Vagal Neural Crest Cells Research Articles

Sacral Neural Crest-Independent Origin of the Enteric Nervous System in Mouse

The enteric nervous system (ENS), the gut's intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases buthas been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3-dimensional reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further used coculture assays to determine the specificity of cell migration from different neural tissues into the hindgut. Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but onlypan-NCCs contributed to the ENS. Dual-lineage tracing combined with 3-dimensional reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Coculture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung's disease.

High MEIS3 Expression Indicates a Poor Prognosis for Patients with Stage II/III Colorectal Cancer.

The Wnt/β-catenin signaling pathway plays crucial roles in tumor budding and the epithelial-mesenchymal transition (EMT). Myeloid ecotropic viral insertion site 3 (MEIS3)-a direct target of Wnt/β-catenin-promotes vagal neural crest cell migration into the gut tissue during development; however, its role in cancer progression remains unclear. In this study, the role of MEIS3 in colorectal cancer (CRC) progression was investigated. We analyzed the association between MEIS3 protein expression and the clinical stages of CRC patients, and the effect on tumor cell migration and invasion by wound healing and transwell assays. Finally, we analyzed the association between MEIS3 expression and the disease-free survival (DFS) and overall survival of CRC patients through Kaplan-Meier analysis. We found that MEIS3 expression was strongly associated with CRC progression and could be employed to assess DFS in postoperative patients. MEIS3-positive cells were mainly distributed in the growth front and tumor-stroma interface of the CRC tissues, which contain abundant EMT-active and tumor budding cells dominating cancer metastasis. Moreover, MEIS3 promoted CRC cell migration and invasion by regulating effectors including laminin subunit beta 1, matrix metalloprotein 2, and vimentin. MEIS3 protein expression increased with CRC progression according to the clinical stage, which could be used as a biomarker to stratify CRC patients. The 5-year DFS of MEIS3-high patients was poorer than that of MEIS3-low patients (40.6% vs. 61.7%; p < 0.0001). Moreover, the 5-year DFS of stage II patients with MEIS3-high expression (53.4%) was comparable to that of stage III patients with MEIS3-low expression (49.5%), while the 5-year DFS of MEIS3-high patients in stage III (30.9%) was comparable to that of stage IV patients (29.6%). This study showed that MEIS3 can promote cancer cell metastasis and thus may be a promising biomarker for higher rates of recurrence in postoperative patients with stage II/III CRC.

Essential Role of BMP4 Signaling in the Avian Ceca in Colorectal Enteric Nervous System Development.

The enteric nervous system (ENS) is principally derived from vagal neural crest cells that migrate caudally along the entire length of the gastrointestinal tract, giving rise to neurons and glial cells in two ganglionated plexuses. Incomplete migration of enteric neural crest-derived cells (ENCDC) leads to Hirschsprung disease, a congenital disorder characterized by the absence of enteric ganglia along variable lengths of the colorectum. Our previous work strongly supported the essential role of the avian ceca, present at the junction of the midgut and hindgut, in hindgut ENS development, since ablation of the cecal buds led to incomplete ENCDC colonization of the hindgut. In situ hybridization shows bone morphogenetic protein-4 (BMP4) is highly expressed in the cecal mesenchyme, leading us to hypothesize that cecal BMP4 is required for hindgut ENS development. To test this, we modulated BMP4 activity using embryonic intestinal organ culture techniques and retroviral infection. We show that overexpression or inhibition of BMP4 in the ceca disrupts hindgut ENS development, with GDNF playing an important regulatory role. Our results suggest that these two important signaling pathways are required for normal ENCDC migration and enteric ganglion formation in the developing hindgut ENS.

Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system.

During development, much of the enteric nervous system (ENS) arises from the vagal neural crest that emerges from the caudal hindbrain and colonizes the entire gastrointestinal tract. However, a second ENS contribution comes from the sacral neural crest that arises in the caudal neural tube and populates the post-umbilical gut. By coupling single-cell transcriptomics with axial-level-specific lineage tracing in avian embryos, we compared the contributions of embryonic vagal and sacral neural crest cells to the chick ENS and the associated peripheral ganglia (Nerve of Remak and pelvic plexuses). At embryonic day (E) 10, the two neural crest populations form overlapping subsets of neuronal and glia cell types. Surprisingly, the post-umbilical vagal neural crest much more closely resembles the sacral neural crest than the pre-umbilical vagal neural crest. However, some differences in cluster types were noted between vagal and sacral derived cells. Notably, RNA trajectory analysis suggests that the vagal neural crest maintains a neuronal/glial progenitor pool, whereas this cluster is depleted in the E10 sacral neural crest which instead has numerous enteric glia. The present findings reveal sacral neural crest contributions to the hindgut and associated peripheral ganglia and highlight the potential influence of the local environment and/or developmental timing in differentiation of neural crest-derived cells in the developing ENS.

Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization

The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders.

Loss of autism-candidate CHD8 perturbs neural crest development and intestinal homeostatic balance.

Individuals with mutations in CHD8 present with gastrointestinal complaints, yet the underlying mechanisms are understudied. Here, using a stable constitutive chd8 mutant zebrafish model, we found that the loss of chd8 leads to a reduced number of vagal neural crest cells (NCCs), enteric neural and glial progenitors, emigrating from the neural tube, and that their early migration capability was altered. At later stages, although the intestinal colonization by NCCs was complete, we found the decreased numbers of both serotonin-producing enterochromaffin cells and NCC-derived serotonergic neurons, suggesting an intestinal hyposerotonemia in the absence of chd8 Furthermore, transcriptomic analyses revealed an altered expression of key receptors and enzymes in serotonin and acetylcholine signaling pathways. The tissue examination of chd8 mutants revealed a thinner intestinal epithelium accompanied by an accumulation of neutrophils and the decreased numbers of goblet cells and eosinophils. Last, single-cell sequencing of whole intestines showed a global disruption of the immune balance with a perturbed expression of inflammatory interleukins and changes in immune cell clusters. Our findings propose a causal developmental link between chd8, NCC development, intestinal homeostasis, and autism-associated gastrointestinal complaints.

Multiple Roles of Ret Signalling During Enteric Neurogenesis.

The majority of the enteric nervous system is formed by vagal neural crest cells which enter the foregut and migrate rostrocaudally to colonise the entire length of the gastrointestinal tract. Absence of enteric ganglia from the distal colon are the hallmark of Hirschsprung disease, a congenital disorder characterised by severe intestinal dysmotility. Mutations in the receptor tyrosine kinase RET have been identified in approximately 50% of familial cases of Hirschsprung disease but the cellular processes misregulated in this condition remain unclear. By lineage tracing neural crest cells in mice homozygous for a knock-in allele of Ret (Ret51/51), we demonstrate that normal activity of this receptor is required in vivo for the migration of enteric nervous system progenitors throughout the gut. In mutant mice, progenitors of enteric neurons fail to colonise the distal colon, indicating that failure of colonisation of the distal intestine is a major contributing factor for the pathogenesis of Hirschsprung disease. Enteric nervous system progenitors in the ganglionic proximal guts of mutant mice are also characterised by reduced proliferation and differentiation. These findings suggest that the functional abnormalities in Hirschsprung disease result from a combination of colonic aganglionosis and deficits in neuronal circuitry of more proximal gut segments. The reduced neurogenesis in the gut of Ret51/51 mutants was reproduced in the multilineage enteric nervous system progenitors isolated from these animals. Correction of the molecular defects of such progenitors fully restored their neurogenic potential in culture. These observations enhance our understanding of the pathogenesis of Hirschsprung disease and highlight potential approaches for its treatment.

Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish.

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.

The RET gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and RET mutation results in Hirschsprung's disease.

Enteric neurons and ganglia are derived from vagal and sacral neural crest cells, which undergo migration from the neural tube to the gut wall. In the gut wall, they first undergo rostrocaudal migration followed by migration from the superficial to deep layers. After migration, they proliferate and differentiate into the enteric plexus. Expression of the Rearranged During Transfection (RET) gene and its protein RET plays a crucial role in the formation of enteric neurons. This review describes the molecular mechanism by which the RET gene and the RET protein influence the development of enteric neurons. Vagal neural crest cells give rise to enteric neurons and glia of the foregut and midgut while sacral neural crest cells give rise to neurons of the hindgut. Interaction of RET protein with its ligands (glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), and artemin (ARTN)) and its co-receptors (GDNF receptor alpha proteins (GFRα1-4)) activates the Phosphoinositide-3-kinase-protein kinase B (PI3K-PKB/AKT), RAS mitogen-activated protein kinase (RAS/MAPK) and phospholipase Cγ (PLCγ) signaling pathways, which control the survival, migration, proliferation, differentiation, and maturation of the vagal and sacral neural crest cells into enteric neurons. Abnormalities of the RET gene result in Hirschsprung's disease.

