuits or labeled lines? What are the developmental mechanisms

leading to polymodality of most sensory neurons? Do the transcription factors responsible for the

expression of ion channels and receptors also control the connectivity to central targets in the

spinal cord? For the coding of pain versus itch, what is the neural basis of pain-induced itch

inhibition? In order to answer these questions, I initiated my PhD at Professor Qiufu Ma’s lab,

who has had a longstanding interest in studying the assembly of the somatosensory system.

Specifically, I focused on a homeobox protein, T-cell leukemia 3, or Tlx3, that in Professor Ma’s

laboratory was initially discovered to have an essential role in controlling the development of

glutamatergic excitatory neurons in the spinal cord and in the hindbrain.

36

Recurring to the generation of specific conditional knockouts in the dorsal root ganglia and in the

superficial dorsal horn I aimed:

1. To determine if Tlx3 had a role in the development of a subset of the TrkA lineage

neurons in the dorsal root ganglia involved in nociception, pruriception and nociception.

2. To evaluate what roles Tlx3-dependent spinal excitatory neurons play in processing

somatic sensory information, particularly in pain and itch.

3. To evaluate if glutamate release from a specific population of nociceptors modulates the

sensations of itch and pain, including pain-induced inhibition of itch

37

II . Publications

38

Publication I

Development/Plasticity/Repair

Tlx3 and Runx1 Act in Combination to Coordinate theDevelopment of a Cohort of Nociceptors, Thermoceptors,and Pruriceptors

Claudia Lopes,1,2* Zijing Liu,1* Yi Xu,1* and Qiufu Ma1

1Dana-Farber Cancer Institute and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, and 2Laboratory of Molecular CellBiology, University of Porto, 4200-319 Porto, Portugal

Neurons in the mouse dorsal root ganglia (DRGs) are composed of a variety of sensory modalities, such as pain-related nociceptors,itch-related pruriceptors, and thermoceptors. All these neurons are derived from late-born neurons that are initially marked by theexpression of the nerve growth factor receptor TrkA. During perinatal and postnatal development, these TrkA lineage neurons areglobally segregated into Ret-expressing and TrkA-expressing subtypes, and start to express a variety of sensory receptors and ionchannels. The runt domain transcription factor Runx1 plays a pivotal role in controlling these developmental processes, but it remainsunclear how it works. Here we showed that the homeodomain transcription factor Tlx3, expressed broadly in DRG neurons, is required toestablish most Runx1-dependent phenotypes, including the segregation of TrkA-expressing versus Ret-expressing neurons and theexpression of a dozen of sensory channels and receptors implicated in sensing pain, itch and temperature. Expression of Runx1 and Tlx3is independent of each other at prenatal stages when they first establish the expression of these channels and receptors. Moreover,overexpression of Runx1 plus Tlx3 was able to induce ectopic expression of sensory channels and receptors. Collectively, these studiessuggest that genetically Tlx3 acts in combination with Runx1 to control the development of a cohort of nociceptors, thermoceptors, andpruriceptors in mice.

IntroductionNeurons in the DRGs involved in sensing pain, itch, and temper-ature are derived from embryonic neurons initially marked bythe expression of TrkA, the nerve growth factor receptor (Liu andMa, 2011; Lallemend and Ernfors, 2012). For simplicity, “TrkAlineage neurons” are used here to include all sensory neuronsinitially expressing TrkA. In the past decade, a number of tran-scription factors have been identified that control distinct aspectsof TrkA lineage neuron development (Liu and Ma, 2011; Lalle-mend and Ernfors, 2012). Genesis of TrkA lineage neurons isdependent mainly on the proneural protein Neurog1, but alsopartly on Neurog2 in caudal DRGs (Ma et al., 1998, 1999; Bachyet al., 2011). The homeodomain proteins Brn3a and Islet1 arecritical for making the transition from neurogenesis to neuronal

differentiation, and they also control sensory neuron identities(Eng et al., 2004; Sun et al., 2008; Lanier et al., 2009; Dykes et al.,2010, 2011; Zou et al., 2012). The zinfic protein KLF7 acts syner-gistically with Brn3a to control TrkA expression (Lei et al., 2006).The homeobox protein Cux2 might be involved with the devel-opment of the thinly myelinated A-� subset of TrkA lineage sen-sory neurons (Bachy et al., 2011).

The runt domain protein Runx1 also plays pivotal roles in con-trolling TrkA lineage neuron development (Liu and Ma, 2011).Runx1 is initially expressed in most newly born TrkA-expressingcells, and both Runx1 and TrkA expression then undergoesdynamic changes during perinatal and postnatal development(Liu and Ma, 2011; Lallemend and Ernfors, 2012). Runx1 is re-quired for proper development of �50% of those TrkA lineageneurons that are going to switch off TrkA and activate the Retreceptor tyrosine kinase (Chen et al., 2006; Yoshikawa et al.,2007). Conversely, the other 50% of TrkA lineage neurons retainTrkA and switch off Runx1, most of which become peptidergicneurons marked by the expression of the calcitonin gene-relatedpeptide (CGRP) and substance P (SP) (Chen et al., 2006). Asubset of Runx1-dependent neurons are also peptidergic (Liu andMa, 2011; Lallemend and Ernfors, 2012). In mice with a condi-tional knock-out of Runx1 in sensory precursors, Ret� sensoryneurons switch to become TrkA� neurons (Chen et al., 2006;Yoshikawa et al., 2007). Within Ret� sublineage neurons, Runx1is necessary for the expression of dozens of channels and recep-tors involved in sensing pain, itch, and/or temperature (Chen etal., 2006; Abdel Samad et al., 2010; Liu and Ma, 2011).

Received March 6, 2012; revised May 1, 2012; accepted May 28, 2012.Author contributions: C.L., Z.L., Y.X., and Q.M. designed research; C.L., Z.L., and Y.X. performed research; C.L., Z.L.,

Y.X., and Q.M. analyzed data; C.L., Z.L., and Q.M. wrote the paper.We thank Drs. Nancy Speck and Gary Gilliland for the Runx1 conditional knock-out mice, Rohini Kuner for the

Nav1.8-Cre mice, and David Rowitch for the Wnt1-Cre mice. We also thank Dr. Carmen Birchmeier, Tom Jessell, andLouis Reichardt for the Tlx3, Runx1, and TrkA antibodies, respectively. We also thank the Mouse Gene ManipulationFacility of the Children’s Hospital Intellectual and Developmental Disabilities Research Center (IDDRC) supported byNIH Grant P30-HD18655. The work done in the Ma lab was supported by NIH grants from NIDCR (1R01DE018025)and NINDS (P01NS047572).

*C.L., Z.L., and Y.X. contributed equally to this article.Z. Liu’s present address: Beijing Institute of Biotechnology, 27 Tai-Ping Road, Beijing 100850, China.Correspondence should be addressed to Qiufu Ma, Dana-Farber Cancer Institute and Department of Neurobiol-

ogy, Harvard Medical School, 450 Brookline Avenue, Boston, MA 02115. E-mail: Qiufu_Ma@dfci.harvard.edu.DOI:10.1523/JNEUROSCI.1109-12.2012

Copyright © 2012 the authors 0270-6474/12/329706-10$15.00/0

9706 • The Journal of Neuroscience, July 11, 2012 • 32(28):9706 –9715

However, it remains unclear how Runx1 coordinates suchdiverse phenotypes within the Ret� subset of TrkA lineage neu-rons. The homeodomain protein Tlx3 plays a pivotal role in con-trolling relay somatic sensory neuron development in the dorsalspinal cord and hindbrain (Qian et al., 2002; Cheng et al., 2004; Liet al., 2006; Xu et al., 2008). Here, we report that in DRG neurons,Tlx3 is required to establish most Runx1-dependent sensory phe-notypes, and Runx1 and Tlx3 operate at different developmentalstages to establish distinct sensory modalities.

Materials and MethodsAnimals. The generation and genotyping of Tlx3 complete null mice,Runx1 conditional knock-out mice, Wnt1-Cre mice, and Nav1.8-Cremice have been previously described (Danielian et al., 1998; Shirasawa etal., 2000; Qian et al., 2001; Agarwal et al., 2004; Growney et al., 2005;Chen et al., 2006). Male and female embryos and mice were equally usedfor the analyses. The morning the vaginal plugs were observed was con-sidered as embryonic day 0.5 (E0.5). All animal procedures are withinprotocols reviewed and approved by the Animal Care Committees at theDana Farber Cancer Institute, Harvard Medical School.

Generation of Tlx3 conditional null mice. Schematics in Figure 2 (seebelow) illustrate our strategy to generate mice carrying a conditional nullallele of Tlx3. The targeting vector was constructed partly by using therecombination system developed by Liu et al. (2003). The 5� LoxP DNAsequence (the recognition site for the Cre recombinase) was inserted intothe first intron at the site 0.135 kb 5� to the second exon; a EcoRV site wasadded 5� to this LoxP site, which was used for identification of embryonicstem cell clones with successful gene targeting (see Fig. 2). The FRT-Neo-FRT-LoxP positive selection cassette, which drives the expression of theaminoglycoside phosphotransferase that will confer G418 resistant, wasinserted into the second intron at 0.3 kb 3� to the second exon. This neocassette is flanked with two FRT sites, which can be recognized andcleaved by the Flipase recombinase (Flpe), and ends with the second LoxPsite at the 3� end. The 9.4 kb 5� recombination arm starts at the endoge-nous EcoR1 site at the 5� end and ends right next to the first Loxp site(0.235 kb 3� to the first exon). The 3.1kb 3� recombination arm startsright after the neo cassette (0.3 kb 3� to the second exon) and ends withthe endogenous Sal1 site (see Fig. 2). The DTA cassette, which drives theexpression of the Diphtheria Toxin A (DTA) for negative selection, isplaced 3� to the short recombination arm (see Fig. 2). Upon linearization,this targeting vector was electroporated into the J1 embryonic stem (ES)cell line (derived from 129Sv/J mouse strain), with G418 included in theculture medium for positive selection. Southern hybridization on genomicDNA was performed, using a 0.889 Sal1-Xhol1 genomic fragment (3� to theshort recombination arm; see Fig. 2) as the probe, which produced a 15.3 kbEcoRV fragment for the wild-type allele, and a 10.5 kb fragment for thetargeted allele (see Fig. 2). Greater than 80% of ES clones showed successfultargeting, and the targeted ES cells were injected into the blastocysts derivedfrom C57BL/6J females. Chimeric male mice were mated with C57BL/6Jfemales to generate heterozygous mice carrying the targeted allele. Thesemice were mated with the Flpe deleter mouse line (Rodríguez et al., 2000), toremove the neo cassette, leading to the creation of the mice carrying the Tlx3floxed allele, referred to as Tlx3F (see Fig. 2). Tlx3F mice were then matedwith Nav1.8-Cre mice to remove the second exon, leading to the creation ofthe null allele and generation of conditional knock-out mice (Tlx3F/F;Nav1.8Cre/�) (see Fig. 2). The genotyping for the conditional knock-out miceafter crossing Tlx3F/F mice with Nav1.8-Cre mice was performed with thefollowing set of primers: (1) for Nav1.8-Cre allele, 5-AGACTAATCGCCATCTTCCAGC-3 and 5�-TATCTCACGTACTGACGGTG-3�, and (2) forTlx3 wild-type and floxed allele, 5�-TGTTTCGCCTCCTTTGCTCG-3� and5�-GTTGGATGGAAGCAAAGATAG-3�, with the floxed allele showing alarger DNA band after gel electrophoresis.

In situ hybridization and immunostaining. In situ hybridization and theprobes used in this study (Mrgprd, Mrgpra3, Mrgprb4, Mrgprb5,TRPA1, TRPM8, TRPV1, Nav1.9, Ret, P2X3, Runx1, Tlx3) have beendescribed previously (Ma et al., 1999; Qian et al., 2001; Chen et al., 2006;Liu et al., 2008; Abdel Samad et al., 2010). Collection of E16.5 and post-natal day 0 (P0) embryos was done in ice-cold PBS, fixed overnight in 4%

paraformaldehyde in PBS (PFA-PBS), and saturated in 20% sucrose inPBS, also overnight at 4°C. For P30 mice, after perfusion with 4% PFA-PBS, lumbar and thoracic DRGs were dissected and continued to be fixedin 4% PFA-PBS for 2 h and saturated in 20% sucrose overnight at 4°C.For immunohistochemistry studies, the following antibodies were used:rabbit and guinea pig anti-Tlx3 (a gift from C. Birchmeier, Max DelbruckCenter for Molecular Medicine, Berlin, Germany), rabbit anti-Runx1 (agift from T. Jessell, Columbia University, New York, NY), P2X3 (1:1000,Neuromics), IB4-biotin (10 �g/ml, Sigma), rabbit anti-CGRP (1:500,Peninsula), and rabbit anti-TrkA (1:500, a gift from L. Reichardt, Uni-versity of California, San Francisco). Ret in situ hybridization combinedwith IB4 immunostaining was performed as previously described (AbdelSamad et al., 2010).

Cell counting. To quantify neurons expressing different molecularmarkers, L4 and L5 (or T12 when mentioned) DRGs were dissected from3 different mice, and these DRGs were sectioned into eight adjacent setswith 12 �m in thickness. Each set was used for in situ hybridization witha specific probe or for immunostaining with a specific antibody. For eachmolecular marker, at least three DRGs from different mice were in-cluded. Only cells showing clear nuclei were counted. Average and SEMwere calculated, and the difference between wild-type and mutant micewas subjected to a Student’s t test, with p � 0.05 considered significant.

Electroporation on spinal cord explants. Full-length mouse Runx1 andTlx3 cDNAs were subcloned into the replication-competent retroviralvector RCASBP (Morgan and Fekete, 1996), with resulting plasmids re-ferred to as RCASBP-Tlx3 and RCASBP-Runx1 and used for electropo-ration. The parental empty vector RCASBP was used for control. E12.5mouse embryonic spinal cords were dissected, and cut into 4-mm-longpieces along the rostrocaudal axis. The dorsal midline was cut to makeflat open-book explants as shown below in Figure 6. The explants wereplaced onto the filter membrane (Millipore catalog #AABP02500). Plas-mid (1 �l) was added onto the surface of each spinal explant, and theexplants were then electroporated with BTX #ECM 830 electroporator.Electroporation conditions were as follows: 15 V, 50 ms, 5 pluses. Forsingle plasmid electroporation, 1 �l of (2 �g/�l) of the plasmid was used.For Runx1 plus Tlx3 electroporation, 1 �g/�l of each plasmid was used.After electroporation, the explants were cultured on the floating filtermembranes, using the neural basal medium (Invitrogen 21103049) sup-plemented with the nerve growth factor NGF (Invitrogen 13257-019) at25 ng/ml, 1� Glutamax (Invitrogen 35050-061), and 1� B27 (Invitro-gen 17504). After 3 d of culture, the explants were fixed with 4% PFA-PBS and then processed for in situ hybridization.

ResultsBroad Tlx3 expression in TrkA lineage DRG neuronsTlx3 is expressed broadly in developing sensory neurons in DRGsat embryonic stages (Shirasawa et al., 2000; Qian et al., 2002), andits expression was also detected at adult stages (Fig. 1A). In lum-bar DRGs at P30, Tlx3 was expressed in a majority of small andmedium diameter neurons (Fig. 1A, arrows), but was excludedfrom a subset of large and small diameter neurons (Fig. 1A, ar-rowheads). The TrkA lineage neurons involved with pain, itch,and/or temperature sensations are divided into two subpopula-tions, the peptidergic neurons marked by persistent expression ofTrkA and the neuropeptide CGRP, and nonpeptidergic neuronsmarked by the expression of Ret and partly by the binding of theisolectin IB4, although a small subset of Ret� neurons is alsopeptidergic (Snider and McMahon, 1998; Bachy et al., 2011; Liuand Ma, 2011). In P30 lumbar DRGs, Tlx3 was detected in 78%(319/408) of TrkA� neurons, 76% (170/225) of CGRP� neu-rons, and in 92% (532/577) of IB4� neurons, albeit at variablelevels (Fig. 1B–D). Nearly all neurons that persistently expressedRunx1 also coexpressed Tlx3 (99%, 612/618) (Fig. 1E), andRunx1 was expressed in a subset of Tlx3� neurons (Fig. 1E).Thus, persistent Tlx3 expression is broadly associated with bothpeptidergic and nonpeptidergic neurons within the TrkA lineageneurons.

Lopes et al. • Tlx3 Controls Sensory Subtype Specification J. Neurosci., July 11, 2012 • 32(28):9706 –9715 • 9707

We have reported previously that in the dorsal spinal cord,Tlx3 and its related protein Tlx1 determine the excitatory overthe inhibitory neuronal cell fate, by activating VGLUT2, the ve-sicular transporter that packages the glutamate into excitatoryvesicles, and suppressing GABAergic neuron markers (Cheng etal., 2004). Mechanistically, Tlx3 acts to suppress the activity of thehomeobox protein Lbx1; in the genetic background lacking Lbx1,Tlx3 would be no longer required for VGLUT2 expression(Cheng et al., 2005). DRG neurons are glutamatergic (Yoshimuraand Jessell, 1990; Brumovsky et al., 2007; Lagerstrom et al., 2010;Liu et al., 2010). Consistent with a lack of Lbx1 expression inDRG neurons (Gross et al., 2002; Muller et al., 2002), Tlx3 andTlx1 are not involved in controlling glutamatergic differentiationin DRG neurons, as indicated by normal VGLUT2 expressionand no derepression of GABAergic neuron markers in mice lack-ing both Tlx3 and Tlx1 (data not shown).

Generation of Tlx3 conditional knock-out miceTlx3 conventional null mice die at birth (Shirasawa et al., 2000),which precludes their usage for a detailed analysis of Tlx3 func-tions in controlling sensory neuron development and matura-tion. To overcome this, we have generated mice carrying aconditional null allele of Tlx3, referred to as Tlx3F, in which thesecond exon encoding part of the homeobox domain was flankedwith two loxP sites and can thereby be removed by the Cre DNArecombinase (Fig. 2A–E). We next crossed Tlx3F mice withNav1.8-Cre mice, with the resulting conditional null mice re-

ferred to as Tlx3F/F;Nav1.8Cre/� (Fig. 2F,G). Nav1.8-Cre was ex-pressed selectively in 81% of total DRG neurons (Liu et al., 2010),including IB4� and CGRP� subsets of TrkA lineage neurons(Agarwal et al., 2004). Consistently, Tlx3 expression in DRGs wasmarkedly reduced in Tlx3F/F;Nav1.8Cre/� mice, but not com-pletely eliminated (Fig. 2G). The Tlx3F/F;Nav1.8Cre/� mice sur-vived to adulthood, allowing us to examine the roles of Tlx3 incontrolling sensory neuron development and maturation, as de-scribed below.

Impaired segregation of TrkA � versus Ret � sensory neuronsA key event in TrkA lineage neuron development is perinatal/postnatal segregation of these neurons into 1) CGRP� peptider-gic neurons that retain TrkA expression versus 2) IB4� neuronsthat switch off TrkA and activate Ret (Bennett et al., 1996; Mol-liver et al., 1997; Chen et al., 2006; Luo et al., 2007). We found thatin P30 lumbar DRGs of Tlx3F/F;Nav1.8Cre/� conditional knock-out mice, the levels of Ret expression in IB4� neurons were re-duced, but not fully lost (Fig. 3A, data not shown), with IB4�

neurons expressing high level Ret reduced from 69% (272/397) incontrol littermates (Tlx3F/F or Tlx3F/� mice that did not carryNav1.8-Cre) to 18% (64/365) in Tlx3F/F;Nav1.8Cre/� mice. Mean-while, TrkA expression failed to be extinguished in IB4� neurons(Fig. 3B), with IB4� cells expressing TrkA increased from 13%(56/435) in wild-type mice to 92% (532/577) in Tlx3F/F;Nav1.8Cre/� mice. Moreover, for those double-positive cells,TrkA expression levels in IB4� neurons were low in wild-typemice, but medium or high in Tlx3F/F;Nav1.8Cre/� mice (Fig. 3B,arrows). In contrast to the dramatic expansion of TrkA expres-sion in IB4� neurons, CGRP expression was, however, onlymodestly expanded (Fig. 3C), with percentages of IB4� neuronsexpressing CGRP increased from 7.7% (49/637) in wild-typemice to 22% (118/506) in Tlx3F/F;Nav1.8Cre/� mice. Thus, Tlx3 isrequired to elevate Ret expression and to switch off TrkA, eventhough most of these neurons (100 - 22 � 78%) retained thenonpeptidergic identity.

This change in molecular identities in IB4� neurons could befurther visualized in the dorsal spinal cord. In wild-type controlmice, CGRP� and IB4� sensory afferents project to distinct lam-inae, with 1) CGRP� fibers mainly in the lamina I and the outerlayer of lamina II (IIo), and 2) IB4� fibers in the inner layer oflamina II (IIi) (Fig. 3D) (Snider and McMahon, 1998). We foundthat lamina-specific innervations by CGRP� and IB4� fiberswere largely unchanged in Tlx3F/F;Nav1.8Cre/� mice (Fig. 3D).TrkA immunostaining, which labeled CGRP� fibers in laminae Iand IIo in control mice (Fig. 3E), was expanded ventrally to co-label IB4� fibers in lamina IIi in Tlx3F/F;Nav1.8Cre/� mice (Fig.3E), thereby confirming the failed extinguishment of TrkA inIB4� neurons. The vesicular glutamate transporter VGLUT1 wasenriched in mechanoreceptors that innervate in laminae III-V,ventral to IB4� fibers in control mice (Fig. 3F), and the sameinnervation pattern was observed in Tlx3F/F;Nav1.8Cre/� mice(Fig. 3F), further suggesting that primary afferent innervations inthe dorsal spinal cord is largely unchanged in Tlx3F/F;Nav1.8Cre/�

mice.

Loss of Runx1-dependent ion channels and sensory receptorsin Tlx3F/F;Nav1.8Cre/� miceThe impaired segregation of TrkA� versus Ret� neurons inTlx3F/F;Nav1.8Cre/� mice is reminiscent of the mutant phenotypeobserved in Runx1 conditional knock-out mice (Chen et al.,2006; Yoshikawa et al., 2007). Runx1 is additionally required forthe expression of dozens of sensory channels and receptors (Chen

Figure 1. Broad Tlx3 expression in small and medium diameter DRG neurons. A, Tlx3 immu-nostaining on a lumbar DRG section of a P30 wild-type mouse. Note detectable Tlx3 expressionin small and medium diameter neurons (small and large arrows, respectively), but not in somelarge and small neurons (large and small arrowheads, respectively). B–E, Double immunostain-ing of Tlx3 (green) with indicated markers (red). Arrows indicate double-positive cells. Arrow-heads in B and C indicate neurons expressing TrkA or CGRP, but not Tlx3, and the arrowhead inE indicates cells expressing Tlx3 but not Runx1. Scale bars, 50 �m.

