Differentiation Of Enteric Neurons Research Articles

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

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 invivo has remained largely unclear. 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. 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. 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.

Enteric neuronal cell therapy reverses architectural changes in a novel diphtheria toxin-mediated model of colonic aganglionosis

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Short communication: Tryptic β-casein hydrolysate modulates enteric nervous system development in primary culture

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic β-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic β-casein hydrolysate and compared with untreated controls or to cultures treated with native β-casein or a Prolyve β-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic β-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic β-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native β-casein or Prolyve β-casein hydrolysate did not. Additionally, treatment with tryptic bioactive β-casein hydrolysate increased the expression of the glial marker glial fibrillary acidic protein and induced profound modifications of enteric glial cells morphology. Finally, expression of BMP2 and BMP4 and activation of Smad1/5 was altered after treatment with tryptic bioactive β-casein hydrolysate. Our data suggests that this milk-derived bioactive hydrolysate modulates ENS maturation through the regulation of BMP/Smad-signaling pathway. This study supports the need for further investigation on the influence of milk-derived bioactive peptides on ENS and intestinal maturation in vivo.

Enteric neural differentiation in innervated, physiologically functional, smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells.

The enteric nervous system (ENS) controls gastrointestinal (GI) functions, including motility and digestion, which are impaired in ENS disorders. Differentiation of enteric neurons is mediated by factors released by the gut mesenchyme, including smooth muscle cells (SMCs). SMC-derived factors involved in adult enteric neural progenitor cells (NPCs) differentiation remain elusive. Furthermore, physiologically relevant in vitro models to investigate the innervations of various regions of the gut, such as the pylorus and lower oesophageal sphincter (LES), are not available. Here, neural differentiation in bioengineered innervated circular constructs composed of SMCs isolated from the internal anal sphincter (IAS), pylorus, LES and colon of rabbits was investigated. Additionally, SMC-derived factors that induce neural differentiation were identified to optimize bioengineered construct innervations. Sphincteric and non-sphincteric bioengineered constructs aligned circumferentially and SMCs maintained contractile phenotypes. Sphincteric constructs generated spontaneous basal tones. Higher levels of excitatory and inhibitory motor neuron differentiation and secretion of bone morphogenic protein 2 (BMP2) were observed in bioengineered, innervated, sphincteric constructs compared to non-sphincteric constructs. The addition of BMP2 to non-sphincteric colonic SMC constructs increased nitrergic innervations, and inhibition of BMP2 with noggin in sphincteric constructs decreased functional relaxation. These studies provide: (a) the first bioengineered innervated pylorus and LES constructs; (b) physiologically relevant models to investigate SMCs and adult NPCs interactions; and (c) evidence of the region-specific effects of SMCs on neural differentiation mediated by BMP2. Furthermore, this study paves the way for the development of innervated bioengineered GI tissue constructs tailored to specific disorders and locations within the gut. Copyright © 2015 John Wiley & Sons, Ltd.

Appearance of interstitial cells of Cajal in the human midgut

Several subtypes of the interstitial cells of Cajal (ICC) form networks that play a role in gastrointestinal motor control. ICC express c-kit and depend on signaling via Kit receptors for development and phenotype maintenance. At 7-8 weeks of development, c-kit-immunoreactive (c-kit-IR) cells are present in the human oesophagus, stomach and proximal duodenum wall. In the remaining small and large bowel, c-kit-IR cells appear later. The object of the present study is to determine the timing of the appearance of c-kit-IR ICC in the parts of the digestive tube originating from the midgut (distal duodenum, jejunum, ileum and proximal colon). Specimens were obtained from eight human embryos and 11 fetuses at 7-12 weeks of gestational age. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. The differentiation of enteric neurons and smooth muscle cells was immunohistochemically examined by using anti-PGP9,5 and anti-desmin antibodies, respectively. In the distal duodenum, jejunum and ileum, c-kit-IR cells emerged at week 9 at the level of the myenteric plexus in the form of a thin row of cells encircling the inception of the ganglia. These cells were multipolar or spindle-shaped with two long processes and corresponded to the ICC of the myenteric plexus. In the proximal colon, c-kit-IR cells emerged at week 9-10 in the form of two parallel belts of cells extending at the submucosal plexus and the myenteric plexus levels. We conclude that ICC develop following two different patterns in the human midgut.

