Migration Of Enteric Neural Crest Cells Research Articles

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11–12 (E11–12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12–13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140–144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Suppression of PGE2/EP2 signaling alleviates Hirschsprung disease by upregulating p38 mitogen-activated protein kinase activity.

Hirschsprung disease (HSCR) is a congenital disorder caused bythe failure of enteric neural crest cells (ENCCs) to colonize the distal bowel, resulting in absence of enteric nervous system. While a range of molecules and signaling pathways have been found to contribute to HSCR development, the risk factors and pathogenesis of this disease in many patients remain unknown. We previously demonstrated that increased activity of the prostaglandin E2 (PGE2)/PGE2 receptor subtype EP2 pathway can be a risk factor for HSCR. In this study, an Ednrb-deficient mouse model of HSCR was generated and used to investigate if PGE2/EP2 pathway could be a potential therapeutic target for HSCR. We found that downregulation of PGE2/EP2 signaling by siRNA-mediated ablation of a PGE2 synthase or pharmacologic blockage of EP2 enhanced ENCC colonization in the distal bowel of Ednrb-/- mice and alleviated their HSCR-like symptoms. Furthermore, blockage of EP2 was shown to promote ENCC migration through upregulating p38 mitogen-activated protein kinase activity, which was downregulated in the colon of Ednrb-/- mice and in the distal aganglionic bowel of HSCR patients. These data provide evidence that maternal exposure during embryonic development to an environment with dysregulated activation of the PGE2/EP2 pathway may predispose genetically susceptible offspring to HSCR, and avoidance or early disruption of maternal events (e.g. inflammation) that possibly enhance PGE2/EP2 signaling during pregnancy would reduce the occurrence and severity of this disease. KEY MESSAGES : Knockdown of PTGES alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling alleviates HSCR severity in Ednrb-/- mice. Blockage of EP2-mediated PGE2 signaling promotes ENCC migration via enhancing p38 activity.

Up-Regulation of microRNA-424 Causes an Imbalance in AKT Phosphorylation and Impairs Enteric Neural Crest Cell Migration in Hirschsprung Disease.

Insights into the role of microRNAs (miRNAs) in disease pathogenesis have made them attractive therapeutic targets, and numerous miRNAs have been functionally linked to Hirschsprung disease (HSCR), a life-threatening genetic disorder due to defective migration, proliferation, and colonization of enteric neural crest cells (ENCCs) in the gut. Recent studies have demonstrated that miR-424 strongly inhibits migration in a variety of cell types and its potential target RICTOR is essential for neural crest cell development. We therefore sought to interrogate how miR-424 and RICTOR contribute to the pathogenesis of HSCR. We utilized HSCR cases and human neural cells to evaluate the miR-424-mediated regulation of RICTOR and the downstream AKT phosphorylation. We further developed an ex vivo model to assess the effects of miR-424 on ENCC migration and proliferation. Then, single-cell atlases of gene expression in both human and mouse fetal intestines were used to determine the characteristics of RICTOR and AKT expression in the developing gut. Our findings demonstrate that miR-424 levels are markedly increased in the colonic tissues of patients with HSCR and that it regulates human neural cell migration by directly targeting RICTOR. Up-regulation of miR-424 leads to decreased AKT phosphorylation levels in a RICTOR-dependent manner, and this, in turn, impairs ENCC proliferation and migration in the developing gut. Interestingly, we further identified prominent RICTOR and AKT expressions in the enteric neurons and other types of enteric neural cells in human and mouse fetal intestines. Our present study reveals the role of the miR-424/RICTOR axis in HSCR pathogenesis and indicates that miR-424 is a promising candidate for the development of targeted therapies against HSCR.

More than missing neurons: Intestinal fibrosis in Hirschsprung disease

More than missing neurons: Intestinal fibrosis in Hirschsprung disease

In toto imaging of early enteric nervous system development reveals that gut colonization is tied to proliferation downstream of Ret.

