L1cam acts as a modifier gene during enteric nervous system development

Neurobiology of Disease 40 (2010) 622–633

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i

L1cam acts as a modifier gene during enteric nervous system development Adam S. Wallace a, Claudia Schmidt b, Melitta Schachner c, Michael Wegner b, Richard B. Anderson a,⁎ a b c

Department of Anatomy and Cell Biology, University of Melbourne, 3010, Australia Institut fur Biochemie, Universitat Erlangen-Nurnberg, D-91054, Germany Zentrum fur Molekulare Neurobiologie, University of Hamburg, 20246, Germany

a r t i c l e

i n f o

Article history: Received 14 May 2010 Revised 21 July 2010 Accepted 3 August 2010 Available online 7 August 2010 Keywords: Hirschsprung's disease Neural crest Cell migration Enteric nervous system Development

a b s t r a c t The enteric nervous system is derived from neural crest cells that migrate from the caudal hindbrain and colonise the gut. Failure of neural crest cells to fully colonise the gut results in an “aganglionic zone” that lacks a functional enteric nervous system over a variable length of the distal bowel, a condition in human infants known as Hirschsprung's disease. The variability observed in the penetrance and severity of Hirschsprung's disease suggests a role for modifier genes. Clinical studies have identified a population of Hirschsprung's patients with mutations in L1CAM that also have a common polymorphism in RET, suggesting a possible interaction between L1CAM and RET. Therefore, we examined whether L1cam could interact with Ret, its ligand Gdnf, and a known transcriptional activator of Ret, Sox10. Using a two-locus complementation approach, we show that loss of L1cam in conjunction with a heterozygous loss of Ret or Gdnf did not result in aganglionosis. However, L1cam did interact with Sox10 to significantly increase the incidence of aganglionosis. We show that an interaction between L1cam and Sox10 significantly perturbs neural crest migration within the developing gut, and that neural crest cells undergo excessive cell death prior to gut entry. Finally, we show that Sox10 can regulate the expression of L1cam. Thus, L1cam can act as a modifier gene for the HSCR associated gene, Sox10, and is likely to play a role in the etiology of Hirschsprung's disease. © 2010 Elsevier Inc. All rights reserved.

Introduction The enteric nervous system (ENS) comprises of a large array of neurons and glia located in the wall of the gastrointestinal tract. Arranged as two concentric plexus, its role is to modulate intestinal functions including motility, blood flow and luminal secretions. During development, the ENS is derived exclusively from neural crest cells that predominantly arise from the vagal region of the caudal hindbrain, (Le Douarin and Teillet, 1973; Yntema and Hammond, 1954). After migrating dorsolaterally from the neural tube, vagal neural crest cells enter the foregut of the developing mouse embryo at E9.5 (Anderson et al., 2006b). Within the gut they migrate in a rostro-caudal direction colonising the full length of the mouse gut by E14.5 (Young and Newgreen, 2001). In humans, this equates to weeks four to seven of development (Fu et al., 2003; Wallace and Burns, 2005). Sacral level neural crest cells also contribute a small number of cells to the ENS (Anderson et al., 2006a; Burns and Le Douarin, 1998; Kapur, 2000).

Abbreviations: BrdU, bromodeoxyuridine; E, embryonic day; Ednrb, endothelin receptor B; ENS, enteric nervous system; Et-3, endothelin-3; Gdnf, glial cell line-derived neurotrophic factor; GFP, green fluorescent protein; HSCR, Hirschsprung's disease; PCR, polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction; SEM, standard error of the mean; TGM, tau-EGFP-myc. ⁎ Corresponding author. Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Australia. Fax: + 61 3 9347 5219. E-mail address: [email protected] (R.B. Anderson). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2010.08.006

Defects in the migration of neural crest-derived cells result in an absence of a functional ENS in the terminal region of the gastrointestinal tract, which results in a lack of motility in this “aganglionic zone” (Newgreen and Young, 2002a, b; Roberts et al., 2008). In humans this neurocristopathy is known as Hirschsprung's disease (HSCR; congenital aganglionosis) and affects 1:4000 live births with a 4:1 male predominance (Amiel et al., 2008). An absence of motility patterns from the aganglionic zone results in the accumulation of faecal materials proximal to this region, resulting in the formation of a megacolon. Initially presenting as constipation and abdominal swelling, the condition results in failure to thrive and may be fatal due to malnutrition or rupture of the distended bowel. Treatment of HSCR requires surgery to remove the aganglionic length of bowel and re-anastomosis, however, functional problems often persist with many patients suffering continued faecal soiling (Catto-Smith et al., 2007; Tannuri et al., 2009). HSCR is a multigenic disorder, and the genetics of HSCR are complex and not strictly Mendelian (Belknap, 2002). Mutations in a number of genes have been identified that, when mutated or deleted, result in HSCR (Amiel et al., 2008). The best characterised of these are members of the glial cell line-derived neurotrophic factor (GDNF)/RET signalling pathways. Mutations in RET account for approximately 50% of familial and 20% of spontaneous cases of HSCR (Parisi and Kapur, 2000). Gdnf- or Ret-null mice die shortly after birth, and lack enteric neurons along most of the gastrointestinal tract (Pichel et al., 1996; Schuchardt et al., 1994). Gdnf+/− mice do not exhibit aganglionosis, but have a reduced number of enteric neurons (Gianino et al., 2003). Ret+/− mice have normal neuron numbers and do not exhibit aganglionosis (Gianino et al., 2003).