Fractional models for the migration of biological cells in complex spatial domains

Travelling wave phenomena are observed in many biological applications. Mathematical theory of standard reaction-diffusion problems shows that simple partial differential equations exhibit travelling wave solutions with constant wavespeed and such models are used to describe, for example, waves of chemical concentrations, electrical signals, cell migration, waves of epidemics and population dynamics. However, as in the study of cell motion in complex spatial geometries, experimental data are often not consistent with constant wavespeed. Non-local spatial models are successfully used to model anomalous diffusion and spatial heterogeneity in different physical contexts. We develop a fractional model based on the Fisher--Kolmogoroff equation, analyse it for its wavespeed properties, and relate the numerical results obtained from our simulations to experimental data describing enteric neural crest-derived cells migrating along the intact gut of mouse embryos. The model proposed essentially combines fractional and standard diffusion in different regions of the spatial domain and qualitatively reproduces the behaviour of neural crest-derived cells observed in the caecum and the hindgut of mouse embryos during in vivo experiments. References R. J. Adler, R. E. Feldman and M. S. Taqqu. A practical guide to heavy tails: Statistical techniques and applications. Birkauser, 1998. I. J. Allan and D. F. Newgreen. The origin and differentiation of enteric neurons of the intestine of the fowl embryo. The American Journal of Anatomy , 157, 137--154, 1980. doi:10.1002/aja.1001570203 B. J. Binder, K. A. Landman, M. J. Simpson, M. Mariani and D. F. Newgreen. Modeling proliferative tissue growth: A general approach and an avian case study. Physical Review E (Statistical, Nonlinear, and Soft Matter Physics) , 78(3), 1--13, 2008. doi:10.1103/PhysRevE.78.031912 M. A. Breau, A. Dahmani, F. Broders--Bondon, J. P. Thiery and S. Dufour. $\beta 1$ integrins are required for the invasion of the caecum and proximal hindgut by enteric neural crest cells. Development , 136, 2791--2801, 2009. doi:10.1242/dev.031419 K. Burrage, N. Hale and D. Kay. An efficient implementation of an implicit FEM scheme for fractional-in-space reaction-diffusion equations. SIAM Journal on Scientific Computing , 34(4), A2145--A2172. doi:10.1137/110847007 N. R. Druckenbrod and M. L. Epstein. The patterns of neural crest advance in the cecum and colon. Developmental Biology , 287, 125--133, 2005. doi:10.1016/j.ydbio.2005.08.040 H. Engler. On the speed of spread for fractional reaction-diffusion equations. International Journal of Differential Equations , 2010, Article ID 315421, 2010. doi:10.1155/2010/315421 M. Ilic, F. Liu, I. Turner and V. Anh. Numerical approximation of a fractional-in-space diffusion equation (II)--with nonhomogeneous boundary conditions. Fractional Calculus and Applied Analysis , 9, 333--349, 2006. http://eprints.qut.edu.au/23835/ P. K. Maini, D. L. S. McElwain and D. Leavesley. Travelling waves in a wound healing assay. Applied Mathematics Letters , 17, 575--580, 2004. doi:10.1016/S0893-9659(04)90128-0 R. McLennan, L. Dyson, K. W. Prather, J. A. Morrison, R. E. Baker, P. K. Maini and P. M. Kulesa. Multiscale mechanisms of cell migration during development: theory and experiment. Development , 139, 2935--2944, 2012. doi:10.1242/dev.081471 J. D. Murray. Mathematical Biology I and II. Springer Verlag, 2003. M. J. Simpson, D. C. Zhang, M. Mariani, K. A. Landman and D. F. Newgreen. Cell proliferation drives neural crest cell invasion of the intestine. Developmental Biology , 302, 553--568, 2007. doi:10.1016/j.ydbio.2006.10.017 H. M. Young, A. J. Bergner, R. B. Anderson, H. Enomoto, J. Milbrandt, D. F. Newgreen and P. M. Whitington. Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Developmental Biology , 270, 455--473, 2004. doi:10.1016/j.ydbio.2004.03.015 P. Zhuang, F. Liu, V. Anh and I. Turner. Numerical methods for the variable-order fractional advection-diffusion equation with a nonlinear source term. SIAM Journal on Numerical Analysis , 47(3), 1760--1781, 2009. doi:10.1137/080730597

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.

Expression Level of Hand2 Affects Specification of Enteric Neurons and Gastrointestinal Function in Mice

Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2(+/+) > Hand2(LoxP/+) > Hand2(+/-) > Hand2(LoxP/-). Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase(+) and calretinin(+), but not substance P(+) or vasoactive intestinal peptide(+) neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.

BMP2 promotes differentiation of nitrergic and catecholaminergic enteric neurons through a Smad1-dependent pathway.

The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-beta) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and beta-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP(+)) compared with those cultured with MPP(+) alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-beta signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.