9708 • J. Neurosci., July 11, 2012 • 32(28):9706 –9715 Lopes et al. • Tlx3 Controls Sensory Subtype Specification

et al., 2006; Abdel Samad et al., 2010). To determine whether Tlx3is involved in regulating sensory channels/receptors, we per-formed a series of in situ hybridization on sections through lum-bar or thoracic DRGs from P30 Tlx3F/F;Nav1.8Cre/� and controlmice. Remarkably, expression of virtually every Runx1-dependent gene examined thus far was either eliminated ormarkedly reduced in Tlx3F/F;Nav1.8Cre/� mice (Fig. 4). As de-scribed below, these genes encode sensory channels and receptorsinvolved with pain, itch and/or temperature sensation.

Mrgprd, a mas-related G-protein coupled receptor (GPCR), isa marker for a large group of polymodal nociceptors that repre-sent 30% of total DRG neurons, innervate exclusively the skin

epidermis, and are involved with mechan-ical pain sensation (Zylka et al., 2005;Imamachi et al., 2009; Rau et al., 2009). InP30 Tlx3F/F;Nav1.8Cre/� mice, Mrgprd ex-pression was greatly reduced, but notcompletely eliminated (Fig. 4A). As de-scribed below, a complete loss of Mrgprdexpression was observed in Tlx3 completenull mice. Mrgprd� neurons express a setof other Runx1-dependent genes, includ-ing those encoding the Mrgprd-relatedMrgprb5 and the sodium channel Nav1.9,respectively (Abdel Samad et al., 2010). Ex-pression of Mrgprb5, whose mRNA wassomehow enriched in the nuclei, was elimi-nated (Fig. 4A), and Nav1.9 was largelyeliminated in DRGs of Tlx3F/F;Nav1.8Cre/�

mice (Fig. 4A). The ATP-gated channelP2X3 is expressed in both IB4� and IB4�

neurons, and its expression in IB4� neu-rons is selectively dependent on Runx1 (Ab-del Samad et al., 2010). We found that P2X3expression in lumbar DRG neurons was alsoreduced in Tlx3F/F;Nav1.8Cre/� mice, withIB4� neurons coexpressing P2X3 reducedfrom 50% (346/689) in control mice to 17%(96/566) in Tlx3F/F;Nav1.8Cre/� mice (datanot shown). Thus, Tlx3 is required to estab-lish a set of Runx1-dependent molecularidentities in Mrgprd� polymodal nocicep-tors, including Ret, Mrgprd, Mrgprb5,P2X3, and Nav1.9.

Another Mrgpr class GPCR, Mrgpra3,mediates histamine-independent itch(Liu et al., 2009). Its expression in lumbarDRGs was eliminated in Tlx3F/F;Nav1.8Cre/� mice (Fig. 4B). Expression ofMrgprb4, which marks a group of sensoryneurons that innervate the hairy skin andare hypothesized to mediate pleasanttouch (Liu et al., 2007), was also elimi-nated in P30 Tlx3F/F;Nav1.8Cre/� mice(Fig. 4C). Thus, Tlx3 is necessary for theexpression of most members of Mrgprclass GPCRs, as Runx1 does (Chen et al.,2006; Liu et al., 2008).

The transient receptor potential (TRP)channels are involved with pain, itch, andthermal sensations (Basbaum et al., 2009;Patel and Dong, 2011), whose expressionis partially dependent on Runx1 (Chen et

al., 2006). TRPA1 is involved in sensing chemical pain (Basbaumet al., 2009) and histamine-independent itch (Wilson et al.,2011). In wild-type mice, high level TRPA1 expression(TRPA1 high) was enriched in cervical and thoracic DRGs (datanot shown), whereas a lower level of TRPA1 expression was de-tected in lumbar DRGs (Fig. 4D) (Abdel Samad et al., 2010). Wefound that no TRPA1 expression in lumbar DRGs was detected inP30 Tlx3F/F;Nav1.8Cre/� mice (Fig. 4D), as the situation seen inRunx1 mutant mice (Abdel Samad et al., 2010). TRPA1 expres-sion in thoracic DRGs was also greatly reduced, but not com-pletely eliminated (data not shown). Neurons expressingTRPM8, a cold-sensing receptor (Basbaum et al., 2009), was re-

Figure 2. Generation of Tlx3 conditional null mice. A, The wild-type Tlx3 genomic allele, with solid boxes indicating codingexons (1, 2, and 3). E, EcoR1; RV, EcoRV; S, Sal1. B, The targeting vector, with 9.4 kb long and 3.1 kb short recombination arms. Thefirst LoxP site (left triangle, the recognition site for the Cre recombinase) and an exogenous EcoRV (RV) site were introduced into thefirst intron (between exons 1 and 2). Neo represents the Neo expression cassette for the G418 drug-resistant selection (seeMaterials and Methods), flanked with two FRT sequences (recognition sites for the Flipase recombinase) and the second LoxP site,as indicated in the schematics. DTA, The expression cassette that drives the expression of DTA for negative section. C, The targetedTlx3 allele after successful homologous recombination. D, Genomic Southern hybridization revealing three of four selected embry-onic stem cell clones undergoing successful targeting. Using the 3� probe indicated in C, EcoRV digestion generated a 15.3 kbgenomic fragment for the wild-type allele (see A), and a 10.5 kb fragment for the targeted allele (see C). E, The floxed Tlx3 allele,after removal of the neo cassette by Flipase-mediated DNA recombination (see Materials and Methods). F, The Tlx3 conditional nullallele upon removal of the second coding exon through Cre-mediated DNA recombination. G, Immunostaining showing a loss ofTlx3 expression in most P30 lumbar DRGs in Tlx3 conditional knock-out mice, or Tlx3F/F;Nav1.8Cre/� mice (arrowhead), in compar-ison with that in control littermates (left, arrow). Only few neurons in Tlx3F/F;Nav1.8Cre/� mice retained Tlx3 expression (right,arrow). Scale bars, 50 �m.

Lopes et al. • Tlx3 Controls Sensory Subtype Specification J. Neurosci., July 11, 2012 • 32(28):9706 –9715 • 9709

duced by 45% (Fig. 4D), from 74 � 3.3 per set of DRG sections incontrol mice to 41 � 5.4 in Tlx3F/F;Nav1.8Cre/� mice at P30 (p �0.005). As described below, the reduction of TRPM8 expressionwas more complete in Tlx3 complete null mice.

TRPV1 is a polymodal receptor that senses warm, heat, itch,acid, and other chemicals (Woolf and Ma, 2007; Shim and Oh,2008; Basbaum et al., 2009). Approximately 10% of TRPV1�

neurons express extremely high levels of TRPV1 (TRPV1 high)(Chen et al., 2006; Abdel Samad et al., 2010), and these neuronsselectively innervate the skin epidermis and sense warm and mildheat (Kiasalari et al., 2010). Runx1 is required to establishTRPV1 high expression, but dispensable for low levels of TRPV1expression (Chen et al., 2006). Maintenance of a portion ofTRPV1 high expression is, however, independent of Runx1, as in-dicated by a partial loss of TRPV1 high neurons in late Runx1conditional knock-out mice using Nav1.8-Cre that removedRunx1 around E17 (Abdel Samad et al., 2010). In Tlx3F/F;

Nav1.8Cre/� conditional knock-out mice using the same Nav1.8-Cre, we still observed TRPV1 high neurons (Fig. 4D, arrow),although it remains unclear whether the number was reduced. Asdescribed below, TRPV1 high neurons were, however, nearly com-pletely eliminated in Tlx3 complete null mice. Low or mediumlevels of TRPV1 expression were unaffected in either Tlx3F/F;Nav1.8Cre/� mice at P30 (Fig. 4D, arrowhead) or Tlx3 completenull mice at P0 (see below), as the case seen in early and lateRunx1 knock-out mice (Chen et al., 2006; Abdel Samad et al.,2010).

In Figure 3, we have shown that Tlx3 does not have a positiverole in controlling the expression of two Runx1-independent (oractually Runx1-suppressed) peptidergic neuron markers, TrkAand CGRP. Expression of other Runx1-independent genes wasalso unaffected (if not increased) in Tlx3F/F;Nav1.8Cre/� mice,including DRASIC encoding an acid sensing channel and Tac1(the preprotachykinin 1 gene) encoding the neuropeptide SP (Fig.4E). All together, Tlx3 is required selectively to establish Runx1-dependent molecular phenotypes, despite of its broad expressionin TrkA lineage neurons.

Tlx3 and Runx1 are independently regulatedTo gain insight into how Tlx3 and Runx1 establish sensory phe-notypes, we next analyzed and compared Tlx3 and Runx1 mutantmice at E16.5 or in newly born mice, when the expression of a fewTlx3- and Runx1-dependent receptors has just been establishedin wild-type mice (Chen et al., 2006). For this analysis, the Tlx3complete null mice (Shirasawa et al., 2000) were used such thatthe Tlx3 activity was eliminated from the very beginning. Mean-while, mice with a conditional knock-out of Runx1 in sensoryprecursor using Wnt1-Cre (Runx1F/F;Wnt1Cre/�) were used for acomparison. We had previously reported that expression of Mrg-prd and TRPM8 is eliminated in Runx1F/F;Wnt1Cre/� mice atE16.5 (Chen et al., 2006). Here we found that Mrgprd expressionwas also eliminated in Tlx3 complete null mice at E16.5 (Fig. 5A).Expression of TRPM8 was, however, still detected at E16.5, butgreatly reduced at P0 in Tlx3 null DRGs, suggesting that Tlx3 isrequired to maintain, but not to initiate, TRPM8 expression. Itshould be noted that neurons with weak expression of TRPM8were still observed in Tlx3 null DRGs (Fig. 5A, arrow), indicatingthat at least a portion of TRPM8-expressing neurons survived,which in turn suggests a requirement of Tlx3 for elevated TRPM8expression. For TRPV1, TRPV1 high expression was eliminated inTlx3 null mice (Fig. 5A, arrows), whereas low and medium levelsof TRPV1 expression were unaffected (Fig. 5A, arrowheads), asthe situation seen in early Runx1 knock-out mice (Chen et al.,2006; data not shown).

Importantly, expression of Runx1 itself was grossly unaffectedin Tlx3 null mice at E16.5 (Fig. 5B) or at P0 (data not shown).Conversely, Tlx3 expression was unchanged in Runx1F/F;Wnt1-Cre mice at P0 (Fig. 5C). Thus, during the period when both Tlx3and Runx1 are required to establish Mrgprd and TRPV1 high ex-pression, expression of Tlx3 and Runx1 appears to be indepen-dently regulated, suggesting that genetically, these twotranscription factors might act in combination to control the expres-sion of sensory channels and receptors, although their action modescan be different (see below, Discussion).

Overexpression of Tlx3 and Runx1 was able to inducesensory channels/receptorsWe next asked whether overexpression of Tlx3 and Runx1,singly or in combination, was able to activate sensory channels

Figure 3. Impaired segregation of Ret � versus TrkA � neurons in Tlx3F/F;Nav1.8Cre/� mice.A–C, A combination of IB4 binding (red) with Ret mRNA (A, green) detected by in situ hybrid-ization, or the TrkA protein (B, green), or the CGRP peptide (C, green) detected by immunostain-ing on P30 lumbar DRG sections. Arrows in A indicate high-level Ret expression in both panels,and the arrowhead indicates reduced Ret expression in IB4 � neurons in Tlx3F/F;Nav1.8Cre/�

mice. In B, the arrow and arrowhead in the left panel indicate low and nondetectable TrkAexpression in IB4 � neurons in control mice, and the arrow in the right panel indicates elevatedTrkA expression in Tlx3F/F;Nav1.8Cre/� mice. In C, arrows and arrowheads indicate neurons withdetectable and nondetectable CGRP expression, respectively. D–F, A combination of IB4 bind-ing (red) with immunostaining against CGRP, TrkA, or VGLUT1 (green) on transverse sectionsthrough P30 spinal cords. I/IIo, Lamina I and the outer layer of lamina II; IIi, inner layer of laminaII. Scale bars, 50 �m in length.

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and receptors in heterologous systems. To do this, we per-formed gain-of-function analyses, using electroporation ontothe ventricular zone of the “open-book” explants of the E12.5mouse spinal cord (Fig. 6 A). After electroporation, theexplants were cultured for 3 d on floating membranes withneural basal medium containing the nerve growth factor. Elec-troporation of the Tlx3 or Runx1 expression plasmid led to arobust expression of exogenous Tlx3 (Fig. 6 B) or Runx1 (datanot shown) on the surface of the explants. We then found thatelectroporation of the control vector RCASBP was unable toinduce any of molecules examined (Fig. 6C). Electroporationof the Tlx3 expression vector alone was able to induce Mrg-prA3, but not Mrgprd, TRPM8, P2X3, Ret or Nav1.9. Electro-poration of the Runx1 expression vector was able to induceMrgprA3, Mrgprd, TRPM8, P2X3 and Ret, albeit at variablelevels, but unable to induce Nav1.9 (Fig. 6C). Electroporationof Tlx3 plus Runx1 resulted in robust induction for all thesesix molecules. Thus, when expressed at exceedingly high levelsfollowing electroporation, exogenous Runx1 is able to inducea subset of sensory channels and receptors, but a combinationof Runx1 and Tlx3 is generally more efficient in doing so.However, it needs to be pointed out that when expressed at thephysiological level or in the context of developing DRG neu-rons, both Runx1 and Tlx3 are needed to establish most sen-sory channels and receptors, as indicated by the loss orreduced expression of these molecules in Runx1 or Tlx3 defi-cient mice.

Furthermore, in the absence of the nerve growth factor in thecultured medium, Tlx3 plus Runx1 was unable to induce any of

these ion channels and receptors (data not shown), consistentwith the reported requirement of the TrkA signaling for the es-tablishment of most Runx1-dependent genes (Luo et al., 2007).

DiscussionTlx3 controls proper segregation of Ret � versus TrkA �

subpopulations of somatic sensory neuronsThe homeodomain protein Tlx3 is broadly expressed in somaticsensory neurons, including both Runx1-dependent Ret� non-peptidergic neurons (a majority of them can be labeled by IB4),and Runx1-independent peptidergic neurons marked by the ex-pression of TrkA and CGRP (Fig. 1). Both populations are de-rived from embryonic TrkA-expressing neurons, collectivelyreferred to as TrkA lineage neurons. Despite of the broad expres-sion, the genetic data presented here suggest that Tlx3 appears tobe required selectively for the development of the Runx1-dependent Ret� subset of TrkA lineage neurons.

First, during segregation of the TrkA lineage neurons intoRet�;IB4� versus TrkA�;CGRP� subtypes, both Runx1 andTlx3 are required to drive a high level of Ret expression and toswitch off TrkA, with a knock-out of either Tlx3 or Runx1 leadingto a concurrent reduction of Ret and an expansion of TrkA inIB4� neurons. It should, however, be noted that Tlx3 is onlypartially involved in controlling this segregation process. Whilein Runx1 knock-out mice CGRP is dramatically derepressed inIB4� neurons (Chen et al., 2006; Abdel Samad et al., 2010), onlya modest CGRP derepression occurs in Tlx3 mutant mice (Fig. 3).Furthermore, Runx1 is required for IB4� neurons to innervatelamina IIi in the dorsal spinal cord; in both early (using Wnt1-

Figure 4. Loss of expression of sensory receptors and ion channels in Tlx3F/F;Nav1.8Cre/� mice compared with littermate control. A–E, In situ hybridization with indicated probes on sectionsthrough lumbar DRGs (A, B, D, E) or thoracic DRGs (C), with the exception of TRPV1 in D, which was detected by immunostaining. Arrow in D indicates extremely high-level TRPV1 expression, andarrowheads indicate low or medium levels of expression. Scale bars, 50 �m in length.

Lopes et al. • Tlx3 Controls Sensory Subtype Specification J. Neurosci., July 11, 2012 • 32(28):9706 –9715 • 9711

Cre) and late (using Nav1.8-Cre) Runx1 knock-out mice, thesefibers switch to innervate the more superficial laminae (Chen etal., 2006; Abdel Samad et al., 2010). In contrast, no obviousswitch on lamina-specific innervations was observed in Tlx3 con-ditional knock-out mice (Fig. 3). Thus, Runx1 uses both Tlx3-dependent and Tlx3-independent pathways to control molecularand anatomical segregation of two TrkA lineage sensory neuronsubtypes, Ret�;IB4� versus TrkA�;CGRP�.

Second, Tlx3 is required for the expression of virtually allknown Runx1-dependent sensory receptors and ion channelsthat are enriched in Ret� neurons, such as Ret, the entire Mrgprfamily of GPCRs, P2X3, and Nav1.9. In contrast, expression of aset of Runx1-independent genes that are enriched in TrkA� pep-tidergic neurons, including those encoding TrkA, CGRP, and SP,is either expanded or unchanged in Tlx3 mutants. Notably, Tlx3and Runx1 appear to be independently regulated at perinatal andneonatal stages when they first establish the expression of a set ofsensory channels and receptors, and overexpression of Runx1

plus Tlx3 is sufficient to induce robust ectopic expression of thesemolecules. Collectively, these observations suggest that Runx1acts in combination with Tlx3 to control the molecular identitiesof the Ret� subset of TrkA lineage neurons.

Two other broadly distributed homeobox proteins, Brn3a andIslet1, also play pivotal roles in controlling sensory neuron devel-opment (Huang et al., 1999; Ma et al., 2003; Eng et al., 2004; Lei etal., 2006; Sun et al., 2008; Lanier et al., 2009; Dykes et al., 2010,2011; Zou et al., 2012). Both Brn3a and Islet1 are necessary for theexpression of a set of Runx1/Tlx3-dependent sensory channelsand receptors, and they act partly by controlling Runx1 expres-sion (Sun et al., 2008; Dykes et al., 2010). However, Brn3a andRunx1 can have opposing activities. During segregation of Ret�

versus TrkA� neurons, Brn3a acts to promote TrkA and suppressRet (Ma et al., 2003; Lei et al., 2006; Zou et al., 2012), exactlyopposite to what Runx1 and Tlx3 do: activating Ret and suppress-ing TrkA (Chen et al., 2006; Yoshikawa et al., 2007). How exactlyBrn3a positively and negatively interfaces with Runx1 (and Tlx3)in establishing distinct aspects of sensory neuron identities re-mains to be investigated.

Distinct control modes used by Runx1 and Tlx3 inestablishing distinct somatic sensory modalitiesOne key question in studying sensory neuron development is tounderstand how distinct sensory modalities are specified. Neu-rons expressing Tlx3/Runx1-dependent channels and receptorsare composed of multiple sensory modalities, including 1) pain-related nociceptors (such as Mrgprd� polymodal nociceptors formechanical pain and TRPA1� nociceptors for sensing noxiouschemical and possibly cold pain), 2) thermoceptors (TRPM8�

cold-sensing neurons and TRPV1 high warm/heat-sensing neu-rons), and 3) pruriceptors (MrgprA3� itch-sensing neurons)(Basbaum et al., 2009; Kiasalari et al., 2010; Patel and Dong,2011). Interestingly, Runx1 and Tlx3 appear to use distinct actionmodes in establishing distinct sensory modalities.

First, Runx1 and Tlx3 operate at different times in controllingthe development of Mrgpra3� itch-sensing pruriceptors. Expres-sion of Mrgpra3 was absent in early Runx1 conditional knock-out, in which Runx1 was removed in sensory precursors by usingWnt1-Cre mice (Chen et al., 2006). However, when Runx1 wasremoved at a later stage (around E17) by using the Nav1.8-Cremice, expression of Mrgpra3 was no longer affected (Abdel Sa-mad et al., 2010). In fact, Runx1 makes a switch from acting as anactivator to a repressor in regulating Mrgpra3 expression (in ge-netic terms); as a result, Mrgpra3 expression can only be sus-tained in neurons with transient Runx1 expression (Liu et al.,2008). In contrast, Mrgpra3 expression is still eliminated in Tlx3CKO mice using the same Nav1.8-Cre, suggesting that Tlx3 ac-tivity operates beyond E17 to control the development of thesepruriceptors (Fig. 4). We propose the following temporal controlmodel: Runx1 operates before E17 to establish a competent statefor subsequent Tlx3-mediated activation of Mrgpra3 at perinataland neonatal stages. This control mechanism is analogous to thesequential involvement of Runx1 and Pax5 in controlling mb-1expression in the developing B cells of the immune system (Maieret al., 2004). It also suggests that when two transcription factorsgenetically act in combination, they do not have to form a proteincomplex, but could also operate through sequential events.

Second, the development of TRPM8� cold-sensing thermo-ceptors is also subjected to a complex temporal control. Runx1,but not Tlx3, is required to initiate TRPM8 expression at E16.5,and Tlx3 activity is required to maintain elevated TRPM8 expres-sion after E16.5. Surprisingly, while TRPM8 expression levels are

Figure 5. Changes in sensory receptor expression in Tlx3 complete null mice at embryonic orneonatal stages and independent regulation of Tlx3 versus Runx1. A, B, In situ hybridizationwith indicated probes on sections through E16.5 or P0 lumbar DRGs of wild-type control or Tlx3null mice. For TRPM8 panels, the small arrow indicates neurons with weak TRPM8 expression inTlx3 null mice. For TRPV1 panels, arrows indicate neurons with extremely high levels ofTRPV1 expression in the control DRGs, and such neurons are absent in the Tlx3 null DRGs.Arrowheads indicate neurons with medium or low levels of TRPV1 expression. C, Un-changed Tlx3 expression in lumbar DRGs of P0 control mice versus Runx1 conditionalknock-out mice (Runx1F/F;Wnt1Cre/�). Scale bars, 50 �m.

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greatly reduced in Tlx3 complete null mice at P0, strong TRPM8expression is observed in a conditional knock-out of Tlx3 usingNav1.8-Cre (removing Tlx3 around E17), even though the num-ber of neurons is reduced. This finding suggests that persistentpresence of Tlx3 per se is not required to maintain TRPM8 ex-pression in a subset of TRPM8� sensory neurons; rather Tlx3activity before E17 is required to establish a competent state forother factors to maintain TRPM8 expression at later stages.TRPM8 expression is also absent in Brn3a null mice and partiallylost in Islet1 conditional knock-out mice (Sun et al., 2008), andfurther studies are needed to determine how exactly Runx1, Tlx3,Brn3a, and Islet1 establish and maintain TRPM8 expression.

Third, TRPV1 high neurons have been recently shown to sensewarm and mild heat, but not noxious heat (Kiasalari et al., 2010).TRPV1 high neurons are absent in both Tlx3 complete null mice

(Fig. 5) and early Runx1 knock-out mice using Wnt1-Cre (Chenet al., 2006), but a portion of them is still observed in either lateTlx3 (Fig. 4) or late Runx1 (Abdel Samad et al., 2010) conditionalknock-outs using the same Nav1.8-Cre. Thus, early Runx1 andTlx3 activity is necessary for proper development of TRPV1 high

neurons, but neither Runx1 nor Tlx3 is required to maintain aportion of these thermoceptors. Islet1 appears to play a moreprominent role in controlling TRPV1 expression, as suggested bythe apparent loss of both TRPV1 high and TRPV1 low expression inIslet1 deficient mice (Sun et al., 2008), but it remains unknownhow exactly Islet1 interfaces with Runx1 and Tlx3 in controllingTRPV1 high expression.

