A single rostrocaudal colonization of the rodent intestine by enteric neuron precursors is revealed by the expression of Phox2b, Ret, and p75 and by explants grown under the kidney capsule or in organ culture.

The colonization of the rodent gastrointestinal tract by enteric neuron precursors is controversial due to the lack of specific cellular markers at early stages. The transcription factor, Phox2b, is expressed by enteric neuron precursors (Pattyn et al. Development 124, 4065-4075, 1997). In this study, we have used an antiserum to Phox2b to characterize in detail the spatiotemporal expression of Phox2b in the gastrointestinal tract of adult mice and embryonic mice and rats. In adult mice, all enteric neurons (labeled with neuron-specific enolase antibodies), and a subpopulation of glial cells (labeled with GFAP antibodies), showed immunoreactivity to Phox2b. In embryonic mice, the appearance of Phox2b-immunoreactive cells was mapped during development of the gastrointestinal tract. At Embryonic Days 9.5-10 (E9.5-10), Phox2b-labeled cells were present only in the stomach, and during subsequent development, labeled cells appeared as a single rostrocaudal wave along the gastrointestinal tract; at E14 Phox2b-labeled cells were present along the entire length of the gastrointestinal tract. Ret and p75 have also been reported to label migratory-stage enteric neuron precursors. A unidirectional, rostral-to-caudal colonization of the gastrointestinal tract of embryonic mice by Ret- and p75-immunoreactive cells was also observed, and the locations of Ret- and p75-positive cells within the gut were very similar to that of Phox2b-positive cells. To verify the location of enteric neuron precursors within the gut, explants from spatiotemporally defined regions of embryonic intestine, 0.3-3 mm long, were grown in the kidney subcapsular space, or in catenary organ culture, and examined for the presence of neurons. The location and sequence of appearance of enteric neuron precursors deduced from the explants grown under the kidney capsule or in organ culture was very similar to that seen with the Phox2b, Ret, and p75 antisera. Previous studies have mapped the rostrocaudal colonization of the rat intestine by enteric neuron precursors using HNK-1 as a marker. In the current study, all HNK-1-labeled cells in the gastrointestinal tract of rat embryos showed immunoreactivity to Phox2b, but HNK-1 cells comprised only a small subpopulation of the Phox2b-labeled cells. In addition, in rats, Phox2b-labeled cells were present in advance of (more caudal to) the most caudal HNK-1-labeled cells by 600-700 microm in the hindgut at E15. We conclude that the neural crest cell population that arises from the vagal level of the neural axis and that populates the stomach, midgut, and hindgut expresses Phox2b, Ret, and p75. In contrast, the sacral-level neural crest cells that populate the hindgut either do not express, or show a delayed expression of, all of the known markers of vagal- and trunk-level neural crest cells.

[1]  T. Tsuzuki,et al.  Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. , 1995, Oncogene.

[2]  C. L. Yntema,et al.  The origin of intrinsic ganglia of trunk viscera from vagal neural crest in the chick embryo , 1954, The Journal of comparative neurology.

[3]  F. Costantini,et al.  Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. , 1996, Development.

[4]  Y. Watanabe,et al.  ret Proto-oncogene product is a useful marker of lineage determination in the development of the enteric nervous system in rats. , 1997, Journal of pediatric surgery.

[5]  T. P. Rothman,et al.  Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats. , 1981, Developmental biology.

[6]  X. Morin,et al.  Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. , 1997, Development.

[7]  D. Newgreen,et al.  Migration of enteric neural crest cells in relation to growth of the gut in avian embryos. , 1996, Acta anatomica.

[8]  S. Fraser,et al.  Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos. , 1991, Development.

[9]  X. Morin,et al.  Defects in Sensory and Autonomic Ganglia and Absence of Locus Coeruleus in Mice Deficient for the Homeobox Gene Phox2a , 1997, Neuron.

[10]  M A Teillet,et al.  Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique. , 1974, Developmental biology.

[11]  J. Louis,et al.  GDNF–Induced Activation of the Ret Protein Tyrosine Kinase Is Mediated by GDNFR-α, a Novel Receptor for GDNF , 1996, Cell.

[12]  T. P. Rothman,et al.  Phenotypic expression in the developing murine enteric nervous system , 1982, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[13]  K. Poulsen,et al.  Formation of ganglia in the gut of the chick embryo , 1991, The Journal of comparative neurology.

[14]  F. Costantini,et al.  Expression of the c-ret proto-oncogene during mouse embryogenesis. , 1993, Development.

[15]  T. P. Rothman,et al.  Colonization of the developing murine enteric nervous system and subsequent phenotypic expression by the precursors of peptidergic neurons , 1984, The Journal of comparative neurology.

[16]  I. Black,et al.  Ontogenetic appearance and disappearance of tyrosine hydroxylase and catecholamines in the rat embryo. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[17]  H. Young,et al.  Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse , 1996, Cell and Tissue Research.

[18]  T. Branchek,et al.  Time course of expression of neuropeptide Y, calcitonin gene‐related peptide, and NADPH diaphorase activity in neurons of the developing murine bowel and the appearance of 5‐hydroxytryptamine in mucosal enterochromaffin cells , 1989, The Journal of comparative neurology.

