Pcgf1 gene disruption reveals primary involvement of epigenetic mechanism in neuronal subtype specification in the enteric nervous system.

The enteric nervous system (ENS) regulates gut functions independently from the central nervous system (CNS) by its highly autonomic neural circuit that integrates diverse neuronal subtypes. Although several transcription factors are shown to be necessary for the generation of some enteric neuron subtypes, the mechanisms underlying neuronal subtype specification in the ENS remain elusive. In this study, we examined the biological function of Polycomb group RING finger protein 1 (PCGF1), one of the epigenetic modifiers, in the development and differentiation of the ENS by disrupting the Pcgf1 gene selectively in the autonomic-lineage cells. Although ENS precursor migration and enteric neurogenesis were largely unaffected, neuronal differentiation was impaired in the Pcgf1-deficient mice, with the numbers of neurons expressing somatostatin (Sst+ ) decreased in multiple gut regions. Notably, the decrease in Sst+ neurons was associated with the corresponding increase in calbindin+ neurons in the proximal colon. These findings suggest that neuronal subtype conversion may occur in the absence of PCGF1, and that epigenetic mechanism is primarily involved in specification of some enteric neuron subtypes.

A Single RET Mutation in Hirschsprung Disease Induces Intestinal Aganglionosis Via a Dominant-Negative Mechanism.

BACKGROUND & AIMS: Hirschsprung disease (HSCR) is a congenital disorder characterized by the absence of the enteric nervous system (ENS). HSCR potentially involves multiple gene aberrations and displays complex patterns of inheritance. Mutations of the RET gene, encoding the RET receptor tyrosine kinase, play a central role in the pathogenesis of HSCR. Although a wide variety of coding RET mutations have been identified, their pathogenetic significance in vivo has remained largely unclear. METHODS: We introduced a HSCR-associated RET missense mutation, RET(S811F), into the corresponding region (S812) of the mouse Ret gene. Pathogenetic impact of Ret(S812F) was assessed by histologic and functional analyses of the ENS and by biochemical analyses. Interactions of the Ret(S812F) allele with HSCR susceptibility genes, the RET9 allele and the Ednrb gene, were examined by genetic crossing in mice. RESULTS: RetS812F/+ mice displayed intestinal aganglionosis (incidence, 50%) or hypoganglionosis (50%), impaired differentiation of enteric neurons, defecation deficits, and increased lethality. Biochemical analyses revealed that Ret(S811F) protein was not only kinase-deficient but also abrogated function of wild-type RET in trans. Moreover, the Ret(S812F) allele interacted with other HSCR susceptibility genes and caused intestinal aganglionosis with full penetrance. CONCLUSIONS: This study demonstrates that a single RET missense mutation alone induces intestinal aganglionosis via a dominant-negative mechanism. The RetS812F/+ mice model HSCR displays dominant inheritance with incomplete penetrance and serves as a valuable platform for better understanding of the pathogenetic mechanism of HSCR caused by coding RET mutations.

Uts2b is a microbiota-regulated gene expressed in vagal afferent neurons connected to enteroendocrine cells producing cholecystokinin.

Enteroendocrine cells (EECs) are the primary sensory cells that sense the gut luminal environment and secret hormones to regulate organ function. Recent studies revealed that vagal afferent neurons are connected to EECs and relay sensory information from EECs to the brain stem. To date, however, the identity of vagal afferent neurons connected to a given EEC subtype and the mode of their gene responses to its intestinal hormone have remained unknown. Hypothesizing that EEC-associated vagal afferent neurons change their gene expression in response to the microbiota-related extracellular stimuli, we conducted comparative gene expression analyses of the nodose-petrosal ganglion complex (NPG) using specific pathogen-free (SPF) and germ-free (GF) mice. We report here that the Uts2b gene, which encodes a functionally unknown neuropeptide, urotensin 2B (UTS2B), is expressed in a microbiota-dependent manner in NPG neurons. In cultured NPG neurons, expression of Uts2b was induced by AR420626, the selective agonist for FFAR3. Moreover, distinct gastrointestinal hormones exerted differential effects on Uts2b expression in NPG neurons, where cholecystokinin (CCK) significantly increased its expression. The majority of Uts2b-expressing NPG neurons expressed CCK-A, the receptor for CCK, which comprised approximately 25% of all CCK-A-expressing NPG neurons. Selective fluorescent labeling of Uts2b-expressing NPG neurons revealed a direct contact of their nerve fibers to CCK-expressing EECs. This study identifies the Uts2b as a microbiota-regulated gene, demonstrates that Uts2b-expressing vagal afferent neurons transduce sensory information from CCK-expressing EECs to the brain, and suggests potential involvement of UTS2B in a modality of CCK actions.