Finally, Runx1 and Tlx3 exhibit similar temporal activities incontrolling the development of a large group of polymodal noci-ceptors marked by the coexpression of Mrgprd, Mrgprb5,

Figure 6. Ectopic induction of sensory channels and receptors by Tlx3 and/or Runx1. A, Schematics showing electroporation and culture of embryonic (E12.5) spinal cord explants. f.p., Floor plate;� and �, direction of electric current during electroporation; floating m., filter membrane floating on the surface of the cultured medium; expl., spinal cord explant. B, Electroporation of theRCASBP-Tlx3 expression plasmid (Tlx3 under the line), but not the control RCASBP plasmid (Control), led to robust expression of exogenous Tlx3 mRNA (arrow) detected by in situ hybridization onthe surface of the explant (after 3 d culture). With a short period of color development following in situ hybridization, endogenous Tlx3 mRNA was only weakly detected (arrowhead). C, Mrgpra3,Mrgprd, TRPM8, P2X3, Ret, and Nav1.9 mRNAs were detected by in situ hybridization following electroporation on the explants with the control plasmid (Control), or the plasmid expressing Tlx3(Tlx3) or Runx1 (Runx1), or mixed plasmids expressing both Tlx3 and Runx1 (Tlx3 � Runx1).

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Nav1.9, and P2X3. The development of these neurons is impairedin mice following a conditional knock-out of either Runx1 (AbdelSamad et al., 2010) or Tlx3 (Fig. 4) by using the same Nav1.8-Cremice, suggesting that both Runx1 and Tlx3 operate beyond E17to establish and/or maintain the expression of this set of genes.Collectively, our studies suggest that Tlx3 and Runx1 use distinctaction modes to control the development of distinct somatic sen-sory modalities within the Ret� subset of TrkA lineage neurons.Future studies will be directed to determine how such dynamicRunx1 and Tlx3 activities are regulated during development.

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39

Publication II

Development/Plasticity/Repair

Ontogeny of Excitatory Spinal Neurons Processing DistinctSomatic Sensory Modalities

Yi Xu,1* Claudia Lopes,1* Hagen Wende,2 Zhen Guo,3 Leping Cheng,3 Carmen Birchmeier,2 and Qiufu Ma1

1Dana-Farber Cancer Institute and Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, 2Department of Neuroscience,Max Delbruck Center, 13125 Berlin, Germany, and 3Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai 200031, China

Spatial and temporal cues govern the genesis of a diverse array of neurons located in the dorsal spinal cord, including dI1-dI6, dILA , anddILB subtypes, but their physiological functions are poorly understood. Here we generated a new line of conditional knock-out (CKO)mice, in which the homeobox gene Tlx3 was removed in dI5 and dILB cells. In these CKO mice, development of a subset of excitatoryneurons located in laminae I and II was impaired, including itch-related GRPR-expressing neurons, PKC�-expressing neurons, andneurons expressing three neuropeptide genes: somatostatin, preprotachykinin 1, and the gastrin-releasing peptide. These CKO micedisplayed marked deficits in generating nocifensive motor behaviors evoked by a range of pain-related or itch-related stimuli. Themutants also failed to exhibit escape response evoked by dynamic mechanical stimuli but retained the ability to sense innocuous coolingand/or warm. Thus, our studies provide new insight into the ontogeny of spinal neurons processing distinct sensory modalities.

IntroductionThe dorsal horn of the spinal cord processes diverse somatic sen-sory information, including pain, itch, touch, cold, and warm(Craig, 2003; Todd, 2010). Early electrophysiological and mor-phological studies suggest the existence of modality selectivespinal neurons (Christensen and Perl, 1970; Han et al., 1998),which is further supported by recent cell ablation and behav-ioral studies (Sun et al., 2009; Mishra et al., 2012; Mishra andHoon, 2013). For example, neurons expressing the gastrin-releasing peptide receptor (GRPR) or the natriuretic polypep-tide b receptor (Npra) are required to sense itch, but not pain(Sun et al., 2009; Mishra and Hoon, 2013). However, howmodality-selective spinal neurons emerge during develop-ment is still poorly understood.

Early during development, eight groups of molecularly dis-tinct dorsal horn neurons have been identified, namely, dI1-dI6,dILA, and dILB (Gross et al., 2002; Muller et al., 2002; Helms andJohnson, 2003) (see Fig. 1A). Based on differential expression ofthe homeodomain protein Lbx1, they are divided into (1) Lbx1-negative class A excitatory neurons (dI1–3), which are derived

from progenitors expressing the transcription factor Olig3,and (2) Lbx1-expressing class B neurons, derived from Olig3-negative progenitors (see Fig. 1A) (Gross et al., 2002; Muller et al.,2002, 2005). Class B neurons are further divided into glutamater-gic excitatory neurons (dI5 and dILB) marked by the expressionof the homeobox proteins Lmx1b and Tlx3, and GABAergic/glycinergic inhibitory neurons (dI4, dI6, and dILA) marked by theexpression of the homeobox proteins Pax2 and Lhx2/9 (Gross etal., 2002; Muller et al., 2002; Qian et al., 2002; Helms and John-son, 2003; Cheng et al., 2004, 2005; Glasgow et al., 2005; Rebelo etal., 2010).

Previously, we and others reported that the Tlx3 homeoboxgene is necessary for proper development of dI3, dI5, and dILB

excitatory neurons in the dorsal spinal cord, including specifica-tion of the glutamatergic and peptidergic transmitter phenotypes(Qian et al., 2002; Cheng et al., 2004; Xu et al., 2008; Guo et al.,2012). A recent study shows that class A dI3 neurons are involvedwith sensory motor coordination, such as hand grasp (Bui et al.,2013). The focus of this study, however, is the physiological func-tions of class B dI5 and dILB neurons, which are still unknown.

Genetic fate mapping shows that the majority of Tlx3 lineageneurons are enriched in superficial laminae (I-III), although alsoscattered throughout the ventral laminae (Xu et al., 2008). Be-cause dI3 neurons are enriched in the deep dorsal horn (Helmsand Johnson, 2003; Bui et al., 2013), it can be certain that thoseTlx3 lineage neurons located in the superficial laminae must bederived from dI5 and dILB neurons. Superficial laminae normallyreceive inputs from primary sensory afferents that transmitpain-, itch-, and temperature-related sensory information(Todd, 2010). To determine to what degree dI5 and dILB neuronsprocess these types of somatic sensory information, here we usedLbx1-Cre mice (Sieber et al., 2007) to selectively remove Tlx3 inthese neurons. Subsequent histochemical and behavioral analy-

Received Nov. 29, 2012; revised Aug. 3, 2013; accepted Aug. 7, 2013.Author contributions: Y.X., C.L., Z.G., L.C., and Q.M. designed research; Y.X., C.L., Z.G., L.C., and Q.M. performed

research; H.W. and C.B. contributed unpublished reagents/analytic tools; Y.X., C.L., Z.G., L.C., and Q.M. analyzeddata; Y.X., C.L., L.C., C.B., and Q.M. wrote the paper.

The work done in the Q.M. laboratory was supported by the National Institutes of Health, National Institute ofNeurological Disorders and Stroke Grant R01NS047710. We thank Dr. Silvia Arber for the Tau-loxp-STOP-lox-mGFP-IRES-NLS-LacZ reporter mice; Dr. Jean-Francois Brunet for the Phox2a antibody; and Drs. Clifford Woolf, WendyKnowlton, Fu-Chia Yang, and Bo Duan for critical discussions and comments.

The authors declare no competing financial interests.*Y.X. and C.L. contributed equally to this work.Correspondence should be addressed to Dr. Qiufu Ma, Dana-Farber Cancer Institute and Department of Neuro-

biology, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115. E-mail: Qiufu_Ma@dfci.harvard.edu.DOI:10.1523/JNEUROSCI.5512-12.2013

Copyright © 2013 the authors 0270-6474/13/3314738-11$15.00/0

14738 • The Journal of Neuroscience, September 11, 2013 • 33(37):14738 –14748

ses provide new insight into the ontogeny of spinal neurons pro-cessing distinct types of somatic sensory modalities.

Materials and MethodsAnimals. The generation of mice carrying the Tlx3 conditional null allele(Tlx3F/�), the Lbx1cre mouse line, the Tlx3Cre mice, and the Tau-loxp-STOP-lox-mGFP-IRES-NLS-LacZ reporter line had been described pre-viously (Hippenmeyer et al., 2005; Sieber et al., 2007; Xu et al., 2008;Lopes et al., 2012). In all timed matings, the morning that vaginal plugswere observed was considered to be E0.5. Genotypes were identified byPCR analysis of genomic DNA extracted from mouse tail tissue, and thefollowing PCR primers were used to identify the floxed and deleted Tlx3alleles:5�-TGTTTCGCCTCCTTTGCTCG-3�and5�-GTTGGATGGAAGCAAAGATAG-3�. For histochemical studies, mice at P28 or youngerages were used. For behavioral analyses, mutant and control littermatesat P21-P28 were used. All animal procedures are contained in protocolsreviewed and approved by the Animal Care and Use Committees at theDana-Faber Cancer Institute. Both males and females were used for thestudies.

In situ hybridization and immunostaining. Detailed methods for single-color in situ hybridization and in situ hybridization combined with flu-orescent immunostaining had been described previously (Liu et al.,2010). The following in situ probes were described previously, includingSOM, Tac1, VGLUT2, and GRPR (Cheng et al., 2004; Xu et al., 2008).GRP in situ probe was amplified with gene-specific sets of PCR prim-ers and cDNA template prepared from postnatal day 0 (P0) mousespinal cords with a final fragment size of 0.387 kb. The followingantibodies were used for single or double immunostaining: rabbitanti-Pax2 (1:500, Zymed Laboratories), rabbit anti-VGLUT1 (1:1000,Swant), rabbit anti-phospho-ERK (1:250, Cell Signaling Technol-ogy), rabbit anti-Pkc� (1:500, Santa Cruz Biotechnology), and chickanti-lacZ (1:500, Abcam). The rabbit anti-Tlx3 (1:500) and guinea piganti-Tlx3 (1:500) were acquired from C. Birchmeier at Max-Delbruck-Center for Molecular Medicine, Berlin, Germany, and rab-bit anti-Phox2a was a gift from Dr. Jean-Francois Brunet at CentreNational de la Recherche Scientifique, Paris, France. Sections wereprocessed with immunofluorescence by incubating overnight withprimary antibody and 1 h at room temperature with appropriatefluorescence-conjugated secondary antibodies (Invitrogen).

Counting p-ERK� cells. To count p-ERK � spinal cells induced by heat,four pairs of P28 control and mutant mice were used, and the righthindpaw of each of these mice was dipped into a 50°C water bath for 20 swhile holding the mouse by its neck, tail, and the left paw. Five minuteslater, the mouse was killed and perfused transcardially with 4% parafor-maldehyde in 0.1 M phosphate buffer, pH 7.4. The lumbar spinal cord wasremoved from L3 to L6, and transverse sections (20 �m thickness) werecut and processed for p-ERK immunohistochemistry. Cells with a clearnuclear morphology and staining levels clearly above background werecounted. The total number of p-ERK � cells per set of sections throughthe L3-L6 spinal cord was determined, and values were presented asmean � SD. The differences between control and mutant samples weresubjected to a Student’s t test, with p � 0.05 considered significant.

Behavioral analysis. All pain and itch behavioral tests were performedas previously described (Liu et al., 2010). All animals (Tlx3F//F;Lbx1cre

mutants and Tlx3F/F or Tlx3F/� control littermates) were acclimatized tothe behavioral testing apparatus for at least three ‘‘habituation” sessions.Two days before the injection, the nape of the neck was shaved after briefanesthesia with isoflurane (2% in 100% oxygen). A total of 10 �g ofCompound 48/80 (Sigma-Aldrich), 100 �g of PAR2 agonist SLIGRL-NH2 (Bachem), or 200 �g of chloroquine (Sigma-Aldrich) in 50 �l ofsterile saline were injected intradermally into the nape, and a camcorder(Sony model DCR-SR220) was positioned to video-record the behaviorof mice. The video recording was played back and scratching bouts werecounted. The experimenter was blinded to the genotype of the animals,and only the bouts to the shaved region were counted. To measure heatsensitivity, we placed mice on a hot plate (IITC) and the latency to hind-paw flicking, licking, or jumping was measured. The hot plate was set to50°C with a cutoff time of 60 s, or to 54°C with a cutoff time of 30 s. Allanimals were tested sequentially with a minimum of 5 min between tests.

Capsaicin was intradermally administrated in a dosage of 2.5 �g/10 �linto the right hindpaw, and the duration of lifting, licking, and flinchingwas measured. To measure sensitivity to noxious cold, the cold plate(IITC) was set to 0°C. The mice were video-recorded for 120 s, and thecombined number of hindpaw flicking, licking, or jumping wasmeasured.

For the temperature chamber assay, animals were allowed to exploreadjacent surfaces, with one held at 20°C and the other ranging from 30°Cto 5°C. The mice were video-recorded for 5 min, and the percentages oftime staying at 20°C chamber were determined. The acetone evaporationcooling assay was performed as previously described (Knowlton et al.,2011) with some modifications. Mice were acclimated for 10 min in anelevated wire grid. A syringe with a piece of rubber tubing attached to theend was filled with acetone and the plunger depressed so that a small dropof acetone formed at the top of the tubing. The syringe was raised to thehindpaw from below, depositing the acetone drop on the paw. The testwas repeated for 10 times (5 times for each paw), with intervals of 3 min.Responses were video-recorded, and duration times of lifting, licking,flinching, shacking, and rotating on the torso were determined. To mea-sure punctate mechanical sensitivity, we placed animals on an elevatedwire grid and the lateral plantar surface of the hindpaw stimulated withcalibrated von Frey monofilaments (0.0174 – 4.57 g). The paw with-drawal threshold for the von Frey assay was determined using Dixonsup-down method (Chaplan et al., 1994). To measure dynamic mechan-ical sensitivity, the middle part of the right hindpaw was stimulated bylight stroking with a cotton swab, in the direction from heel to toe. Thetest was repeated three times, with intervals of 10 seconds. For each test,a score of 0 indicates no movement, and a score of 1 is a lifting of thestimulated paw and/or walking way. For each mouse, the accumulativescores of three tests were used to indicate “the dynamic score” shown inFigure 7A.

To measure sensorimotor coordination, we performed the rotarod test.Initially, mice were habituated for 1 min at a rotating speed of 4.0 rpm/s. Forthe actual test, we set the ramp to start at a velocity of 4.0 rpm/s, with acontinuous acceleration of 0.4 rpm/s. The time points when mutant andcontrol mice fell off the ramp were recorded. Data for the different behav-ioral assays are represented as the mean � SE. Statistical significance wasassessed with the Student’s t test, with p � 0.05 considered significant. Theexperimenter was blinded to the genotype of animals.

ResultsGeneration of Tlx3F/F;Lbx1Cre/� conditional knock-out miceTlx3 null mice die at birth (Shirasawa et al., 2000). We recentlygenerated a mouse line carrying a conditional null allele of Tlx3(Lopes et al., 2012), referred to as Tlx3F/�. To study the physio-logical functions of class B dI5 and dILB neurons, we crossedTlx3F/� mice with Lbx1-Cre mice (Sieber et al., 2007) to generateTlx3F/F;Lbx1Cre/� mice, referred to here as conditional knock-out(CKO) mice. As indicated by developmental ontogeny of spinalneurons (Fig. 1A), in these CKO mice, Tlx3 was removed selec-tively in Lbx1-expressing class B dI5 and dILB neurons (Fig. 1A).In wild-type mice, expression of both Tlx3 and Lbx1 is initiatedimmediately in newly formed postmitotic neurons (Gross et al.,2002; Muller et al., 2002; Qian et al., 2002). We found that Tlx3expression in the dorsal spinal cord was still detected at E12.5 inCKO mice (Fig. 1B) but was largely eliminated by E16.5 (data notshown). At P21, persistent Tlx3 expression, which was detected insuperficial dorsal horn laminae in wild-type mice (Fig. 1C, ar-row), was not detected in CKO mice (Fig. 1C), suggesting thatTlx3-persistent neurons belong to Lbx1 lineage neurons. This isconsistent with class B dorsal horn excitatory neurons (dI5/dILB)being defined by the expression of the homeobox protein Lmx1b(Gross et al., 2002; Muller et al., 2002), and persistent Tlx3 beingconfined to these Lmx1b� spinal neurons (Rebelo et al., 2010).

We found that CKO mice survived to postnatal stages (seebelow). In the hindbrain, Tlx3 is expressed transiently in, but

Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738 –14748 • 14739

necessary for proper development of, sev-eral Lbx1-negative class A neurons thatare involved in respiration control, in-cluding noradrenergic (NA) neurons andthe nucleus of the solitary tract (NTS) inthe hindbrain (Qian et al., 2001, 2002;Sieber et al., 2007). Development of theseLbx1-negative neurons was naturally un-affected in CKO mice because Lbx1-Crewas used for making conditional knock-outs, as indicated by the normal expres-sion of (1) the homeobox gene Phox2b, amarker for NTS neurons, and (2) the do-pamine � hydroxylase, a marker for NAneurons (data not shown) (Qian et al.,2001). In Tlx3 complete null mice, the lossof NA and NTS neurons causes a failure incentral respiration control and neonatallethality (Shirasawa et al., 2000; Qian etal., 2001). Accordingly, the preservationof these neurons in CKO mice explainswhy these mutants survive.

Selective impairment of lamina I/IIneurons in CKO miceTlx3 is expressed persistently in neurons lo-cated in the superficial dorsal horn, buttransiently in more ventral laminae (Xu etal., 2008). With the preservation of earlyTlx3 expression in CKO mice, we hypothe-sized that the development of Tlx3-persistent neurons might be preferentiallyimpaired. Before we tested this hypothesis,we first examined which laminae containTlx3-persistent neurons. VGLUT1 is thevesicular glutamate transporter expressedin peripheral low threshold myelinatedmechanoreceptors and proprioceptors thatterminate in the inner layer of lamina II andmore ventral laminae (Li et al., 2003). Dou-ble immunostaining showed that the ven-tral portion of Tlx3-persistent neurons wasintermingled with most dorsally localizedVGLUT1� terminals (Fig. 2A), and as de-scribed below, corresponded to PKC��

neurons that represent the inner layer oflamina II (Neumann et al., 2008). Thus,Tlx3-persistent neurons are confined tolaminae I and II.

We then found that development of lamina I/II neurons wascompromised in CKO mice. The somatostatin neuropeptidegene (SOM) is expressed in both excitatory and inhibitory neu-rons, with Tlx3-dependent SOM� excitatory neurons located inthe superficial laminae (Xu et al., 2008). Double staining of SOMmRNA and VGLUT1 showed that SOM expression was elimi-nated in the P21 CKO superficial dorsal horn but still detected inmore ventral laminae (Fig. 2B). Loss of SOM expression occurredby E16.5 (data not shown) and at both lumbar (Fig. 2B) andcervical (Fig. 2C) levels at P21. The preprotachykinin gene (Tac1)encodes two neuropeptides: substance P and neurokinin A (Hok-felt, 1991). There are two waves of Tac1 expression in the dorsalspinal cord. We reported previously that the early wave is estab-lished at embryonic stages and enriched in the deep dorsal horn

(Fig. 2D, arrowheads) (Xu et al., 2008). Here, we found a latewave of Tac1� neurons that emerged during postnatal develop-ment and were located in lamina II (Fig. 2D, arrow). In CKOmice, whereas the late wave of Tac1 expression in lamina II waseliminated (Fig. 2D, arrows), the early wave in deep laminae wasat least partially preserved (Fig. 2D, arrowheads). Thus, theSOM� and Tac1� subsets of excitatory neurons that are locatedat laminae I and II are selectively compromised in CKO mice.

Because Tac1 expression is eliminated in Tlx3 complete nullmice (Xu et al., 2008), the preservation of a subset of Tac1�

neurons in CKO mice suggests that the development of someTlx3-transient deep dorsal horn is unaffected in CKO mice. Tofurther support this, we examined additional markers. A subset ofTlx3-transient dI5 neurons is marked by the expression of

Figure 1. Ontogeny of spinal dorsal horn neurons and generation of CKO mice. A, Schematics showing the ontogeny of eightdistinct groups of dorsal horn neurons (dI1– 6, dILA, dILB). Tlx3 �(T), Neurons expressing Tlx3 transiently; Tlx3 �(T/P), neuronsexpressing Tlx3 transiently or persistently. B, C, Tlx3 immunostaining on transverse spinal sections at indicated stages and geno-types. At E12.5, Tlx3 protein expression was comparable between control (Ctrl ) and CKO embryos (B, arrows). At P21, persistentTlx3 expression was observed in the superficial dorsal horn of Ctrl mice (C, arrow) but abolished in CKO mice (C).

14740 • J. Neurosci., September 11, 2013 • 33(37):14738 –14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

Phox2a (Qian et al., 2002). We again found that these Phox2a�

neurons showed normal ventral migration in CKO mice at E16.5(Fig. 2E) and at P0 (data not shown). Development of Lbx1-negative class A spinal neurons is naturally unaffected. Forexample, dI1 neurons, marked by the expression of BarhL1(Bermingham et al., 2001; Ding et al., 2012), and dI3 neurons,marked by the expression of Islet1 (Gross et al., 2002; Muller etal., 2002; Qian et al., 2002), all showed normal ventral migra-tion at E16.5 and P0 in CKO mice compared with controls(data not shown).

The incomplete loss of SOM � and Tac1 � neurons, andthe normal development of Phox2a � neurons, does raise aquestion as to whether or not these neurons originate fullyfrom Lbx1 � class B neurons. To address this, we crossed Lbx1-Cre mice (Sieber et al., 2007) with the Tau-loxp-STOP-lox-mGFP-IRES-NLS-LacZ reporter mice (Hippenmeyer et al.,2005) to generate mice referred to here as Tau-LSL-nlacZ;Lbx1-Cre. In these mice, the Lbx1 lineage neurons are markedby the expression of the nuclear lacZ protein, regardless ofpersistent or transient Lbx1 expression. Double stainingshowed that most, but not all, SOM � neurons coexpressednLacZ (Fig. 2F, arrow vs arrowhead), suggesting that SOM �

neurons originate mainly from Lbx1 � class B neurons, butalso to a lesser degree from Lbx1 � class A neurons. In addi-tion, most, if not all, Tac1 � neurons, as well as all Phox2a �

neurons coexpressed nlacZ (Fig. 2G,H ), suggesting that theseneurons are also derived from Lbx1 lineage class B neurons.This finding suggests that the normal development of theseTlx3-transient deep dorsal horn neurons in CKO mice is theresult of the preservation of transient Tlx3 expression in classB neurons, rather than a separate developmental origin fromLbx1 � class A neurons.