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In the ENS, the expression of Ret is regulated by the transcription factor, Sox10 (Lang and Epstein, 2003). In humans, mutations in SOX10 have been linked to syndromic and non-syndromic forms of HSCR (SanchezMejias et al., 2010). Mice lacking Sox10 are devoid of enteric neurons throughout the entire gastrointestinal tract (Southard-Smith et al., 1998). The majority of Sox10+/− mice do not show an ENS phenotype, but some do develop aganglionosis (Maka et al., 2005). Interactions between known HSCR-associated genes have been shown to increase the incidence and severity of the condition (Barlow et al., 2003; Cantrell et al., 2004; Carrasquillo et al., 2002; Lang et al., 2000; Lang and Epstein, 2003; McCallion et al., 2003; Stanchina et al., 2006; Zhu et al., 2004). However, mutations in known HSCR-associated genes account for less than 50% of all cases of Hirschsprung's disease (Parisi and Kapur, 2000). Moreover, incomplete penetrance and intrafamilial variation are commonly observed in Hirschsprung's disease. There is now increasing evidence to suggest that the phenotypic variability and incomplete penetrance of HSCR is due to complex interactions between known HSCR susceptibility genes and modifier genes (Amiel et al., 2008; Parisi and Kapur, 2000). Genome wide screens and familial studies have identified a number of putative HSCR modifier genes (Bolk et al., 2000; Gabriel et al., 2002; Garcia-Barcelo et al., 2009; Owens et al., 2005). However, very few of these suspected modifier genes have actually been shown to be involved in HSCR (de Pontual et al., 2009; Maka et al., 2005). Mutations in the human L1CAM gene, which is located on the X chromosome and encodes the cell adhesion molecule L1, result in congenital developmental defects including corpus callosum agenesis, mental retardation, adducted thumbs, spastic paraplegia, and hydrocephalus, known collectively as CRASH syndrome (Kenwrick et al., 2000). L1CAM has been implicated in human HSCR cases associated with X-linked hydrocephalus (Griseri et al., 2009; Hofstra et al., 2002; Okamoto et al., 2004; Parisi et al., 2002). Interestingly, some of these HSCR patients have been reported to possess a common RET polymorphism that is over-represented in populations with HSCR (Griseri et al., 2009; Parisi et al., 2002). This has led to the suggestion that L1CAM may interact with RET. Animal model studies have shown that L1cam is required for neural crest migration, but loss of L1cam on its own is not sufficient to produce aganglionosis, suggesting that L1cam may act as a modifier gene (Anderson et al., 2006c). In this study, we examined whether L1cam could act as a modifier gene for Ret, its ligand Gdnf, and a known transcriptional activator of Ret, Sox10. Using a two-locus complementation approach we showed that loss of L1cam in conjunction with a heterozygous loss of Ret or Gdnf did not result in aganglionosis. However, loss of L1cam in conjunction with a heterozygous loss of Sox10 significantly increased the incidence of aganglionosis. We also showed that the timetable by which neural crest-derived cells colonise the gut is significantly perturbed in L1cam−/y;Sox10+/lacZ embryos, and that neural crest cells undergo excessive cell death prior to entering the gut. Finally, using a doxycycline inducible system, we showed that Sox10 can regulate the expression of L1cam. Materials and methods Animals As L1cam is X-linked, the generation of L1cam null mutant mice (L1cam−/y mice) was obtained from mating female L1cam+/− mice. L1cam heterozygous female mice were crossed with either RetTGM (Enomoto et al., 2001), Gdnf (Pichel et al., 1996) or Sox10lacZ (Britsch et al., 2001) heterozygous male mice. Embryos were genotyped by PCR as previously described (Anderson et al., 2006c; Britsch et al., 2001; Enomoto et al., 2001; Pichel et al., 1996). Pregnant females were killed by cervical dislocation and the embryos dissected. L1cam, Ret and Gdnf mice were maintained on a C57/Black6 background and Sox10 mice were maintained on a C3Fe background.

