Interactions Between Sox10, Edn3 and Ednrb During Enteric Nervous System and Melanocyte Development

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Developmental Biology 295 (2006) 232–249 www.elsevier.com/locate/ydbio

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

Laure Stanchina a,c, Viviane Baral a,c, Fabienne Robert b,c, Veronique Pingault b,c, ⁎ Nicole Lemort a,c, Vassilis Pachnis d, Michel Goossens a,c, Nadege Bondurand a,c,

a INSERM, U654, Bases moléculaires et cellulaires des maladies génétiques, Hôpital Henri Mondor, Creteil, F-94000, France b INSERM, U654, Equipe Avenir, Hôpital Henri Mondor, Creteil, F-94000, France c Université Paris 12, Faculté de Médecine, IFR10, Créteil, F-94000, France d Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Received for publication 18 November 2005; revised 20 March 2006; accepted 21 March 2006 Available online 3 April 2006

Abstract

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg–Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations. © 2006 Elsevier Inc. All rights reserved.

Keywords: Sox10; Hirschsprung; Waardenburg; Enteric nervous system; Melanocyte; Endothelin; Ednrb; Edn3; High-mobility group; Neural crest

Introduction development are a significant cause of human disease. The term neurocristopathies collectively refers to these neural crest The embryonic neural crest gives rise to a population of disorders, among which are Hirschsprung disease (HSCR, pluripotent cells which, upon neural tube closure, delaminates MIM142623) and Waardenburg syndrome (WS) (Amiel and in a rostrocaudal wave and migrates to diverse locations Lyonnet, 2001; Bolande, 1974; Read and Newton, 1997). throughout the embryo. As they reach their final locations, HSCR is the main cause of congenital intestinal obstruction neural crest cells have the ability to differentiate into several cell with an incidence of 1/5000 live births and is characterised by types, including adrenomedullary cells, most of the craniofacial the absence of enteric ganglia along a variable length of the skeletal tissue, glia and some neurons of the peripheral nervous intestine (Amiel and Lyonnet, 2001). WS, on the other hand, is a system, enteric neurons and glia, and melanocytes of the skin, rare disorder (1/40,000) with sensorineural deafness and hair and inner ear (Le Douarin, 1999). Defects in neural crest pigmentation defects caused by the absence of melanocytes in the skin and stria vascularis of the inner ear (Read and Newton, 1997; Tachibana, 1999). The association of HSCR and WS ⁎ Corresponding author. INSERM U654, Hôpital Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010, Creteil, France. Fax: +33 148 993 features has been described in rare individuals and is referred to 345. as Shah–Waardenburg syndrome, Waardenburg–Hirschsprung E-mail address: [email protected] (N. Bondurand). disease or Waardenburg syndrome type 4 (WS4; MIM277580).