Tlx3 is known to determine glutamatergic over GABAergicneurotransmitter phenotypes, and VGLUT2 expression in thelumbar superficial dorsal horn is eliminated in Tlx3 null mice(Cheng et al., 2004). Interestingly, with the preservation of earlyTlx3 expression in CKO mice, VGLUT2 expression was largelyunaffected at E16.5 (Fig. 2I). As a comparison, SOM expressionin superficial laminae was already eliminated by E16.5 (data notshown). Thus, transient Tlx3 expression is sufficient to establishthe glutamatergic transmitter phenotype, which is in contrastwith the requirement of Tlx3 activity beyond E12.5 in establish-ing other molecular identities, such as the expression of SOM andTac1 in superficial laminae.

Figure 2. Selective loss of markers in laminae I/II of CKO mice. A, Double immunostaining of Tlx3 protein in spinal neurons (green) with VGLUT1 protein in primary afferents (red) on a transverselumbar spinal section from a P21 wild-type mouse. B, Double staining of the VGLUT1 protein in primary afferent terminals (green) and cytoplasmic SOM mRNA in spinal neurons (pseudo-red, by insitu hybridization) on transverse P21 lumbar spinal cord sections of control (Ctrl ) and CKO mice. There is a selective loss of SOM mRNA in lamina I/II dorsal to VGLUT1 � terminals (arrow). C, SOM insitu hybridization on transverse P21 spinal sections at the cervical level. Arrow indicates SOM expression in the superficial laminae. D, Tac1 in situ hybridization on transverse P21 lumbar spinal cordsections. Arrows and arrowheads indicate Tac1 expression in superficial and deep laminae, respectively. E, Phox2a in situ hybridization on E16.5 lumbar spinal cord sections. F–H, Double stainingof the nLacZ protein (green) and SOM mRNA (F, red), Tac1 mRNA (G, red), or Phox2a protein (H, red) on P21 or P0 lumbar spinal cord sections from Tau-LSL-nlacZ;Lbx1-Cre fate mapping mice. Arrowsand arrowheads indicate double or singular staining, respectively. I, VGLUT2 in situ hybridization on E16.5 lumbar spinal sections with indicated genotypes.

Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738 –14748 • 14741

Reduced innervation by primary sensory afferents inCKO miceDorsal horn laminae I and II are mainly innervated by sensoryneurons detecting pain-, itch-, and temperature-related sensoryinformation (Lallemend and Ernfors, 2012). These sensory neu-rons, located in the DRG, are divided into two main subtypes,peptidergic and nonpeptidergic neurons. Peptidergic neuronsexpress the calcitonin gene-related peptide (CGRP) and the neu-rotrophin receptor TrkA, and innervate lamina I and the outerlayer of lamina II. Many nonpeptidergic neurons can be labeledby the binding of isolectin B4 (IB4), coexpress the neurotrophinreceptor Ret, and innervate the dorsal portion of the inner layerof lamina II. Deep dorsal horn laminae (III-V) are innervatedmainly by myelinated mechanoreceptors marked by the expres-sion of various neurotrophin receptors, including TrkC, TrkB,and Ret (Lallemend and Ernfors, 2012), as well as by the expres-sion of VGLUT1 (Li et al., 2003). In CKO mice, DRG neurondevelopment and survival were unaffected, as suggested by thegrossly normal expression of TrkA, TrkB, TrkC, CGRP, and Ret(Fig. 3A–E). This is consistent with the lack of Lbx1 expression inDRG (Gross et al., 2002; Muller et al., 2002), and unaffected Tlx3expression in Tlx3F/F;Lbx1Cre/� CKO mice (data not shown).

Despite normal differentiation of DRG neurons, we foundthat there are much fewer CGRP� and IB4� nerve terminals inCKO dorsal spinal cord, although their relative dorsoventral pro-jection pattern remained (Fig. 3F). In contrast, innervation ofVGLUT1� low threshold mechanoreceptors to the deep dorsalhorn and to the ventral motor neurons was largely unaffected(data not shown). Thus, developmental impairment of a largesubset of Tlx3-dependent excitatory neurons in the superficial

dorsal horn results in reduced innervation by CGRP� and IB4�

afferents, but the basic organization of these terminals in thedorsal horn is preserved in CKO mice.

Impaired behavioral responses evoked by pain-related stimuliin CKO miceCKO mice can only survive for 1–2 months. These mutant miceshowed overgrowth of teeth, possibly leading to malocclusionthat may affect feeding (Fig. 4A), but the underlying cause is notknown. Nonetheless, CKO mice at 3– 4 weeks of age grosslylooked healthy, although their body sizes were smaller than wild-type littermates (data not shown). We therefore performed allbehavioral analyses at these ages, with Tlx3F/F or Tlx3F/� litter-mates as controls.

CKO mice showed normal proprioception and sensorimotorcoordination, as suggested by proper clustering of the forelimbsand the extension of hindlimbs when suspended by the tail (Fig.4B). Furthermore, after one round of training, CKO and controllittermates remained on an accelerating rotarod for similaramounts of time (Fig. 4C).

We next applied a mechanical stimulus to the plantar surfaceof the hindpaw using von Frey fibers and determined the thresh-old leading to hindpaw lifting and flinching. In comparison withcontrols, CKO mice showed a marked increase in withdrawalthresholds, suggesting a deficit in generating proper reflex behav-ior in response to noxious mechanical stimuli (Fig. 4D).

To measure behavioral response to noxious heat, we placedmice on a hot plate set to 50°C or 54°C and found that the latencyof hindpaw lifting/flinching increased markedly in CKO mice,suggesting a defect in processing heat-related sensory informa-tion (Fig. 4E,F). Heat pain is mediated by DRG neurons express-ing the transient receptor potential channel TRPV1 (Cavanaughet al., 2009; Mishra and Hoon, 2010), which is also the receptorfor the chili pepper ingredient capsaicin (Caterina et al., 1997).Consistently, intraplantar capsaicin injection, which evoked ro-bust licking and flinching in controls, failed to elicit responses inCKO mice (Fig. 4G).

To more directly assess how spinal neurons responded topainful stimuli in CKO mice, we took advantage of the previousfinding that noxious painful stimuli, such as 50°C heat, activatethe ERK protein kinase through phosphorylation (p-ERK) selec-tively in lamina I/II neurons (Ji et al., 1999). We found that, after50°C heat stimulation of the CKO hindpaw for a short period (20s), the number of p-ERK� neurons in superficial laminae of thelumbar spinal cord was greatly reduced compared with controls,from 190 � 44 per set of sections in control mice to 31 � 9 inCKO mice (n � 4, p � 0.05), an 84% reduction (Fig. 4H, I).Together, these data suggest that Tlx3-dependent dI5 and/or dILb

neurons are required to process mechanical and heat pain-relatedsensory information.

Reduced scratching response evoked by pruritic compoundsin CKO miceTo measure itch-related behavior, we performed nape injectionsof itch-inducing compounds and monitored scratching re-sponse. Compound 48/80 activates a histamine-dependent itchpathway (Sugimoto et al., 1998), and the number of scratch boutsevoked by this compound was reduced, but not completely elim-inated, in CKO mice compared with controls (Fig. 5A). Both themalaria drug chloroquine and the Par2 agonist peptide SLIGRL-NH2 evoke histamine-independent itch by activating theG-protein coupled receptors Mrgpra3 and Mrgprc11, respec-tively (Liu et al., 2009, 2011; Wilson et al., 2011). Nape injection

Figure 3. Normal DRG neuron development and reduced central innervation in CKO mice.A–E, In situ hybridization with indicated probes on transverse sections through lumbar DRG ofP21 control (Ctrl ) and CKO mice. F, Double staining on transverse P21 lumbar spinal cord sec-tions with CGRP immunostaining (red) and IB4 binding (green).

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of these compounds evoked robust scratching responses in con-trol littermates, but little in CKO mice. Thus, Tlx3-dependent dI5and/or dILb class B excitatory neurons are required to processitch-related information (Fig. 5A).

Dorsal horn neurons expressing GRPR, the receptor for theGRP, have been implicated in itch sensing (Sun et al., 2009). Wefound that expression of both GRPR and GRP was eliminated inCKO mice (Fig. 5B).

Double staining on spinal sections of Tau-LSL-nlacZ;Lbx1-Cre fate-mapping mice showed that neurons with detectableGRPR or GRP mRNA coexpressed nLacZ (Fig. 5C,D), suggestingthat GRPR� and GRP� neurons represent subsets of Lbx1� classB neurons. To further investigate the ontogeny of GRPR� neu-rons, we crossed Tlx3-Cre mice with the Tau-LSL-nLacZ reportermice, with Tlx3 lineage spinal neurons permanently marked bythe expression of nlacZ (Hippenmeyer et al., 2005; Xu et al.,2008). Double staining showed that GRPR� neurons are con-fined to Tlx3 lineage excitatory neurons (Fig. 5E). Consistently,GRPR� neurons did not express Pax2 (Fig. 5F), which is amarker for inhibitory neurons (Cheng et al., 2004). Collectively,these studies suggest that Tlx3 may autonomously control thedevelopment of itch-related GRPR� neurons.

Impairment of touch-evoked escape responses in CKO miceIn response to the dynamic mechanical stimulus of stroking thehindpaw with a cotton swab, control mice responded by liftingthe hindpaw and walking away. Interestingly, this touch-evoked“escape” response was virtually abolished in CKO mice (Fig. 6A).To assess the potential cause for this behavioral deficit, we nextexamined neurons expressing the � isoform of PKC� in the innerlamina II, which respond to dynamic mechanical stimuli (Mirau-court et al., 2007; Neumann et al., 2008) and belong to excitatoryneurons (Polgar et al., 1999). We found that PKC� expression inthe inner lamina II was eliminated in CKO mice (Fig. 6B). As apositive control, PKC�� fibers in the dorsal funiculus were stilldetected in both control and mutant mice (Fig. 6B).

Consistently, double staining on spinal sections of Tau-LSL-nlacZ;Lbx1-Cre fate-mapping mice showed that all PKC�� neu-rons coexpressed nlacZ (Fig. 6C), suggesting that they representLbx1 lineage class B neurons. Furthermore, many, although notall, PKC�� neurons showed persistent Tlx3 expression (Fig. 6D).The location of PKC�� neurons delineates the ventral border ofTlx3-persistent neurons (Fig. 6D). We hypothesized that the im-paired development of PKC�� mechanoresponsive cells mightunderlie the loss of the escape response evoked by dynamic me-

Figure 4. Long teeth, normal sensorimotor coordination and impaired nocifensive behaviors in CKO mice. A, Malocclusion in a P28 CKO mouse (arrows) compared with a control (Ctrl ). B, P28 CKOmice exhibiting normal sensorimotor coordination. C, Rotarod assay. No significant difference was found between Ctrl and CKO mice on time (seconds) staying on accelerating rotarod. Ctrl, n � 10,45.6 � 5.5 s; CKO, n � 10, 39.4 � 5.2 s. p � 0.44. D, von Frey test. CKO mice showed a higher withdrawal threshold than Ctrl mice. Ctrl, n � 7, 0.29 � 0.04 g; CKO, n � 7, 1.36 � 0.05 g. ***p �0.001. E, F, Hot plate assay. CKO mice showed longer latency (seconds) in response to both 50°C and 54°C stimuli compared with Ctrl mice. E, For 50°C: Ctrl, n � 8, 23.4 � 1.2 s; CKO, n � 8, 56.5 �1.0 s. ***p � 0.001. F, For 54°C: Ctrl, n � 6, 7.6 � 1.0 s; CKO, n � 6, 26.8 � 1.3 s. **p � 0.01. G, Capsaicin hindpaw injection assay: measuring the duration (seconds) of licking and flinching.Abolished response in CKO mice. Ctrl, n � 9, 31.8 � 3.2 s; CKO, n � 9, 0.0 � 0.0 s. H, p-ERK immunostaining on P28 lumbar spinal transverse sections after heat stimulation. Arrows indicatesuperficial laminae. I, Quantitative analysis of p-ERK � neurons per set of sections through L3–L6. Ctrl, n � 4, 190 � 44; CKO, n � 4, 31 � 9. *p � 0.05. Error bars indicate SEM.

Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738 –14748 • 14743

chanical stimuli in CKO mice, although future studies are neededto test this hypothesis.

CKO mice are able to sense innocuous cooling and/or warm,but not noxious coldWe next examined how mutants responded to cold stimuli. Wefirst placed controls and mutants onto the cold plate set to 0°C for2 min. We found that most control mice exhibited paw lifting andlicking, but these nocifensive responses evoked by noxious coldwere virtually abolished in CKO mice (Fig. 7A). We next per-formed the acetone evaporation assay, which cools skin temper-ature down to 14 –18°C, around the transition zone of noxiousversus innocuous cold temperatures (Colburn et al., 2007). Wefound that both CKO mice and control littermates showed sim-ilar responses to the evaporative cooling as measured by the du-ration of paw shaking, lifting, and licking (Fig. 7B), suggestingthat Tlx3 CKO mice appear to retain the ability to sense innocu-ous or mild noxious cold.

To further explore whether CKO mice were able to sense in-nocuous cold, we performed the temperature chamber selectionassay (Bautista et al., 2007; Colburn et al., 2007; Dhaka et al.,2007; Knowlton et al., 2010). We found that mutant and controlmice were indistinguishable in choosing the 30°C over the roomtemperature (�20°C) chamber, or the 20°C chamber over the

15°C or 5°C chamber (Fig. 7C). The normal selection of 30°Cover 20°C chambers suggests that CKO mice are able to senseinnocuous cooling and/or warm. In the 20°C versus 5°C assay,some investigators used the avoidance of the 5°C chamber as away of measuring noxious cold sensation (Bautista et al., 2007;Colburn et al., 2007; Dhaka et al., 2007; Knowlton et al., 2010).However, both mutant and control mice spent �94% of time inthe 20°C chamber (Fig. 7C): the moment they reached the 5°Cchamber, they immediately withdrew to the warmer side (datanot shown). Such momentary exposure to the cold chamber un-likely drops the skin temperature to the noxious cold range. Thus,the normal selection of 20°C over 5°C by CKO mice most likelyreflects a normal sense of innocuous cooling, rather than coldpain, particularly considering that these mice fail to generatepain-suggestive responses at 0°C (Fig. 7A). Collectively, thesefindings suggest that CKO mice fail to respond to extreme coldbut retain the ability to sense innocuous cooling and/or warm.

DiscussionTemporal control of dorsal horn excitatoryneuron phenotypesTlx3 and its related gene Tlx1 act as selector genes that coordinatethe development of a diverse array of dorsal horn excitatory neu-rons by specifying glutamatergic and peptidergic transmitter

Figure 5. Impaired scratching response evoked by pruritic compounds in CKO mice. A, Scratching response was examined in animals at 3 to 4 weeks old. Nape injections of compound 48/80 (10�g) evokedreduced scratching bouts in CKO mice compared with controls (Ctrl ). Ctrl, n�6, 149�74; CKO, n�6, 21�19. **p�0.01. Injection of the PAR2 agonist SLIGRL-NH2 (100�g) evoked virtually no scratchingin CKO mice. Ctrl, n�6, 134�111; CKO, n�6, 1�2. *p�0.05. Injection of chloroquine injection (200 �g) also did not induce scratching in mutants. Ctrl, n�4, 152�70; CKO, n�4, 0�0. *p�0.05.Error bars indicate SEM. B, In situ hybridization on P28 lumbar spinal cord sections with indicated probes. C, D, GRPR � and GRP � neurons are derived from Lbx1 lineage neurons. Double staining of the nLacZprotein (green) and indicated mRNA (pseudo-red) on P21 lumbar spinal cord sections from Tau-LSL-nlacZ;Lbx1-Cre fate mapping mice. Arrows indicate colocalization. E, GRPR � neurons are derived from Tlx3lineage neurons. Double staining of the nLacZ protein (green) and GRPR mRNA (pseudo-red) on P0 lumbar spinal cord sections of Tau-LSL-nlacZ;Tlx3-Cre fate mapping mice. Arrows indicate colocalization. F,Double staining of the nuclear Pax2 protein (green) and GRPR mRNA (pseudo-red) on an E16.5 wild-type spinal section, showing no colocalization (arrowhead).

14744 • J. Neurosci., September 11, 2013 • 33(37):14738 –14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

phenotypes as well as controlling transmitter receptor expression(Qian et al., 2002; Cheng et al., 2004; Xu et al., 2008; Guo et al.,2012). Tlx1 is expressed in a subset of excitatory neurons of thecervical/thoracic spinal cord, and its expression is switched offonce postmitotic neurons migrate out of the ventricular zone(Qian et al., 2002). In our CKO mice, Tlx3 expression was de-tected normally at E12.5 but largely eliminated by E16.5. That is,expression of both Tlx1 and Tlx3 becomes transient throughoutthe spinal cord in CKO mice.

A comparison of phenotypes of Tlx3 null versus CKO miceprovides insight into the temporal control of dorsal horn excit-atory neuron phenotypes. We found that transient Tlx3 expres-sion retained in CKO mice is sufficient to activate VGLUT2 in thedorsal horn, in contrast to complete loss of VGLUT2 expressionat the lumbar superficial dorsal horn in Tlx3 null mice (Cheng etal., 2004). The early determination of the glutamatergic transmit-ter phenotype is consistent with that VGLUT2 expression is ini-tiated shortly after birth of neurons and that transient Tlx1expression in the cervical/thoracic dorsal horn is able to compen-sate Tlx3 loss in establishing VGLUT2 expression (Cheng et al.,2004). In contrast, expression of SOM, Tac1, GRP, GRPR, andPKC� in laminae I/II is eliminated in CKO mice, suggesting a

requirement of Tlx3 activity beyond E12.5 in establishing this setof molecular identities. Transient Tlx1 expression is unable tocompensate this late Tlx3 activity, resulting in a loss of SOM andother markers at both lumbar and cervical levels (Fig. 2). Thus,early and late Tlx protein activities establish distinct molecularidentities in dorsal horn excitatory neurons.

Ontogeny of spinal neurons processing distinctsensory modalitiesThe dorsal horn excitatory neurons are divided into Lbx1 � classA neurons (dI1-dI3) and Lbx1� class B neurons (dI5 and dILB)(Figs. 1A and 8). Our studies and others show that this molecularand developmental subdivision is correlated with distinct sensorymodalities processed by these spinal neurons. Two class A neu-rons (dI1 and dI3) are located in the deep dorsal horn laminaeand involved with sensory-motor coordination, with dI1 neu-rons necessary for proprioception (Bermingham et al., 2001) anddI3 neurons involved with hand grasp performance (Bui et al.,2013). The studies described here show that Tlx3-dependent classB excitatory neurons (dI5 and/or dILB) are required to processpain-related and itch-related sensory information and to gener-ate the escape response in response to dynamic mechanical stim-uli (Fig. 8).

Class B neurons can be further divided into two categories (Iand II), based on how their development is affected in Tlx3F/F;Lbx1Cre/� CKO mice. The first category (Fig. 8, “I”) includesthose whose development is unaffected in CKO mice, such as the

Figure 6. Impairment of touch-evoked escape responses and Pkc� � neurons in CKO mice.A, The cotton wipe assay. The dynamic score, used to measure touch-evoked escapes, wasreduced in CKO mice compared with control littermates (Ctrl ). Ctrl, n � 7, 2.71 � 0.18; CKO,n � 7, 0.14 � 0.14. ***p � 0.05. B, Immunostaining of Pkc on P21 lumbar spinal cord sectionsat indicated genotypes. Insets, Pkc staining within the dorsal funiculus. C, Pkc�� neurons arederived from Lbx1 lineage neurons. Double staining of the nLacZ protein (green) and Pkc� (red)on P21 lumbar spinal cord sections from Tau-LSL-nlacZ;Lbx1-Cre fate mapping mice. D, Doublestaining of Pkc protein (red) with Tlx3 protein (green) on a P21 wild-type transverse lumbarspinal cord section. Arrow indicates colocalization; arrowhead indicates Pkc neurons lackingTlx3.

Figure 7. Cold behavior analyses. A, The 0°C cold plate assay. The numbers of licking/flinch-ing (“L/F”) were counted. The CKO mice failed to respond to noxious cold (Ctrl, n � 20, 7.45 �0.13; CKO, n � 5, 1.27 � 0.08). ***p � 0.001. B, The acetone evaporation assay. No differencebetween control and CKO mice (Ctrl, n � 7, 8.73 � 0.97; CKO, n � 5, 6.40 � 0.57). p � 0.05.C, The temperature chamber selection assay. The percentages of time staying at the 20°C cham-ber were determined. No differences between control and CKO mice were observed at fourdifferent sets of temperatures. For 20°C versus 20°C selection chamber: control, n � 6, 52.8 �6.8; CKO, n � 6, 52.9 � 56.0. p � 0.8. For 20°C versus 30°C: control, n � 6, 18.6 � 6.4; CKO,n � 6, 18.4 � 14.5. p � 0.9. For 20°C versus 15°C: control, n � 6, 76.9 � 11.0; CKO, n � 6,78.8 � 7.4. p � 0.9. For 20°C versus 5°C: control, n � 6, 94.0 � 4.5; CKO, n � 6, 93.5 � 2.9.p � 0.4.

Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities J. Neurosci., September 11, 2013 • 33(37):14738 –14748 • 14745

Phox2a� subset of dI5 neurons and a portion of Tac1 � neuronslocated in deep laminae. Genetic fate mapping shows that bothPhox2a� and Tac1� neurons do represent Lbx1� class B neu-rons (Fig. 2). We reported previously that Tlx3 expression isswitched off soon after the genesis of these neurons at E11.5 orE12.5 (Qian et al., 2002; Xu et al., 2008). Accordingly, the pres-ervation of transient Tlx3 expression in CKO mice explains anormal development of these neurons. Because the CKO miceretain the ability to sense innocuous cold and/or warm, we spec-ulate that this type of somatic sensory information might be pro-cessed by Type I category of class B excitatory neurons or byLbx1-negative class A neurons, whose development is also unaf-fected in CKO mice (Fig. 8, dashed arrows).

The second category (Fig. 8, “II”) of class B excitatory neuronsare those whose development is compromised in CKO mice, suchas SOM�, Tac1�, GRP�, GRPR�, and PKC�� neurons locatedin laminae I and II. Genetic fate mapping shows that all theseneurons represent Lbx1 lineage neurons, with the exception of asmall subset of SOM� neurons. Our behavioral analyses showthat this category of Tlx3-dependent neurons are specialized toprocess pain-related and itch-related information, as well as togenerate the touch-evoked escape response. An involvement ofTlx3-dependent spinal neurons in sensing pain is consistent withthat mice lacking DRG11/Prrxl1, a Tlx3-dependent gene (Qian etal., 2002), exhibited marked deficits in generating nocifensivebehavior in response to painful stimuli (Chen et al., 2001), al-though the interpretation is complicated by a latter report show-

ing a concurrent requirement of DRG11/Prrxl1 for DRG neurondevelopment (Rebelo et al., 2006). Among type II class B neurons,GRPR� and GRP� neurons have already been implicated in pro-cessing itch (Sun et al., 2009; Mishra and Hoon, 2013). The iden-tities of spinal neurons processing pain-related information arestill poorly understood, but SOM� and/or Tac1� neurons, rep-resenting two large subsets of excitatory neurons located in lam-inae I/II, could be attractive candidates. PKC�� neurons respondto dynamic mechanical stimuli (Miraucourt et al., 2007; Neu-mann et al., 2008); their developmental impairment might con-tribute to the loss of the touch-evoked escape response in CKOmice.