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Immunohistochemistry and histochemistry Immunohistochemistry was performed as previously described (Anderson, 2010). The following primary antibodies were used: rabbit anti-phosphohistone H3 (1:1000; Auspep), rabbit anti-activated caspase-3 (1:1000; R&D Systems), goat anti-Sox10 (1:250; Santa Cruz), rabbit anti-p75 (1:1000; Promega) and sheep anti-PGP9.5 (1:2000; Ultraclone). Secondary antibodies used were: donkey antiRabbit Alexa488 (1:200; Molecular Probes), donkey anti-Rabbit Alexa 594 (1:400; Molecular Probes), anti-Rabbit Alexa 647 (1:400; Molecular Probes) and donkey anti-sheep FITC (1:100; Jackson Immunoresearch). 16 μm cryostat sections were immunostained as previously described (Anderson et al., 2007). β-galactosidase staining was performed as previously described (Stewart et al., 2003). BrdU labeling BrdU was injected intraperitoneally into pregnant females 2 h before killing, as previously described (Turner et al., 2009). Embryonic gut was processed for immunohistochemistry using goat anti-Sox10 (1:250; Santa Cruz) and rat anti-BrdU (1:50; Bioclone) antibodies. Secondary antibodies used were: donkey anti-sheep FITC (1:100; Jackson Immunoresearch) and donkey anti-rat Alexa 594 (1:100; Molecular Probes). Timetable of migration The gut of E14.5 and E18.5 embryos were immunostained using the neuronal marker, PGP9.5. The length of the colon was defined as the distance from the ileocaecal junction to the anus. Colonisation and aganglionosis was calculated as a percentage of total colonic length. The distance migrated by crest-derived cells at E11.5 was determined by measuring the distance between the ileocaecal junction and most caudal cell, as previously described (Anderson, 2010; Anderson et al., 2006c). Results are presented as mean±standard error of the mean (SEM). Cell counts Randomly chosen fields within four defined regions of E18.5 gut (rostral and caudal small intestine, rostral and caudal colon) were imaged. The total number of neurons present in each image was counted and the area of gut measured. E11.5 gut was immunostained using the neural crest marker, Sox10. To determine the proportion of cells that were undergoing cell death or proliferation, the number of Sox10+ and activated caspase-3+, phosphohistone H3+ or BrdU+ cells was counted. A minimum of four preparations of each genotype were examined, with a minimum of 100 cells per preparation counted. The results are presented as mean± SEM. Cell culture and transfections Sox10 cDNA was inserted into pMPTRE behind the tetracyclineregulatable promoter. Stable Sox10 transfections of N2A cells were performed using calcium phosphate precipitates and hygromycin selection. The resulting transfectants were maintained in DMEM containing 10% fetal calf serum, G418 (400 μg/ml; Gibco) and hygromycin (150 μg/ml; Roche). RNA preparation and RT-PCR To induce expression of Sox10, 2.5 μg/ml doxycycline was added to the cells. Three hours later, total RNA was isolated using TRIZOL reagent (Gibco) and reverse transcribed into cDNA. For quantitation, 2 μl of cDNA was amplified with primers specific for Sox10 and glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The following pairs were used: Sox10 (5'-CCCTACACCCACCATCAAGT-3' and 5'-

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TGCCCTGCTCTCCACCTCAT-3’, yielding a 1.0-kb product) and Gapdh (5'-GCCATCAA(C/T)GACCCCTTCATT-3' and 5'-CGCCTGCTTCACCACCTTCTT-3', yielding a 0.7-kb product). Amplification products obtained after 17, 20, and 23 cycles were separated on a 4% polyacrylamide gel and quantitated by densitometry. Results Loss of L1cam does not cause aganglionosis or hypoganglionosis in Ret+/TGM mice Some patients with HSCR and X-linked hydrocephalus have been reported to carry a common polymorphism in RET (Griseri et al., 2009; Parisi et al., 2002), suggesting an interaction between L1CAM and RET. To examine whether L1cam interacts with Ret to influence ENS development, we performed a two-locus complementation screen using L1cam heterozygous female mice and male mice that carry a heterozygous mutation in Ret (Ret+/TGM), and analysed whether a loss of L1cam in Ret+/TGM mice results in aganglionosis or hypoganglionosis. Since a functional ENS is required at birth, we examined the development of the ENS just prior to birth. At E18.5, all genotypes were present in the expected Mendelian ratio (Chi-squared, P=0.94). PGP9.5+ neurons were present along the entire length of the gastrointestinal tract of L1cam+/+;Ret+/+ (n=29), L1cam+/−;Ret+/+ (n=12) and L1cam−/y; Ret+/+ (n=17) embryos (Fig. 1A–I). Neurons were also observed in all regions of the gastrointestinal tract examined in L1cam+/+;Ret+/TGM (n=30), L1cam+/−;Ret+/TGM (n=11) and L1cam−/y;Ret+/TGM (n=12) embryos (Fig. 1J–R). This suggests that loss of L1cam does not cause aganglionosis in Ret+/TGM mice. To examine whether there were any changes in the number of neurons along the length of the gastrointestinal tact in any of the six genotypes, we quantified the density of enteric neurons at four different regions (rostral and caudal small intestine, rostral and caudal colon) of the E18.5 gastrointestinal tract. No significant difference was detected in the density of PGP 9.5+ neurons between any of the genotypes examined in all four regions (one-way ANOVA; data not shown). This suggests that loss of L1cam does not cause hypoganglionosis in Ret+/TGM mice. Loss of L1cam does not cause aganglionosis or hypoganglionosis in Gdnf+/− mice RET is the signalling receptor for GDNF. We examined whether L1cam could interact with Gdnf, by analysing whether a loss of L1cam in Gdnf+/− mice resulted in aganglionosis or hypoganglionosis. At E18.5, all genotypes were present in the expected Mendelian ratio (Chi-squared, P = 0.92). All embryos, irrespective of their genotype, contained neurons along the entire length of the gastrointestinal tract (Supplementary Fig. 1). No significant difference was detected in the density of PGP9.5+ neurons between L1cam+/+;Gdnf+/+ (n = 26), L1cam+/−;Gdnf+/+ (n = 14) or L1cam−/y;Gdnf+/+ (n = 16) embryos in any of the four regions examined (one-way ANOVA). However, we did observe a significant reduction in the density of neurons in L1cam+/+; Gdnf+/− (n = 27), L1cam+/−;Gdnf+/− (n = 14) and L1cam−/y;Gdnf+/− (n = 10) embryos compared to Gdnf+/+ embryos (one-way ANOVA; Supplementary Fig. 1), which is consistent with previously published studies (Gianino et al., 2003). There was no significant difference in neuron density between L1cam+/+;Gdnf+/−, L1cam+/−;Gdnf+/− or L1cam−/y;Gdnf+/− embryos in any of the four regions examined (oneway ANOVA). These data suggest that loss of L1cam does not cause aganglionosis or increase the hypoganglionosis in Gdnf+/− mice.