Neuron-Glia Interaction in the Developing and Adult Enteric Nervous System.

The enteric nervous system (ENS) constitutes the largest part of the peripheral nervous system. In recent years, ENS development and its neurogenetic capacity in homeostasis and allostasishave gained increasing attention. Developmentally, the neural precursors of the ENS are mainly derived from vagal and sacral neural crest cell portions. Furthermore, Schwann cell precursors, as well as endodermal pancreatic progenitors, participate in ENS formation. Neural precursors enherite three subpopulations: a bipotent neuron-glia, a neuronal-fated and a glial-fated subpopulation. Typically, enteric neural precursors migrate along the entire bowel to the anal end, chemoattracted by glial cell-derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) molecules. During migration, a fraction undergoes differentiation into neurons and glial cells. Differentiation is regulated by bone morphogenetic proteins (BMP), Hedgehog and Notch signalling. The fully formed adult ENS may react to injury and damage with neurogenesis and gliogenesis. Nevertheless, the origin of differentiating cells is currently under debate. Putative candidates are an embryonic-like enteric neural progenitor population, Schwann cell precursors and transdifferentiating glial cells. These cells can be isolated and propagated in culture as adult ENS progenitors and may be used for cell transplantation therapies for treating enteric aganglionosis in Chagas and Hirschsprung’s diseases.

Mesenteric Neural Crest Cells Are the Embryological Basis of Skip Segment Hirschsprung's Disease.

Background & AimsDefective rostrocaudal colonization of the gut by vagal neural crest cells (vNCCs) results in Hirschsprung's disease (HSCR), which is characterized by aganglionosis in variable lengths of the distal bowel. Skip segment Hirschsprung’s disease (SSHD), referring to a ganglionated segment within an otherwise aganglionic intestine, contradicts HSCR pathogenesis and underscores a significant gap in our understanding of the development of the enteric nervous system. Here, we aimed to identify the embryonic origin of the ganglionic segments in SSHD.MethodsIntestinal biopsy specimens from HSCR patients were prepared via the Swiss-roll technique to search for SSHD cases. NCC migration from the neural tube to the gut was spatiotemporally traced using targeted cell lineages and gene manipulation in mice.ResultsAfter invading the mesentery surrounding the foregut, vNCCs separated into 2 populations: mesenteric NCCs (mNCCs) proceeded to migrate along the mesentery, whereas enteric NCCs invaded the foregut to migrate along the gut. mNCCs not only produced neurons and glia within the gut mesentery, but also continuously complemented the enteric NCC pool. Two new cases of SSHD were identified from 183 HSCR patients, and Ednrb-mutant mice, but not Ret-/- mice, showed a high incidence rate of SSHD-like phenotypes.ConclusionsmNCCs, a subset of vNCCs that migrate into the gut via the gut mesentery to give rise to enteric neurons, could provide an embryologic explanation for SSHD. These findings lead to novel insights into the development of the enteric nervous system and the etiology of HSCR.

Pluripotent stem cell derived intestinal organoids with an enteric nervous system.

The use of human pluripotent stem cells (hPSCs) and differentiation techniques offer new ways to generate specific tissue. It is now possible to differentiate hPSC into human intestinal organoids that include an enteric nervous system. Using step-wise differentiation processes, we generate innervated intestinal organoids that form three-dimensional structures bearing an epithelium, neurons and glial cells embedded in a supporting mesenchyme. Innervated organoids further develop to a complex structure with similar organization and cellular differentiation as the developing intestine. These tools open up new fields of application in the study of the development and pathophysiology of enteric neuropathies. Herein, we describe the generation of both human intestinal organoids and vagal neural crest cells from hPSC and their combination into an innervated organoid. We also discuss technical considerations for these experiments, and highlight advantages and limitations of the system.

BMP2 Is Related to Hirschsprung's Disease and Required for Enteric Nervous System Development.