Among spinal neurons responding to low threshold mechan-ical stimuli, PKC�� neurons are unique in terms of their locationin the inner layer of lamina II (Neumann et al., 2008), rather thanin the more conventional laminae III-V (Kandel et al., 2000; Lal-lemend and Ernfors, 2012). Here, we showed that these neuronsare derived from Lbx1� class B neurons, rather than from Lbx1�

class A neurons that mainly settle in laminae III-V (Helms andJohnson, 2003). Moreover, upon central disinhibition after pe-ripheral nerve injury, PKC�� neurons are part of the circuit thatmediates pain evoked by low threshold mechanical stimuli(Takazawa and MacDermott, 2010). It should also be noted thattouch-evoked escape is a conserved behavior seen in worms, in-sects, fishes, and mammals and likely evolves for animals to es-cape from predators in the natural environment. Thus, theshared developmental ontogeny of the type II class B excitatory

Figure 8. Ontogeny of spinal neurons processing distinct sensory modalities. I and II are referred to as two categories of Lbx1 � class B excitatory neurons. Type I includes neurons whosedevelopment is unaffected in CKO mice, whereas Type II category is composed of a cohort of neurons whose development is impaired in CKO mice. Type II neurons are located in laminae I/II and arerequired to generate nocifensive motor behaviors evoked by pain-related or itch-related stimuli, as well as to generate escape response evoked by dynamic mechanical stimuli. Lbx1 � class Aneurons mediate proprioceptive and mechanoreceptive sensory information. However, it is not clear whether the sense of innocuous cold or warm, which is preserved in CKO mice, is mediated byLbx1 � class A neurons or by the Type I category of Lbx1 � class B excitatory neurons (dashed arrows). SOM �/S and Tac1 �/S, SOM � and Tac1 � neurons at laminae I/II, or the superficial (“s”)laminae.

14746 • J. Neurosci., September 11, 2013 • 33(37):14738 –14748 Xu, Lopes et al. • Excitatory Spinal Neurons and Somatic Sensory Modalities

neurons appears to be correlated with the shared roles in sensingenvironmental danger and generating proper behavior to avoidnoxious stimuli or to escape from predators (Fig. 8).

In 1905, Henry Head performed nerve injury studies on hisown arm (Head, 1905); and based on differential speeds ofsensory fiber regeneration and sequential recovery of distinctsensory modalities, he discovered two constituents of cutane-ous sensibility: (1) the “protopathic” system, responding topainful stimuli and to the extremes of heat and cold, the kindof stimuli that evoke strong reflex but are poorly localized; and(2) the “epicritic” system, responding to innocuous stimula-tions permitting fine discriminations of temperature andtouch. In our Tlx3 CKO mice, processing of “protopathic”-like sensory modalities (e.g., the senses of pain and itch) ap-pears to be selectively compromised, whereas processing of“epicritic” sensory information (e.g., proprioception and thesense of innocuous cold or warm) is preserved (summarized inFig. 8). Thus, Henry Head’s distinction between epicritic andprotopathic peripheral sensory modalities might be mapped atthe level of the spinal cord as well. Our studies thereby providenovel insight into the ontogeny of spinal neurons processingdistinct somatic sensory modalities.

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40

Publication III

Neuron

Article

VGLUT2-Dependent Glutamate Releasefrom Nociceptors Is Requiredto Sense Pain and Suppress ItchYang Liu,1,4 Omar Abdel Samad,1,4 Ling Zhang,3 Bo Duan,1 Qingchun Tong,2,5 Claudia Lopes,1 Ru-Rong Ji,3

Bradford B. Lowell,2 and Qiufu Ma1,*1Dana-Farber Cancer Institute and Department of Neurobiology, Harvard Medical School, 1 Jimmy Fund Way, Boston, MA 02115, USA2Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School,99 Brookline Avenue, Boston, MA 02215, USA3Pain Research Center, Department of Anesthesiology, Brigham and Women’s Hospital and Harvard Medical School, Boston,

MA 02115, USA4These authors contributed equally to this work5Present address: Center for Diabetes and Obesity Research of the Brown Foundation Institute of Molecule Medicine

and Division of Endocrine of Department of Internal Medicine, University of Texas, Houston, TX, 77030, USA

*Correspondence: qiufu_ma@dfci.harvard.edu

DOI 10.1016/j.neuron.2010.09.008

SUMMARY

Itch can be suppressed by painful stimuli, but theunderlying neural basis is unknown. We generatedconditional null mice in which vesicular glutamatetransporter type 2 (VGLUT2)-dependent synapticglutamate release from mainly Nav1.8-expressingnociceptorswas abolished. Thesemice showeddefi-cits in pain behaviors, including mechanical pain,heat pain, capsaicin-evoked pain, inflammatorypain, and neuropathic pain. The pain deficits wereaccompanied by greatly enhanced itching, as sug-gested by (1) sensitization of both histamine-depen-dent and histamine-independent itch pathways and(2) development of spontaneous scratching andskin lesions. Strikingly, intradermal capsaicin injec-tion promotes itch responses in these mutantmice, as opposed to pain responses in control litter-mates. Consequently, coinjection of capsaicin wasno longer able to mask itch evoked by pruritogeniccompounds. Our studies suggest that synapticglutamate release from a group of peripheral noci-ceptors is required to sense pain and suppressitch. Elimination of VGLUT2 in these nociceptorscreates a mouse model of chronic neurogenic itch.

INTRODUCTION

Itch and pain represent two distinct sensations. Moreover, it has

been long recognized that there is an antagonistic relationship

between pain and itch (Ikoma et al., 2006; Schmelz, 2010). Over

80 years ago, Lewis et al. first reported that itch sensation evoked

by histamine injection in humans can be blocked by electrical

stimuli (Lewis et al., 1927). Other painful stimuli, such as noxious

heat and noxious chemicals (mustard oil or capsaicin), can also

suppress itch (Brull et al., 1999; Graham et al., 1951; Ward

et al., 1996). Electrophysiological studies show that the firing of

spinal itch relay neuronscanbesuppressedby inputsof pain-pro-

cessing neurons (Andrew andCraig, 2001; Davidson et al., 2009).

Conversely, a blockage of pain can induce or enhance itch. For

example, pain inhibition by anesthetic compounds can enhance

itch evoked by histamine (Atanassoff et al., 1999), and intrathecal

injection of opioid analgesics is often associated with itch side

effects (Ikoma et al., 2006; Schmelz, 2010).

Several theories have been proposed to explain itch suppres-

sion by pain. The population coding hypothesis, also called

selectivity hypothesis, proposes that the senses of itch and

pain are processed along specific neural circuits or labeled lines,

but the activation of pain-sensing fibers can dominantly mask

itch, even if the stimuli activate both pain-sensing and itch-

sensing fibers (Handwerker, 2010; McMahon and Koltzenburg,

1992; Schmelz, 2010; Wood et al., 2009). The existence of

itch-specific neurons was supported initially by electrophysio-

logical studies in humans and cats (Andrew and Craig, 2001;

Schmelz et al., 1997) and subsequently by genetic and cell abla-

tion studies in mice (Liu et al., 2009; Sun and Chen, 2007; Sun

et al., 2009). For example, sensory neurons expressing the

G-protein coupled receptor Mrgpra3, which represent 4%–5%

of neurons in dorsal root ganglia (DRG) (Liu et al., 2008), are

necessary for itch evoked by chloroquine but dispensable for

pain (Liu et al., 2009). Spinal neurons expressing the gastrin-

releasing peptide receptor (GRPR) are also dedicated to itch

(Sun and Chen, 2007; Sun et al., 2009). The spatial contrast

theory, however, proposes that pain and itch can be encoded

without having itch-specific and pain-specific neurons: itch is

evoked when a minority of nociceptive fibers are activated in

a receptive field, whereas pain is evoked when a majority of

fibers are activated (Johanek et al., 2008; LaMotte et al., 2009;

Namer et al., 2008; Schmelz, 2010; Sikand et al., 2009).

However, this view seems to conflict with the actual existence

of itch-specific circuits, as mentioned above.

Thus, the coding of pain versus itch may be best explained by

the population-coding hypothesis that highlights both the

Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc. 543

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

existence of itch-specific and pain-specific sensory neurons, as

well as a dominant suppression of itch by pain. However, while

important progress has been made in identifying itch-specific

sensory neurons (Andrew and Craig, 2001; Liu et al., 2009;

Schmelz et al., 1997; Sun and Chen, 2007; Sun et al., 2009),

the neural basis underlying itch suppression by painful stimuli

has not yet been characterized.

Vesicular glutamate transporter type 2 (VGLUT2) and the

related proteins, VGLUT1 and VGLUT3, belong to a family of

transporters that package glutamate into synaptic vesicles and

are necessary for most fast excitatory synaptic transmission in

the vertebrate nervous system (Fremeau et al., 2004). These

three proteins are expressed in a partially overlapping manner

in peripheral sensory neurons in DRG (Brumovsky et al., 2007;

Seal et al., 2009). By generating and analyzing Vglut2 conditional

knockout mice, here we found that VGLUT2-dependent synaptic

glutamate release from mainly Nav1.8-expressing nociceptors

represents a neuronal component that is critical for pain sensa-

tion and itch suppression. Removal of VGLUT2 in these nocicep-

tors leads to (1) marked pain deficits, (2) sensitization of multiple

itch pathways, and (3) spontaneous development of excessive

scratching and skin lesions. Moreover, capsaicin is able to acti-

vate a normally hidden itch pathway in these mutant mice and

fails to suppress itch evoked by pruritogenic compounds. These

studies provide insight into the neural basis underlying an antag-

onistic interaction between pain and itch.

RESULTS

Generation of Vglut2 CKO MiceIn this study, we made Vglut2 conditional knockout mice by

using the Nav1.8Cre transgenic mice made by the Kuner group

(Agarwal et al., 2004). It had been reported that in Nav1.8Cre

transgenic mice, Cre activity is detected in DRG but not in the

central nervous system (CNS) (Agarwal et al., 2004). To further

determine Cre specificity, we crossed Nav1.8Cre mice with

Cre-dependent TauGFP reporter mice (Hippenmeyer et al.,

2005) in which Nav1.8Cre-active neurons were marked by the

expression of green fluorescent protein (GFP) and can be

detected by GFP immunostaining (Figure S1, available online).

A double staining of GFP with the panneural marker SCG10

(Stein et al., 1988) showed that 81.0% (884/1091) of lumbar

DRG neurons expressed GFP. Additional double staining

showed that GFP was expressed in all Nav1.8-expressing

neurons but, surprisingly, also in a small subset of Nav1.8-nega-

tive DRG neurons (Figure S1), which is different from another

Nav1.8Cre line made by the Wood group that drives reporter

expression only in Nav1.8-expressing neurons (Stirling et al.,

2005). We had also crossed Nav1.8Cre with ROSARFP reporter

mice (Madisen et al., 2010), with the resulting heterozygous

mice referred to as ROSARFP;Nav1.8Cre, in which Nav1.8Cre-

active neurons can be directly visualized with the expression of

red fluorescent protein (RFP) without the involvement of immu-

nostaining (Figure S2). As described below, ROSARFP;Nav1.8Cre

mice were used to examine the expression of other markers in

Nav1.8Cre-active DRG neurons.

To determine the distribution of VGLUT2 expression in DRG,

we performed VGLUT2 immunostaining in lumbar DRG of

544 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

ROSARFP;Nav1.8Cre mice. We found that all RFP-expressing,

and thereby Nav1.8Cre-active, neurons coexpressed VGLUT2,

albeit at heterogeneous expression levels (Figure 1A). Eighty-

eight percent and twelve percent of VGLUT2-expressing

neurons are RFP positive (Nav1.8Cre active) and RFP negative

(Nav1.8Cre negative), respectively (Figure 1A).

To make a Vglut2 conditional knockout in the DRG, we

crossed mice carrying a conditional null allele of Vglut2 (Vglut2F)

with Nav1.8Cre transgenic mice (Agarwal et al., 2004; Tong et al.,

2007), with the resulting homozygous conditional knockout mice

(Vglut2F/F;Nav1.8Cre) referred to as CKO mice (Figure S3A). The

Vglut2F/F littermates were referred to as control mice. Consistent

with the observation that 88% of VGLUT2-expressing neurons

are Nav1.8Cre active, VGLUT2 expression was greatly reduced

in CKOmice (Figure S3B). Double staining showed that in control

mice, VGLUT2 expression was expressed in all nonpeptidergic

nociceptors marked by the binding of the isolectin B4

(IB4; Figure 1B) and peptidergic nociceptors marked by the

expression of calcitonin-gene-related peptide (CGRP;

Figure 1C), albeit at various expression levels; in CKO mice,

VGLUT2 expression in these two groups of neuronswas reduced

to 0.88% and 12.3%, respectively (Figure 1). VGLUT2 expres-

sion was detected in 94.2% of TRPV1-expressing neurons in

control mice but reduced to 3.88% in CKO mice (Figure 1D).

Thus, in Vglut2 CKO mice, VGLUT2 expression is eliminated in

a majority of classical nociceptors.

To determine to what degree the expression of VGLUT1 or

VGLUT3 can compensate for the loss of VGLUT2, we examined

VGLUT1 and VGLUT3 expression in wild-type and CKO mice.

Using ROSARFP;Nav1.8Cre fate-mapping mice, we found that

15.7% of RFP-positive (Nav1.8Cre-active) neurons in lumbar

DRG at postnatal day 30 (P30) coexpressed VGLUT1, and these

neurons represent 38.0% of VGLUT1-expressing neurons

(Figure 2A). Additional double staining showed that VGLUT1

expression was detected in 25.5% of CGRP-expressing

neurons, 2.66% of IB4-positive neurons, and virtually no

TRPV1-expressing neurons (Figure 2B). Importantly, the

numbers of VGLUT1-expressing neurons in P30 lumbar DRG

were unchanged between control and Vglut2 CKO mice

(Figure 2C). With VGLUT3-GFP reporter mice, it was shown

that VGLUT3 was expressed in �10% of lumbar DRG neurons

(Seal et al., 2009). However, VGLUT3 was expressed at levels

too low to be detected by our nonradioactive in situ hybridization

(ISH). We then measured VGLUT3 expression levels in lumbar

DRG by quantitative real-time RT-PCR and found that there

was no significant change in CKO mice (data not shown).

Thus, there is no compensatory increase in VGLUT1 or VGLUT3

expression in Vglut2 CKO mice.

Based on these expression analyses, we concluded that

a majority of Nav1.8Cre-active neurons express VGLUT2 but

not VGLUT1 or VGLUT3; glutamate release from these neurons

should be eliminated in Vglut2 CKO mice. Synaptic glutamate

release in the remaining Nav1.8Cre-active neurons was expected

to be attenuated (because of a loss of VGLUT2) but not to be fully

eliminated (because of VGLUT1 and/or VGLUT3 expression).

Finally, synaptic glutamate release from Nav1.8Cre-negative

neurons, representing 19.0% of total DRG neurons, was

unaffected (summarized in Figure 2D).

Figure 1. Distribution of VGLUT2 Expres-

sion in Control and CKO Lumbar DRG

(A) Double staining of VGLUT2 protein (green) and

the RFP reporter (red) on a section through

a lumbar DRG of the RosaRFP;Nav1.8Cre fate-

mapping mice (described in Figure S2). Note

that VGLUT2 was expressed mainly in RFP-posi-

tive (Nav1.8Cre-active) neurons (arrow) but also

in a subset of GFP-negative (Nav1.8Cre-negative)

neurons (arrowhead). Quantitative data are shown

to the right.

(B–D) Double staining of VGLUT2 protein (green)

with IB4 (red) in (B), CGRP (red) in (C), or TRPV1

protein (red) in (D) on sections through P30 lumbar

DRG of control mice (top) and mutant (CKO) mice

(bottom). Quantitative data are shown to the right.

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VGLUT2 Is Required for Itch Suppression by Pain

Attenuation of Excitatory Synaptic TransmissionThe loss of VGLUT2 in a large subset of DRG neurons was

expected to cause reduced excitatory glutamatergic transmis-

sion from DRG neurons onto the dorsal spinal cord. To test

this hypothesis, we recorded spontaneous excitatory postsyn-

aptic currents (sEPSCs) from neurons in lamina II ex vivo in

isolated spinal cord slices from control and CKO mice

(Figure 3A). We indeed found that the frequency of sEPSC was

reduced by 47% in CKO mice (4.22 ± 0.63 spikes/s, n = 11) in

comparison with that in control mice (7.86 ± 1.16 spikes/s,

Neuron 68, 543–556, N

n = 9, p < 0.05) (Figure 3B). Since

VGLUT2 was eliminated in the DRG but

not in the CNS, the reduction of sEPSC

in mutant dorsal horn neurons should

be caused exclusively by an impairment

of excitatory glutamatergic transmission

by VGLUT2-dependent primary sensory

afferents, irrespective of direct or indirect

connections between recorded dorsal

horn neurons and primary sensory fibers.

The synaptic transmission defect was

further supported by a change of c-Fos

induction in specific groups of spinal

neurons after intraplantar injection of

capsaicin (see below).

We next askedwhether the attenuation

of excitatory synaptic transmission led

to molecular and anatomical changes

in DRG and the dorsal spinal cord.

The number of total DRG neurons, deter-

mined by the expression of the pan-

neuronal marker SCG10, and the

numbers of IB4-positive andCGRP-posi-

tive neurons were unchanged in lumbar

DRG between CKO and control mice

(Figure S3), suggesting that the develop-

ment and survival of DRG neurons are

unaffected. Furthermore, calcium-im-

aging studies showed that responses to

capsaicin and histamine by DRGneurons

were unchanged (Figure S4). Lamina-

specific projections of IB4-positive, CGRP-positive, and

VGLUT1-positive sensory fibers in the dorsal horn of the spinal

cord were also unaffected (Figure 3C). Furthermore, molecular

identities of dorsal horn neurons were also unaffected (Fig-

ure 3D). For example, expression of somatostatin, which is en-

riched in excitatory neurons in the superficial dorsal horn, and

the expression of dynorphin or enkephalin, which are enriched

in inhibitory neurons, were grossly unaffected in CKO mice in

comparison with control mice (Figure 3D) (Brohl et al., 2008;

Huang et al., 2008; Xu et al., 2008). Thus, the molecular and

ovember 4, 2010 ª2010 Elsevier Inc. 545

Figure 2. Distribution of VGLUT1 Expression in Lumbar DRG

(A and B) Double staining on lumbar DRG. Arrows indicate coexpression and arrowheads indicate singular VGLUT1 expression. Quantitative data are shown to

the right. (A) Double staining of VGLUT1 protein (green) and the RFP reporter (red) on a lumbar DRG section of the RosaRFP;Nav1.8Cre fate-mapping mice

(described in Figure S2). Note that VGLUT1 was expressed mainly in RFP-negative (Nav1.8Cre-negative) neurons (arrowheads) but also in a small subset of

RFP-positive (Nav1.8Cre-active) neurons (arrow). (B) Double staining of VGLUT1 protein (green) with CGRP mRNA (red, top), IB4 (red, middle), or VGLUT1

mRNA (green) with TRPV1 protein (red, bottom) on sections through wild-type lumbar DRG.

(C) Graph showing the total number of VGLUT1-expressing neurons per set of sections through adult lumbar DRG of control and CKO mice. Note that no differ-

ence in the number of VGLUT1-expressing neurons was detected (control: 470 ± 27; CKO: 468 ± 29; p > 0.05). Error bars represent SEM.

(D) Schematic showing expected loss of VGLUT2-dependent synaptic glutamate release in a subset of Nav1.8Cre-active neurons (Nav1.8Cre-act.) that only

express VGLUT2. Glutamate release was partially or fully retained in a subset of Nav1.8Cre-active neurons that express VGLUT1 and/or VGLUT3 (indicated

as VGLUT1,3) or in Nav1.8Cre-negative neurons (Nav1.8Cre-neg.) that express one or multiple VGLUT proteins (indicated as VGLUT1,2,3).

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

anatomical features of DRG and dorsal spinal cord neurons are

grossly unaffected in Vglut2 CKO mice.

Impaired Acute and Chronic PainWe next assessed how pain behaviors were affected in CKO

mice, by using Vglut2F/F littermates as a control. In comparison

with control mice, CKO mice showed similar paw withdrawal

546 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

latencies on a 50�C hot plate but increased latency at 52�Cand 55�C (Figure 4A). We also observed a small but significant

increase in withdrawal latencies in the Hargreaves radiant heat

test (Figure 4B). Increased heat pain deficit at higher tempera-

tures is analogous to the mutant phenotype seen in TRPV1 null

mice (Caterina et al., 2000; Davis et al., 2000). Consistently,

acute pain induced by capsaicin injection, which is TRPV1

Figure 3. Attenuation of Excitatory Synaptic Transmission and Unchanged Molecular or Anatomical Features in CKO Mice

(A) sEPSCs recorded from neurons in lamina II of superficial dorsal horn of control (left) and CKO mice (right).

(B) The frequency of sEPSC was reduced by 47% in CKO mice (4.22 ± 0.63 spikes/s, n = 11) in comparison with that in control mice (7.86 ± 1.16 spikes/s, n = 9,

*p < 0.05). Error bars represent SEM.

(C) Double staining of IB4 (red) and CGRP (green) (top) and immunostaining of VGLUT1 (bottom) on P30 dorsal horn sections of control and CKO mice.

(D) ISH with the indicated probes on sections through P30 lumbar (L4-L5) dorsal horn of control and CKO mutant mice.

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

dependent (Caterina et al., 2000; Davis et al., 2000), was again

markedly impaired in CKO mice (see below).

The withdrawal threshold in response to von Frey filaments

was unchanged in CKO mice (Figure 4C), implying that pain

evoked by light noxious mechanical stimuli remains intact. In

contrast, responses to intense noxious mechanical stimuli deliv-

ered by the Randall-Selitto apparatus were markedly attenuated

(Figure 4D).

We next examined two types of chronic pain: inflammatory

and neuropathic. Inflammatory pain was induced by intraplan-

tar injection of complete Freund’s adjuvant (CFA). CFA-induced

mechanical hypersensitivity, measured by reduced mechanical

threshold in eliciting painful withdrawal responses, was signifi-

cantly attenuated in CKO mice (Figure 4E). CFA-induced heat

hypersensitivity, measured by reduced latency in response to

radiant heat, was largely abolished in CKO mice (Figure 4F).