interact with Sox10, we crossed L1cam+/− mice with mice that carry a heterozygous mutation in Sox10 (Sox10+/lacZ), and analysed whether a loss of L1cam could increase the incidence and/or severity of aganglionosis in Sox10+/lacZ mice. At E18.5, all genotypes were present in the expected Mendelian ratio (Chi-squared, P = 0.56). PGP9.5+ neurons were present along the entire length of the gastrointestinal tract of L1cam+/+;Sox10+/+ (n = 26), L1cam+/−;Sox10+/+ (n = 11) and L1cam−/y;Sox10+/+ (n = 15) embryos (Fig. 2A–I, S). In the majority of cases (n = 24/28), neurons were present in both the small intestine and colon of L1cam+/+;Sox10+/lacZ embryos (Fig. 2J–L, S). However, consistent with previous findings (Cantrell et al., 2004; Maka et al., 2005; Stanchina et al., 2006), a small proportion of L1cam+/+;Sox10+/lacZ embryos (n = 4/28) lacked neurons in the distal-most region of the colon (Fig. 2S). In most L1cam+/−; Sox10+/lacZ embryos (n = 6/10), neurons were present along the entire length of the gastrointestinal tract (Fig. 2M–O, S). In contrast, neurons were absent from the distal-most region of the colon in the majority of L1cam−/y;Sox10+/lacZ embryos (n = 17/19; Fig. 2P–S). PGP9.5+ extrinsic fibres were present within the aganglionic caudal colon (Fig. 2R). Statistical analysis revealed that the incidence of aganglionosis was significantly higher in L1cam−/y;Sox10+/lacZ embryos compared to embryos that had a mutation in Sox10 alone (Fishers exact, P b 0.0001). To determine whether a loss of L1cam increased the extent of aganglionosis in Sox10+/lacZ mice, aganglionosis was calculated as a percentage of total colonic length. No significant difference was detected in the overall length of the colon between any of the genotypes examined (data not shown). A mutation in Sox10 alone (L1cam+/+;Sox10+/lacZ) resulted in 4/28 embryos exhibiting aganglionosis. The average length of colon that was affected by aganglionosis in these four embryos was not significantly different from that observed in L1cam+/−;Sox10+/lacZ (n = 4) and L1cam−/y; Sox10+/lacZ (n = 17) embryos (one-way ANOVA). These data demonstrate that loss of L1cam significantly increases the penetrance of the aganglionic phenotype observed in Sox10+/lacZ embryos. Loss of L1cam results in hypoganglionosis in the distal colon of Sox10+/lacZ mice In addition to aganglionosis, L1cam−/y;Sox10+/lacZ embryos appeared to have fewer neurons than control genotypes. Therefore, the density of enteric neurons was quantified in E18.5 mice. No significant difference was detected in the density of neurons in either the rostral (data not shown) or caudal small intestine between any of the genotypes examined (one-way ANOVA; Fig. 3B). In the rostral colon, Sox10+/lacZ (L1cam+/+;Sox10+/lacZ, L1cam+/−;Sox10+/lacZ and L1cam−/y;Sox10+/lacZ) embryos had a significantly lower density of neurons compared to Sox10+/+ (L1cam+/+;Sox10+/+, L1cam+/−; Sox10 +/+ or L1cam−/y;Sox10+/+) embryos (one-way ANOVA, P b 0.05; Fig. 3C). However, no significant difference was detected in the density of enteric neurons between L1cam+/+;Sox10+/lacZ, L1cam+/−;Sox10+/lacZ or L1cam−/y;Sox10+/lacZ embryos (one-way ANOVA). In the caudal colon, no significant difference was detected in the density of neurons between Sox10+/+ (L1cam+/+;Sox10+/+, L1cam+/−;Sox10+/+ or L1cam−/y;Sox10+/+) embryos (one-way ANOVA; Fig. 3D). No significant difference was observed in L1cam+/+; Sox10+/lacZ and L1cam+/−;Sox10+/lacZ embryos (one-way ANOVA), but the density of neurons in the caudal colon of L1cam−/y;Sox+/lacZ embryos was significantly lower than in L1cam+/+;Sox10+/lacZ embryos (one-way ANOVA, P b 0.05; Fig. 3D).