The enteric nervous system (ENS) is derived from neural crest cells (NCCs). Defects in ENS NCCs colonizing in the intestines lead to an absence of enteric ganglia in the colon and results in Hirschsprung’s disease (HSCR). Bone morphogenetic proteins (BMPs) play diverse roles in the proliferation, migration and survival of ENS NCCs; however, whether BMPs are involved in HSCR and the underlying mechanism remains largely unknown. In this study, we found that BMP2 expression is significantly decreased in HSCR patients. Further experiments demonstrated that BMP2 is involved in the regulation of NCC proliferation, migration and differentiation. In a detailed analysis of the role of BMP2 in HSCR development in vivo, we demonstrated that BMP2b regulates the proliferation, migration and differentiation of vagal NCCs in zebrafish and that BMP2b is required for intestinal smooth muscle development. In addition, we showed that BMP2b is involved in regulating the expression of glial cell line-derived neurotrophic factor (GDNF) in the intestine, which mediates the regulation of ENS development by BMP2b in zebrafish. These results highlight a central role of the BMP-GDNF cascade in intestinal patterning and ENS development. Our results further demonstrate the key role of BMP2 in the etiology of HSCR in vitro and in vivo.

Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages.

The enteric nervous system (ENS) predominantly originates from vagal neural crest cells (VNC) that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally-specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies.

PROX1 is a specific and dynamic marker of sacral neural crest cells in the chicken intestine.

The enteric nervous system (ENS) is a complex network constituted of neurons and glial cells that ensures the intrinsic innervation of the gastrointestinal tract. ENS cells originate from vagal and sacral neural crest cells that are initially located at the border of the neural tube. In birds, sacral neural crest cells (sNCCs) first give rise to an extramural ganglionated structure (the so-called Nerve of Remak [NoR]) and to the pelvic plexus. Later, sNCCs enter the colon mesenchyme to colonize and contribute to the intrinsic innervation of the caudal part of the gut. However, no specific sNCC marker has been described. Here, we report the expression pattern of prospero-related homeobox 1 (PROX1) in the developing chick colon. PROX1 is a homeobox domain transcription factor that plays a role in cell type specification in various tissues. Using in situ hybridization and immunofluorescence techniques, we showed that PROX1 is expressed in sNCCs localized in the NoR and in the pelvic plexus. Then, using real-time quantitative PCR we found that PROX1 displays a strong and highly dynamic expression pattern during NoR development. Moreover, we demonstrated using in vivo cell tracing, that sNCCs are the source of the PROX1-positive cells within the NoR. Our results indicate that PROX1 is the first marker that specifically identifies sNCCs. This might help to better identify the role of the different neural crest cell populations in distal gut innervation, and consequently to improve the diagnosis of diseases linked to incomplete ENS formation, such as Hirschsprung's disease.

Fine scale differences within the vagal neural crest for enteric nervous system formation

The enteric nervous system is mostly derived from vagal neural crest (NC) cells adjacent to somites (s)1–7. We used in ovo focal fluorescent vital dyes and focal electroporation of fluorophore-encoding plasmids in quail embryos to investigate NC cell migration to the foregut initially and later throughout the entire gut. NC cells of different somite-level origins were largely separate until reaching the foregut at about QE2.5, when all routes converged. By QE3.5, NC cells of different somite-levels became mixed, although s1-s2 NC cells were mainly confined to rostral foregut. Mid-vagal NC-derived cells (s3 and s4 level) arrived earliest at the foregut, and occurred in greatest number. By QE6.5 ENS was present from foregut to hindgut. Mid-vagal NC-derived cells occurred in greatest numbers from foregut to distal hindgut. NC-derived cells of s2, s5, and s6 levels were fewer and were widely distributed but were never observed in the distal hindgut. Rostro-vagal (s1) and caudo-vagal (s7) levels were few and restricted to the foregut. Single somite levels of quail neural tube/NC from s1 to s8 were combined with chick aneural ChE4.5 midgut and hindgut and the ensemble was grown on the chorio-allantoic membrane for 6 days. This tests ENS-forming competence in the absence of intra-segmental competition between NC cells, of differential influences of segmental paraxial tissues, and of positional advantage. All vagal NC-levels, but not s8 level, furnished enteric plexuses in the recipient gut, but the density of both ENS cells in total and neurons was highest from mid-vagal level donors, as was the length colonised. We conclude that the fate and competence for ENS formation of vagal NC sub-levels is not uniform over the vagal level but is biased to favour mid-vagal levels. Overviewing this and prior studies suggests the vagal region is, as in its traditional sense, a natural unit but with complex sub-divisions.

Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells

The enteric nervous system arises from neural crest cells that migrate as chains into and along the primitive gut, subsequently differentiating into enteric neurons and glia. Little is known about the mechanisms governing neural crest migration en route to and along the gut in vivo. Here, we report that Retinoic Acid (RA) temporally controls zebrafish enteric neural crest cell chain migration. In vivo imaging reveals that RA loss severely compromises the integrity and migration of the chain of neural crest cells during the window of time window when they are moving along the foregut. After loss of RA, enteric progenitors accumulate in the foregut and differentiate into enteric neurons, but subsequently undergo apoptosis resulting in a striking neuronal deficit. Moreover, ectopic expression of the transcription factor meis3 and/or the receptor ret, partially rescues enteric neuron colonization after RA attenuation. Collectively, our findings suggest that retinoic acid plays a critical temporal role in promoting enteric neural crest chain migration and neuronal survival upstream of Meis3 and RET in vivo.

Enteric nervous system formation: disproportionate stochastic clonal expansion of a few initiating vagal neural crest cells

Enteric nervous system formation: disproportionate stochastic clonal expansion of a few initiating vagal neural crest cells

The Role of Rho/ROCK Pathway on the Sacral Neural Crest Cell Migration in the Mouse Gut

The enteric nervous system (ENS) originates from neural crest cells (NCCs) at two different levels along the neuraxis: vagal (somites 1–7) and sacral (caudal to somite 24 in the mouse) levels. While the development of vagal NCCs has been widely studied, little has been known about sacral NCCs, particularly in mammals. Our objective was to test whether inhibition of small GTPases such as ROCK (an effector of Rho) affects sacral NCC migration in the mouse gut.First, cultured sacral NCCs from pelvic ganglia were treated with different concentrations of Y‐27632 to suppress active ROCK in vitro, and results showed that inhibition of ROCK significantly decreased neurite outgrowths and migration distances of sacral NCCs. Immunofluorescent localization demonstrated that the expression of cofilin which is a downstream protein of Rho/ROCK pathway remained unchanged while its phosphorylated form p‐cofilin (inactive state) was increased significantly. Expression of phalloidin which is a marker of stress fibers decreased after Y‐27632 treatment. Time‐lapse live cell imaging also showed that the speed and persistence of sacral NCC migration decreased, and the directional migration was disrupted after Y‐27632 treatment. Interestingly, expression of both phalloidin and p‐cofilin in sacral NCCs was restored to normal levels by Rho activator treatment.Next, we established an embryonic gut explant culture, where a pelvic ganglion (serving as a source of sacral NCCs) was recombined with a recipient hindgut segment ex vivo. Whole‐mount preparation and cryosection staining showed that the migration and differentiation of endogenous vagal NCCs in the hindgut explant cultured ex vivo was similar to those in the hindgut developed from E12.5 to E16.5 in vivo. Hence the gut culture explant system could support the normal development of the hindgut explant ex vivo. The recipient hindgut segment into which sacral NCCs from the pelvic ganglion had migrated 2 day after cultured ex vivo was then treated with different concentrations of Y‐27632. It was found that Y‐27632 reduced the migration distance, cell number and axonal extension of sacral NCCs within the hindgut segment. Moreover, time‐lapse live cell imaging also suggested that the speed and persistence of axonal extension of sacral NCCs decreased, and directional migration was disrupted after Y‐27632 treatment.In summary, these observations provide evidence that Rho/ROCK pathway regulates stress fiber formation and directional migration during sacral NCC migration.Support or Funding InformationThe work was supported by grants from RGC General Research Fund (Project Reference: 14102214) and RGC Theme‐based Research Scheme (Project Reference: T12C‐714/14‐R).

Ancient evolutionary origin of vertebrate enteric neurons from trunk-derived neural crest.

The enteric nervous system (ENS) of jawed vertebrates arises primarily from vagal neural crest cells that migrate to the foregut and subsequently colonize and innervate the entire gastrointestinal tract. To gain insight into its evolutionary origin, we examined ENS development in the basal jawless vertebrate, the sea lamprey. Surprisingly, we found no evidence for the existence of a vagally-derived enteric neural crest population in the lamprey. Rather, DiI labeling showed that late-migrating cells, originating from the trunk neural tube and associated with nerve fibers, differentiated into neurons within the gut wall and typhlosole. We propose that these trunk-derived neural crest cells are homologous to Schwann cell precursors (SCPs), recently shown in mammalian embryos to populate post-embryonic parasympathetic ganglia1,2, including enteric ganglia3. Our results suggest that neural crest-derived SCPs made an important contribution to the ancient ENS of early jawless vertebrates, a role that was largely subsumed by vagal neural crest cells in early gnathostomes.