To assess neuropathic pain, we used the spared nerve injury

(SNI) model (Decosterd and Woolf, 2000). In control mice,

SNI caused a profound mechanical hypersensitivity, as indi-

cated by a marked reduction in withdrawal threshold in

response to mechanical stimuli (Figure 4G). Such SNI-induced

mechanical hypersensitivity was largely abolished in CKO mice

(Figure 4G).

Thus, a loss of VGLUT2-dependent synaptic glutamate

release from Nav1.8Cre-active neurons results in deficits in

a range of acute and chronic pain, including intense mechanical

pain, intense heat pain, capsaicin-induced spontaneous pain

(see below), CFA-induced inflammatory pain, and SNI-induced

neuropathic pain.

Sensitization of Multiple Itch Pathways in CKO MiceThe most noticeable mutant phenotype was that by the time

Vglut2 CKO mice reached two months old, a vast majority of

them had developed skin lesions (Figure 5A). The lesions

were most frequently observed around the neck and ears of

mice with brown hairs but also in other parts of the body. In

contrast, none of the control mice showed such lesions (data

not shown). We postulated that the skin lesions were caused

by itch-induced scratches. Therefore, we monitored sponta-

neous scratching at the time when CKO mice showed the

earliest sign of hair loss. We found that CKO mice showed

6-fold more spontaneous scratch bouts than control littermates

Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc. 547

Figure 4. Pain Deficits in Vglut2 CKO Mice

(A) Hot plate assay.While no statistically significant difference between control

and CKOmice was observed at 50�C (control, n = 8, 17.5 ± 2.0 s; CKO, n = 11,

22.2 ± 1.8 s; p > 0.05), a significant delay in response in CKO versus control

mice was observed at both 52�C (control, n = 8, 13.2 ± 0.7 s; CKO, n = 9,

19.7 ± 1.0 s; *p < 0.05) and 55�C (control, n = 8, 8.4 ± 0.8 s; CKO, n = 9,

13.4 ± 1.2 s; **p < 0.01).

(B) The Hargreaves radiant heat test. CKO mice showed a delay in withdrawal

latency (control, n = 9, 8.6 ± 0.4 s; CKO, n = 12, 10.0 ± 0.3 s; *p < 0.05).

(C) The von Frey assay. No difference in withdrawal thresholds (control, n = 6,

0.27 ± 0.03 g; CKO, n = 9, 0.29 ± 0.04 g; p > 0.05).

(D) The Randall-Selitto assay. CKO mice showed higher resistance to noxious

mechanical stimulation (control, n = 9, 65 ± 5 g; CKO, n = 11, 115 ± 7 g;

***p < 0.001).

(E and F) CFA-induced inflammatory pain. (E) Mechanical sensitivity (von Frey).

While control animals show a strong drop inwithdrawal threshold with von Frey

filaments after CFA injection (ANOVA; n = 6; p < 0.001), CKO mice showed no

such significant decrease (ANOVA; n = 8; p > 0.05). Importantly, a strong differ-

ence was observed between control and CKO mice (p < 0.01, two-way

repeated ANOVA). (F) Thermal sensitivity (Hargreaves). Similar to the mechan-

ical sensitivity, control animals showed a strong drop in withdrawal threshold

after CFA injection (ANOVA; n = 6; p < 0.001), while CKO mice showed no

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

548 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

(Figure 5B). In other words, excessive scratching preceded the

development of overt skin lesions.

To determine whether itching pathways were sensitized in

Vglut2 CKO mice, we injected low dosages of pruritogenic

agents and monitored scratching responses. The experiments

were done on 1-month-old mice; at this age, most CKO mice

had not yet developed excessive spontaneous scratching

behavior (Figure 5C–5H). Compound 48/80 activates a hista-

mine-dependent itching pathway (Sugimoto et al., 1998). Nape

injection of 2 mg of compound 48/80, as opposed to 100 mg

used in other studies (Sun et al., 2009), induced 2-fold increase

in scratching responses in CKO mice from that in control mice

(Figure 5C). Injection of 10 mg of compound 48/80 also induced

more scratching in CKO mice (Figure 5D). The protease-acti-

vated receptor 2 (PAR2) is a G-protein coupled receptor and

activates a histamine-independent itch pathway (Shimada

et al., 2006; Tsujii et al., 2008). Injection of a low dosage (20 mg

as opposed to 100 mg [Sun et al., 2009]) of the PAR2 agonist

SLIGRL-NH2 induced modest scratching responses in control

mice but induced 3-fold more scratch bouts in CKO mice

(Figure 5E). A serotonin derivative, a-Me-5-HT, evokes pure itch-

ing responses in mice (Imamachi et al., 2009). Injection of 30 mg

of a-Me-5-HT also induced more scratching responses in CKO

mice (841 bouts) than in control mice (377 bouts) (Figure 5F).

There was an exception; chloroquine, a compound used to treat

malaria in humans, evoked histamine-independent itch through

activating Mrgpra3 (Liu et al., 2009). We found that chloroquine

evoked similar degrees of scratching responses in Vglut2 CKO

mice and in control littermates at both low and high dosages

(Figure 6G and 6H). These observations suggested that the

loss of VGLUT2 in Nav1.8Cre-active neurons in CKOmice results

in sensitization of multiple, but not all, itch pathways.

Capsaicin Evokes Itch in CKO MiceCapsaicin activates the TRPV1 transient receptor potential ion

channel, and capsaicin-responsive neurons are essential for

the senses of both pain and itch (Basbaumet al., 2009; Imamachi

et al., 2009; Liu et al., 2009; Lynn, 1992; Shim and Oh, 2008;

Simone et al., 1989). To determine how capsaicin-evoked pain

or itch was affected in CKO mice, we used a recently developed

behavioral assay that clearly distinguishes pain versus itch:

intradermal capsaicin injection in the cheek induces pain-indica-

tive wiping by the forelimb, whereas injection of itching

compounds in the cheek induces scratching by the hind limb

(Shimada and LaMotte, 2008). Again, 1-month-old control and

CKOmicewere used; at this age,mutantmice had not yet shown

any significant difference in baseline scratching or wiping

(Figure 6). After injection of 20 mg of capsaicin, CKO mice

significant decrease (ANOVA; n = 8; p > 0.05). Importantly, a strong difference

was observed between control and CKO mice (p < 0.001, two-way repeated

ANOVA).

(G) SNI-induced neuropathic pain. After SNI, CKO mice showed no significant

decrease over time in withdrawal thresholds after SNI with von Frey filaments

(n = 6, ANOVA, p > 0.05), while control mice showed a strong drop in with-

drawal thresholds (n = 6, ANOVA, p < 0.001). Importantly, a strong difference

was observed between control and CKO (two-way repeated ANOVA,

p < 0.001). Error bars represent SEM.

Figure 5. Itch Sensitization in CKO Mice

(A) Skin lesions in the neck or body of CKO mice.

(B) Increased spontaneous scratch bouts in 2-

month-old CKO mice (control: 56 ± 13; CKO:

371 ± 64; n = 6; **p < 0.01).

(C–H) Itching responses were examined in 1-

month-old control and CKO mice before CKO

mice developed excessive spontaneous scratch-

ing. (C) Increased scratch bouts evoked by

compound 48/80 (2 mg) in CKO mice (control,

from 17 ± 6 baseline scratch bouts to 91 ± 19 after

injections; CKO, from 34 ± 11 to 290 ± 40; n = 6;

**p < 0.01). (D) Increased scratch bouts evoked

by 10 mg of compound 48/80 in CKO mice

(control, from 30 ± 9 baseline scratch bouts to

260 ± 26 after injections; CKO, from 38 ± 9 scratch

bouts to 448 ± 35; n = 8; **p < 0.01). (E) Increased

scratch bouts evoked by PAR2 agonist SLIGRL-

NH2 (20 mg) in CKO mice (control, from 22 ± 4

baseline scratch bouts to 54 ± 15 after injections;

CKO, from 47 ± 14 to 240 ± 48; n = 7; **p < 0.01).

(F) Increased scratch bouts evoked by a-Me-5-HT

(30 mg) in CKO mice (control, from 18 ± 5 scratch

bouts to 377 ± 49; CKO, from 23 ± 11 to 841 ± 78;

n = 3; **p <0.01). (G) Unchanged scratch bouts

evoked by chloroquine (25 mg) in CKO mice

(control, from 17 ± 5 scratch bouts to 126 ± 23;

CKO, from 44 ± 6 to 151 ± 18; n = 4; p > 0.05).

(H) Unchanged scratch bouts evoked by 200 mg

of chloroquine in CKO mice (control, from 16 ± 4

scratch bouts to 328 ± 53; CKO, from 34 ± 13 to

279 ± 79; n = 7; p >0.05). Error bars represent

SEM.

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

exhibited a 56% reduction in the number of wipes compared

to control mice, suggesting an attenuation of capsaicin-evoked

pain (Figure 6A and 6B). More strikingly, capsaicin induced

robust scratching responses in CKO mice, as opposed to

minimal responses in control mice (Figure 6C and 6D). Injection

of capsaicin at a lower dosage (5 mg) also induced more

scratching bouts in CKO mice than control littermates

(Figure 6E), although this low dosage evoked fewer scratching

bouts in comparison with the higher dosage of capsaicin

Neuron 68, 543–556, N

(Figure 6E versus Figure 6C). This

dramatic behavioral switch suggested

that VGLUT2-dependent synaptic gluta-

mate release is necessary for capsaicin-

evoked pain, and its removal allows

capsaicin to activate a normally masked

itch pathway.

One interpretation of this behavioral

switch is that capsaicin-evoked pain

can dominantly inhibit itch evoked

by capsaicin-sensitive pruriceptors, and

this inhibition is attenuated in CKO

mice. To further explore this possibility,

we asked whether capsaicin can sup-

press itch evoked by a-Me-5-HT, a

potent pruritogenic compound (Fig-

ure 5F) that acts through capsaicin-

sensitive neurons (Imamachi et al., 2009). In control mice, cheek

injection of a-Me-5-HT evoked robust scratching responses

(Figure 6F); strikingly, a coinjection of a-Me-5-HT and capsaicin

almost completely inhibited scratching response. In contrast,

a coinjection of a-Me-5-HT and capsaicin in CKO mice still

showed a significant scratching response (Figure 6F), with the

amount of scratch bouts comparable to that evoked by capsa-

icin alone in CKO mice (Figure 6C). These studies suggested

that a strong pain-inducing stimulus such as capsaicin can

ovember 4, 2010 ª2010 Elsevier Inc. 549

Figure 6. Scratching and Wiping Behaviors

Evoked by Capsaicin

One-month-old control and CKOmice, prior to the

development of excessive spontaneous scratch-

ing in CKO mice, were used.

(A and C) Decreased total wipes in CKO mice (A)

(control, 73 ± 15 wipes; CKO, 32.2 ± 6.7; n = 10;

*p < 0.05) and increased scratching bouts in

CKO mice (C) (control, 3.8 ± 2.0 bouts; CKO,

52.3 ± 11.3; n = 10; **p < 0.01) after capsaicin

(20 mg) injection into the cheek. Prior to capsaicin

injections, there were no differences in baseline

wiping in a 10 min period (control, 1.2 ± 0.4 wipes;

CKO, 1.3 ± 0.7; p > 0.05) or scratching (control,

1.5 ± 0.6 bouts; CKO, 4.3 ± 1.6; p > 0.05).

(B and D) Time course of capsaicin-induced

wiping and scratching (�10 to +20 min) in control

mice (blue) and CKO mice (purple). Injections

occurred at time = 0. The mean numbers of bouts

or wipes per minute were plotted.

(E) Increased scratching bouts in CKO mice

(control, 9.9 ± 2.6 bouts; CKO, 33.9 ± 8.7; n = 7;

*p < 0.05) after capsaicin (5 mg) injection. Note

that, compared to (C), lower dosage of capsaicin

evoked fewer scratching bouts in CKO mice.

(F) Capsaicin (20 mg) failed to suppress the

scratching responses evoked by a-Me-5-HT

(10 mg) in CKO mice. Note that in control mice,

itch evoked by injection of a-Me-5-HT (10 mg)

was completely suppressed by coinjection of

capsaicin (20 mg), with scratch bouts reduced

from 112.5 ± 25.4 scratch bouts (a-Me-5-HT

alone) to 3.4 ± 1.6 (a-Me-5-HT plus capsaicin)

(n = 5; *p < 0.05). In CKO mice, however, coinjec-

tion of a-Me-5-HT (10 mg) and capsaicin (20 mg)

still caused significant scratching responses

(49.8 ± 11.9 scratch bouts; n = 5; *p < 0.05), which

is comparable to that evoked by singular injection

of capsaicin (20 mg) in CKO mice.

Error bars in (A), (C), (E), and (F) represent SEM.

The abbreviation Caps. means capsaicin.

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

dominantly mask a strong itch signal such as a-Me-5-HT, and

this pain-induced itch inhibition is markedly attenuated in CKO

mice.

Loss of VGLUT2 in Mrgpra3-Expressingand GRP-Expressing Neurons in Vglut2 CKO MiceThe enhanced or normal itching responses seen in Vglut2 CKO

mice suggested that VGLUT2 expression in Nav1.8Cre-active

sensory neurons is dispensable for itch. To further look into

this issue, we examined how VGLUT2 expression was affected

in established or putative pruriceptors. Mrgpra3-expressing pru-

riceptors mediate itch evoked by chloroquine (Liu et al., 2009).

We found that these pruriceptors expressed VGLUT2

(Figure 7A), but not VGLUT1 (Figure 7B), in control mice, and

VGLUT2 expression in these neurons was eliminated in Vglut2

CKO mice (Figure 7A). Sensory neurons expressing the

gastrin-releasing peptide (GRP) are putative pruriceptors (Sun

and Chen, 2007). In control mice, GRP was expressed at high

levels in a subset of small diameter neurons (Figure 7C, arrows),

as reported previously (Sun and Chen, 2007). Low levels of GRP

550 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

expression were detected in many small and large DRG neurons

(Figure 7C, arrowheads), consistent with a more recent report

(Liu et al., 2009). In GRPhigh neurons, VGLUT2 expression was

detected in control mice but was eliminated in Vglut2 CKO

mice (Figure 7C, arrows). Again, no VGLUT1 expression was

detected in GRPhigh neurons (Figure 7D). With enhanced or unaf-

fected itching responses observed in Vglut2 CKO mice, these

data suggest that under the Vglut2 CKO genetic background,

glutamate release from Mrgpra3-expressing and GRPhigh

neurons appears to be dispensable for itch.

A Change in Spinal Neuron Activation after IntraplantarCapsaicin Injection in Vglut2 CKO MiceSpinal inhibitory neurons have recently been suggested to

play a role in preventing itch sensitization (Ross et al., 2010). It

has also been known for a while that noxious stimuli, such as

intraplantar capsaicin injection, were able to activate spinal

inhibitory neurons (Binshtok et al., 2007; Zou et al., 2002). We

therefore asked whether such activation was affected in Vglut2

CKO mice. We used the induction of c-Fos to identify

Figure 7. Loss of VGLUT2 and Lack of

VGLUT1 in Mrgpra3-Expressing Neurons

and GRPhigh DRG Neurons

(A and C) Double staining of VGLUT2 protein

(green) with Mrgpra3 mRNA (red) in (A) or with

GRP (red) in (C) on sections through control (top)

and CKO (bottom) lumbar DRGs. Arrows and

arrowheads in (C) indicate neurons with high and

low levels of GRP expression, respectively. Note

that in control mice, Mrgpra3 or GRPhigh was

coexpressed with VGLUT2. In CKO mice,

VGLUT2 expression was eliminated in all

Mrgpra3-expressing (A) or GRPhigh neurons (C,

arrows), whereas a subset of GRPlow neurons re-

tained VGLUT2 (C, arrowheads). Quantitative

data are shown to the right.

(B and D) Double staining of VGLUT1 protein

(green) with Mrgpra3 mRNA (red) in (B) or with

GRP (red) in (D) on sections through lumbar

wild-type DRG. Note that VGLUT1 was not ex-

pressed in GRPhigh neurons (D, arrows). Quantita-

tive data are shown to the right.

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

capsaicin-responsive spinal neurons (Gao and Ji, 2009; Hunt

et al., 1987; Zou et al., 2002). Previous studies showed that

spinal inhibitory neurons can be identified by the expression of

a set of neuropeptides, including neuropeptide Y (NPY) and

enkephalin (ENK) (Brohl et al., 2008; Huang et al., 2008; Xu

et al., 2008). We found that, after capsaicin injection in hindpaw,

c-Fos expression was detected in 40.1% of NPY-expressing

neurons at the ipsilateral side of control mice but reduced to

11.9% in Vglut2 CKO mice (Figure 8A), a 70.3% reduction.

The reduction of c-Fos-positive neurons was not due to a loss

of NPY-expressing neurons per se, as suggested by the normal

numbers of NPY-expressing neurons in CKO versus control

mice (data not shown). Interestingly, c-Fos induction in ENK-ex-

pressing neurons was unaffected in CKO mice (Figure 8B).

These data suggest that VGLUT2-dependent synaptic gluta-

mate release from Nav1.8Cre-active neurons is required for

capsaicin to activate a specific (NPY-expressing) subset of

spinal inhibitory neurons.

Neuron 68, 543–556, N

GRPR-expressing spinal neurons are

required to process itching information

(Sun et al., 2009). With the finding that

capsaicin evokes itch, rather than pain,

in CKO mice, we next asked whether

capsaicin injection could differentially

activate GRPR-expressing neurons in

control versus CKO mice. We found

that after intraplantar capsaicin injec-

tion, there was indeed a significant,

albeit modest, increase of c-Fos induc-

tion in GRPR-expressing neurons from

18.5% ± 0.4% in control mice to

27.2% ± 1.5% in CKO mice (p < 0.05),

a 47% increase (Figure 8C). All together,

these studies show that upon removal

of VGLUT2 in Nav1.8Cre-active neurons,

capsaicin on one hand fails to activate NPY-expressing spinal

inhibitory neurons but, on the other hand, causes an increase

of activation of GPRP-expressing spinal itch relay neurons.

DISCUSSION

VGLUT2-Dependent Glutamate Release from DRGNeurons Is Necessary for Acute and Chronic PainIn this study, we have generated Vglut2 CKO mice in which

Vglut2 was removed from Nav1.8Cre-active DRG neurons.

The loss of VGLUT2 in these neurons leads to impaired glutama-

tergic transmission, as indicated by reduced sEPSC frequency

in postsynaptic dorsal horn neurons, although additional

electrophysiological recording is needed to determine exact

deficits in synaptic transmission in these mice. Behavioral

studies show that VGLUT2-dependent glutamate release from

Nav1.8Cre-active DRG neurons is required to sense a range of

acute and chronic pain, including intense mechanical pain,

ovember 4, 2010 ª2010 Elsevier Inc. 551

Figure 8. Changes of c-Fos Induction in Spinal Neurons after Intradermal Capsaicin Injection in the Hindpaw

(A) Transverse sections through dorsal spinal cord at L4-L5 levels of P30 control mice and CKO littermates after capsaicin injection into one hindpaw. Double

staining of c-Fos protein by immunostaining (green) and NPY mRNA by ISH was shown. The bright-field ISH signal (left) was converted into pseudofluorescent

signal (red, middle and right). Arrows indicate colocalization of NPY and c-Fos, and arrowheads indicate singular NPY expression. (Right) Graph shows the

percentage of NPY-expressing neurons that coexpressed c-Fos in the ipsilateral superficial spinal cords of P30 control (open bars) and CKO (gray bars)

mice. Error bars represent SEM. Note that c-Fos induction in NPY-expressing neurons was reduced in CKO mice, from 40.1% ± 3.5% in control mice to

11.9% ± 2.4% in CKO mice (***p < 0.001).

(B and C) are the same as in (A), except that NPY expression was replaced by that of ENKmRNA in (B) and GRPRmRNA in (C). Note that c-Fos induction in ENK-

expressing neurons in the ipsilateral superficial spinal cords after capsaicin injection was unchanged (11.5% ± 1.6% in control mice and 10.9% ± 1.1% in CKO

mice, p > 0.05). In contrast, c-Fos induction in GRPR-expressing neurons was increased in CKO mice, from 18.5% ± 0.4% in control mice to 27.2% ± 1.5% in

CKO mice (*p < 0.05).

The abbreviation Ipsi means ipsilateral to the injection side. Error bars represent SEM.

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VGLUT2 Is Required for Itch Suppression by Pain

capsaicin-evoked pain, intense heat pain, and inflammatory and

neuropathic pain. It has been previously reported that ablation of

Nav1.8-expressing neurons causes similar deficits in mechan-

ical and inflammatory pain but, surprisingly, without affecting

neuropathic pain or heat pain (Abrahamsen et al., 2008). How

could we explain the deficits in neuropathic and heat pain after

the loss of VGLUT2 in Nav1.8Cre-active neurons? This discrep-

ancy could be caused by the use of two different Nav1.8Cre

mice. The Nav1.8Cre mice used for cell ablation were made by

the knockin strategy (Abrahamsen et al., 2008) and drove

reporter expression that matches Nav1.8 expression in DRG

(Stirling et al., 2005). However, the Nav1.8Cre mice used in this

study were made through a transgenic approach (Agarwal

et al., 2004) and drove reporter expression not only in all

Nav1.8-expressing neurons but also in a small subset of

Nav1.8-negative neurons (Figure S1). It is possible that the trans-

genic approach might drive Cre expression at higher levels than

the knockin approach, such that neurons normally expressing

low levels of Nav1.8 might contain sufficient Cre activity.

552 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

The loss of VGLUT2 expression in these extra Nav1.8-negative

neurons in Vglut2 CKOmice might then contribute to the impair-

ment of neuropathic and heat pain. Alternatively, compensatory

mechanisms may develop after ablation of Nav1.8-expressing

neurons but not after removal of VGLUT2.

Neuropathic pain is also impaired in conventional Vglut2

heterozygous null mice (Leo et al., 2009; Moechars et al.,

2006). Because VGLUT2 is broadly expressed in the CNS,

including the dorsal horn of the spinal cord, we cannot be certain

of the underlying cellular basis of neuropathic pain deficit in

those heterozygous null mice. By analyzing Vglut2 CKO mice,

we have now clearly shown that VGLUT2-dependent synaptic

glutamate release fromDRG neurons is essential for neuropathic

pain; this association is also consistent with the report that

VGLUT2 expression is elevated in a subset of nociceptors

following nerve injury (Brumovsky et al., 2007).

VGLUT3-expressing DRG neurons represent low-threshold

mechanoreceptors (Seal et al., 2009). Interestingly, mice lacking

Vglut3 also show a partial defect in sensing intense mechanical

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

pain and a marked deficit in developing persistent mechanical

hypersensitivity following inflammation or nerve injury (Seal

et al., 2009), suggesting that glutamate release from both

VGLUT3-expressing and VGLUT2-expressing neurons is

involved in sensing mechanical pain. Meanwhile, the expression

patterns of VGLUT1, VGLUT2, and VGLUT3 in DRG neurons

could explain why inflammatory heat hypersensitivity, which is

known to be dependent on TRPV1 (Caterina et al., 2000; Davis

et al., 2000), is abolished in Vglut2 CKO mice but not in Vglut3

mutant mice (Seal et al., 2009). Most TRPV1-expressing neurons

show detectable expression of VGLUT2 (Figure 1) but not

VGLUT1 (Figure 2) or VGLUT3 (Seal et al., 2009). In Vglut2

CKO mice, only 4% of TRPV1-expressing neurons retain

VGLUT2 expression. In other words, excitatory glutamatergic

synaptic transmission is eliminated from most TRPV1-express-

ing neurons in Vglut2 CKO mice, which may render them func-

tionally silent, thereby explaining the impaired inflammatory

heat hyperalgesia in these mice.