Loss of L1cam results in the increase of aganglionosis in Sox10+/lacZ mice

The timetable of neural crest migration is perturbed in L1cam−/y;Sox10+/lacZ embryos

In the ENS, the expression of Ret has been shown to be regulated by Sox10 (Lang and Epstein, 2003). To examine whether L1cam could

To determine whether the increased incidence of aganglionosis in E18.5 L1cam−/y;Sox10+/lacZ embryos is due to a disruption in the

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Fig. 1. Loss of L1cam does not cause aganglionosis in Ret+/TGM mice. Confocal images of E18.5 gut from caudal small intestine (A, D, G, J, M, P), rostal colon (B, E, H, K, N, Q) and caudal colon (C, F, I, L, O, R). Enteric neurons were present in the small intestine and colon of all genotypes examined (A–R). Scale bar = 500 μm.

normal timetable by which neural crest-derived cells colonise the developing gut, we examined E14.5 and E11.5 embryos. E14.5 PGP9.5+ neurons were present along the entire length of the gastrointestinal tract of all L1cam+/+;Sox10+/+ (n = 9), L1cam+/−; Sox10+/+ (n = 3) and L1cam−/y;Sox10+/+ (n = 4) embryos (Fig. 4A–I,

S). Neurons were present along the entire length of the colon in only 2/ 7 L1cam+/+;Sox10+/lacZ embryos (Fig. 4J–L, S). In the remaining 5/7 L1cam+/+;Sox10+/lacZ embryos, neurons were absent from only the distal-most region of the colon (Fig. 4S). Enteric neurons were not observed in the caudal colon in any of the L1cam+/−;Sox10+/lacZ or L1cam−/y;Sox10+/lacZ embryo examined (n = 6 and n = 8 respectively; Fig. 4M–R, S). The caudal-most neuron in L1cam+/−;Sox10+/lacZ

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and L1cam−/y;Sox10+/lacZ embryos was often located only midway along the colon. Statistical analysis revealed a significant difference in the location of the caudal-most neuron in the colon of L1cam+/−;Sox10+/lacZ and L1cam−/y;Sox10+/lacZ embryos compared to L1cam+/+;Sox10+/lacZ embryos (one-way ANOVA, P b 0.05). E11.5 The distance migrated by enteric neural crest-derived cells was determined by measuring the distance between the ileocaecal junction and the most caudal p75+ cell. In L1cam+/+;Sox10+/+ (n = 21) and L1cam+/−;Sox10+/+ (n = 10) embryos, the most caudal p75+ cell was located in the rostral hindgut (Fig. 5A–B, G). However, consistent with previous findings (Anderson et al., 2006c), a significant difference in the location of the most caudal p75+ cell in L1cam−/y;Sox10+/+ embryos (n = 10) was observed compared to L1cam+/+;Sox10+/+ embryos (one-way ANOVA, P b 0.05; Fig. 5C and G). Neural crest-derived cell migration has previously been shown to be perturbed in Sox10+/lacZ embryos (Maka et al., 2005). Consistent with this, we found that the location of the most caudal p75+ cell in L1cam+/+;Sox+/lacZ embryos (n = 15) was significantly more rostral than in L1cam +/+ ;Sox10 +/+ embryos (one-way ANOVA, P b 0.05; Fig. 5D and G). No significant difference was detected in the location of the most caudal p75+ cell between L1cam+/−;Sox10+/lacZ and L1cam+/+;Sox10+/lacZ embryos (oneway ANOVA, P N 0.05; Fig. 5D–G), but the most caudal p75+ cell in L1cam−/y;Sox10+/lacZ embryos (n = 12) was significantly more rostral compared to L1cam+/+;Sox10+/lacZ embryos (one-way ANOVA P b 0.05; Fig. 5F–G). Increased neural crest cell death in L1cam−/y;Sox10+/lacZ embryos To determine whether the ENS defects observed in L1cam−/y; Sox10+/lacZ embryos were associated with changes in the rate of apoptosis, immunohistochemistry was performed on E11.5 gut using antibodies against the neural crest marker, Sox10, and activated caspase-3, a marker of apoptotic cells. No significant difference was detected in the percentage of Sox10+ cells that were activated caspase-3+ in any of the genotypes examined (Fig. 6A). We then examined whether the ENS defects were associated with changes in the proliferation rate of crest-derived cells, using an antibody against phosphohistone H3, which detects cells from late G2 to telophase. No significant difference was detected in the percentage of Sox10+ cells that were phosphohistone H3+ between any of the genotypes examined (Fig. 6B). To confirm these observations, we also examined the proliferation of Sox10+ cells in the gut using BrdU and found no significant difference in the percentage of Sox10+ cells that were BrdU+ between any of the genotypes examined (data not shown). Therefore, the disruption in the migration of neural crest-derived cells observed in L1cam−/y;Sox10+/lacZ embryos does not appear to be due to altered rates of apoptosis or cell proliferation within the developing gut. To determine whether the ENS defects observed in L1cam−/y; Sox10+/lacZ embryos were associated with a reduction in the number of neural crest cells en route to the foregut, apoptosis was examined in sections of E9.5 embryos. No activated caspase-3+ cells were detected in the stream of vagal neural crest cells en route to the foregut in L1cam+/+;Sox10+/+ or L1cam−/y;Sox10+/+ embryos (Fig. 6C and D). However, consistent with previous findings (Maka