Enhanced enteric neurogenesis by Schwann cell precursors in mouse models of Hirschsprung disease.

Hirschsprung disease (HSCR) is characterized by congenital absence of enteric neurons in distal portions of the gut. Although recent studies identified Schwann cell precursors (SCPs) as a novel cellular source of enteric neurons, it is unknown how SCPs contribute to the disease phenotype of HSCR. Using Schwann cell-specific genetic labeling, we investigated SCP-derived neurogenesis in two mouse models of HSCR; Sox10 haploinsufficient mice exhibiting distal colonic aganglionosis and Ednrb knockout mice showing small intestinal aganglionosis. We also examined Ret dependency in SCP-derived neurogenesis using mice displaying intestinal aganglionosis in which Ret expression was conditionally removed in the Schwann cell lineage. SCP-derived neurons were abundant in the transition zone lying between the ganglionated and aganglionic segments, although SCP-derived neurogenesis was scarce in the aganglionic region. In the transition zone, SCPs mainly gave rise to nitrergic neurons that are rarely observed in the SCP-derived neurons under the normal condition. Enhanced SCP-derived neurogenesis was also detected in the transition zone of mice lacking RET expression in the Schwann cell lineage. Increased SCP-derived neurogenesis in the transition zone suggests that reduction in the vagal neural crest-derived enteric neurons promotes SCP-derived neurogenesis. SCPs may adopt a neuronal subtype by responding to changes in the gut environment. Robust SCP-derived neurogenesis can occur in a Ret-independent manner, which suggests that SCPs are a cellular source to compensate for missing enteric neurons in HSCR.

Live visualization of a functional RET-EGFP chimeric receptor in homozygous knock-in mice.

The GDNF Family Ligands (GFLs) regulate neural development and kidney organogenesis by activating the RET receptor tyrosine kinase. Many RET-dependent developmental processes involve long-distance cell-cell communications or cell polarity, which includes cell migration and axon guidance. This suggests that spatiotemporally regulated subcellular localization of RET protein and appropriate propagation of RET signaling in cells are essential for the physiological function of the GFLs. Little is known, however, about the dynamics of RET protein in cells. Addressing this issue requires development of a system that allows visualization of RET in living cells. In this study, we report generation of a novel knock-in mouse line in which the RET-EGFP chimeric receptor is expressed under the Ret promoter. Unlike Ret-deficient mice that die after birth due to the absence of the enteric nervous system (ENS) and kidneys, RetRET-EGFP/RET-EGFP mice were viable and grew to adulthood with no overt abnormality, which indicated that RET-EGFP exerts function comparable to RET. In neurons and ENS progenitors, RET-EGFP signals were detected both on the cell membrane and in the cytoplasm, the latter of which appeared as a punctate pattern. Time-lapse imaging of cultured neural cells and embryos revealed active transport of RET-EGFP puncta in neuronal axons and cell bodies. Immunohistochemical analyses detected RET-EGFP signals in early and recycling endosomes, indicating that RET-EGFP is trafficked via the endocytic pathway. RetRET-EGFP/RET-EGFP mice enable visualization of functional RET protein in vivo for the first time and provide a unique platform to examine the dynamics and physiology of RET trafficking.