WS4 is a genetically heterogeneous condition. Indeed, homo- equally well (for reviews see Kurihara et al., 1999; Sakurai et zygous or heterozygous mutations in the genes encoding al., 1992). Analysis of spontaneous and targeted mutations in endothelin-3 (EDN3) or endothelin-B receptor (EDNRB), as the genes encoding endothelin-3 and EDNRB established the well as heterozygous mutations in SOX10 have been reported requirement for this signalling pathway in melanocytes and (Edery et al., 1996; Hofstra et al., 1996; Inoue et al., 2004; ENS development (Baynash et al., 1994; Hosoda et al., 1994 Pingault et al., 1998; Pingault et al., 2001; Puffenberger et al., and for reviews see Dupin and Le Douarin, 2003; Kurihara et 1994). Identification and functional characterisation of muta- al., 1999; Pla and Larue, 2003). Animals homozygous for the tions within these 3 genes in human, as well as analysis of spontaneous piebald lethal mutation, in which the Ednrb gene is available animal models, have been the main source of deleted (Ednrbsl) and Ednrb knock-out animals present with information for our understanding of WS4 genetics. megacolon and an almost complete loss of pigmentation SOX10, a high-mobility group box containing transcription (Hosoda et al., 1994; Lee et al., 2003). Similar defects result factor of the Sox family, belongs to a separate subgroup along from targeted disruption or spontaneous mutation of the with two closely related proteins, SOX9 and SOX8 (Bowles et endothelin-3 gene (lethal spotting, Edn3ls)(Baynash et al., al., 2000; Wegner, 1999). The expression profile of Sox10 in 1994). However, in this mutant, hypopigmentation is less severe different species and the phenotypes of mouse and zebrafish compared to Ednrb knock-out, a feature which could result from Sox10 mutants pointed out the essential function of this factor in local molecular compensation by other endothelins. Finally, various steps of neural crest development. Two mutations for another spontaneous Ednrb mutant, piebald (Ednrbs), also Sox10 have been examined so far in mice: a null mutation shows a milder phenotype with white spotting limited to about generated by targeted deletion and a spontaneous mutation, the 20% of the coat; it almost never manifests megacolon and is Dominant megacolon (Sox10Dom)(Britsch et al., 2001; considered as a hypomorphic mutation (Hosoda et al., 1994). Herbarth et al., 1998; Lane and Liu, 1984; Southard-Smith et Over the past decade, several studies addressed the issue of al., 1998). At the heterozygous state, both mouse models temporal requirement of endothelin-3/EDNRB function in ENS present with a phenotype similar to WS4. Thus, a large development and melanogenesis. Experiments with an inducible percentage of Sox10Dom/+ or Sox10LacZ/+ animals lack enteric allele of Ednrb have shown that its function is required between ganglia from a variable length of the colon and exhibit E10 and E12.5 when melanoblasts are dispersing along the pigmentation defects (white belly spot and white paws). dorsolateral pathway (Shin et al., 1999). Furthermore, mutant Homozygous mutants on the other hand have severe deficits rescue experiments, analysis of the effects of endothelin-3 in culture in several neural crest derivatives, including complete lack of and fate analysis of EdnrblacZ mutant cells in vivo and in vitro ENS and melanocytes (Britsch et al., 2001; Herbarth et al., suggested that EDNRB is necessary for the migration and/or 1998; Kapur, 1999; Southard-Smith et al., 1998). proliferation and differentiation of mouse melanoblasts, but not for Several in vitro and in vivo studies indicated that Sox10 their determination in the migrating staging area (MSA) (Hou et al., function is crucial for the survival and the maintenance of 2004; Lahav et al., 1996; Lee et al., 2003; Opdecamp et al., 1998; pluripotency of migrating neural crest progenitors and also Pla and Larue, 2003; Reid et al., 1996). As far as ENS development influences fate decision at later stages (Aoki et al., 2003; is concerned, endothelin-3/EDNRB seem to be required between Britsch et al., 2001; Dutton et al., 2001; Honore et al., 2003; E10.5 and E12.5, during the migration of precursor cells along the Kapur, 1999; Kim et al., 2003; McKeown et al., 2005; gut (Shin et al., 1999; Sidebotham et al., 2002; Woodward et al., Paratore et al., 2001 and for reviews see Hong and Saint- 2000). Experiments performed in mouse and chick have shown that Jeannet, 2005; Mollaaghababa and Pavan, 2003; Wegner, endothelin-3 inhibits neuronal differentiation (Hearn et al., 1998; 1999; Wegner, 2005). During melanocyte development in Nagy and Goldstein, 2006; Wu et al., 1999). Moreover, a particular, SOX10 was shown to play a role in specification permissive effect of endothelin-3 on proliferation of enteric crest and differentiation of the melanocyte lineage by regulating the cells has been supported by some (Barlow et al., 2003; Nagy and MITF/Mitf and TRYP2/Dct (Dopachrome tautomerase) genes Goldstein, 2006) and refuted by others (Kruger et al., 2003; (Bondurand et al., 2000; Elworthy et al., 2003; Jiao et al., Woodward et al., 2003; Wu et al., 1999). 2004; Lee et al., 2000; Ludwig et al., 2004; Potterf et al., Here, we investigate the interaction and crosstalk between 2000; Potterf et al., 2001; Verastegui et al., 2000). In the endothelin signalling pathway and SOX10 in ENS and Sox10LacZ heterozygous embryos, neural-crest-derived enteric melanocyte development. To this end, we analysed the progenitors colonise the proximal intestine and are unaffected phenotype (enteric, coat pigmentation and melanocytes of in their survival capacity. However, unlike their wild-type the inner ear) of mutant mice carrying various combinations counterparts, they are unable to maintain their progenitor of mutant Sox10, Edn3 and Ednrb alleles, and we status and acquire preneuronal traits, resulting in the reduction investigated the cellular origin of the ENS defects observed. of the progenitor pool size (Paratore et al., 2002). Endothelins 1, 2 and 3 are a family of 21-amino-acid Materials and methods vasoactive peptides, which bind to G-protein-coupled heptahe- lical receptors (Yanagisawa et al., 1988). Two types of receptors Animals and genotyping have been identified in mammals, endothelin receptor A and B The generation and genotyping of Sox10LacZ have been described previously (EDNRA and EDNRB). EDNRA displays a preferential affinity (Britsch et al., 2001). The Sox10Dom arose spontaneously on a C57BL/6JXC3H/ for endothelin-1, whereas EDNRB binds all three peptides HheOuJ hybrid background at the Jackson laboratory (Lane and Liu, 1984) and 234 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 was maintained for several years in this mixed background. Sox10Dom is a (rabbit, 1:1000 dilution, kindly provided by Dr. J.F. Brunet; Pattyn et al., 1997), frameshift mutation which leads to a truncated protein. Mice were genotyped by B-FABP (rabbit, 1:5000 dilution, kindly provided by Dr. T. Müller; Kurtz et al., PCR digestion making use of the previously described intragenic A to T 1994), anti-β-galactosidase (goat, 1:1000 dilution, Biotrend). Incorporated transversion at position 32 of the Sox10 ORF (Herbarth et al., 1998). This BrdU was detected after 2 M HCl treatment for 30 min at 37°C using BrdU polymorphism, shown to be linked to the Sox10 mutation, disrupts an AluI antibody (mouse, 1:40 dilution, Becton Dickinson). Secondary antibodies used restriction site. The primer pair used for amplification was 5′-GACATGGCC- were as follows: anti-mouse FITC-conjugated (1:150 dilution, Sigma), anti- GAGGAACAAGA-3′ and 5′-CGGATGCACACGGGGAACTT-3′. After PCR mouse alexafluor568 (1:500 dilution, Molecular probes), anti-rabbit FITC- amplification and AluI digestion, the wild-type allele segregates with a 196-bp conjugated (1:150 dilution, Sigma), anti-rabbit Cy3 (1:150 dilution, Sigma), product whereas the Sox10Dom allele segregates with a 230-bp product. anti-guinea pig Cy3 (1:200 dilution, Dianova), anti-goat FITC (1:200 dilution, Sox10LacZ and Sox10Dom mice were backcrossed on C3HeB/FeJ for at least 4 Jackson Immunoresearch). Alexafluor488-phalloidin was used at a 1:1000 generations before use. dilution (Molecular probes). Preparations were mounted directly in Vectashield SSL/LeJ and LS/LeJ breeding pairs were obtained from the Jackson containing DAPI (Vector laboratories). X-Gal staining followed standard laboratory. The SSL/LeJ inbred strains carries both the piebald (Ednrbs) and procedures. Sections, guts or cultures were examined with a Leica DMR piebald lethal (Ednrbsl) mutations. Ednrbs/s and Ednrbs/+ mice were generated microscope equipped with a Hamamatsu digital camera or Olympus SZH10 and by crossing the Ednrbs;Ednrbsl mice. Piebald is considered as a hypomorphic Leica MZ6 stereo microscope coupled to remote capture program. mutation as Ednrbs/s homozygous mutants still express 30% of structurally intact receptor (Hosoda et al., 1994; Roix et al., 2001). Mice were genotyped as previously described (Kumagai et al., 1998). The LS/LeJ inbred strain Results carried both the lethal spotting (Edn3ls) and the black and tan coat colour (at) ls mutations. Crosses allowed us to segregate the Edn3 mutation, which prevents Sox10;Edn3 and Sox10;Ednrb double mutants show increased conversion of the inactive precursor of endothelin-3 to its active form (Baynash et al., 1994). Mice were genotyped as previously described (Rice et al., 2000). postnatal mortality The sex of Sox10Dom/+;Edn3ls/+ animals was determined by PCR amplification as previously described (McClive and Sinclair, 2001). Recent reports demonstrated a genetic interaction between Sox10 and Ednrb during ENS development (Cantrell et al., Pups, embryos, tissue collection and acute culture 2004; Zhu et al., 2004). Indeed, by crossing mice carrying the Dom sl ls/+ s/+ Sox10 and two different Ednrb mutations (Ednrb ; i.e. To generate embryos and pups, Edn3 and Ednrb mice were crossed tm1Ywa Dom/+ LacZ/+ piebald lethal and Ednrb ), Cantrell et al. recently reported with Sox10 , Sox10 or double heterozygotes respectively. Embryos Dom/+ sl/sl Dom/+ from E10 to E15.5 were obtained from staged pregnancies and genotyped as that ENS defects of Sox10 ;Ednrb and Sox10 ; tm1Ywa/+ described above. The day of vaginal plug was considered to be E0.5. For BrdU Ednrb double mutants are significantly more severe labelling, pregnant mice were injected intraperitoneally with 100 μg BrdU than those observed in single mutants at birth (Cantrell et al., (Sigma) per gram of animal weight 1 h before embryos collection. 2004). Here, we tested a possible cooperative activity between After paraformaldehyde fixation, 10–15 μm frozen sections of genotyped embryos were generated and used for immunohistochemistry. Alternatively, Sox10 and the ligand of Ednrb, endothelin-3. Moreover, we dissected guts were used for whole-mount immunohistochemistry or whole- attempted to decipher the cellular mechanisms underlying the mount X-Gal staining. combined activity of SOX10 and endothelin signalling. To this Acute cultures of dissociated stomach were performed as described (Barlow end, we first generated animals carrying different combinations et al., 2003). Briefly, stomachs were dissected from E11.5 or E13.5 embryos of of the Sox10Dom or Sox10LacZ alleles and the lethal spotting different genotypes and incubated with dispase/collagenase (0.5 mg/ml in 1× ls s PBS, Roche) for 5 min at room temperature. Digested tissues were washed twice (Edn3 ) or the piebald (Ednrb ) mutations and examined their with 1× PBS, dissociated by repeated pipetting and plated on fibronectin-coated postnatal survival (for details regarding the various mutations, (20 μg/ml) SONIC-SEAL wells (VWR) in optiMEM (Life technologies). After a see Materials and methods). Dom/Dom few hours in an atmosphere of 5% CO2, most cells were adherent and no In contrast to Sox10 homozygous animals that die differential cell adherence was observed between the genotypes as assessed by during embryogenesis or at birth, Sox10Dom/+ mice survive counting the number of cells before and after plating (DAPI staining). Cultures were fixed 10 min in 4% paraformaldehyde at 4°C and used for postnatally although a large fraction of them develop megacolon immunohistochemistry. and die within the first 3 weeks of age (Herbarth et al., 1998; The pigmentation phenotype was observed on P5–P10 or P20–30 mice of Southard-Smith et al., 1999). A similar, albeit milder, phenotype different genotypes. Observation of melanocytes within the inner ear was is also described in Edn3ls/ls animals, whereas Edn3ls/+ – performed on newborn animals (P0 P2). To this end, temporal bones were heterozygotes are unaffected (Baynash et al., 1994). We dissected in 1× PBS. The bone surrounding vestibular structures was removed to Dom/+ ls/+ allow visualisation of internal structures and melanocytes. After general intercrossed Sox10 and Edn3 single mutants and observation and photography, the cochlea was dissected and the stria vascularis analysed the mortality of each specific genotype within the separated from the organ of Corti. Again, presence of melanocytes was recorded progeny. At birth, all genotypes are represented in the expected by general observation using a Leica MZ6 stereo microscope. Alternatively, the Mendelian ratio. Moreover, 33% of Sox10Dom/+ animals died organ of Corti was dissected out of the temporal bone in 1× PBS, fixed 1 h with within the first 6 weeks. In contrast, 71% of double heterozygotes 4% paraformaldehyde at 4°C and used for phalloidin staining. died within the same period (Table 1). The few Sox10Dom/+; ls/+ ls/+ Immunostaining and X-Gal staining Edn3 animals that survived were intercrossed with Edn3 animals in order to analyse the survival of Sox10Dom/+;Edn3ls/ls Immunostaining was performed as previously described (Barlow et al., mice. Of the 10 animals collected, none survived more than 10 2003; Bondurand et al., 2003). The following primary antibodies were used in days (Table 1). Therefore, partial or complete removal of various combinations: TuJ1 (mouse, 1:1000 dilution, Eurogentec), PGP9.5 endothelin-3 activity in Sox10Dom/+ animals significantly (rabbit, 1:400 dilution, Biogenesis), phospho-histone H3 (PH3, rabbit, 1:500 decreases the survival rate of these animals. dilution, Upstate), activated-caspase-3 (casp3, rabbit, 1:200 dilution, Cell Dom s signalling), Sox10 (rabbit, 1:200 dilution, Chemicon), Sox10 (guinea pig, Next, we analysed the survival rate of Sox10 ;Ednrb Dom/+ s/+ 1:2000 dilution, kindly provided by Dr. M. Wegner; Maka et al., 2005), Phox2b double mutants. Sox10 ;Ednrb animals had a mortality L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 235