The enteric nervous system is a vast intrinsic network of neurons and glia within the gastrointestinal tract and is largely derived from enteric neural crest cells (ENCCs) that emigrate into the gut during vertebrate embryonic development. Study of ENCC migration dynamics and their genetic regulators provides great insights into fundamentals of collective cell migration and nervous system formation, and these are pertinent subjects for study due to their relevance to the human congenital disease Hirschsprung disease (HSCR). For the first time, we performed in toto gut imaging and single-cell generation tracing of ENCC migration in wild type and a novel ret heterozygous background zebrafish (retwmr1/+) to gain insight into ENCC dynamics in vivo. We observed that retwmr1/+ zebrafish produced fewer ENCCs localized along the gut, and these ENCCs failed to reach the hindgut, resulting in HSCR-like phenotypes. Specifically, we observed a proliferation-dependent migration mechanism, where cell divisions were associated with inter-cell distances and migration speed. Lastly, we detected a premature neuronal differentiation gene expression signature in retwmr1/+ ENCCs. These results suggest that Ret signaling may regulate maintenance of a stem state in ENCCs.

Transplanted neural crest cells migrate toward Auerbach's plexus layer instead of the colon surface in recipient colon pretreated with collagenase and fibronectin

The enteric nervous system (ENS) regulates gastrointestinal motility, secretion, and absorption. Developmental ENS dysplasia causes intestinal ganglion dysfunction, including Hirschsprung's disease. Given their potential ability to replenish insufficient neurons, transplantation of enteric neural cells provides the prospect of a cure. In this study, we used an ex vivo mouse colon transplant model to demonstrate that treatment with collagenase and fibronectin altered the migration of transplanted cells from the direction of the colon surface toward the lumen. Collagenase-treated colons exhibited enhanced expression of type III and VI collagens, which inhibited fibronectin-induced enteric neural crest cell (ENCC) migration. Invasion of neurospheres into colon was dependent on preoperative treatment of recipient colon with collagenase and fibronectin, which enhanced neurosphere motility towards the direction of colon lumen. Infiltration of transplanted ENCCs into the colon increased proportionally to the degree of dedifferentiation of surrounding smooth muscle cells, which was induced in a neurosphere-dependent manner in collagenase-treated colon. Furthermore, induction of GDNF expression, a Ret ligand that promotes enteric neural cell migration, was observed in treated colons. Our results suggest that the environment provided by the extracellular matrix of the colon surface affects the direction of transplanted ENCC migration. Moreover, these findings demonstrating that ENCCs can be accepted by the recipient colon will help to refine current strategies for cell therapy.

Avian ceca are indispensable for hindgut enteric nervous system development.

The enteric nervous system (ENS), which is derived from enteric neural crest cells (ENCCs), represents the neuronal innervation of the intestine. Compromised ENCC migration can lead to Hirschsprung disease, which is characterized by an aganglionic distal bowel. During the craniocaudal migration of ENCCs along the gut, we find that their proliferation is greatest as the ENCC wavefront passes through the ceca, a pair of pouches at the midgut-hindgut junction in avian intestine. Removal of the ceca leads to hindgut aganglionosis, suggesting that they are required for ENS development. Comparative transcriptome profiling of the cecal buds compared with the interceca region shows that the non-canonical Wnt signaling pathway is preferentially expressed within the ceca. Specifically, WNT11 is highly expressed, as confirmed by RNA in situ hybridization, leading us to hypothesize that cecal expression of WNT11 is important for ENCC colonization of the hindgut. Organ cultures using embryonic day 6 avian intestine show that WNT11 inhibits enteric neuronal differentiation. These results reveal an essential role for the ceca during hindgut ENS formation and highlight an important function for non-canonical Wnt signaling in regulating ENCC differentiation.

Peptide Derived from AHNAK Inhibits Cell Migration and Proliferation in Hirschsprung's Disease by Targeting the ERK1/2 Pathway.