et al., 2005; Stanchina et al., 2006) a small number of Sox10+ cells in L1cam+/+;Sox10+/lacZ embryos were activated caspase-3+ (Fig. 6E). In L1cam−/y;Sox10+/lacZ embryos, numerous activated caspase-3+ cells were detected in the stream of vagal neural crest cells en route to the foregut, particularly in the region dorsolateral to the foregut prior to the medial turn (Fig. 6F). Sox10 regulates L1cam expression Sox10 has been shown to regulate the expression of genes involved in ENS development and HSCR (Lang and Epstein, 2003; Zhu et al., 2004). To determine whether Sox10 can regulate the expression of L1cam, we used a doxycycline inducible Sox10 cell line to determine whether the induction of Sox10 alters the endogenous level of L1cam transcripts. Doxycycline treatment of mock-transfected N2A cells did not cause activation of endogenous L1cam expression (data not shown). RT-PCR analysis showed that un-induced N2A cells showed very low levels of endogenous L1cam transcripts (Fig. 7). Exposure to doxycycline resulted in a significant increase in the level of L1cam transcripts detected in N2A cells (Fig. 7). Using quantitative PCR, a 14.5 fold increase was detected in the intensity of the L1cam-specific signal in N2A-induced cells compared to un-induced cells. These results suggest that Sox10 can regulate the endogenous expression of L1cam. Discussion Some humans with HSCR and X-linked hydrocephalus have been reported to have a common RET polymorphism (Griseri et al., 2009; Parisi et al., 2002), suggesting a possible interaction between L1CAM and RET. In this study, we show that L1cam interacts with Sox10, but not Ret and Gdnf, to influence ENS development and the incidence of aganglionosis. Our data show that L1cam can act as a modifier gene for some HSCR susceptibility genes. L1cam is a modifier gene for Sox10 The transcription factor, Sox10, has been the focus of several studies assessing the complex multigenic nature of HSCR (Cantrell et al., 2004; Maka et al., 2005; Stanchina et al., 2006). Interactions between Sox10 and Et-3, Ednrb or Sox8 have been shown to increase the penetrance and severity of the aganglionic phenotype observed in Sox10 heterozygous mice (Cantrell et al., 2004; Maka et al., 2005; Stanchina et al., 2006). In the current study, we showed that a loss of L1cam significantly increased the penetrance of the aganglionic phenotype observed in Sox10+/lacZ embryos. We also observed a significant increase in the frequency of aganglionosis in embryos that had a heterozygous mutation in both L1cam and Sox10. Similar observations were reported for interactions between Sox10 and Sox8 (Maka et al., 2005). However, an increase in the penetrance of aganglionosis in double heterozygotic embryos is not observed for all HSCR-related genes. When Ednrbsl/+ mice were crossed to Sox10Dom/ + mice, the resulting double heterozygotic progeny did not exhibit an increase in the penetrance of aganglionosis (Cantrell et al., 2004), suggesting that Ednrb expression must be reduced by more than 50% in order to modify the Sox10Dom/+ phenotype. Using Ednrbs/s mice, Stanchina et al. (2006) showed that a threshold level of between 30% and 50% of Ednrb expression was required in order to significantly

Fig. 2. Loss of L1cam increases the penetrance of aganglionosis in Sox10+/lacZ mice. Confocal images of E18.5 gut from caudal small intestine (A, D, G, J, M, P), rostal colon (B, E, H, K, N, Q) and caudal colon (C, F, I, L, O, R). Enteric neurons were present in the small intestine and colon in L1cam+/+;Sox10+/+ (A–C), L1cam+/−;Sox10+/+ (D–F), L1cam−/y;Sox10+/+ (G– I) and the majority of L1cam+/+;Sox10+/lacZ (J–L) and L1cam+/−;Sox10+/lacZ (M–O) embryos. L1cam−/y;Sox10+/lacZ embryos contained neurons in the small intestine (P) and rostral colon (Q) but not in the caudal colon (R). The staining observed in the caudal colon is due to the presence of extrinsic fibres. (S) Schematic representation of the gut. The arrows represent the location of the most caudal neuron in each sample. The number of embryos with the most caudal neuron in that location is indicated above or beside the arrow if greater than one. S.I. small intestine. Scale bar = 500 μm.