Table 1 findings, we observed a migration delay in the majority of Dom ls Genotypes distribution and viability analysis among the Sox10 ;Edn3 and Sox10Dom/+ single mutants (Fig. 1C1–4) (Herbarth et al., Sox10Dom;Ednrbs crosses 1998; Kapur, 1999; Puliti et al., 1996; Southard-Smith et al., Crosses Genotypes Number Number %of 1998). Indeed, in the majority of the guts analysed, TuJ1 of animals of mortality positive cells were present in apparently normal number and genotyped animals dead distribution within the small intestine, but not within the between caecum or colon (TuJ1 staining stopped before the caecum P0 and 6 entry in 6 of 19 guts, or halfway through the caecum in 9 of weeks 19 guts; Fig. 1G). More importantly, we observed a marked Sox10+/+;Edn3+/+ 16 0 0 Dom/+ ls/+ Sox10Dom/+;Edn3+/+ decrease of TuJ1 positive cells within Sox10 ;Edn3 Sox10+/+;Edn3ls/+ 17 2 11 × guts compared to single mutants. Indeed, no TuJ1 positive Sox10Dom/+;Edn3+/+ 21 7 33 Sox10+/+;Edn3ls/+ cells were observed after the ileocaecal junction of the gut of Sox10Dom/+;Edn3ls/+ 28 20 71 these animals (22/22), and in the large majority of cases (15/ Sox10Dom/+;Edn3ls/+ × Sox10Dom/+;Edn3ls/ls 10 10 100 22), the small intestine was only partially populated (see Fig. Sox10+/+;Edn3ls/+ 1D1–4 and G). Strikingly, we found almost no TuJ1 positive Sox10+/+;Ednrb+/+ 18 0 0 cells in the small intestine of 3 animals. As previously Dom/+ +/+ Sox10 ;Ednrb Sox10+/+;Ednrbs/+ 13 0 0 described, the number of neurons in the small intestine of × Sox10Dom/+;Ednrb+/+ 11 3 27 Edn3ls/ls embryos was apparently similar to that of wild-type +/+ s/+ Dom/+ s/+ Sox10 ;Ednrb Sox10 ;Ednrb 13 4 31 controls, but TuJ1 cells were detected only in the proximal 1/ Dom/+ s/+ 3–1/2 of the colon or halfway through the caecum (Barlow et Sox10 ;Ednrb Dom/+ s/s × Sox10 ;Ednrb 18 18 100 al., 2003, and Fig. 1E1–4 and G). Finally, analysis of Sox10+/+;Ednrbs/+ Sox10Dom/+;Edn3ls/ls guts indicated that TuJ1 staining was Pups mortality was quantified by identifying all animals post-mortem or at virtually absent from the intestine and was restricted to the weaning and counting the number of animals dead between P0 and 6 weeks. Sex – Dom/+ ls/+ stomach wall (5/5; Fig. 1F1 4 and G). Similar results were ratio (r) was similar in Sox10 ;Edn3 animals presenting with severe LacZ (r = 0.60) or mild (r = 0.61) phenotype, showing that gender does not influence obtained using the Sox10 allele (data not shown). Taken viability of these animals. together, these results indicate that removal of endothelin-3 activity in Sox10 heterozygous animals results in an ENS phenotype that is much more severe than that of single similar to that of Sox10Dom/+ mice (27% versus 31%, Table 1). mutants, highlighting a strong genetic interaction between In contrast, all (18/18) Sox10Dom/+;Ednrbs/s mutants died within these two genes during ENS development. the first 10 days (Table 1). Therefore, partial removal of Using whole-mount TuJ1 immunostaining, we also observed a EDNRB function in Sox10Dom/+ animals reduced drastically similar pattern of neurogenesis in the gut of E13.5 Sox10Dom/+ postnatal survival, a finding that extends previous observations and Sox10Dom/+;Ednrbs/+ embryos (Fig. 2, compare B1 to B4 and obtained using Ednrbtm1Ywa mutant allele (Cantrell et al., 2004). C1 to C4). Indeed, in both cases, TuJ1 positive cells were present in apparently normal number and distribution within the small Sox10;Edn3 and Sox10;Ednrb double mutants show severe intestine, but no TuJ1 positive cells were detected after the caecum ENS defects at E13.5 area (Fig. 2F). When Ednrbs/s guts were analysed, a normal number of neurons were observed up to 1/3–2/3 of the colon (Fig. To determine the cause of the high mortality observed in 2D1–4 and F). These results are consistent with a small delay of Sox10Dom/+;Edn3ls/+, Sox10Dom/+;Edn3ls/ls and Sox10Dom/+; migration that could be compensated afterwards as these animals Ednrbs/s double mutants animals, we compared the ENS almost never manifest megacolon. In contrast to these mutants, development of embryos with these genotypes relative to single analysis of guts from Sox10Dom/+;Ednrbs/s embryos showed TuJ1 heterozygous (control) littermates. In mouse embryos, vagal positive cells almost exclusively within the stomach wall and only neural crest cells invade the foregut at E9–E9.5. By E11.5, occasionally were neurons present within the proximal small enteric progenitors have colonised the entire small intestine and intestine (5/5; Fig. 2E1–4 and F). Similar results were obtained reached the caecum, while colonisation of the colon is using the Sox10LacZ allele (data not shown). Thus, partial removal completed by E13.5–14.5 (Barlow et al., 2003; Kapur, 1999; of EDNRB function in Sox10 heterozygous animals results in an Kapur et al., 1992; Young et al., 1998). As distribution of enteric ENS phenotype that is much more severe than that of single neurons along the gut reflects the progress of migration and mutants, confirming a strong genetic interaction between these differentiation of ENS progenitors, we first compared neuro- two genes during ENS development. genesis in the gut of mutant embryos (ranging from E13.5– 14.5) bearing different combinations of mutant alleles using The ENS phenotype of E13.5 double mutants results from whole-mount immunostaining with TuJ1. absence of enteric progenitor cells beyond the stomach As expected, we observed no significant difference in the intensity or distribution of TuJ1 immunostaining between To determine if the deficits in enteric neurogenesis E13.5 wild-type and Edn3ls heterozygous guts (Fig. 1, observed in the guts of the double mutants studied are due compare panels A1–4 and B1–4). Consistent with previous to defects in neuronal differentiation or to an absence of 236 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 enteric progenitor cells, we took advantage of the LacZ gut of double mutants relative to single mutant littermates. In reporter present in the Sox10LacZ allele. As enteric progenitor particular, the wave front of migrating enteric neural crest cells cells express Sox10, X-Gal staining was used to visualise the appeared severely delayed in all Sox10LacZ/+;Edn3ls/+ embryos distribution of these cells along the guts of mice with various (n = 6; compare Fig. 4A4 and A2; note that 2/6 embryos combinations of mutant alleles (Maka et al., 2005; Young et presented with TuJ1 positive cells in the stomach only), while al., 2003). To this end, Sox10LacZ/+;Edn3ls/+ or Sox10LacZ/+; reduced numbers of TuJ1 positive cells were detected only Ednrbs/+ double heterozygotes were crossed with Edn3ls/+ or within the proximal stomach of Sox10LacZ/+;Edn3ls/ls (3/3, Fig. Ednrbs/+ animals, respectively, and whole-mount X-Gal 4A5) and Sox10LacZ/+;Ednrbs/s (6/6; Fig. 4C4). Whole-mount staining was carried out on guts collected from the resulting X-Gal staining confirmed these results. Indeed, β-gal positive embryos at E13.5–15.5. When staining of guts from cells were detected within the stomach and proximal part of the Sox10LacZ/+;Edn3ls/+ and Sox10LacZ/+;Edn3ls/ls embryos was small intestine of Sox10LacZ/+;Edn3ls/+ (3/3),andinthe compared to Sox10LacZ/+ littermates, a strong colonisation proximal part of the stomach of Sox10LacZ/+;Edn3ls/ls (4/4) delay was observed in most cases. Indeed, in a pattern similar and Sox10LacZ/+;Ednrbs/s embryos only (4/5; compare Fig. 4B1 to the TuJ1 immunostaining previously described, the wave to B2–3 and D1 to 2). front of migrating enteric neural crest cells appeared less To further examine this issue, sections of E11.5 wild-type, advanced in Sox10LacZ/+;Edn3ls/+ compared to Sox10LacZ/+ Sox10LacZ/+, Ednrbs/s and Sox10LacZ/+;Ednrbs/s embryos were (12 of 17 embryos, compare Fig. 3A and B, note that 80% of labelled with Phox2b and TuJ1 antibodies in order to visualise Sox10LacZ/+ and the remaining Sox10LacZ/+;Edn3ls/+ showed enteric progenitors and their progeny from the oesophagus no enteric defects), while β-gal positive cells were only onwards. We confirmed the presence of enteric progenitors cells detected within the stomach of Sox10LacZ/+;Edn3ls/ls embryos and derivatives in the stomach of wild-type, Sox10LacZ/+ and (3/3, Fig. 3C). Comparison of X-Gal staining of Sox10LacZ/+ Ednrbs/s embryos and the reduced number of cells within the and Sox10LacZ/+;Ednrbs/+ guts showed no differences (Fig. stomach region of Sox10LacZ/+;Ednrbs/s embryos (data not 3D, E, data not shown). In contrast, β-gal positive cells were shown). Furthermore, these experiments indicated apparently absent from the entire intestine of Sox10LacZ/+;Ednrbs/s normal staining in the oesophagus of all embryos, including embryos but were found only within the stomach of all guts Sox10LacZ/+;Ednrbs/s (Fig. 4E1–4). Therefore, in E11.5 Sox10- analysed (5/5, see Fig. 3F). Therefore, X-Gal histochemical ;Ednrbs/s embryos, cells seem to be able to reach the staining is consistent with TuJ1 immunostaining, suggesting oesophagus but fail to colonise the entire stomach and more that the enteric defects observed in Sox10LacZ/+;Edn3ls/+, distal parts of the gastrointestinal tract. Sox10LacZ/+;Edn3ls/ls and Sox10LacZ/+;Ednrbs/s are due to the absence of enteric progenitors along a variable length of the Cellular mechanism at the origin of the ENS defects in mice intestine, depending on the genotype analysed. with Sox10 and Ednrb deficiencies