Hirschsprung's disease (HSCR) is characterized by the lack of ganglion cells in the distal part of the digestive tract. It occurs due to migration disorders of enteric neural crest cells (ENCCs) from 5 to 12 weeks of embryonic development. More and more studies show that HSCR is a result of the interaction of multiple genes and the microenvironments, but its specific pathogenesis has not been fully elucidated. Studies have confirmed that many substances in the intestinal microenvironment, such as laminin and β1-integrin, play a vital regulatory role in cell growth and disease progression. In addition to these high-molecular-weight proteins, research on endogenous polypeptides derived from these proteins has been increasing in recent years. However, it is unclear whether these endogenous peptides have effects on the migration of ENCCs and thus participate in the occurrence of HSCR. Previously, our research group found that compared with the normal intestinal tissue, the expression of AHNAK protein in the stenosed intestinal tissue of HSCR patients was significantly upregulated, and overexpression of AHNAK could inhibit cell migration and proliferation. In this study, endogenous peptides were extracted from the normal control intestinal tissue and the stenosed HSCR intestinal tissue. The endogenous polypeptide expression profile was analyzed by liquid chromatography-mass spectrometry, and multiple peptides derived from AHNAK protein were found. We selected one of them, "EGPEVDVNLPK", for research. Because there is no uniform naming system, this peptide is temporarily named PDAHNAK (peptide derived from AHNAK). This project aims to clarify the potential role of PDAHNAK in the development of HSCR and to further understand its relationship with its precursor protein AHNAK and how they contribute to the development of HSCR.

Inhibition of ROCK signaling pathway accelerates enteric neural crest cell-based therapy after transplantation in a rat hypoganglionic model.

Hirschsprung's disease (HSCR) is a congenital gastrointestinal disorder, characterized by enteric ganglia absence in part or entire of the colon, due to abnormal colonization and migration of enteric neural crest cells (ENCCs) during development. Currently, besides surgery which is the main therapy for HSCR, the potential of stem cell-based transplantation was investigated as an alternative option. Although promising, it has limitations, including poor survival, differentiation, and migration of the grafted cells. We hypothesized that modulation of extracellular factors during transplantation could promote ENCCs proliferation and migration, leading to increased transplantation efficiency. Considering that the RhoA/ROCK pathway is highly involved in cytoskeletal dynamics and neurite growth, our study explored the effect of inhibition of this pathway to improve the success of ENCCs transplantation. Enteric neural crest cells were isolated from rat embryos and labeled with a GFP-tag. Cell viability, apoptosis, differentiation, and migration assays were performed with and without RhoA/ROCK inhibition. Labeled ENCCs were transplanted into the muscle layer of an induced hypoganglionic rat model followed by intraperitoneal injections of ROCK inhibitor. The transplanted segments were collected 3weeks after for histological analysis. Our results showed that inhibition of ROCK increased viable cell number, differentiation, and migration of ENCCs in vitro. Moreover, transplantation of labeled ENCCs into the hypoganglionic model showed enhanced distribution of grafted ENCCs, upon treatment with ROCK inhibitor. ROCK inhibitors influence ENCCs growth and migration in vitro and in vivo, and should be considered to improve the efficiency of ENCCs transplantation.

Collagen VI suppresses fibronectin-induced enteric neural crest cell migration by downregulation of focal adhesion proteins

The enteric nervous system (ENS) is a network of neurons and glia that are derived from enteric neural crest cells (ENCCs) and essential for regulating peristaltic activity of the colon. ENCCs migrate along the gastrointestinal tract to form the ENS, and disruption of ENCC motility leads to ENS disorders, such as Hirschsprung's disease. Previous ENCC-transplant experiments show that ENCCs can invade into isolated mouse intestines by age E13.5, but not after E15.5. We hypothesized that altered age-specific micro-environments in the intestine are responsible for ENCC invasion/migration. Here, we compared gene expression in the intestine between at E11.5 and E15.5 and identified 1355 differentially expressed transcripts. Among these, we found that genes encoding extracellular matrix (ECM) proteins were enriched. Notably, collagen VI (ColVI) family members were upregulated in the E15.5 mouse intestine at the mRNA and protein levels, whereas fibronectin (FN) was downregulated; however, both proteins showed colocalization at E15.5. To understand the mechanisms of ColVI and FN in ENCC migration, we examined neurosphere or individual ENCC-adherence capabilities toward the ECM. ColVI suppressed FN-induced ENCC spreading/migration, whereas ColVI induced morphologically narrow ENCC spreading and weak stress-fiber formation as compared with those with FN. Additionally, in ENCCs cultured on plates containing ColVI, the expression and phosphorylation of p130Cas, a members of focal adhesion complexes, was reduced. These data indicated an inhibitory role of ColVI in ENCC migration and suggested that ColVI suppression in the intestine might represent a novel therapeutic strategy for aganglionic colonic diseases.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET+/- and RET-/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs invitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can be used to identify disease-associated mutations and determine how they affect cell functions and contribute to pathogenesis.