Increased RET Activity Coupled with a Reduction in the RET Gene Dosage Causes Intestinal Aganglionosis in Mice.

Mutations of the gene encoding the RET tyrosine kinase causes Hirschsprung's disease (HSCR) and medullary thyroid carcinoma (MTC). Current consensus holds that HSCR and MTC are induced by inactivating and activating RET mutations, respectively. However, it remains unknown whether activating mutations in the RET gene have adverse effects on ENS development in vivo We addressed this issue by examining mice engineered to express RET51(C618F), an activating mutation identified in MTC patients. Although Ret51(C618F)/51(C618F) mice displayed hyperganglionosis of the ENS, Ret51(C618F)/- mice exhibited severe intestinal aganglionosis because of premature neuronal differentiation. Reduced levels of glial cell-derived neurotrophic factor (GDNF), a RET-activating neurotrophic factor, ameliorated the ENS phenotype of Ret51(C618F)/- mice, demonstrating that GDNF-mediated activation of RET51(C618F) is responsible for severe aganglionic phenotype. The RET51(C618F) allele showed genetic interaction with Ednrb gene, one of modifier genes for HSCR. These data reveal that proliferation and differentiation of ENS precursors are exquisitely controlled by both the activation levels and total dose of RET. Increased RET activity coupled with a decreased gene dosage can cause intestinal aganglionosis, a finding that provides novel insight into HSCR pathogenesis.

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene.

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (RetM919T , RetN396K and RetY792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. RetM919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in RetM919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.

Generation of conditional ALK F1174L mutant mouse models for the study of neuroblastoma pathogenesis.

Neuroblastoma, an embryonal tumor arising from the sympathetic ganglia and adrenal medulla, is among the most intractable pediatric cancers. Although a variety of genetic changes have been identified in neuroblastoma, how they contribute to its pathogenesis remains largely unclear. Recent studies have identified alterations of the anaplastic lymphoma kinase (ALK) gene in neuroblastoma; ALK F1174L (a phenylalanine-to-leucine substitution at codon 1174) represents one of the most frequent of these somatic mutations, and is associated with amplification of the MYCN gene, the most reliable marker for the poor survival. We engineered the mouse Alk locus so that ALK F1174L is expressed by its endogenous promoter and can be induced in a spatiotemporally controlled fashion using Cre-loxP system. Although expression of ALK F1174L resulted in enhanced proliferation of sympathetic ganglion progenitors and increased the size of the sympathetic ganglia, it was insufficient to cause neuroblastoma. However, lethal neuroblastoma frequently developed in mice co-expressing ALK F1174L and MYCN, even in a genetic background where MYCN alone does not cause overt tumors. These data reveal that physiological expression of ALK F1174L significantly potentiates the oncogenic ability of MYCN in vivo. Our conditional mutant mice provide a valuable platform for investigating the pathogenesis of neuroblastoma.

Technologies for Live Imaging of Enteric Neural Crest-Derived Cells.

Time-lapse imaging of gut explants from embryonic mice in which neural crest-derived cells express fluorescent proteins allows the behavior of enteric neural crest cells to be observed and analyzed. Explants of embryonic gut are dissected, mounted on filter paper supports so the gut retains its tubular three-dimensional structure, and then placed in coverglass bottom culture dishes in tissue culture medium. A stainless steel ring is placed on top of the filter support to prevent movement. Imaging is performed using a confocal microscope in an environmental chamber. A z series of images through the network of fluorescent cells is collected every 3, 5, or 10 min. At the end of imaging, the z series are projected.

Mice conditionally expressing RET(C618F) mutation display C cell hyperplasia and hyperganglionosis of the enteric nervous system.

Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine-rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand-independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock-in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret-null and express mCherry by Cre-loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain-of-function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.

Catenins Steer Cell Migration via Stabilization of Front-Rear Polarity.