Sox10;Edn3 and Sox10;Ednrb double mutants show severe To determine the cellular mechanism underlying the ENS ENS defects at E11.5 defects observed in E11.5 and E13.5 embryos with combined Sox10 and Edn3 or Sox10 and Ednrb mutations, we analysed We also examined the migration and neuronal differentiation the proliferation, survival and differentiation capacities of of enteric progenitors earlier during development, using whole- enteric neural crest cells within the stomach region. Given that mount preparations of E11.5 guts from wild-type, single reduction in the activity of endothelin-3 or its receptor in (Sox10LacZ/+, Edn3ls/ls, Ednrbs/s) and double (Sox10LacZ/+; Sox10Dom or Sox10LacZ heterozygous animals has a similar Edn3ls/+, Sox10LacZ/+;Edn3ls/ls, Sox10LacZ/+;Ednrbs/s) mutant effect and the better survival of Sox10Dom/+;Ednrbs/+ and embryos stained with TuJ1 antibodies or X-Gal histochemistry Sox10LacZ/+;Ednrbs/+ animals, we performed our analysis on (Fig. 4A–D). TuJ1 staining revealed a small colonisation delay stomach of embryos generated from crosses between Sox10 in Sox10LacZ/+ and Edn3ls/ls embryos compared to wild-type and Ednrb mutants. We first quantified cell apoptosis on controls (Fig. 4A1–3, C1–2 and Barlow et al., 2003). We also cultures of acutely dissociated stomach at E11.5 by counting observed a different migration of enteric cells in the gut of the fraction of SOX10 positive cells co-labelled by activated Ednrbs/s embryos. This defect was variable, with some embryos caspase-3. No significant increase in apoptosis was observed (2/5) showing normal colonisation and others (3/5) presenting in Sox10LacZ/+;Ednrbs/s embryos compared to other genotypes with fewer TuJ1 positive cells in the distal part of the stomach (Fig. 5A). The same experiment was performed on E13.5 and along the gut (Fig. 4C3). More importantly, we observed a stomach cultures and gave very similar results. Indeed, the dramatic reduction in the number of TuJ1 positive cells in the percentage of apoptosis in wild-type, Sox10LacZ/+, Ednrbs/s and

Fig. 1. Genetic interaction between Sox10Dom and Edn3ls alleles controls ENS development. Guts were dissected from E13.5 mouse embryos of appropriate genotype and immunostained with neuron-specific tubulin (TuJ1) antibody as whole-mount gut preparations. This marker tracks the migration of neural-crest-derived cells along the rostrocaudal axis of the gut. Panels A1 to F1, A2 to F2, A3 to F3 and A4 to F4 show staining in the distal stomach, small intestine, caecum region and colon, respectively. (G) Schematic representation of the gut. Above this scheme, shaded areas represent the regions of the gut shown in panels A–F. Below this scheme, lines and perpendicular arrows indicate the extent of colonisation for each class of genotyped embryos. The number of embryos presenting with a defined defect is indicated on the left of each arrow. Note the severe colonisation delay in the double heterozygous (D1–4) and Sox10Dom/+;Edn3ls/ls animals (F1–4) compared to single mutants (B1–4, C1–4 and E1–4). s: stomach; si: small intestine; ce: caecum; co: colon. L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 237 238 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249

Fig. 2. Genetic interaction between Sox10Dom and Ednrbs alleles controls ENS development. Whole-mount TuJ1 immunohistochemistry was performed on E13.5 guts from wild-type (A), Sox10Dom/+ (B), Sox10Dom/+;Ednrbs/+ (C), Ednrbs/s (D) or Sox10Dom/+;Ednrbs/s (E) embryos. Panels A1–E1, A2–E2, A3–E3 and A4–E4 show staining in the distal stomach, proximal small intestine, caecum region and colon, respectively. (F) Schematic presentation of the gut. Above this scheme, shaded areas represent the regions of the gut shown in panels A–E. Below this scheme, lines and perpendicular arrows indicate the extent of colonisation for each class of genotyped embryos. The numbers on the left of each arrow indicate embryos presenting with a defined defect. Note the severe colonisation delay in the Sox10Dom/+;Ednrbs/s animals (E1–4) compared to single mutants (B1–4, D1–4) or double heterozygotes (C1–4). s: stomach; si: small intestine; ce: caecum; co: colon.

Sox10LacZ/+;Ednrbs/s cultures was 0.6 ± 0.4%, 1 ± 0.7%, phospho-histone H3. No significant decrease in proliferation 0.6 ± 0.5% and 0.4 ± 0.3%, respectively, suggesting that the was observed in Sox10LacZ/+;Ednrbs/s embryos compared to more severe ENS defects in Sox10LacZ/+;Ednrbs/s embryos are single mutants (Fig. 5B). To confirm these observations, we also not due to increased apoptosis of progenitor cells within the performed a quantification of cell proliferation by analysing the stomach wall. We next attempted to quantify cell proliferation of in vivo incorporation of the nucleotide analogue bromodeox- neural crest derivatives at E11.5 by counting the fraction of yuridine (BrdU) into Sox10 positive cells present in the stomach SOX10 positive cells co-labelled by the proliferation marker cultures of E11.5 embryos of different genotypes. The fraction of L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 239

Fig. 3. ENS phenotype of double mutants results from an absence of enteric progenitor cells. Whole-mount X-Gal staining was performed at E13.5–14 on guts from Sox10LacZ/+ (A, D, E), Sox10LacZ/+;Edn3ls/+ (B), Sox10LacZ/+;Edn3ls/ls (C) and Sox10LacZ/+;Ednrbs/s (F). Note i) the phenotype variation among Sox10LacZ/+ embryos (compare A, D, E), ii) the absence of X-Gal staining halfway through the small intestine of Sox10LacZ/+;Edn3ls/+ embryos (B), iii) the absence of X-Gal staining in the entire intestine of Sox10LacZ/+;Edn3ls/ls and Sox10LacZ/+;Ednrbs/s embryos (C, F). s: stomach; si: small intestine; ce: caecum; co: colon. White arrows indicate the caecum region.

BrdU positive cells among the Sox10 positive population was TuJ1+ cells expresses lower levels of TuJ1, has very few short similar in all the genotypes (including Sox10LacZ/+;Ednrbs/s; axons and is likely to correspond to Ret- and Mash1-positive Fig. 5C), suggesting that the more severe ENS defects in committed enteric neuroblasts described previously (Fig. 5F2) Sox10LacZ/+;Ednrbs/s embryos are not due to decreased (Barlow et al., 2003; Bondurand et al., 2003). To quantify the proliferation of progenitor cells within the stomach wall. two cell populations, we determined the percentage of “TuJ1- Finally, we examined the possibility that the absence of Low” cells among the total population of neurons. Interestingly, enteric neural crest cells within the small intestine of we found a significant increase in the relative proportion of Sox10LacZ/+;Ednrbs/s or Sox10Dom/+;Ednrbs/s embryos could “TuJ1-Low” cells in Sox10LacZ/+;Ednrbs/s embryos compared result from an increase in overall differentiation of these cells. to other genotypes, suggesting that terminal neuronal differen- To test this possibility, we determined the percentage of enteric tiation could be specifically affected in mice with combined cells specified to a neuronal fate by calculating the fraction of Sox10 and Ednrb mutations (Fig. 5G). In parallel, we also Phox2b positive cells (identifying enteric neural crest progeni- analysed the percentage of E13.5 ENS cells that were specified tors and derivatives; Young et al., 2003) co-labelled with the to a glial fate by determining the fraction of Sox10 positive cells neuronal marker TuJ1. We first attempted to analyse overall co-labelled with the early glial marker B-FABP. We found no neuronal differentiation in E11.5 embryos of different geno- significant change in the percentage of double positive cells types. We found no significant change in the percentage of in Sox10LacZ/+;Ednrbs/s compared to other genotypes, indi- double positive (Phox2b+TuJ1+) cells in Ednrbs/s compared to cating that glial differentiation in the stomach of double wild-type. In agreement with previous report, an overall mutants proceeds normally (Fig. 5H). increase in neuronal differentiation was observed in Sox10- single mutants (about 15% relative to wild-type) (Paratore et Vagal neural crest cells defects in E10 Sox10;Ednrb double al., 2002). Interestingly, the same increase was detected in mutants Sox10LacZ/+;Ednrbs/s embryos (Fig. 5D). Although not specific of double mutants, this small increase in differentiation could To determine the origin of the ENS defects observed in partially contribute to the more severe ENS defect observed in Sox10;Edn3 and Sox10;Ednrb double mutants, we analysed the double mutants. We next performed similar experiments on behaviour of vagal neural crest cells around the time of invasion acute cultures from stomach of E13.5 embryos of different of the foregut (E9.5–E10). Sections of E10 wild-type, genotypes. No significant difference in the percentage of double Sox10LacZ/+, Ednrbs/s and Sox10LacZ/+;Ednrbs/s embryos were positive (Phox2b+TuJ1+) cells was observed between all labelled with Sox10 in order to visualise vagal neural crest cells. genotypes, including Sox10LacZ/+;Ednrbs/s (Fig. 5E). However, Sections were double stained with activated caspase-3 to detect careful analysis of TuJ1 staining at E13.5 allowed us to identify apoptosis. In wild-type and Ednrbs/s embryos, cells were two types of TuJ1 positive cell populations. One of these visualised migrating within the foregut, and few caspase-3 expresses high level of TuJ1 and has long axonal processes (Fig. positive cells were detected within or outside the gut (Fig. 6B1, 5F1) and is likely to correspond to postmitotic neurons B2, C1, C2). As previously described, we observed a higher expressing high levels of Ret. The second population of number of apoptotic cells in the stream of migrating neural crest 240 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249