Corrigendum: How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration.

Scientific Reports 6: Article number: 20927; published online: 18 February 2016; updated: 23 September 2016 The original version of this Article contained a typographical error in the spelling of the author E. Gazquez, which was incorrectly given as E. Gazguez. This has now been corrected in the PDFand HTML versions of the Article.

Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin

We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration.We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

How Tissue Mechanical Properties Affect Enteric Neural Crest Cell Migration.

Neural crest cells (NCCs) are a population of multipotent cells that migrate extensively during vertebrate development. Alterations to neural crest ontogenesis cause several diseases, including cancers and congenital defects, such as Hirschprung disease, which results from incomplete colonization of the colon by enteric NCCs (ENCCs). We investigated the influence of the stiffness and structure of the environment on ENCC migration in vitro and during colonization of the gastrointestinal tract in chicken and mouse embryos. We showed using tensile stretching and atomic force microscopy (AFM) that the mesenchyme of the gut was initially soft but gradually stiffened during the period of ENCC colonization. Second-harmonic generation (SHG) microscopy revealed that this stiffening was associated with a gradual organization and enrichment of collagen fibers in the developing gut. Ex-vivo 2D cell migration assays showed that ENCCs migrated on substrates with very low levels of stiffness. In 3D collagen gels, the speed of the ENCC migratory front decreased with increasing gel stiffness, whereas no correlation was found between porosity and ENCC migration behavior. Metalloprotease inhibition experiments showed that ENCCs actively degraded collagen in order to progress. These results shed light on the role of the mechanical properties of tissues in ENCC migration during development.

Analysis of Enteric Neural Crest Cell Migration Using Heterotopic Grafts of Embryonic Guts.

Hirschsprung disease (HSCR), also named aganglionic megacolon, is a severe congenital malformation characterized by a lack of enteric nervous system (ENS) in the terminal regions of the bowel (Bergeron et al., 2013). As the ENS notably regulates motility in the whole gastrointestinal track, the segment without neurons remains tonically contracted, resulting in functional intestinal obstruction and accumulation of fecal material (megacolon). HSCR occurs when enteric neural progenitors of vagal neural crest origin fail to fully colonize the developing intestines. These "enteric" neural crest cells (ENCCs) have to migrate in a rostro-caudal direction during a fixed temporal window, which is between embryonic day (e) 9.5 and e14.5 in the mouse (Obermayr et al., 2013). Recently, our group generated a new HSCR mouse model called Holstein in which migration of ENCCs is impaired because of increased collagen VI levels in their microenvironment (Soret e al., 2015). Here, we describe the method that allowed us to demonstrate the cell-autonomous nature of this migration defect. In this system adapted from a previously described heterotopic grafting approach (Breau et al., 2006), the donor tissue is a fully colonized segment of e12.5 midgut while the host tissue is an aneural segment of e12.5 hindgut. Extent of ENCC migration in host tissue is assessed after 24 h of culture and is greatly facilitated when donor tissue has a transgenic background such as the Gata4-RFP (Pilon et al., 2008) that allows endogenous labeling of ENCCs with fluorescence. Depending of the genetic background of donor and host tissues, this approach can allow evaluating both cell-autonomous and non-cell-autonomous defects of ENCC migration.

Covering the Cover

Covering the Cover

The relationship between prenatal exposure to BP-3 and Hirschsprung's disease.