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Vagal neural crest defects in L1−/y;Sox10+/lacZ embryos Changes in apoptosis or proliferation of neural crest-derived cells have been associated with disruption to the colonisation of the gut (Maka et al., 2005; Simpson et al., 2007; Southard-Smith et al., 1998; Uesaka et al., 2008). In the current study, we detected no significant difference in the rate of proliferation or cell death of neural crest-derived cells within gut. However, we did observe an increase in the number of vagal neural crest undergoing apoptosis en route to the foregut in L1cam−/y;Sox10+/lacZ embryos, suggesting that crest cells are more susceptible to the loss of Sox10 and L1cam prior to entering the gut than after they have entered it. Thus, the ENS defects observed in L1cam−/y;Sox10+/lacZ embryos are likely to be due to the reduced survival of vagal neural crest cells prior to entering the gut. This would result in fewer cells entering the gut, and may explain the disruption in migration observed in E11.5 and E14.5 L1cam−/y;Sox10+/lacZ embryos. However, we cannot exclude the possibility that additional factors may also affect the migration of crest-derived cells along the gut of L1cam−/y;Sox10+/lacZ embryos. For example, enteric neural crest-derived cells are known to migrate in chains (Anderson et al., 2006c; Young et al., 2004). Blocking the activity of L1 has been shown to disrupt neural crest-derived cell–cell interactions in vitro, resulting in a delay in migration (Anderson et al., 2006c). It has also been shown that as the gut mesenchyme matures, the local environment becomes less permissive for cell migration (Druckenbrod and Epstein, 2009), suggesting that there may be only a small window of opportunity for cells to successfully colonise the gut. Thus, the reduced number of vagal neural crest cells entering the gut combined with altered cell–cell interactions within the gut, may slow migration sufficiently such that the window of opportunity to colonise the gut passes, resulting in colonic aganglionosis in L1cam−/y;Sox10+/lacZ embryos.

Sox10 regulates the expression of L1cam

Fig. 3. Hypoganglionosis in the colon of Sox10+/lacZ mice. Quantification of neuron density along the gastrointestinal tract (A). No significant difference was observed in the density of neurons between the genotypes in the caudal small intestine (B). A significant reduction in the density of neurons was detected in rostral colon in all three Sox10+/lacZ genotypes (P b 0.05) (C). In the caudal colon a significant decrease in the density of neurons was only detected in L1cam−/y;Sox10+/lacZ embryos (P b 0.05) (D).

increase the penetrance of aganglionosis in Sox10+/Dom mice. This suggests that the migration of neural crest-derived cells is more sensitive to reduced levels of L1cam than to that of Ednrb, when combined with a heterozygous mutation in Sox10.

The transcription factor, Sox10, has been shown to directly regulate genes involved in ENS development and HSCR. For example, Sox10 has been shown to form a transcriptional complex with Pax3 to regulate the expression of Ret (Lang and Epstein, 2003). It has also been shown to bind to an ENS-specific enhancer region of the Ednrb promoter that directly regulates the expression of Ednrb (Zhu et al., 2004). In the current study, we show that Sox10 can also regulate the expression of L1cam. Using a doxycycline inducible Sox10 cell line, which has previously been shown to identify direct targets of Sox10 regulation (Peirano et al., 2000), we detected a 14.5 fold increase in the level of L1cam transcripts in cells exposed to doxycycline. The significant increase in the expression of L1cam following doxycycline induced transcription of Sox10, suggests that L1cam is a direct target of Sox10. The fact that Sox10 is required for neural crest survival prevents us from examining whether Sox10 is required for L1cam expression in vivo, as mice lacking Sox10 are devoid of enteric neural crest-derived cells throughout the entire gastrointestinal tract (Kapur, 1999; Southard-Smith et al., 1998). However, L1 is expressed by migrating enteric neural crest cells in Sox10 heterozygous mice (ASW and RBA unpublished data). Interestingly, the L1cam promoter contains numerous putative Sox10 binding sites. Further studies are required to determine which of the Sox10 binding sites are required to regulate the expression of L1cam in the ENS.