Fig. 4. ENS phenotype of E11.5 Sox10;Edn3 and Sox10;Ednrb double mutants. (A1–5 and C1–4) Whole-mount TuJ1 immunohistochemistry and (B1–3 and D1, D2) whole-mount X-Gal staining of mouse embryonic guts of indicated genotypes. Note the severe migration delay in Sox10LacZ/+;Edn3ls/+, Sox10LacZ/+;Edn3ls/ls (compare A4–B2 and A5–B3 to A1–3, B1) and Sox10LacZ/+;Ednrbs/s embryos (compare C4 and D2 to C1–3 and D1). Insets in panels B1–3, D1 and D2 show a side view of the stomach. (E) Immunohistochemistry on sections (oesophagus region) of wild-type (E1), Sox10LacZ/+ (E2), Ednrbs/s (E3) and Sox10LacZ/+;Ednrbs/s (E4) embryos, using Phox2b (red) and TuJ1 (green) antibodies. s: stomach; ce: caecum. White arrows indicate the oesophagus region. cells of Sox10LacZ/+ embryos, but caspase-3 positive cells agreement with previous observations, apoptotic cells reached were localised outside the gut only (Fig. 6D1, D2 and Maka their maximum in Sox10 deficient embryos, resulting in a et al., 2005). Interestingly, the number of vagal neural crest complete absence of Sox10 positive cells within the gut (Fig. cells migrating along the foregut was severely reduced in 6F1, F2 and Maka et al., 2005). Thus, the more severe ENS Sox10LacZ/+;Ednrbs/s embryos compared to controls (wild-type defects observed in Sox10LacZ/+;Ednrbs/s animals could at least and single mutants) (Fig. 6E1, E2). Indeed, the average number partially be due to increased cell death of vagal neural crest cells of Sox10 positive cells detected along the foregut of double prior to their invasion of the foregut. mutants was reduced by 42–86% compared to Sox10LacZ/+ single mutants and by 54–90% compared to wild-type or Sox10;Ednrb but not all Sox10;Edn3 double mutants manifest Ednrbs/s (differences to the wild-type, Ednrbs/s and Sox10LacZ/+ extended white spotting were statistically significant for all sections of Sox10LacZ/+; Ednrbs/s analysed, P < 0.05). Moreover, we observed a small In addition to ENS deficits, Sox10, Edn3 and Ednrb increase in the number of caspase-3 positive cells compared to mutations result in variable white spotting (hypopigmenta- Sox10LacZ/+ embryos (compare Fig. 6E1, E2 to D1, D2). tion) due to defects in melanocyte development. Progenitors Again, apoptotic cells were visualised only outside of the gut. In of melanocytes arise from the dorsal aspect of the neural L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 241

Fig. 5. Cellular origin of the ENS defects in E11.5 and E13.5 Sox10;Ednrb double mutants. (A) Quantification of the fraction of Sox10 positive cells co-labelled with activated caspase-3 in acute dissociated stomach cultures of E11.5 embryos of different genotypes. On average, 300 to 600 cells from 4 different embryos of each genotype were counted. (B and C) Proliferation capacities of E11.5 enteric progenitor cells. (B) Quantification of the fraction of Sox10 positive cells co-labelled with phospho-histone H3. On average, 500 to 1000 cells from 3 different embryos of each genotype were counted. (C) Fraction of Sox10-positive cells co-labelled by BrdU. On average, 200 to 800 cells from 3 different embryos of each genotype were counted. (D–E) Quantification of the fraction of Phox2b positive cells co-labelled with TuJ1, reflecting the overall neuronal differentiation on acute stomach cultures of E11.5 (D) and E13.5 (E) embryos of different genotypes. On average, 300 to 800 cells from 3 to 5 different embryos of each genotype were counted. Differences to the wild-type were statistically significant for Sox10LacZ/+ and Sox10LacZ/+;Ednrbs/s E11.5 embryos as determined by the Student's t test (P = 0.001 and P = 0.02, respectively). (F) Careful examination of TuJ1 staining in E13.5 cultures revealed two types of TuJ1 cell populations. One of these expresses high levels of TuJ1 and has long axons (“TuJ1-High”, F1). The second population expresses lower levels of TuJ1 and has very few short axons (“TuJ1-Low”, F2). (G) Quantification of TuJ1-Low cells over the total population of TuJ1 positive cells in embryos of each genotype. Note the significant increased percentage of TuJ1-Low cells in Sox10LacZ/+;Ednrbs/s stomach cultures compared to controls (wild-type, P < 0.006; and single mutants, P < 0.05). (H) Quantification of the fraction of SOX10 positive cells co-labelled with the early B-FABP glial marker, reflecting glial differentiation. On average, 300 cells from 3 different embryos of each genotype were counted. tube at E9.5 and accumulate for approximately 24 h lateral between SOX10 and the endothelin-3/EDNRB signalling to the neural tube in the MSA. At E10.5, melanoblasts start pathway during melanocyte development, we analysed the their migration along the dorsolateral pathway in a temporal extent of white spotting in mice generated from Sox10Dom rostrocaudal gradient. After migration, melanoblasts enter the and Edn3ls or Ednrbs crosses. epidermis where they proliferate extensively (between E12.5 As previously described, Edn3ls/+ did not show any and E13.5) before they differentiate into pigment-producing pigmentation anomalies, but Edn3ls/ls animals exhibited a melanocytes shortly after birth. To test a cooperative activity white patch of hair on the head, ventral hypopigmentation and 242 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249

cantly worsen the pigmentary phenotype. Due to the early mortality of Sox10Dom/+;Edn3ls/ls animals (all die before P10 as a result of severe megacolon), we analysed the severity of their hypopigmentation between P7 and P10. We observed a clear increase in the extent of white spotting in 30% of these animals (5/13; Fig. 7D). Of the remaining pups, 6/13 presented with hypopigmentation affecting the head region only (Fig. 7E) and 2/13 displayed a pigmentation pattern similar to that of Edn3ls/ls pups (Fig. 7F). These results suggest that severe reduction of endothelin-3 activity in Sox10Dom/+ animals increases the severity of white spotting in some cases but not all. Similar experiments were carried out using the Sox10LacZ mice and produced identical results (data not shown). However, in addition to endothelin-3, the EDNRB receptor can be activated by the other endothelin peptides during melanocyte development. Therefore, we analysed the extent of white spotting in mice generated from Sox10Dom and Ednrbs crosses. As expected, Ednrbs/+ mice did not show pigmentation abnormalities, but Ednrbs/s presented with a white patch of hair on the head, a hypopigmented patch on the abdomen, which could extent down to the hindlimbs, and variable albeit moderate (10–30%) white patches on the dorsal surface (Fig. 7G1, G2; Hosoda et al., 1994). No significant differences could be seen between Sox10Dom/+ and Sox10Dom/+;Ednrbs/+ mice (Fig. 7, compare H1-2 to I1-2). Both exhibited a white ventral spot in most cases (Sox10Dom/+: 8/11 and Sox10Dom/+; Ednrbs/+: 18/21; the remaining 3 Sox10Dom/+ animals were normally pigmented while the remaining 3 Sox10Dom/+; Ednrbs/+ presented with a slightly increased hypopigmentation pattern, with a larger white belly spot extending down to the hindlimbs). However, these crosses also produced progeny with extensive hypopigmentation, affecting up to 70% of their dorsal coat. These mice were severely runted, died before P10 and, upon genotyping, they were all identified as Sox10Dom/+; Ednrbs/s (Fig. 7J, arrow indicates a double mutant). Thus, while in Ednrbs/s homozygotes white spotting does not exceed 30% of the dorsal coat (and in 25/33 animals it is less that 20%), the majority of Sox10Dom/+;Ednrbs/s animals Fig. 6. Vagal neural crest cells anomalies in E10 Sox10;Ednrb double mutants. (10/13) presented extensive white spotting covering 50–70% (A) Schematic of the area shown in panels B–F. Immunohistochemistry on s/s LacZ/+ LacZ/+ of their coat. Similar experiments were performed using sections of E10 wild-type (B), Ednrb (C), Sox10 (D), Sox10 ; LacZ Ednrbs/s (E) and Sox10LacZ/LacZ (F) embryos, using Sox10 (red) and caspase-3 Sox10 mice and produced very similar results (data not (green) or β-galactosidase (green) and caspase-3 (red) antibodies in the shown). Therefore, combined deficits of SOX10 function and oesophagus tracheal (B1–F1) and foregut (B2–F2) regions. White dotted lines EDNRB signalling cause profound defects on pigmentation indicate the oesophagus tracheal and foregut regions. and argue for a cooperative activity or a successive requirement of these two molecules during coat melanocyte development. variable albeit moderate (5–30%) dorsal white spotting (Fig. 7A1, A2 and Rice et al., 2000). Most Sox10Dom/+ mice, on the Sox10;Ednrb double mutants lack melanocytes in the inner ear other hand, presented with a variable white ventral spot only (Fig. 7B1, B2). Moreover, no significant difference in the extent The interrelation between pigmentation and hearing has of white spotting was observed between Sox10Dom/+ and been known for more than a century, as indicated by the Sox10Dom/+;Edn3ls/+ (Fig. 7C1, C2). Thus, 17/20 Sox10Dom/+ requirement of melanocytes (intermediate cells) for normal and 7/12 Sox10Dom/+;Edn3ls/+ animals analysed exhibited a development and function of the cochlea. Thus, absence of similar size abdominal spot (of the remaining animals, 3 double melanocytes in the inner ear results in the loss of heterozygotes showed a larger white area, and other single and endolymphatic potential and causes endolymphatic collapse double mutants showed no pigmentary defects). Thus, reducing and sensory hair cell degeneration in the organ of Corti endothelin-3 activity in Sox10Dom/+ animals did not signifi- (Tachibana, 1999; Tachibana et al., 2003). Little is known L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 243