Hirschsprung's disease (HSCR) is neonatal intestinal abnormality which derived from the faliure of enteric neural crest cells migration to hindgut during embryogenesis from 5 to 12 weeks. Currenly, the knowledge of environmental factors contributing to HSCR is still scarce. Benzophenone-3 (BP-3) is one of the most widely used UV filters, and has weak estrogen and strong anti-androgenic effects. In order to examine the effect of maternal BP-3 exposure on development of offspring and explore the potential mechanism, we conducted case and control study and in vitro study. In this work, BP-3 concertrations in maternal urine was detected by ultra-high performance liquid chromatography. Besides, we investigated the cytotoxicity and receptor tyrosine kinase (RET) expression in cells exposed to BP-3. The results showed that maternal BP-3 exposure was associated with offspring's HSCR in the population as well as inhibited migration of 293T and SH-SY5Y cells. What's more, we discovered dose-response relationship between RET expression and BP-3 exposure dose, and miR-218 and some other genes involved in SLIT2/ROBO1-miR-218-RET/PLAG1 pathway were also related to BP-3 exposure. Therefore, we deduced that BP-3 influenced cell migration via SLIT2/ROBO1-miR-218-RET/PLAG1 pathway. Our study firstly revealed the relationship between maternal BP-3 exposure and HSCR as well as its potential mechanism.

Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice

Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice.

Gut Epithelium‐derived Sonic Hedgehog Regulates the Extracellular Matrix During Formation of the Intestinal Nervous System

The enteric nervous system (ENS) is principally derived from vagal-level neural crest cells that migrate rostrocaudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of vagal-derived enteric neural crest cells (ENCCs) leads to Hirschsprung disease (HD), a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the distal intestine. Inductive interactions between gut epithelium and mesenchyme have been suggested to regulate the migration and differentiation of ENCCs. However, little is known about the function of epithelial derived factors, such as Sonic hedgehog (Shh), how they influence hindgut mesenchyme derived factors and how they regulate extracellular matrix expression during ENS development. Hindgut from 6 day old chicken embryo was cultured in the presence of Shh protein or Shh overexpressing RCAS-virus. In presence of Shh the hindgut is aganglionic, while in the presence of Shh inhibitor large, ectopic ganglia developed. Shh treatment strongly induced the expression of extracellular matrix versican and collagen type IX, whereas cyclopamine reduced the expression pattern of these inhibitory matrix molecules. These results indicate that versican and collagen IX is a candidate for mediating the effects of Shh on ENCC migration. Shh also inhibited the proliferation and promoted the differentiation of ENCCs. Abnormalities of NCC migration and extracellular pattern formation are characteristic of two human intestinal disorders, HD and intestinal neuronal dysplasia. Our results support an essential role for epithelial-mesenchymal interactions in these aspects of ENS development.

Sox10 and Itgb1 interaction in enteric neural crest cell migration

SOX10 involvement in syndromic form of Hirschsprung disease (intestinal aganglionosis, HSCR) in humans as well as developmental defects in animal models highlight the importance of this transcription factor in control of the pool of enteric progenitors and their differentiation. Here, we characterized the role of SOX10 in cell migration and its interactions with β1-integrins. To this end, we crossed the Sox10lacZ/+ mice with the conditional Ht-PA::Cre; beta1neo/+ and beta1fl/fl mice and compared the phenotype of embryos of different genotypes during enteric nervous system (ENS) development. The Sox10lacZ/+; Ht-PA::Cre; beta1neo/fl double mutant embryos presented with increased intestinal aganglionosis length and more severe neuronal network disorganization compared to single mutants. These defects, detected by E11.5, are not compensated after birth, showing that a coordinated and balanced interaction between these two genes is required for normal ENS development. Use of video-microscopy revealed that defects observed result from reduced migration speed and altered directionality of enteric neural crest cells. Expression of β1-integrins upon SOX10 overexpression or in Sox10lacZ/+ mice was also analyzed. The modulation of SOX10 expression altered β1-integrins, suggesting that SOX10 levels are critical for proper expression and function of this adhesion molecule. Together with previous studies, our results strongly indicate that SOX10 mediates ENCC adhesion and migration, and contribute to the understanding of the molecular and cellular basis of ENS defects observed both in mutant mouse models and in patients carrying SOX10 mutations.