VGLUT2-Dependent Glutamate Release fromNav1.8Cre-Active Neurons Is Required to Suppress ItchOur studies provide insight into the coding of pain versus itch by

showing that VGLUT2-dependent glutamate release from

Nav1.8Cre-active peripheral nociceptors represents a neuronal

component that is required to sense pain and suppress itch.

Removal of this component leads to (1) marked pain deficits,

(2) sensitization of both histamine-dependent and histamine-

independent itch pathways, (3) spontaneous development of

excessive scratching and eventual skin lesions, (4) a failure of

capsaicin to dominantly mask a strong itch signal, and (5) direct

paradoxical promotion of itch by capsaicin. Our studies also

suggest that neurons that retain glutamate release in

Vglut2 CKO mice, including Nav1.8Cre-negative neurons plus

Nav1.8Cre-active neurons that express VGLUT1 and/or VGLUT3,

are insufficient to prevent itch sensitization, even though they

are sufficient to mediate light mechanical pain (measured by

von Frey assay) and light heat pain (measured by a hot plate

at 50�C).Our studies raise an intriguing question regarding the role of

synaptic glutamate release from peripheral pruriceptors in pro-

cessing itching information. In Vglut2 CKO mice, VGLUT2 was

eliminated in 81% of DRG neurons. The loss was not just

confined to pain-sensing neurons, but also occurred in

Mrgpra3-expressing pruriceptors and in putative pruriceptors

marked by GRPhigh expression (Figure 7) (Liu et al., 2009;

Sun and Chen, 2007; Sun et al., 2009). Moreover, Mrgpra3-ex-

pressing and GRPhigh neurons do not express VGLUT1

(Figure 7) and may also not express VGLUT3 since most of

these neurons coexpressed CGRP (Sun and Chen, 2007)

(data not shown), and CGRP-expressing neurons do not

express VGLUT3 (Seal et al., 2009). With the loss of VGLUT2

and a lack of VGLUT1 and VGLUT3 expression in these estab-

lished and putative pruriceptors, it is surprising to observe that

itch evoked by a range of pruritogenic compounds is either

enhanced or unaffected in Vglut2 CKO mice, including chloro-

quine-evoked itch that is mediated by Mrgpra3-expressing

pruriceptors. We envision the following two possibilities. First,

glutamate release from pruriceptors might be dispensable for

itch. In other words, pruriceptors may use other transmitters

such as GRP to mediate itch. Consistent with this, intrathecal

injection of GRP is sufficient to evoke scratching response

and histamine-independent itch is markedly impaired in

GRPR mutant mice (Sun and Chen, 2007). Alternatively, gluta-

mate release from pruriceptors may normally act to remove

tonic itch inhibition by pain-processing neurons (Andrew and

Craig, 2001). In Vglut2 CKO mice, the loss of pain may have

already removed such inhibition; as a result, glutamate release

from pruriceptors becomes dispensable for the transmission of

itching information.

How do the pain loss and itch sensitization observed in Vglut2

CKO mice fit into current itch theories? The spatial contrast

hypothesis (Johanek et al., 2008; Namer et al., 2008; Schmelz,

2010) proposes that itch is coded when a small subset of noci-

ceptor fibers is activated in a receptive field, whereas pain is

encoded when more nociceptor fibers are activated. The loss

of VGLUT2 in 81% of DRG neurons in Vglut2 CKO mice will

certainly lead to a great reduction in the density of functional

nociceptor fibers in the skin, and, according to this theory,

such reduction should result in enhanced itching. However, the

argument of the spatial contrast theory that pain and itch can

be coded without having pain-specific and itch-specific fibers

conflicts with the actual existence of itch-specific sensory

neurons, such as Mrgpra3-expressing DRG neurons and

GRPR-expressing spinal neurons (Andrew and Craig, 2001;

Liu et al., 2009; Schmelz et al., 1997; Sun and Chen, 2007; Sun

et al., 2009). Moreover, a reduction in density of functional noci-

ceptor fibers in the skin is not automatically linked with itch

sensitization. For example, ablation of Mrgprd-positive polymo-

dal nociceptors that densely innervate the skin epidermis has no

impact on itch (Imamachi et al., 2009; Rau et al., 2009; Zylka

et al., 2005). Consistently, in Vglut2 CKO mice, a lower dosage

of capsaicin, which is supposed to activate fewer capsaicin-

sensitive fibers and should enhance itch according to the spatial

contrast hypothesis, actually causes reduced scratching

responses in comparison with a higher dosage of capsaicin

(Figure 6).

Thus, with increasing evidence arguing against the spatial

contrast theory, several investigators suggested that the coding

of pain versus itch may be best explained by the population-

coding hypothesis, which emphasizes both the existence of

itch-specific and pain-specific neural components and the

dominant suppression of itch by pain (Handwerker, 2010;

McMahon and Koltzenburg, 1992; Wood et al., 2009). According

to this hypothesis, VGLUT2-dependent glutamate release from

Nav1.8Cre-active DRG neurons should represent a neural

component that is necessary for pain sensation and itch

suppression. For example, in the absence of this component in

Vglut2 CKO mice, a coinjection of a strong pain-inducing

compound (capsaicin) is no longer able to mask itch evoked

by a strong pruritogenic compound (Figure 6). Moreover,

because many itch-sensing neurons respond to capsaicin

(Imamachi et al., 2009; Liu et al., 2009; Lynn, 1992; Shim and

Oh, 2008), the loss of pain-induced inhibition of itch may explain

why capsaicin is able to activate a normally hidden itch path-

way in these mutant mice (Figure 6). The enhanced itching

responses observed in Vglut2 CKO mice do raise the following

Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc. 553

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

question: why do itching compounds fail to evoke maximum

amount of itch in wild-type mice? It should be noted that most

itching compounds (such as histamine) in fact activate both

itch-sensing and pain-sensing neurons (Atanassoff et al.,

1999), and the amount of itch evoked by a pruritogenic

compound is normally attenuated by this pain component. For

example, anesthetic blockage of pain can greatly enhance hista-

mine-evoked itch in humans (Atanassoff et al., 1999). In Vglut2

CKOmice, the loss of pain (and itch inhibition by pain) maymimic

anesthetic treatment in humans, thereby allowing itching

compounds to evoke enhanced responses.

How could painful stimuli suppress itch? It was proposed that

painful stimuli may activate inhibitory neurons in the dorsal spinal

cord, which in turn inhibit itch-processing neurons (Andrew and

Craig, 2001; Davidson et al., 2009; Handwerker, 2010). This

hypothesis is strongly supported by a recent study showing

that Bhlhb5-dependent spinal inhibitory neurons are involved in

itch suppression, and their developmental impairment leads to

sensitization of multiple itch pathways (Ross et al., 2010).

Intriguingly, in Vglut2 CKO mice, capsaicin injection fails to

activate NPY-expressing inhibitory neurons in the spinal cord;

moreover, there is a modest increase in activation of GRPR-ex-

pressing itch relay neurons. Future studies will be warranted to

determine whether capsaicin-responsive pain fibers connect

with NPY-expressing inhibitory neurons to suppress itch and

whether the development of these inhibitory neurons is depen-

dent on Bhlhb5.

Concluding Remarks and a Mouse Modelof Neurogenic ItchOur studies provide insight into the coding of pain versus itch.

First, VGLUT2-dependent glutamate release from Nav1.8Cre-

active neurons is required to sense pain and suppress itch.

The itch sensitization observed in Vglut2 CKO mice is probably

caused by a loss of pain-induced inhibition of itch. Alternatively,

there are two separate populations of VGLUT2-dependent DRG

neurons: one for pain sensation and one for itch suppression.

Second, synaptic glutamate release from established

(Mrgpra3-expressing) and putative (GRPhigh) pruriceptors

appears to be dispensable for the transmission of itching infor-

mation in the Vglut2 CKO background, as indicated by the loss

of VGLUT2 expression and a lack of VGLUT1/3 expression in

these neurons. This observation raises the possibility that pruri-

ceptors may use other transmitters such as GRP tomediate itch.

Third, removal of VGLUT2 from Nav1.8Cre-active DRG neurons

creates a mouse model of chronic neurogenic itch, as indicated

by sensitization of multiple itch pathways and development of

excessive spontaneous scratching and skin lesions. Importantly,

the mutant phenotypes seen in Vglut2 CKO mice are similar to

the symptoms seen in human patients suffering from chronic

itch. In such patients, painful stimuli do not just fail to suppress

itch but, paradoxically, promote itch (Hosogi et al., 2006; Ikoma

et al., 2004; Ishiuji et al., 2008; Schmelz, 2010), exactly analo-

gous to capsaicin-evoked itch seen in Vglut2 CKO mice. The

creation of a mouse model of chronic itch that shares key

features with human patients could be invaluable for future

mechanistic and intervention studies.

554 Neuron 68, 543–556, November 4, 2010 ª2010 Elsevier Inc.

EXPERIMENTAL PROCEDURES

Animals

The generation of mice carrying the floxed Vglut2 allele, Nav1.8Cre transgenic

mice, RosaRFP mice, and the Tau-lox-STOP-lox-mGFP-IRES-nlsLacZ-neo

mice has been described previously (Agarwal et al., 2004; Hippenmeyer

et al., 2005; Madisen et al., 2010; Tong et al., 2007). For histochemical studies,

mice at P30 were used. For behavioral analyses, 1- to 2-month-oldmutant and

control littermates were used. All behavioral test protocols were approved by

the Institutional Animal Care and Use Committee at Dana-Farber Cancer

Institute.

ISH and Immunostaining

ISH procedures and the probes (CGRP, Nav1.8, SCG10, TrkB, parvalbumin,

somatostatin, dynorphin, and enkephalin) have been described previously

(Chen et al., 2006; Cheng et al., 2004, 2005; Ma et al., 1999; Xu et al., 2008).

Immunohistochemistry (IHC) using rabbit anti-VGLUT1 (1/1000, Swant,

Switzerland), guinea pig anti-VGLUT2 (1/200, Frontier Institute Co., Japan),

chicken anti-GFP (1/1000, Invitrogen, USA), rabbit anti-TRPV1 (1/1000, Ab-

Cam, USA), mouse anti-NF200 (1/200, Sigma, USA), rabbit anti-c-Fos

(1/1000, Santa Cruz, USA), rabbit anti-GRP (1/1000, Immunostar, USA; diluted

in 0.3% of triton x-100, 0.2% of BSA, and 5% of goat serum in PBS), or

IB4-biotin (10 mg/ml, Sigma, USA) was carried out as previously described

(Chen et al., 2006). The ISH/IHC double staining was performed as previously

described (Liu et al., 2008). For RFP/IHC double staining, the RFP fluorescent

signal was directly photographed followed by performance of single IHC. The

fluorescent IHC signals were then merged with the RFP signal.

Cell Counting

L4-L5 lumbar DRG were dissected from two to three pairs of mutant and

control mice. Three to four mutant or control DRG were used to prepare eight

adjacent sections at 12 mm thickness. Each set was processed for immunos-

taining or used for ISH with the gene of interest. Only cells containing nuclei

and showing levels of expression or staining clearly above background were

counted. Averages and standard errors of the mean (SEM) were calculated

and the difference between control and mutant samples was subjected to

a Student’s t test, with p < 0.05 considered significant.

Patch-Clamp Recordings in Spinal Slices

Patch-clamp recording in spinal slices was done as previously reported

(Kawasaki et al., 2008) with L4-L5 from Vglut2F/F; Nav1.8Cre CKO, and

Vglut2F/F control littermates (3–5 weeks old). More detailed procedures are

provided in Supplemental Experimental Procedures.

Surgery

The SNI model for neuropathic pain was performed on adult mice (P30 to P60)

as described for rats (Decosterd and Woolf, 2000) and as we previously did in

mice (Chen et al., 2006). More detailed procedures are provided in Supple-

mental Experimental Procedures.

Pain and Itch Behavioral Test

Vglut2F/F; Nav1.8Cre CKO, and Vglut2F/F control littermates of 1–2 months of

age were used. All pain and itch behavioral tests were performed as previously

described (Chen et al., 2006; Shimada and LaMotte, 2008) with minor modifi-

cations. More detailed procedures are provided in Supplemental Experimental

Procedures.

Double Staining ofCapsaicin-Induced c-Foswith Neuropeptides and

GRPR

One-month-old Vglut2F/F; Nav1.8Cre CKO, and Vglut2F/F control littermates

were given a 2.5 mg/10 ml intraplantar injection of capsaicin under anesthesia.

Two hours later, the L4-L5 spinal cords were dissected and treated as

previously described (Liu et al., 2008). c-Fos immunostaining used 30 min

incubation at room temperature for the primary and secondary antibodies,

respectively, followed by ISH with NPY, enkephalin, or GRPR probe as previ-

ously described (Liu et al., 2008). Three to five pairs of control and CKO mice

were used for quantitative analysis.

Neuron

VGLUT2 Is Required for Itch Suppression by Pain

Statistical Analyses of Pain and Itch Behaviors

For itching behaviors, the mean number of scratching bouts or wipes and SEM

during the period were calculated for each group. The difference between the

mutant and control group was subjected to a Student’s t test (two-sample

assuming unequal variance), with p < 0.05 considered significant. For acute

mechanical and heat pain, data were calculated as the average of two inde-

pendent tests performed on two consecutive days and subjected to the

Student’s t test. For CFA-induced inflammatory and SNI-induced neuropathic

pain, time-course measurements were analyzed by both analysis of variance

between groups (ANOVA) (within each group) and two-way repeated

ANOVA (R, R Development Core Team, Austria) (to compare groups), with

p < 0.05 accepted as statistically significant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures

and four figures and can be found with this article online at doi:10.1016/j.

neuron.2010.09.008.

ACKNOWLEDGMENTS

We thank Dr. Rohini Kuner for the Nav1.8Cre mice, Dr. Silvia Arber for the

TauGFP reporter mice, and Dr. Zhou-Feng Chen for the GRPR probe. We thank

Drs. Clifford Woolf, Sang-Kyou Han, Charles Stiles, and Fu-chia Yang for crit-

ical comments on the manuscript. The work is supported by the National Insti-

tutes of Health National Institute of Dental and Craniofacial Research (Grant

R01DE018025) and National Institute of Neurological Disorders and Stroke

(Grants R01NS047710 and P01NS047572).

Accepted: August 18, 2010

Published: November 3, 2010

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III. Discussion

42

Discussion

A. Generation of primary somatic sensory neuron diversity

The great diversity of cell types that composes the nervous system was first appreciated

by the founder of modern neuroscience (Ramon Y Cajal) more than one hundred years ago. Ever

since, one of the fascinating goals in developmental neuroscience has been to understand how

this diversity is created. In recent years several principles have emerged that help to understand

how this diversity is created. Extrinsic signals pattern the neural tube, giving rise to stereotyped

neurons under restricted spatial and temporal cues. This spatial and temporal patterning leads to

expression of intrinsic transcription factors in specific populations of neural precursors and in

postmitotic neurons. These transcription factors then control the differentiation and maturation of

specific neuronal cell types. In the first part of my thesis, I have gained insight into how

transcription factors act in combination to control the molecular identity and axonal central

targeting of the TrkA lineage of somatosensory neurons, thereby helping to understand how

sensory circuits processing distinct sensory modalities are assembled.

A1. Runx1 and Tlx3 are selector-like factors that act in combination to

control the development of the Ret+ subset of TrkA lineage sensory

neurons.

TrkA lineage neurons in DRG include nociceptors, thermoceptors, mechanoceptors, and

pruriceptors. Globally, the TrkA lineage neurons are segregated during perinatal and postnatal

ages into two populations: one that retains TrkA expression and another where TrkA is

downregulated and Ret begins to be expressed. The former population is characterized by the

expression of the neuropetides CGRP and SP, referred to as peptidergic and the latter by the

binding of isolectin B4 and the expression of Ret, referred to as non-peptidergic population.

Before I started my thesis work, our lab and others had shown that the runt-domain transcription

factor Runx1 plays a pivotal role in controlling the specification of the non-peptidergic Ret

positive neurons over the TrkA+ peptidergic neurons, by 1) regulating the expression of

neurotrophin receptors, neuropeptides, and a large cohort of ion channels and receptors, as well

as 2) establishing specific afferent central target selections (Chen et al., 2006; Kramer et al.,

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2006; Marmigere et al., 2006). Because it controls such a plethora of phenotypes, the functional

versatility of Runx1 could be somewhat comparable to the function of hox genes in the

development of distinct types of cells, tissues and organs (Mann and Carrol, 2002). In line with

this, Runx1 activity resembles a selector gene, capable of activating a large molecular program

associated with Ret+ nonpeptidergic neurons and repressing another large molecular program

associated with TrkA+ peptidergic neurons (Liu and Ma, 2011). My thesis work shows that

Runx1 needs to act together with Tlx3 to control the segregation of these two populations of

somatic sensory neurons.

Tlx3 is broadly expressed in DRG neurons from embryonic to adult stages, including

both TrkA+ peptidergic and Ret+ non-peptidergic populations. By analyzing mice with a

conditional knockout of Tlx3 in DRG, we showed that Tlx3 is required for proper segregation of

Ret+/IB4+ versus TrkA+ neurons, as indicated by a dramatic decrease in Ret expression and a

concurrent expansion of TrkA in prospective IB4+ non-peptidergic neurons. However, the cell

fate switch is incomplete, as indicated by no concurrent expansion of CGRP; furthermore,

lamina specific innervation of these neurons to the spinal cord is grossly normal. In comparison,

Runx1 knockout shows a more complete fate switch (Chen et al., 2006). Importantly, Runx1 and

Tlx3 expression is independent of each other. Thus, Runx1 and Tlx3 act in combination to

activate Ret and suppress TrkA, but Runx1 also uses Tlx3-independent pathways to suppress

CGRP and to control central afferent innervations.

Within Ret+/IB4+ non-peptidergic neurons, Tlx3 is also required for the expression of

multiple sensory channels and receptors, including the TRP channels for pain and temperature

senses, ATP-gated channels for pain sense, Mrgpr class GPRRs for itch sense, and others.

Strikingly, virtually all Tlx3-dependent receptors and channels are also dependent on Runx1.

Gain-of-function studies further show that a combination of Runx1 and Tlx3 is sufficient to

induce ectopic expression of these channels and receptors. These findings suggest that the

broadly expressed Tlx3 serves as an essential competent factor that allows Runx1 to specify the

molecular identities of a cohort of Ret+ non-peptidergic nociceptors, thermoceptors and

pruriceptors.

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The finding that Runx1 and Tlx3 control many molecular and anatomical features of the

TrkA lineage neurons also consolidates the idea that relatively few transcription factors are used

to coordinate the terminal differentiation of a specific group of neurons. Such selector-like

control mechanisms are evolutionally conserved, occurring in organisms including C elegans,

flies, and mammals (Hobert O, 2008 & 2010). Besides Runx1 and Tlx3, other examples in

mammals include the homeodomain transcription factor Crx that controls rod and cone

photoreceptor differentiation (Blacksaw et al., 2001;Corbo et al., 2010; Hsiau et al, 2007;

Livesey and Cepko 2001; Swaroop et al., 2010), the Pet-1 ETS domain transcription factor that

drives serotonergic neuron differentiation ( Hendricks et al 1999& 2003), and the Nurr1 and

Pitx3 transcription factors that control midbrain dopaminergic neuron differentiation (Smidt and

Burbach, 2009). Besides controlling primary sensory neuron development, Tlx3 and its related

protein Tlx1 also coordinate the development of a large cohort of excitatory neurons in the dorsal

spinal cord by regulating virtually most known features in these neurons (Cheng et al., 2004; Xu

et al., 2008). It should be noted that selector proteins are often defined by directly regulating a

large terminal differentiation program (Mann and Carrol, 2002; Chen et al., 2006; Hobert O.,

2008; Cheng et al., 2004; Samad et al., 2010). To further consolidate the idea that Runx1 and

Tlx3 are selector genes, chromatin immunoprecipitation will be needed to prove that these two

transcription factors directly control terminal features of nociceptors, thermoceptors, and

pruriceptors.

A2. How do Runx1 and Tlx3 form a combinatorial code?

The process of neuronal cell subtype specification that is controlled by a combination of

transcription factors has been known for decades (Zhou et al., 2002; Lee and Pfaff, 2001; Jessell,

2000). How exactly Runx1 and Tlx3 act in combination is still largely unknown. However,

distinct control modes have been demonstrated in other model systems.

Firstly, the Lim-homeodomain proteins and their cofactors form a hexameric complex to

control the choice between motoneurons versus interneurons in mice (Thaler et al. 2002).

Similarly, in C. elegans, the LIM- and POU-homeodomain proteins, such as MEC-3 and UNC-

86, interact genetically and physically to regulate touch sensory neuron differentiation

(Rockelein et al., 2000, Xue et al., 1993). Interestingly, the mammalian homologues of MEC-3

45

and UNC-86, Islet1 and Brn3a, respectively, are involved in somatic sensory neuron

development, partly by controlling Runx1 expression (Dykes et al., 2011). It will be interesting

to determine if Brn3a and Islet1, or Runx1 and Tlx3, also form complexes.

Secondly, transcription factors may operate sequentially in regulating the same target

gene. In C. elegans, the Hobert group shows that in establishing the bilaterally symmetrical but

functionally distinct pair of gustatory sensory identities, the transcription factor tbx37/38

operates at the four-cell stage to establish a competent chromatin state that is later necessary for

another transcription factor CHE-1 to activate the target gene, lsy-6, whose differential

expression determines the asymmetry in left and right gustatory neurons (Cochella and Hobert.,

2012). In early B cell lymphocytes, Runx1 is required to modify the chromatin states at early

precursors, which is required for Pax5 to activate the mb1 target gene at late stages (Maier et al.,

2004). My thesis work suggests that Runx1 and Tlx3 may also use this control mode to establish

the expression of MrgprA3 in pruriceptors. Runx1 activity before E17 and Tlx3 activity beyond

E17 are required for Mrgrpa3 expression. However, it is not clear how exactly they work

together to control MrgprA3 expression. It is possible that Tlx3 cannot bind target promoters

unless the chromatin is opened by Runx1 at an earlier stage. Hence these two transcription

factors would work sequentially in order to control MrgprA3 neuronal identity.

A3. How are different submodalities further segregated?

The finding that Runx1 and Tlx3 are required for proper development of a large cohort of

functionally distinct somatic sensory neurons raises a question as to how these modalities are

further segregated during development. A few studies, mainly done by colleagues in Ma lab,

have provided some insight into this important question.