Fig. 7. Sox10;Ednrb but not all Sox10;Edn3 double mutants manifest extended white spotting. (A–F) Extent of white spotting in mice generated from Sox10Dom and Edn3ls crosses. Typical dorsal and ventral pigmentation defects observed in Edn3ls/ls (A1–2), Sox10Dom/+ (B1–2) and Sox10Dom/+;Edn3ls/+ (C1–2) at 3–4 weeks. (D– F) Typical dorsal spotting of Sox10Dom/+;Edn3ls/ls pups at P5–7. Note the severe increased of hypopigmentation in panel D and white spotting affecting the head region only in panel E. Black arrows indicate the double mutants. (G–J) Extent of white spotting in mice generated from Sox10Dom and Ednrbs crosses. Typical dorsal and ventral pigmentation defects observed in Ednrbs/s (G1–2), Sox10Dom/+ (H1–2) and Sox10Dom/+;Ednrbs/+ (I1–2) at 3–4 weeks. (J) Comparison of pigmentation defects of, from left to right, Sox10Dom/+;Ednrbs/+ (2 animals), Ednrbs/s, Sox10Dom/+;Ednrbs/s and Sox10Dom/+ at P6. The black arrow indicates the Sox10Dom/+;Ednrbs/s mutant. Note the increased white spotting in Sox10Dom/+;Ednrbs/s compared to single mutants. about endothelin-3/EDNRB and SOX10 function on interme- control (wild-type, Sox10Dom/+, and Ednrbs/s) mice at birth. To diate cells development. Preliminary reports have suggested observe melanocytes within the vestibular region, we first that the cochlea of JF1 mice (a mouse model phenotypically dissected the temporal bone of newborn pups and removed the similar to Ednrbs/s) showed endolymphatic collapse with no bone surrounding the vestibule. At that stage of dissection, a intermediate cells in the stria vascularis (Tachibana et al., high density of melanocytes was clearly visible in the 2003). However, no such deficits have been described in endolymphatic duct and the utricule of wild-type (3/3), Sox10Dom/+ mice (Tachibana et al., 2003). Considering the Ednrbs/+ (7/7), Sox10Dom/+ (2/2)orSox10Dom/+;Ednrbs/+ extensive white spotting observed in Sox10Dom/+;Ednrbs/s,we double heterozygotes (6/6) (Fig. 8A and B). Moreover, about have explored the possibility of a cooperative activity between half the Ednrbs/s mutants presented less melanocytes in the SOX10 and the endothelin-3/EDNRB signalling pathway for vestibular region (4 of 6, Fig. 8C). Strikingly, we observed a the development of melanocytes within the inner ear. For this, severe reduction and a total absence of melanocytes in 2/6 we observed the presence of melanocytes within the vestibular and 4/6 of the Sox10Dom/+;Ednrbs/s mutants, respectively region and the stria vascularis of Sox10Dom/+;Ednrbs/s and (Fig. 8D). Next, we dissected the stria vascularis and 244 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 examined melanocytes (intermediate cells) within this region. Discussion We observed similar results, with a reduced number or absence of melanocytes in the Ednrbs/s (half of the pups Genetic interactions between SOX10 and the analysed in each case), and a total absence of melanocytes in endothelin-3/EDNRB signalling pathway during all the Sox10Dom/+;Ednrbs/s double mutants (6/6; Fig. 8, ENS development compare E–F and G–H). These results are consistent with previous data showing an absence of intermediate cells in the A requirement for SOX10 and endothelin-3/EDNRB stria of JF1 mice but not in Sox10Dom/+ mutants and clearly signalling pathway in the development of the peripheral nervous demonstrate that the melanocyte defects observed in double system in general, and the ENS in particular, is well established mutants are significantly more severe relative to those (Wegner, 1999; Pla and Larue, 2003). In addition, over the past described for the corresponding single mutants. In JF1 mice, several years, the identification and functional characterisation absence of melanocytes is hypothesised to be responsible for of mutations affecting SOX10, EDN3 and EDNRB genes in the degeneration of the organ of Corti (Tachibana et al., 2003). human and studies on the developmental defects observed in the However, the facts that i) the agenesis of the organ of Corti respective spontaneous or induced mouse mutants have been has also been described in Sox10Dom/+ animals in which the main source of information for our understanding of WS4 melanocytes are present and ii) Sox10 is widely expressed in and HSCR genetics (Amiel and Lyonnet, 2001). Variability in the sensory epithelium of the cochlea prompted us to analyse the severity of the phenotypes was reported among patients and the organ of Corti in embryos (E18) and neonates (up to P4) in the different animal models. In mouse mutants, this to determine whether hair cells develop normally prior to variability is ranging from no enteric phenotype in almost all degeneration. In all genotypes analysed, including Sox10Dom/+ Ednrbs/s mutants to total intestinal aganglionosis observed in and Sox10Dom/+;Ednrbs/s, phalloidin staining showed the Sox10Dom/Dom or Sox10LacZ/LacZ animals. Other genotypes such presence of actin in apparently normal hair cells bundles, as Edn3ls/ls, Sox10Dom/+ or Sox10LacZ/+ mice show partial organised in one row of inner hair cells and three rows of aganglionosis of the colon and are thus considered as models for outer hair cells (Fig. 8I–L). Therefore, in all animals HSCR. With regard to SOX10, incomplete penetrance and examined, including Sox10Dom/+ mice, hair cells are generated phenotype variability pointed to the existence and influence of before the degeneration of the organ of Corti. modifier genes. In the search of such genes, Cantrell et al.