Cell migration plays a pivotal role in morphogenetic and pathogenetic processes. To achieve directional migration, cells must establish a front-to-rear axis of polarity. Here we show that components of the cadherin-catenin complex function to stabilize this front-rear polarity. Neural crest and glioblastoma cells undergo directional migration in vivo or in vitro. During this process, αE-catenin accumulated at lamellipodial membranes and then moved toward the rear with the support of a tyrosine-phosphorylated ß-catenin. This relocating αE-catenin bound to p115RhoGEF, leading to gathering of active RhoA in front of the nucleus where myosin-IIB arcs assemble. When catenins or p115RhoGEF were removed, cells lost the polarized myosin-IIB assembly, as well as the capability for directional movement. These results suggest that, apart from its well-known function in cell adhesion, the ß-catenin/αE-catenin complex regulates directional cell migration by restricting active RhoA to perinuclear regions and controlling myosin-IIB dynamics at these sites.

Dual origin of enteric neurons in vagal Schwann cell precursors and the sympathetic neural crest.

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated.

FANTOM5 CAGE profiles of human and mouse samples.

In the FANTOM5 project, transcription initiation events across the human and mouse genomes were mapped at a single base-pair resolution and their frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled with single-molecule sequencing. Approximately three thousands of samples, consisting of a variety of primary cells, tissues, cell lines, and time series samples during cell activation and development, were subjected to a uniform pipeline of CAGE data production. The analysis pipeline started by measuring RNA extracts to assess their quality, and continued to CAGE library production by using a robotic or a manual workflow, single molecule sequencing, and computational processing to generate frequencies of transcription initiation. Resulting data represents the consequence of transcriptional regulation in each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE profiles, approximately 200,000 and 150,000 peaks for the human and mouse genomes, were identified and annotated to provide precise location of known promoters as well as novel ones, and to quantify their activities.

Striatal hypodopamine phenotypes found in transgenic mice that overexpress glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) positively regulates the development and maintenance of in vitro dopaminergic neurons. However, the in vivo influences of GDNF signals on the brain dopamine system are controversial and not fully defined. To address this question, we analyzed dopaminergic phenotypes of the transgenic mice that overexpress GDNF under the control of the glial Gfap promoter. Compared with wild-type, the GDNF transgenic mice contained higher levels of GDNF protein and phosphorylated RET receptors in the brain. However, there were reductions in the levels of tyrosine hydroxylase (TH), dopamine, and its metabolite homovanillic acid in the striatum of transgenic mice. The TH reduction appeared to occur during postnatal development. Immunohistochemistry revealed that striatal TH density was reduced in transgenic mice with no apparent signs of neurodegeneration. In agreement with these neurochemical traits, basal levels of extracellular dopamine and high K+-induced dopamine efflux were decreased in the striatum of transgenic mice. We also explored the influences of GDNF overexpression on lomomotor behavior. GDNF transgenic mice exhibited lower stereotypy and rearing in a novel environment compared with wild-type mice. These results suggest that chronic overexpression of GDNF in brain astrocytes exerts an opposing influence on nigrostriatal dopamine metabolism and neurotransmission.

Retrograde transport of neurotrophic factor signaling: implications in neuronal development and pathogenesis.

Neurotrophic factors and their receptors play a central role in neuronal survival. Since neurons have a highly polarized morphology, target-derived neurotrophic factor signaling is transported retrogradely along the axon to the cell body. A body of evidence suggests that retrograde transport of the neurotrophic factors and their receptors is required for signal propagation. Retrograde transport of neurotrophic factor signaling is crucial not only for neuronal development, but also for preventing neuronal degeneration. Thus, elucidating the mechanism of retrograde transport will lead to insights into the developmental mechanisms of the nervous system as well as contribute to the establishment of novel therapies for neurodegenerative diseases. In this article, we will review the recent progress made in research of retrograde trafficking and discuss its physiological significance.

Development of the intrinsic and extrinsic innervation of the gut.

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract.

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System.

Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues. SIGNIFICANCE STATEMENT: Elucidating the cellular identity of neuronal precursors provides novel insights into development and function of the nervous system. The enteric nervous system (ENS) is innervated richly by extrinsic nerve fibers, but little is known about the significance of extrinsic innervation to the structural integrity of the ENS. This report reveals that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate and differentiates into specific neuronal subtypes. SCP-specific ablation of the Ret gene leads to colonic oligoganglionosis, demonstrating a crucial role of SCP-derived neurogenesis in ENS development. Cross-lineage differentiation capacity in SCPs suggests their potential involvement in the development and pathology of a wide variety of neural crest-derived cell types.

The neurotrophic factor receptor RET drives haematopoietic stem cell survival and function.

Haematopoiesis is a developmental cascade that generates all blood cell lineages in health and disease. This process relies on quiescent haematopoietic stem cells capable of differentiating, self renewing and expanding upon physiological demand. However, the mechanisms that regulate haematopoietic stem cell homeostasis and function remain largely unknown. Here we show that the neurotrophic factor receptor RET (rearranged during transfection) drives haematopoietic stem cell survival, expansion and function. We find that haematopoietic stem cells express RET and that its neurotrophic factor partners are produced in the haematopoietic stem cell environment. Ablation of Ret leads to impaired survival and reduced numbers of haematopoietic stem cells with normal differentiation potential, but loss of cell-autonomous stress response and reconstitution potential. Strikingly, RET signals provide haematopoietic stem cells with critical Bcl2 and Bcl2l1 surviving cues, downstream of p38 mitogen-activated protein (MAP) kinase and cyclic-AMP-response element binding protein (CREB) activation. Accordingly, enforced expression of RET downstream targets, Bcl2 or Bcl2l1, is sufficient to restore the activity of Ret null progenitors in vivo. Activation of RET results in improved haematopoietic stem cell survival, expansion and in vivo transplantation efficiency. Remarkably, human cord-blood progenitor expansion and transplantation is also improved by neurotrophic factors, opening the way for exploration of RET agonists in human haematopoietic stem cell transplantation. Our work shows that neurotrophic factors are novel components of the haematopoietic stem cell microenvironment, revealing that haematopoietic stem cells and neurons are regulated by similar signals.

Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states.

The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing "As model," stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFRα1+ cells and find that, during steady-state spermatogenesis, the entire GFRα1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFRα1+ cells during steady state and postinsult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis.

Colonizing while migrating: how do individual enteric neural crest cells behave?

BACKGROUND: Directed cell migration is essential for normal development. In most of the migratory cell populations that have been analyzed in detail to date, all of the cells migrate as a collective from one location to another. However, there are also migratory cell populations that must populate the areas through which they migrate, and thus some cells get left behind while others advance. Very little is known about how individual cells behave to achieve concomitant directional migration and population of the migratory route. We examined the behavior of enteric neural crest-derived cells (ENCCs), which must both advance caudally to reach the anal end and populate each gut region. RESULTS: The behavior of individual ENCCs was examined using live imaging and mice in which ENCCs express a photoconvertible protein. We show that individual ENCCs exhibit very variable directionalities and speed; as the migratory wavefront of ENCCs advances caudally, each gut region is populated primarily by some ENCCs migrating non-directionally. After populating each region, ENCCs remain migratory for at least 24 hours. Endothelin receptor type B (EDNRB) signaling is known to be essential for the normal advance of the ENCC population. We now show that perturbation of EDNRB principally affects individual ENCC speed rather than directionality. The trajectories of solitary ENCCs, which occur transiently at the wavefront, were consistent with an unbiased random walk and so cell-cell contact is essential for directional migration. ENCCs migrate in close association with neurites. We showed that although ENCCs often use neurites as substrates, ENCCs lead the way, neurites are not required for chain formation and neurite growth is more directional than the migration of ENCCs as a whole. CONCLUSIONS: Each gut region is initially populated by sub-populations of ENCCs migrating non-directionally, rather than stopping. This might provide a mechanism for ensuring a uniform density of ENCCs along the growing gut.