Firstly, Runx1-dependent neurons are subdivided into two large groups, based on

persistent and transient Runx1 expression. Runx1 transient neurons include MrgprA3+

pruriceptors and Mrgprb4+ c-mechanoreceptors associated with pleasant touch. It turns out that

genetically Runx1 first acts as an activator at early embryonic stages, but later switches to

become a repressor for MrgprA3 and Mrgprb4. As a result, persistent MrgprA3 and Mrgprb4

expression can only be maintained in Runx1 transient neurons (Luo et al., 2008). Runx1-

46

persistent neurons include MrgprD+ mechanical nociceptors, VGLUT3+ c-mechanoreceptors,

and TRPM8+ cold-sensitive thermoceptors (Chen et al., 2006; Luo et al., 2009; Lou et al., 2013;

Samad et al., 2010). It is possible that Runx1 likely serves as a constitutive activator to maintain

the expression of these sensory features. Thus, dynamic expression and activity provides a way

to segregate tow populations of Runx1-dependent neurons.

Secondly, many Runx1-dependent neurons innervate specific targets. For example,

Mrgprd+ neurons innervate the skin epidermis, whereas MrgrpA3+ and Mrgrpb4+ neurons

innervate only hairy skin (Zylka et al., 2005;Vrontou et al.,2013;Liu and Ma, 2011). Both our lab

and several other labs showed that target-derived signals can contribute to the activation of

specific sensory channels and receptors. For example, TrkA signaling, besides being pivotal for

axons to reach their target, is also required for expression of most Runx1 dependent genes (Luo

et al., 2007). Ret mediated signaling is necessary for the expression of a subset of Runx1

dependent genes, such as Trpa1, MrgprA3 and MrgprB4, (Luo et al., 2007). Met signaling is

required for the expression of a portion of TRPA1 and TRPV1 high expressing neurons (Gascon

et al., 2010) and Smad-4 is selectively required for the expression of MrgpdB4 (Liu et al., 2008).

Collectively, intrinsic factors, such as Runx1 and Tlx3, may act in combination with target

signals to control sensory subtype specification.

A4. A summary of sensory neuron subtype specification and their Implication

on sensory coding

In overall, the development of TrkA lineage sensory neurons is subjected to a hierarchical

control. Firstly, these neurons are broadly segregated into Runx1/Tlx3-dependent non-

peptidergic neurons that mainly innervate the skin, and Runx1/Tlx3-independent peptidergic

neurons that innervate throughout the body. Secondly, Runx1-dependent neurons are next

segregated into Runx1-persistent neurons that include TRPM8+ thermoceptors, MrgprD+

mechanical nociceptors, VGLUT3+ c-mechanoreceptors, and others, and Runx1-transient

neurons that include MrgprA3+ pruriceptors and Mrgprb4+ c-mechanoreceptors. Finally, target-

derived signals most likely contribute to the further segregation of both Runx1-transient and

Runx1-persistent neurons into distinct subtypes (Figure 5).

47

This progressive segregation of somatic sensory neurons and coordinated regulation of

many sensory channels and receptors by the same transcription factors has important

implications on sensory coding. Firstly, functionally distinct neurons may share some common

features established at earlier developmental stages. This may explain why most sensory neurons

are polymodal as they are activated by multiple stimulus modalities (Perl, 1996; Cain et al.,

2001). The basis for the polymodal nature of neurons occurs when multiple receptors are

expressed in the same neuron. This is the case for TRPV1 and TRPM8 colocalizing in a subset of

TRPM8 cold sensitive neurons (Hjerling et al., 2007). In this case, a neuron expressing both

receptors has the ability to respond to both heat and cool. Similarly, both itch-related pruriceptors

and pain-related nociceptors coexpress TRPV1. Secondly, this polymodal nature of many

somatic sensory neurons raises a big question as to how activation of sensory fibers could lead to

specific perceptions. For example, intradermal injection of capsaicin will activate both pain and

itch fibers, but only burning pain is evoked in healthy human subjects. Thus the application of a

given stimulus does not always have a direct correlation with the percept. A population coding

Figure 5: Runx1 and Tlx3 act in combination with target derived signals to control subtype specification. Note: *Met signaling is required for a portion of TrpA1 and TrpV1 high expression. And even though Ret is not essential for MrgprD expression, it is required for the axons to reach their targets.

48

hypothesis has been proposed to solve this coding puzzle. This hypothesis contains the following

features.

Firstly a specific somatic sensory afferent connects to a specific sensory labeled line, i.e.,

a specific neural circuit and network. Under normal conditions the specific activation of a

somatic sensory afferent will reflect the generation of a specific sensation. Secondly, labeled

lines are interconnected through local inhibitory and excitatory interneurons that can modulate or

provide gate control over another labeled line once activated. Thirdly, due to the polymodality of

most sensory fibers, when a stimulus is presented to the skin, it often activates multiple sensory

labeled lines. Hence in order to determine what sensory labeled line will prevail, crosstalk among

them is needed to generate a dominant sensation. Fourthly and under pathological conditions,

when the antagonistic relationship between sensory labeled lines is disrupted, i.e., after central

disinhibition, normally masked neuronal pathways become activated and new sensations arise.

An example would be in the case of neuropathic pain when pain is evoked by innocuous

mechanical or thermal stimuli or itch is evoked by painful stimuli. Altogether this suggests that

further knowledge is required to understand the complex neural circuits processing somatic

sensory information; this knowledge is not only critical to understand how a specific percept is

formed but also, in the case of pathological conditions, to design new therapies that will restore

the lost anti-pain and anti-itch systems.

B. Developmental ontogeny of spinal neurons processing distinct

sensory modalities

The second part of my thesis addresses how spinal neurons processing distinct somatic

sensory modalities emerge during development. The dorsal horn of the spinal cord is divided into

different lamina and populated by intrinsically diverse neurons both molecularly and

functionally.

49

The development of spinal cord development is subject to strict temporal and spatial

control that will ultimately divide it into eight molecularly distinct groups of neurons, dI1-dI6,

dILA and dILB ( Gross et al., 2002; Muller et al., 2002; Helms and Johnson., 2003). These

dorsal horn neurons are further distinguished into class A and class B neurons according to

differential expression of the homeodomain protein Lbx1 (Gross et al., 2002; Muller et al., 2002

& 2005). Class A neurons lack Lbx1 expression, are derived from Olig3 precursors and their

specification requires roof plate signaling. They encompass dI1, dI2 and dI3 neuronal

populations that arise during early embryonic development from E10.5 through E12.5.

Meanwhile class B neurons are derived from Olig3-negative precursors, do not depend on roof

plate signaling for their specification, and are defined by Lbx1 expression. There are five distinct

populations of class B neurons that arise in two distinct waves. dI4, dI5 and dI6 are formed first,

between E10.5 and E 11.5, and dILA and dILB are formed at a later stage during E11.5 and

E13.5. These neurons can be further distinguished in terms of neurotransmitter phenotype. While

class A neurons are all glutamatergic/ excitatory neurons, class B neurons can be further divided

into glutamatergic excitatory neurons and GABAergic/glycinergic inhibitory neurons. dI5 and

dILB neurons are glutamatergic/excitatory neurons and are marked by the expression of the

homeobox proteins Tlx3 and Lmx1b while dI4, dI6 and dILA populations are inhibitory and

marked by the expression of the homeobox proteins Pax2 and Ptf1a (Huang et al., 2008; Guo et

al., 2012; Glasgow et al., 2005; Burrill et al, 1997). Tlx3 has been given the role of a selector-

like protein, specifying most aspects of class B excitatory neurons, including glutamatergic and

peptidergic phenotype (Chen et al., 2004, Xu et al., 2008; Guo et al., 2012). In the inhibitory

neurons dI4, dI6 and dILA, the activities of the transcription factors Pax2 and Ptf1a specify not

only the GABAergic/glycinergic phenotype but also the peptidergic transmitter phenotype

(Gross et al., 2002; Muller et al., 2002; Qian et al., 2002; Helms and Johnson , 2003; Cheng et

al., 2004 &2005; Glasgow et al., 2005; Rebelo et al., 2010).

Recent studies are starting to link the molecularly and developmentally distinct dorsal

horn neurons to their physiological function. In a study from Bermingham et al., it was shown

that Class A dI1 neurons are involved in propioception (Bermingham et al, 2001), and more

recently Bui et al., showed that dI3 neurons are involved in hand grasp control (Bui et al., 2013).

This is not surprising considering that class A neurons settle in the deep dorsal horn, a region that

50

is known for receiving both mechanoreceptor and proprioceptor input. In the second part of this

thesis, I helped to gain important insight into the physiological function of dI5 and dILB class B

neurons.

We generated a conditional knockout mouse in which Tlx3 is removed selectively in dI5

and dILB class B neurons, which led to developmental impairment of a subset of excitatory

neurons located in lamina I and II. These neurons included itch related GRPR expressing

neurons and PKCγ-expressing neurons, as well as neurons expressing the neuropeptides

somatostatin (SOM), preprotachykinin 1 (Tac1), and gastrin-releasing peptide (GRP). I should

stress that development of subset of Tlx3-transient dI5 and/or dILB neurons was not affected,

including Phox2a+ and some Tac1+ neurons in deep laminae. Based on these findings, we can

subdivide class B excitatory neurons into category I and category II. Hence category I neurons

refers to the neurons whose development is normal in Tlx3 conditional knockouts, such as

Phox2a+ and Tac1+ neurons in the deep dorsal laminae. Category II refers to those neurons

whose development is compromised in Tlx3 conditional knockouts, such as SOM+, Tac1+ ,

GRP+, GRPR+ and PKCγ+ neurons. Our genetic fate mapping studies show that both category I

and category II are derived from Lbx1+ class B neurons, with a minor exception of a small

subset of somatostatin + neurons.

The impairment of category II class B neurons in Tlx3 conditional knockouts translates

into marked deficits in processing somatic sensory information. These mice failed to generate

proper reflex behavior in response to noxious mechanical, heat and cold pain as well as reduced

scratching behavior after injection of pruritic compounds. Moreover, these conditional knockouts

also exhibited an impairment of touch-evoked responses after being presented with dynamic

mechanical stimuli. However, these mice kept the ability to sense innocuous cool and warm

sensations (figure 6). Thus, category II Tlx3-dependent class B neurons are selectively required

to process pain, itch and touch-evoked escape response, thereby crucial for sensing

environmental danger.

Among Tlx3-dependent category II neurons, GRP+ and GRPR + class B category II

neurons have a role in processing itch (Sun et al., 2007&2009; Koga et al., 2011; Fleming et al.,

2012). An involvement of Tlx3 dependent spinal neurons in processing pain is consistent with

51

earlier reports showing that Prrxl1 deficient mice, a Tlx3-dependent gene, also exhibited marked

defects in generating nocifensive behavior in response to painful stimuli (Chen et al., 2001).

SOM+ and Tac1+ neurons represent two abundant sub-populations of excitatory neurons located

in superficial laminae, and could represent the candidates processing pain. PkCγ+ neurons

respond to dynamic mechanical stimuli (Miracourt et al., 2007, Neumann et al., 2008), and they

link Aβ mechanoreceptors to lamina I output neurons that mediate injury induced mechanical

allodynia (Lu et al., 2013;Miraucourt et al, 2007). We speculate that these neurons could be

involved in the loss of touch evoked response in our conditional knockout mice. Innocuous cold

or warm could be processed by category I of class B excitatory neurons, such as Phox2a+

neurons and a subset of Tac1+ neurons, or by class A neurons, whose development is unaffected

in conditional knockout mice.

In 1905, Henry Head proposed the protopathic versus epicritic sub-systems of primary

sensory afferents. The protopathic system responds to painful stimuli and the extremes of heat

and cold, and the epicritic system responds to innocuous stimulation permitting fine

discriminations of temperature and touch. Our studies suggest that Heads’s principle might be

applied to the spinal cord, where category II Tlx3-dependent neurons are selectively required for

the processing of protopathic modalities, like pain and itch, but dispensable for the processing of

epicritic information, referring to proprioception and innocuous cool and warm.

Hence, our own studies and those done previously by others have provided insight into

the developmental ontogeny of spinal neurons processing somatic sensory information. So, while

class A neurons, process sensorimotor information, category II class B excitatory neurons are

involved in pain and itch processing and also touch evoked escape response.

52

C. Unsolved problems and future directions

The population coding hypothesis suggests the existence of specific circuits that process specific

sensory modalities, such as pain versus itch, as well as local inhibitory circuits involved with

cross talk among modality-specific circuits, such as the dominant inhibition of itch by pain.

While my thesis work reveals a critical role of Tlx3 in controlling the development of primary

and spinal sensory neurons that are critical for the perception of pain and itch, many questions

remain unsolved.

Firstly, although Tlx3-dependent GRPR+ spinal neurons are critical for itch sensation,

spinal neurons specific for pain (and different forms of pain) are not known. Also unclear is the

exact ontogeny of spinal neurons processing cold or warm.

Figure 6: Ontogeny of spinal neurons processing distinct sensory modalities. Note:P, stands for persistent and T, transient.

?

53

Secondly, to assemble a specific circuit, the primary sensory neurons have to find a way

to connect with specific postsynaptic targets in the spinal cord. We so far still know very little

about how this happens. In DRG, pain related neurons (such as Mrgrpd+ neurons for mechanical

pain) are distinguished from itch-related neurons (such as MrgprA3+ neurons) by the virtue of

persistent or transient Runx1 expression. In principle, such differential expression of

transcription factors might allow these functionally distinct sensory neurons to connect with

distinct spinal neurons.

Thirdly, the spinal microcircuits involved with sensory modality crosstalk, such as itch

inhibition by pain, are only beginning to be understood. As described in Chapter three, my thesis

work contributes to a study showing that VGLUT2-dependent glutamate release from Nav1.8-

expressing DRG neurons is critical for pain sensation and itch inhibition; this glutamate release

likely activates an unknown group of spinal inhibitory neurons that inhibit itch. Ross et al

discovered that inhibitory neurons whose development is dependent on Bhlhb5 transcription

factor are critical for itch inhibition (Ross et al., 2010). However, it is not known if VGLUT2-

dependent glutamate release operates through activating Bhlhb5-dependent inhibitory neurons to

suppress itch. Nor do we know about the genetic programs that control the assembly of these

spinal microcircuits. Addressing these questions could represent an important research area in

understanding the development and processing of somatic sensory information.

54

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IV. Summary and Conclusions

72

My thesis work focused on genetic programs that control the development of somatic

sensory circuits. Toward this goal, I found that the homeodomain transcription factor Tlx3 is

pivotal to the assembly of neural circuits associated with the senses of pain, itch and temperature.

From previous studies in the lab it had been demonstrated that another transcription

factor, Runx1, controls the expression of dozens of sensory channels and receptors in the dorsal

root ganglia (DRG), including TRP channels for temperature and pain sensations, and Mrgpr

class G-protein coupled receptors for itch sensation. I found that Tlx3, which is expressed

broadly in DRG neurons, serves as a competent factor that is required to establish most Runx1

dependent sensory phenotypes. Moreover, gain-of-function studies showed that co-expression of

Tlx3 and Runx1 is able to induce the expression of many of those TRP channels and Mrgpr

receptors in heterologous systems. Thus, these studies contributed to the identification of a

transcriptional program that is both necessary and sufficient to drive the expression of sensory

channels and receptors critical for pain, itch and temperature sensations (Publication I).

Tlx3 is additionally expressed in the dorsal spinal cord, where it coordinates the

development of a subset of excitatory neurons located in the laminae I and II. However, the

physiological functions of these Tlx3-dependent spinal excitatory neurons are not known. In

publication number II we further explored how modality selective neurons emerged during

development and helped to narrow the gap between developmental molecular signature of dorsal

horn neurons and their physiology in the mature central nervous system. In order to address this

we made conditional knockout mice in which we deleted Tlx3 in Class B, Lbx1 excitatory

lineage neurons, DI5 and DILB. Through molecular analysis we showed that these conditional

knockout mice had a compromised development in lamina I and lamina II neurons, including,

itch sensing GRP and its cognate receptor GRPR positive neurons, the mechanoresponsive cells

PKCγ and the neuropeptides somatostatin and Substance P. Furthermore we showed through

behavioral testing that these mice displayed a defect in generating nocifensive motor behaviors,

for both pain and itch related essays and touch evoked responses generated by dynamic

mechanical stimuli, while innocuous cool and warm sensations and body position were

unaffected. Hence we can conclude that Tlx3-dependent neurons are required for the sense of

pain and itch, as well as for generating touch evoked responses, but dispensable for the

sensations of warm and cold.

73

Thus, our studies provide a linkage between developmental ontogeny and physiological

functions, revealing that Tlx3-dependent neurons are evolved to selectively sense environmental

danger.

Lastly while my thesis work contributed to better understand the development of somatic

sensory circuits, I contributed to a study on the neural basis of somatic sensory coding, namely

on the coding of the sensation of pain versus itch (Publication III).

In publication number III we showed that glutamatergic excitatory transmission within

Nav1.8 expressing neurons is critical for pain sensation and itch inhibition. These mice showed

overt behavioral deficits in all acute and chronic pain behaviors tested, a concomitant itch

sensitization in multiple pathways and skin lesions consequential of spontaneous scratching. This

study supports the possibility that there is a central inhibitory mechanism activated by pain

mediated neurons that blocks itch transmission in a pain pathway. Indeed Ross et al., discovered

that inhibitory neurons whose development is dependent on Bhlhb5 transcription factor are

critical for itch inhibition (Ross et al., 2010). However it is unknown if VGLUT2-dependent

glutamate release operates through activating the Bhlhb5-dependent inhibitory neurons to

suppress itch. Also unknown, is how these spinal microcircuits that are involved with sensory

modality crosstalk, are assembled. Addressing these questions would be of utmost importance to

understand the processing of somatic sensory information.

Altogether the studies conducted in this thesis have given Tlx3 a critical role in

controlling the development of primary and relay sensory neurons, critical for the perception of

pain and itch.

74

V. Resumo e Conclusões

75

O meu trabalho de tese foi focado em estudar os programas genéticos que controlam o

desenvolvimento dos circuitos somáticos sensoriais. Para tal, eu descobri que um factor de

transcrição de homeodominio, Tlx3, é crucial para a construção de circuitos neuronais associados

com as sensações de dor, prurido e temperatura.

Estudos anteriormente efetuados no laboratório demonstraram que outro fator de

transcrição Runx1, controla a expressão de vários canais iónicos sensoriais e recetores no gânglio

dorsal raquidiano (DRG), incluindo canais TRP para as sensações de temperatura e dor e

recetores Mrgpr, importantes na deteção do prurido. Em estudos conducentes a esta tese,

descobrimos que o Tlx3, que é amplamente expresso nos neurónios do gânglio raquidiano, serve

como um fator competente que é necessário para estabelecer a maioria dos fenótipos sensoriais

dependentes de Runx1.

Para alem disso, estudos de ganho de função (GOF) mostraram que a co-expressão de

Tlx3 e Runx1 induz a expressão de muitos dos canais TRP e Mrgpr em sistemas heterologos.

Assim, estes estudos contribuíram para a identificação de um programa transcricional que é

necessário e suficiente para conduzir a expressão de canais sensoriais e recetores críticos para as

sensações de dor, prurido e temperatura, (Publicação I).

Tlx3 é adicionalmente expresso na espinhal medula dorsal, onde coordena o

desenvolvimento de um subconjunto de neurónios excitatórios na lamina I e II. No entanto, as

funções fisiológicas destes neurónios excitatórios espinhais Tlx3-dependentes é desconhecida.

Na publicação II explorámos como neurónios de modalidade seletiva emergem durante o

desenvolvimento e ajudámos a estreitar a fenda entre a assinatura molecular de neurónios do

corno dorsal que surgem durante o desenvolvimento e a sua fisiologia no sistema nervoso central

adulto.

Para desenvolver este estudo, fizemos um ratinho Knockout condicional, no qual

eliminamos a expressão do Tlx3 nos neurónios excitatórios derivados de Lbx1-Class B: DI5 e

DILB.

Através de análise molecular demonstrámos que nestes ratinhos condicionais há um

defeito no desenvolvimento nos neurónios da lamina I e II, incluindo em neurónios que detectam

prurido, como é o caso dos neurónios GRP positivos e do seu recetor cognato, neurónios GRPR

positivos, neurónios Pkcγ positivos que respondem a estímulo mecânico, e dos neuropeptidos

Somatostatina e Substancia P.

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Para além disso demonstrámos através de testes comportamentais que estes ratinhos

apresentam defeitos em gerar comportamento nocifensivo motor, tanto em testes de dor como de

prurido, e também em testes de respostas evocadas por toque geradas a partir de estímulo

dinâmico mecânico. Sensações inócuas de frio e de quente, tal como de posição corporal

mantiveram-se intactas.

Assim sendo, concluímos que neurónios Tlx3-dependentes são necessários para as

sensações de dor e prurido, assim como para gerar respostas evocadas por toque, mas

dispensáveis para sensações de frio e quente.

Assim sendo, os nossos estudos fornecem uma ponte entre a ontogenia de

desenvolvimento e funções fisiológicas, revelando que neurónios Tlx3-dependentes evoluíram

para seletivamente detetarem o perigo no ambiente envolvente.

Por ultimo enquanto que o meu trabalho de tese contribuiu para melhor entender o

desenvolvimento dos circuitos sensoriais somáticos, eu também contribuí para um estudo que

serviu de base para melhor esclarecer a base neuronal do código somático sensorial,

nomeadamente na codificação das sensações de dor e de prurido (Publicacao III) .

Na Publicacao III, nós demonstramos que a transmissão excitatória glutamatérgica nos

neurónios Nav 1.8 positivos é crítica para a sensação de dor e para a inibição do prurido.

Estes ratinhos demonstraram defeitos comportamentais em todos os testes de dor aguda e

crónica, uma concomitante sensibilização ao prurido em múltiplos “ pathways” e lesões de pele

como consequência de “ spontaneous scratching”. Este estudo sustenta a possibilidade de que há

um mecanismo central inibitório ativado por neurónios que detetam a dor , que bloqueia a

transmissão do prurido num “pain pathway”.

De fato, Ross e tal., descobriu que neurónios inibitórios cujo desenvolvimento é

dependente do fator de transcrição Bhlhb5, são críticos na inibição do prurido (Ross e tal., 2010).

No entanto, é desconhecido se a libertação de glutamato dependente de VGLUT2, opera através

da activação de neurónios inibitórios dependentes de Bhlhlb5 para suprimir o prurido. Também

desconhecido, é como estes microcircuitos espinhais envolvidos com a “crosstalk” de

modalidades sensoriais são construídos.

Responder a estas questões será de importância máxima para entender como se dá o

processamento da informação sensorial somática.

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Em conjunto os estudos conduzidos nesta tese deram ao Tlx3 um papel crítico no controle do

desenvolvimento de neurónios sensoriais primários e de “ relay”, críticos na perceção da dor e

prurido.

.