Fig. 8. Inner ear defects of Sox10;Ednrb double mutants. Observation of melanocytes in the vestibular region and stria vascularis of wild-type (A and E), Sox10Dom/+ (B and F), Ednrbs/s (C and G) and Sox10Dom/+;Ednrbs/s (D and H) animals at birth. The black arrow indicates melanocytes within the endolymphatic duct, arrowhead indicates melanocytes within the utricule region, and insets in panels E–H represent higher magnification of the respective boxed areas. Note the presence of melanocytes in Sox10Dom/+, the reduced number of melanocytes in the vestibular region of Ednrbs/s and the total absence of melanocytes in the vestibular region and the stria vascularis of Sox10Dom/+;Ednrbs/s mutants. Whole-mount immunostaining on organ of Corti dissected from wild-type (I), Sox10Dom/+ (J), Ednrbs/s (K) and Sox10Dom/+;Ednrbs/s (L) newborn animals using phalloidin staining. Note the presence of actin in apparently normal hair cells bundles organised in one row of inner hair cells (IHC) and three rows of outer hair cells (OHC) in all animals. scc: semi-circular canals; c: cochlea. L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 245 recently reported that interactions between Sox10 and Ednrb enteric neural crest cells within the stomach regions of E11.5 influence the severity of aganglionosis in the Sox10Dom model and E13.5 embryos of various genotypes. Our results suggest (Cantrell et al., 2004). Crosses between Ednrb and Sox10 that the ENS defects observed in Sox10LacZ/+;Ednrbs/s are not mutants allowed them to corroborate this gene interaction as due to a reduction of the pool of enteric progenitor cells by double mutant progeny exhibited a significantly more severe apoptosis, which would be expected to reduce the number of aganglionosis at birth. Here, we tested a possible cooperative cells with migratory capacities. The rate of proliferation was activity between Sox10 and the functional ligand of EDNRB in also very similar in enteric neural crest cells of the different the gut, endothelin-3, and attempted to decipher the cellular genotypes. Furthermore, we failed to detect an overall increase mechanisms underlying the combined activity of SOX10 and in neuronal or glial differentiation in double mutants compared endothelin signalling in ENS development. Our results extend to single mutants. Nevertheless, it should be mentioned that, in a previous studies by demonstrating a genetic interaction between manner similar to Sox10LacZ/+ single mutants, we observed an Sox10 and Ednrb and between Sox10 and Edn3. overall increase in the percentage of neurons within the stomach The Ednrb mutation we used in our crosses (Ednrbs)isa region of E11.5 double mutants (about 15% compared to wild- hypomorph mutant. Homozygous animals still express 30% of type littermates). Although not specific to double mutants, this functional receptor and present no obvious enteric phenotype after small increase in differentiation could contribute to the more birth. In contrast, Cantrell et al. crossed Sox10Dom mice with two severe ENS defect observed in Sox10LacZ/+;Ednrbs/s embryos. different Ednrb mutants: Ednrbsl and Ednrbtm1Ywa.TheEdnrbsl This overall increase was not found in cultured neural-crest- allele corresponds to a 1 cM deletion spanning the Ednrb gene and derived cells from the stomach region of older embryos (E13.5). possible regulatory regions (Roix et al., 2001). The Ednrbtm1Ywa In contrast, careful examination of neuronal markers in E13.5 mutation has been specifically engineered to replace a 4.2-kb gene Sox10Dom/+;Ednrbs/s acute cultures suggested that terminal fragment carrying exon 3 with a neomycin selection cassette, neuronal differentiation and axonogenesis could be specifically causing premature termination and complete loss of Ednrb affected. Interestingly, a recent report has demonstrated that the expression (Hosoda et al., 1994). Strikingly, the increased severity direction in which neurons projected is correlated with the of the aganglionosis was only observed in Sox10Dom/+;Ednrbs/s direction of cell migration during ENS development (Young et (this study), Sox10Dom/+;Ednrbsl/sl and Sox10Dom/+;Ednrbtm1Ywa/+ al., 2002). Therefore, the reduction in the number of axons animals, but not in Sox10Dom/+;Ednrbsl/+ double heterozygotes observed in enteric neurons of Sox10Dom/+;Ednrbs/s embryos (Cantrell et al., 2004). We also performed crosses using Sox10Dom could alter cell migration and thus, at least partially, explain the and Ednrbsl mice and observed similar results (our unpublished anomalies observed in these double mutants. However, we results).Wesuggestthattherequirementofaminimallevel(30– cannot exclude the possibility that the reduced number of axons 50%) of EDNRB activity accounts for the ENS defects we observed at E13.5 results from secondary effects. Alternatively, observed in Sox10Dom/+;Ednrbs/s animals and thus define a aganglionosis in double mutants could be due to an unrelated minimum level requirement of EDNRB for normal ENS migration defect. development. The differences observed between Sox10Dom/+; We also extended our analysis to earlier times of ENS Ednrbsl/+ and Sox10Dom/+;Ednrbtm1Ywa/+ might simply result development and analysed the behaviour of vagal neural crest from genetic background variations or from other defects specific cells around the time of invasion of the foregut (E9.5–E10). to the Ednrbsl strain, such as the deletion of adjacent regulatory These studies revealed a severe reduction in the number of sequences or genes within the 1 cM deletion. Sox10 positive cells migrating within the foregut region and increased apoptosis of vagal neural crest cells outside of the gut Origin of the ENS defects in double mutant mice of Sox10LacZ/+;Ednrbs/s double mutants. Although not as strong as the Sox10LacZ/LacZ phenotype where apoptosis of vagal crest We analysed the ENS development of Sox10;Edn3 and cells was so high that cells did not even reach the foregut region Sox10;Ednrb double mutants relative to control (wild-type and (Fig. 6 and Maka et al., 2005), these results suggest that reduced single mutants) littermates between E10 and E15.5 using survival of vagal neural crest cells before/at the time they enter various molecular markers. First, we observed a severe the gut (E10) could at least partially explain the more severe migration delay within the intestine of E13.5 Sox10Dom/+; ENS phenotype observed in Sox10;Ednrb double mutants. We Edn3ls/+ and Sox10LacZ/+;Edn3ls/+ embryos, while enteric cannot exclude that the reduced number of Sox10 positive cells progenitor cells (identified by expression of Sox10) and neurons observed in the foregut of E10 Sox10LacZ/+;Ednrbs/s embryos were only detected within the stomach of Sox10Dom/+;Ednrbs/s, could also result from additional migration, proliferation or Sox10LacZ/+;Ednrbs/s, Sox10Dom/+;Edn3ls/ls and Sox10LacZ/+; differentiation anomalies. Further experimentations will be Edn3ls/ls embryos. Moreover, analysis of E11.5 Sox10LacZ/+; necessary to explore these issues. Edn3ls/+, Sox10LacZ/+;Edn3ls/ls and Sox10LacZ/+;Ednrbs/s At the molecular level, the interaction between SOX10 and mutants showed that colonisation of the gut by enteric neural the endothelin-3/EDNRB pathways can be partially explained crest cells was significantly retarded, implying that the by the transcriptional regulation taking place between SOX10 anomalies observed at early times were never corrected at and Ednrb in ENS (Zhu et al., 2004). Indeed, Zhu et al. later developmental stages. recently identified a temporally regulated enteric-specific To decipher the cellular basis of these defects, we analysed Ednrb enhancer containing multiple SOX binding sites, the proliferation, survival and differentiation capacities of which are necessary and sufficient for normal spatio-temporal 246 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 expression of Ednrb. Based on their observations, Zhu et al. of additional pigmentation defects when combined with Sox10 suggested that regulation of this enhancer by SOX10 could be mutations. Extensive white spotting was observed in only 30% critical for Ednrb function when enteric neurons precursors of the Sox10Dom/+;Edn3ls/ls or Sox10LacZ/+;Edn3ls/ls animals colonise the colon (after E11.5) (Zhu et al., 2004). However, with the remaining presenting with an almost complete loss of the partial or complete absence of enteric progenitors within pigment cells over the head region or with a pigmentation the small intestine of Sox10;Ednrb and Sox10;Edn3 double pattern similar to Edn3ls/ls mice. The reasons for this variability mutants at E11.5, and the vagal neural crest cells anomalies are currently unclear, but it is possible that local compensation observed in E10 Sox10;Ednrb double mutants argues for a by other endothelins may occur and account for the relatively possible cooperative activity between SOX10 and the mild phenotype in a fraction of these animals. This idea is endothelin-3/EDNRB signalling pathway earlier during de- consistent with detection of endothelin-1 in the skin and the velopment. We suggest that this “earlier” interaction could be ability of EDNRB to bind endothelin-1 and -3 with similar explained by another regulatory loop taking place between affinities. Nevertheless, the pigmentation patterns observed in endothelin-3 and Sox10, hypothesis in line with the genetic Sox10Dom/+;Edn3ls/ls animals suggest that, if such a compen- interaction observed between these two genes. Indeed, sation mechanism takes place, it is mainly effective at a endothelin-3, through EDNRB activation, could directly or relatively late stage of melanocyte development as head indirectly maintain Sox10 expression or modulate SOX10 melanocytes, which are the first to arise, are missing in almost protein activity. The reduction of Edn3 in combination with all these double mutants (Yoshida et al., 1996). The cellular and Sox10 haploinsufficiency could decrease the level or the molecular mechanisms which could explain the defects number of cells expressing Sox10 in double mutants, observed in Sox10Dom/+;Edn3ls/ls, Sox10LacZ/+;Edn3ls/ls, enabling the precursor population to persist long enough to Sox10Dom/+;Ednrbs/s and Sox10LacZ/+;Ednrbs/s mutants are colonise the gut. currently unknown. Further investigations are required to determine if they are directly or indirectly linked to recent SOX10 and endothelin-3/EDNRB cooperative activity or observations highlighting i) a transient lack of Dct expression in successive requirement in coat melanocyte development Sox10 heterozygous animals, and ii) a requirement of Ednrb for mouse melanoblasts (Dct positive cells) migration, proliferation In addition to ENS deficits, SOX10, EDN3 and EDNRB and differentiation (Lee et al., 2003; Opdecamp et al., 1998; Pla mutations result in variable pigmentation abnormalities due and Larue, 2003; Potterf et al., 2001). to defects in melanocyte development. Upon testing for cooperative activity between SOX10 and the endothelin-3/ SOX10 and endothelin-3/EDNRB cooperative activity during EDNRB signalling pathway in this cell type, we observed no inner ear melanocyte development differences between the pigmentation phenotype of Sox10Dom/+, Sox10Dom/+;Edn3ls/+ or Sox10Dom/+;Ednrbs/+ mice. Indeed, Patients and mouse mutants with SOX10, EDN3 and EDNRB animals with these various genotypes presented with a white mutations also present with hearing impairment, which is mainly belly spot of similar size. Crosses performed with other mutants described as a consequence of an absence of melanocytes within such as Sox10LacZ and Ednrbsl produced similar results (our the inner ear. Absence of these cells within the stria vascularis unpublished results and Cantrell et al., 2004; Hakami et al., results in the loss of endolymphatic potential and causes 2006). In contrast, we observed severe hypopigmentation in endolymphatic collapse and sensory hair cell degeneration in Sox10Dom/+;Ednrbs/s animals compared to single mutants. the organ of Corti. Melanocytes are also found within the While in the Ednrbs/s mice dorsal white spotting did not exceed vestibular region, but their function remains poorly understood. 