Cell adhesion molecule L1 affects the rate of differentiation of enteric neurons in the developing gut

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into and along the developing gut. As the neural crest-derived cells migrate within the gut, a subpopulation begins to differentiate into enteric neurons. Here, we show that the differentiation of neural crest-derived cells into enteric neurons is delayed in L1-deficient mice, compared with littermate controls. However, glial cell differentiation is not affected in L1-deficient mice. These mice also show a delay in the differentiation of a neurotransmitter-specific subtype of enteric neuron within the gastrointestinal tract. Together, these results suggest a role for the cell adhesion molecule, L1, in the differentiation of neural crest-derived cells into enteric neurons within the developing enteric nervous system.

Hand2is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia

Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. We now report that enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2(-/-) bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. We suggest that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage.

Bone morphogenetic proteins-2 and -4 regulate the differentiation of enteric neurons and glia from post-migratory neural crest-derived cells

Bone morphogenetic proteins-2 and -4 regulate the differentiation of enteric neurons and glia from post-migratory neural crest-derived cells

Serotonin and the 5-HT2BReceptor in the Development of Enteric Neurons

We tested the hypothesis that 5-HT promotes the differentiation of enteric neurons by stimulating a developmentally regulated receptor expressed by crest-derived neuronal progenitors. 5-HT and the 5-HT(2) agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine(.)HCl (DOI) enhanced in vitro differentiation of enteric neurons, both in dissociated cultures of mixed cells and in cultures of crest-derived cells isolated from the gut by immunoselection with antibodies to p75(NTR). The promotion of in vitro neuronal differentiation by 5-HT and DOI was blocked by the 5-HT(1/2) antagonist methysergide, the pan-5-HT(2) antagonist ritanserin, and the 5-HT(2B/2C)-selective antagonist SB206553. The 5-HT(2A)-selective antagonist ketanserin did not completely block the developmental effects of 5-HT. 5-HT induced the nuclear translocation of mitogen-activated protein kinase. This effect was blocked by ritanserin. mRNA encoding 5-HT(2A) and 5-HT(2B) receptors was detected in the fetal bowel (stomach and small and large intestine), but that encoding the 5-HT(2C) receptor was not. mRNA encoding the 5-HT(2B) receptor and 5-HT(2B) immunoreactivity were found to be abundant in primordial [embryonic day 15 (E15)-E16] but not in mature myenteric ganglia. 5-HT(2B)-immunoreactive cells were found to be a subset of cells that expressed the neuronal marker PGP9.5. These data demonstrate for the first time that the 5-HT(2B) receptor is expressed in the small intestine as well as the stomach and that it is expressed by enteric neurons as well as by muscle. It is possible that by stimulating 5-HT(2B) receptors, 5-HT affects the fate of the large subset of enteric neurons that arises after the development of endogenous sources of 5-HT.

Morphological classifications of enteric neurons -- 100 years after Dogiel

The first differentiation of enteric neurons into three morphological types was done by the russian histologist A. S. Dogiel on the basis of the different shapes and lengths of their dendrites. Although a number of authors considered his results during the following decades, only a division into two types withstood time: type I neurons had one long and several short processes, whereas type II neurons were characterized by several long processes. Some further structural features were discussed but substantial progress was not made until the late 1970s. This stagnation was due to some inaccuracies in Dogiel's descriptions, to the fact that most histologists in this field followed the reticular concept of the nervous system, to the idea that enteric neurons represent no more than a vegetative, postganglionic relay station between the central nervous system and the periphery, and to methodological difficulties. With the application of modern neuroanatomical techniques it was realized that the enteric nervous system contains a considerable number of neuronal subpopulations. The search for morphological correlates of the chemical diversity of enteric neurons was done mainly in the pig and the guinea-pig. In the pig, additional structural features such as axonal projection, distribution of neurons within ganglia, within different plexuses and along the length of the gut, blood supply etc. were included as criteria for further refining neuronal classification. Most of our knowledge about functional features of enteric neurons, e.g. chemical coding, neuronal connectivity, electrophysiological behaviour, was derived from studies in the guinea-pig small intestine. In light of interspecies differences, comparison of findings from different species is mandatory. The search for morphological and functional peculiarities of human enteric neuronal circuitry has to consider all methodological and conceptual advances made within the past 100 years since the pioneering work of Dogiel.