Fig. 4. Enteric nervous system development at E14.5. Confocal images of E14.5 gut from caudal small intestine (A, D, G, J, M, P), rostal colon (B, E, H, K, N, Q) and caudal colon (C, F, I, L, O, R). Enteric neurons were present along the gut in all L1cam+/+;Sox10+/+ (A–C), L1cam+/−;Sox10+/+ (D–F) and L1cam−/y;Sox10+/+ (G–I) embryos and some L1cam+/+;Sox10+/lacZ embryos (J–L). Neurons were present in the small intestine and rostral colon of L1cam+/−;Sox10+/lacZ (M–O) and L1cam−/y;Sox10+/lacZ (P–R) embryos, but were never observed in the caudal colon. (S) Schematic representation of the gut. The arrows represent the location of the most caudal neuron in each sample. The number of embryos with the most caudal neuron in that location is indicated above or beside the arrow if greater than one. S.I. small intestine. Scale bar = 500 μm.

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Fig. 5. Enteric nervous system development at E11.5. Confocal images of E11.5 gut immunostained using the neural crest marker p75 (A–F). The arrows indicate the position of the most caudal crest-derived cell. (G) Quantification of the location of the most caudal cell relative to the ileocaecal junction. Significant differences were detected between L1cam+/+; Sox10+/+ and L1cam−/y;Sox10+/+ embryos, between Sox10+/lacZ and Sox10+/+ embryos and between L1cam+/+;Sox10+/lacZ and L1cam−/y;Sox10+/lacZ embryos. Scale bar = 500 μm.

L1cam does not interact with members of the Gdnf/Ret signalling pathway Previous studies using Ret+/− mice have reported interactions between Ret and Ednrb (Carrasquillo et al., 2002; McCallion et al., 2003) and Ret and retinoic acid (Fu et al., 2010). Based on human clinical studies, it has been proposed that L1CAM may act as a modifier gene for RET (Griseri et al., 2009; Parisi et al., 2002). However, our data suggest that L1cam does not interact with members of the Gdnf/Ret signalling

pathway during ENS development, as we did not detect aganglionosis or increased hypoganglionosis in L1cam−/y;Ret+/TGM and L1cam−/y;Gdnf+/ − mice. However, we cannot exclude the possibility of an interaction between L1cam and Ret, since mice appear to be more tolerant of reduced levels of Ret activity than humans. RET mutations in humans act dominantly to give rise to HSCR, whereas ENS development appears to be normal in Ret heterozygous mice (Gianino et al., 2003). In fact, a loss of around 60-70% of Ret expression in mice is required in order to mimic the aganglionic phenotype observed in humans (Uesaka et al., 2008).

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Fig. 6. Proliferation and cell death analysis. Quantification of cell death in E11.5 gut. No significant difference was detected in the percentage of crest cells that were activated caspase3+ between the genotypes (A). Quantification of cell proliferation in E11.5 gut. No significant difference was detected in the percentage of crest cells that were phosphohistone H3+ between the genotypes (B). (C–F) Confocal images of L1cam+/+;Sox10+/+ (C), L1cam−/y;Sox10+/+ (D), L1cam+/+;Sox10+/lacZ (E) and L1cam−/y;Sox10+/lacZ (F) embryos immunostained using antibodies against activated caspase-3 (red) and Sox10 (C and D; green) or β-galactosidase (D and E; green) showing vagal neural crest cells en route to the foregut at E9.5. The dotted lines indicate the region of the foregut mesenchyme. Scale bar = 100 μm.

Therefore, it would be interesting to examine whether interactions with L1cam can be shown in Ret51/51 (de Graaff et al., 2001) or RetS697A/S697A (Asai et al., 2006) mice, which exhibit aganglionosis in the distal colon and more closely resemble human HSCR. L1CAM mutations in humans Our data demonstrates that L1cam can act as a modifier gene for the HSCR-associated gene Sox10 in mice. However, it is not yet known whether this is the case in humans. The estimated incidence of HSCR among patients with mutations in the L1CAM gene is around 3% (Okamoto et al., 2004). However, the incidence of HSCR in patients with X-linked hydrocephalus could be significantly higher, since many infants with X-linked hydrocephalus die soon after birth and analysis of the distal colon for aganglionosis is not generally performed (Parisi et al., 2002). Screening for mutations in all known

HSCR associated genes are not routinely performed on patients with HSCR and X-linked hydrocephalus. Therefore, screening individuals that have HSCR and X-linked hydrocephalus for mutations in known HSCR associated genes, such as SOX10, and the human L1CAM promoter region for polymorphisms within a putative ENS specific enhancer region may help provide important insights to our understanding of the genetics underlying HSCR. Conclusions There is increasing evidence to suggest that the phenotypic variability and incomplete penetrance of HSCR is due to complex interactions between known HSCR associated genes and modifier genes (Amiel et al., 2008). Our study shows for the first time that the X-linked gene, L1cam, can act as a modifier gene for some HSCR associated genes, and is likely to play a role in the etiology of HSCR.

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Fig. 7. Sox10 regulates the endogenous expression of L1cam. RT-PCR analysis of cDNA obtained from a doxycycline inducible Sox10 cell line. Levels of L1cam transcript were compared by quantitative PCR using increasing numbers of amplification cycles. A significant increase in the level of L1cam transcripts was detected in doxycyclineinduced cells compared to un-induced cells.

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