30%, the majority of Sox10Dom/+;Ednrbs/s animals (10/13) Upon testing possible interactions between SOX10 and the presented extensive white spotting covering 50–70% of their endothelin/EDNRB signalling pathways for the development of dorsal coat. As Sox10Dom/+ animals present with a white belly these cells, we observed an apparently normal distribution of spot only, these results demonstrate that combination of SOX10 melanocytes in the endolymphatic duct, utricule and stria and EDNRB signalling defects has profound effects on vascularis of wild-type, Sox10Dom/+ and Sox10Dom/+;Ednrbs/+. pigmentation. We believe that the severe hypopigmentation In contrast, about half of the Ednrbs/s animals presented with less observed in Sox10Dom/+;Ednrbs/s animals cannot be explained melanocytes within the vestibular region and reduced number or by concomitant and independent functions of SOX10 and absence of melanocytes within the stria vascularis. More EDNRB and thus argues for a cooperative activity or for a strikingly, we observed a total absence of melanocytes in the successive requirement of these two factors during early coat Sox10Dom/+;Ednrbs/s embryos, both in the vestibular region and melanocyte development. These results highlight unique in the stria vascularis, highlighting a cooperative activity features of the Ednrbs allele relative to other Ednrb mutants between SOX10 and EDNRB during melanocytes inner ear (such as Ednrbsl or knock-out mice) in testing for gene development. In parallel, we analysed hair cell formation using interactions. More specifically, we believe that the hypomorphic phalloidin staining and found no anomalies in all mutants, nature of the Ednrbs allele allows us to detect further influences including Sox10Dom/+ and Sox10Dom/+;Ednrbs/s suggesting on the development of pigment cells upon combination with normal commitment and development of hair cells at early other mutants while the complete loss of melanocytes in other stages, prior to their degeneration. The broad expression profile Ednrb homozygous single mutants would preclude the analysis of Sox10 during inner ear development also prompted us to L. Stanchina et al. / Developmental Biology 295 (2006) 232–249 247 examine the general morphology of the inner ear at embryonic signalling pathway. Although further work will be necessary to day E13.5 and E15.5. For this, we compared X-Gal staining of distinguish between these possibilities, the data presented here Sox10LacZ/+, Sox10LacZ/+;Ednrbs/+, and Sox10LacZ/+;Ednrbs/s contribute to the further understanding of the molecular basis of embryos and found normal morphological development of the enteric, pigmentation and hearing defects observed in mouse endolymphatic duct, semicircular canals and cochlear duct in mutants and patients affected by SOX10, EDN3 and EDNRB embryos of all genotypes (our unpublished observations). Taken mutations. together, these results suggest a cooperative activity between SOX10 and the endothelin-3/EDNRB specifically during inner Acknowledgments ear melanocyte development. We thank Dr. M. Wegner, Dr. J.F. Brunet and Dr. T. Müller SOX10 and endothelin-3/EDNRB interactions during for providing antibodies, Dr. M. Wegner for the Sox10 knock- development of neural crest derivatives out mice, Mohamed Bouaziz for animal husbandry and Cyrille Sage for help with BrdU experiments. We also thank Benedicte Our results show that a coordinate and balanced interrelation Duriez for comments on the manuscript and helpful discussions between SOX10 and the endothelin-3/EDNRB signalling during the course of this work. This work was supported by pathway is required for normal ENS and melanocyte develop- AFM and GIS-Institut Maladies rares. ment. It should be mentioned that Sox10 and Ednrb are also co- expressed in other neural crest derivatives such as cranial References ganglia, dorsal root ganglia (drg), as well as in the sympathetic ganglia chain, and in the peripheral nerves (Britsch et al., 2001; Amiel, J., Lyonnet, S., 2001. Hirschsprung disease, associated syndromes, and Herbarth et al., 1998; Lee et al., 2003). Both factors have been genetics: a review. J. Med. Genet. 38, 729–739. described as key regulators of glial cells in general and Schwann Aoki, Y., Saint-Germain, N., Gyda, M., Magner-Fink, E., Lee, Y.H., Credidio, cells in particular. Indeed, SOX10 is crucial for the survival and C., Saint-Jeannet, J.P., 2003. Sox10 regulates the development of neural – maintenance of pluripotency of migrating neural crest cells but crest-derived melanocytes in Xenopus. Dev. Biol. 259, 19 33. Barlow, A., de Graaff, E., Pachnis, V., 2003. Enteric nervous system progenitors also influences survival and glial fate decision at later stages are coordinately controlled by the G protein-coupled receptor EDNRB and (Britsch et al., 2001 and for reviews see Mollaaghababa and the receptor tyrosine kinase RET. Neuron 40, 905–916. Pavan, 2003; Wegner, 1999; Wegner, 2005). The survival and Baynash, A.G., Hosoda, K., Giaid, A., Richardson, J.A., Emoto, N., Hammer, R. progression of Schwann cell precursors to Schwann cells is also E., Yanagisawa, M., 1994. Interaction of endothelin-3 with endothelin-B regulated by the endothelin/EDNRB signalling pathways receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79, 1277–1285. (Brennan et al., 2000; Jessen and Mirsky, 2002; Woodhoo et Bolande, R.P., 1974. The neurocristopathies: a unifying concept of disease al., 2004). Based on these findings, we have examined the arising in neural crest maldevelopment. Hum. Pathol. 5, 409–429. formation of other neural crest derivatives in E11.5 Sox10Dom; Bondurand, N., Pingault, V., Goerich, D.E., Lemort, N., Sock, E., Caignec, C.L., Ednrbs double mutants relative to single mutants. Immunos- Wegner, M., Goossens, M., 2000. Interaction among SOX10, PAX3 and taining on sections using Sox10, Phox2b, B-FABP or TuJ1 MITF, three genes altered in Waardenburg syndrome. Hum. Mol. Genet. 9, 1907–1917. antibodies revealed no changes in the distribution of Schwann Bondurand, N., Natarajan, D., Thapar, N., Atkins, C., Pachnis, V., 2003. Neuron cell precursors along nerves and no obvious defects in the and glia generating progenitors of the mammalian enteric nervous system formation and differentiation of cells within drg. In addition, the isolated from foetal and postnatal gut cultures. Development 130, sympathetic chain ganglia seemed to form normally (see Fig. 6387–6400. 4E1–4). Thus, the phenotype of mice with combined Sox10; Bowles, J., Schepers, G., Koopman, P., 2000. Phylogeny of the SOX family of developmental transcription factors based on sequence and structural Ednrb mutations suggests a specific cooperative requirement for indicators. Dev. Biol. 227, 239–255. the development of the ENS and melanocytic neural crest cell Brennan, A., Dean, C.H., Zhang, A.L., Cass, D.T., Mirsky, R., Jessen, K.R., lineage. The requirement for lower levels of Ednrb in some 2000. Endothelins control the timing of Schwann cell generation in vitro and neural crest derivatives could explain the differences observed. in vivo. Dev. Biol. 227, 545–557. The use of the hypomorph Ednrbs mutation could therefore Britsch, S., Goerich, D.E., Riethmacher, D., Peirano, R.I., Rossner, M., Nave, K.A., Birchmeier, C., Wegner, M., 2001. The transcription factor preclude the visualisation of defects within the peripheral Sox10 is a key regulator of peripheral glial development. Genes Dev. nervous system. Moreover, Sox10 and Edn3/Ednrb seem to be 15, 66–78. the key players in ENS development, but the expression of other Cantrell, V.A., Owens, S.E., Chandler, R.L., Airey, D.C., Bradley, K.M., Smith, SOX proteins or other endothelin peptides is reported at J.R., Southard-Smith, E.M., 2004. Interactions between Sox10 and EdnrB different stages of melanocyte and peripheral nervous system modulate penetrance and severity of aganglionosis in the Sox10Dom mouse model of Hirschsprung disease. Hum. Mol. Genet. 13, 2289–2301. development. Therefore, functional redundancy could explain Cheung, M., Chaboissier, M.C., Mynett, A., Hirst, E., Schedl, A., Briscoe, J., the relatively mild phenotype or absence of phenotype in these 2005. The transcriptional control of trunk neural crest induction, survival, neural crest derivatives. In line with this information, Sox9 is and delamination. Dev. Cell 8, 179–192. expressed in melanocytes and peripheral nervous system, and Cook, A.L., Smith, A.G., Smit, D.J., Leonard, J.H., Sturm, R.A., 2005. Co- endothelin-1 is known to play a role in melanocytes (Cheung et expression of SOX9 and SOX10 during melanocytic differentiation in vitro. Exp. Cell Res. 308, 222–235. al., 2005; Cook et al., 2005; Pla and Larue, 2003). On the other Dupin, E., Le Douarin, N.M., 2003. Development of melanocyte precursors hand, our results could be explained by tissue-specific from the vertebrate neural crest. Oncogene 22, 3016–3023. interactions between SOX10 and the endothelin-3/EDNRB Dutton, K.A., Pauliny, A., Lopes, S.S., Elworthy, S., Carney, T.J., Rauch, J., 248 L. Stanchina et al. / Developmental Biology 295 (2006) 232–249

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