V. Genes, lineages, and tissue interactions in the development of the enteric nervous system.

The enteric nervous system is derived from the vagal, rostral-truncal, and sacral levels of the neural crest. Because the crest-derived population that colonizes the bowel contains multipotent cells, terminal differentiation occurs in the gut and is influenced by both the enteric microenvironment and the responsivity of multiple lineages of precursors. Enteric growth factor-receptor combinations, which promote the development of enteric neurons and/or glia in most of the gastrointestinal (GI) tract, include glial cell line-derived neurotrophic factor-GFRalpha-1-Ret, NT-3-TrkC, a still-to-be-identified neuropoietic cytokine-ciliary neurotrophic factor receptor-alpha, serotonin (5-HT)-5-HT2B, and LBP110, a 110-kDa laminin-1 binding protein. A qualitatively different effect is shown by the peptide-receptor combination ET-3-ETB, which inhibits neuronal differentiation and appears to prevent the premature differentiation of enteric neurons before colonization of the GI tract has been completed (resulting in aganglionosis of the terminal colon).

Neuronal and non-neuronal plasticity in the rat following myenteric denervation.

The plasticity of the enteric nervous system in adulthood has always been a field of research of the greatest interest. Many experimental models have been developed, allowing to investigate the importance of the peripheral field of innervation in the control of growth and differentiation of enteric neurons during postnatal life (Filogamo, 1987; Filogamo and Cracco, 1995). Among those, we were particularly interested in the chemical ablation of the myenteric plexus by means of a cationic surfactant, benzalkonium chloride (BAC).KeywordsEnteric Nervous SystemMyenteric PlexusEnteric NeuronBenzalkonium ChlorideSmooth Muscle LayerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Formation of ganglia in the gut of the chick embryo

We have examined the formation of myenteric ganglia in the developing avian enteric nervous system. The monoclonal antibody HNK-1 was used to identify neural-crest-derived cells in whole mounts of fore- and midgut of chick embryos. We find that the crest-derived cells extend processes to their neighbors and form a complex network in the wall of the gut. Formation of this network is an unusual behavior of crest-derived cells and suggests the gut microenvironment is critical to this behavior. This cellular network disappears after ablation of the vagal neural crest, indicating the HNK-1-stained cellular network arises from crest-derived cells. The network is found in the gut wall before the vagal nerve fibers are present. This network is first found in the primordium of the proventriculus, distal to the evagination of the lung buds, and progresses just proximal to the yolk stalk at embryonic day (E) 3.5 and almost to the ileocecal junction at E5.5. The number of cells and the complexity of the network decrease in a rostral-caudal direction down the length of the gut at these stages. The leading edge of the network consists of cells serially arranged in longitudinally running strands. The organization of the network changes with increasing embryonic age; we have focused on network changes in the proventriculus. In the primordium of the proventriculus at E3.5, the network consists of a cluster of one or two adjacent crest-derived cells, which extend processes to a number of neighboring crest-derived cells. At E5.5 large increases in the number of cells per cluster and in the length of cellular connectives between clusters are apparent. At E6.5 a crude meshwork of clusters is seen. At E10.5 the arrangement of cell clusters resembles the pattern of ganglia found in the adult myenteric plexus. This network may provide the environmental cues for the differentiation of enteric neurons and a framework for the pattern of ganglia found in the adult enteric nervous system.

The origin and differentiation of enteric neurons of the intestine of the fowl embryo.

AbstractNeuroblasts were identified histologically in the intestine of the fowl embryo of stages 25 to 35 (Hamburger and Hamilton, ′51). These cells occurred rostral to the umbilicus at stage 26, and appeared caudally in a wave that reached the coprodeum at stage 34. The presence of histologically unidentifiable neuronal precursors was studied by grafting onto the chorio‐allantoic membrane (CAM) short lengths of gut from embryos of stage 24 to 34. After growth in isolation on the CAM, the resultant gut grafts were examined histologically for enteric neurons. In these grafts, neuronal differentiation occurred in the gut as a wave moving caudally at about 40 ḿm/hr. This wave preceded by less than 12 hours the normal appearance of neuroblasts at any particular level of the intestine.The findings are discussed with regard to previous reports on the origin of enteric neurons. The most likely conclusion is that all enteric neurons in the fowl are derived from vagal neural crest. The results obtained with grafts containing neurons of Remak's nerve, in addition to intestine, suggest that lumbo‐sacral neural crest may give rise to some enteric supporting cells which, in the absence of normal enteric plexuses, differentiate as pigment cells. If lumbo‐sacral neural crest cells contribute to post‐umbilical enteric neurons, their differentiation requires the presence of vagal cells.A comparison of enteric neuronal density and distribution in short and long colo‐rectal grafts suggests that only the advancing front of the neuronal precursor wave is necessary to populate the remainder of the gut. This high proliferative potential in the wave‐front appears to decline on reaching the most caudal colo‐rectum and coprodeum.