[19]  R. Kapur.  Contemporary approaches toward understanding the pathogenesis of Hirschsprung disease. , 1993, Pediatric pathology.

[20]  D. Newgreen,et al.  The origin and differentiation of enteric neurons of the intestine of the fowl embryo. , 1980, The American journal of anatomy.

[21]  Jonas Frisén,et al.  Renal agenesis and the absence of enteric neurons in mice lacking GDNF , 1996, Nature.

[22]  T. Saito,et al.  Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells. , 1991, Genes & development.

[23]  T. P. Rothman,et al.  Colonization of the bowel by the precursors of enteric glia: Studies of normal and congenitally aganglionic mutant mice , 1986, The Journal of comparative neurology.

[24]  N. L. Le Douarin,et al.  The migration of neural crest cells to the wall of the digestive tract in avian embryo. , 1973, Journal of embryology and experimental morphology.

[25]  David J. Anderson,et al.  MASH1 maintains competence for BMP2-induced neuronal differentiation in post-migratory neural crest cells , 1997, Current Biology.

[26]  R. Palmiter,et al.  Goosecoid and the organizer. , 1992 .

[27]  M. Gershon,et al.  Colonization of the avian hindgut by cells derived from the sacral neural crest. , 1990, Developmental biology.

[28]  R. Palmiter,et al.  Some neuronal cell populations express human dopamine β-hydroxylase-lacZ transgenes transiently during embryonic development , 1991, Neuron.

[29]  T. P. Rothman,et al.  Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus. , 1991, Development.

[30]  D. Anderson,et al.  MASH1 activates expression of the paired homeodomain transcription factor Phox2a, and couples pan-neuronal and subtype-specific components of autonomic neuronal identity. , 1998, Development.

[31]  Frank Costantini,et al.  Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret , 1994, Nature.

[32]  M. Gershon,et al.  Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons. , 1989, Developmental biology.

[33]  I. Fariñas,et al.  Renal and neuronal abnormalities in mice lacking GDNF , 1996, Nature.

[34]  M. Gershon.  Genes and lineages in the formation of the enteric nervous system , 1997, Current Opinion in Neurobiology.

[35]  C. L. Yntema,et al.  Experiments on the origin and development of the sacral autonomic nerves in the chick embryo , 1955 .

[36]  D. Reis,et al.  Transient expression of a noradrenergic phenotype in cells of the rat embryonic gut , 1978, Brain Research.

[37]  David J. Anderson,et al.  Postmigratory neural crest cells expressing c-RET display restricted developmental and proliferative capacities , 1995, Neuron.

[38]  M. Hirsch,et al.  Control of noradrenergic differentiation and Phox2a expression by MASH1 in the central and peripheral nervous system. , 1998, Development.

[39]  A. Buj-Bello,et al.  Characterization of a multicomponent receptor for GDNF , 1996, Nature.

[40]  Karl Theiler,et al.  The House Mouse: Atlas of Embryonic Development , 1972 .

[41]  M. Tiveron,et al.  The Expression Pattern of the Transcription Factor Phox2 Delineates Synaptic Pathways of the Autonomic Nervous System , 1996, The Journal of Neuroscience.

[42]  B. Reese,et al.  Mosaics of Islet-1-Expressing Amacrine Cells Assembled by Short-Range Cellular Interactions , 1997, The Journal of Neuroscience.

[43]  Mart Saarma,et al.  Defects in enteric innervation and kidney development in mice lacking GDNF , 1996, Nature.

[44]  D. Newgreen,et al.  Origin of interstitial cells of Cajal in the mouse intestine. , 1996, Developmental biology.

[45]  S. Ward,et al.  Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine. , 1997, Gastroenterology.

[46]  G. Christie.  Developmental stages in somite and post‐somite rat embryos, based on external appearance, and including some features of the macroscopic development of the oral cavity , 1964, Journal of morphology.

[47]  D. Newgreen,et al.  Extracellular matrix and adhesive molecules in the early development of the gut and its innervation in normal and spotting lethal rat embryos. , 1995, Acta anatomica.

[48]  D. Tibboel,et al.  A model for aganglionosis in the chicken embryo. , 1989, Journal of pediatric surgery.

[49]  J. Thiery,et al.  Pathways of avian neural crest cell migration in the developing gut. , 1986, Developmental biology.

[50]  D. Tibboel,et al.  Formation and malformation of the enteric nervous system in mice: an organ culture study. , 1989, Journal of pediatric surgery.

[51]  W. Webster.  Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. , 1973, Journal of embryology and experimental morphology.

[52]  H. S. Bennett,et al.  Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. , 1976, Stain technology.

[53]  T. P. Rothman,et al.  From neural crest to bowel: development of the enteric nervous system. , 1993, Journal of neurobiology.

[54]  T. Mikawa,et al.  Mapping the origin of the avian enteric nervous system with a retroviral marker , 1994, Developmental dynamics : an official publication of the American Association of Anatomists.

[55]  J. Pintar,et al.  Transiently catecholaminergic (TC) cells in the bowel of the fetal rat: precursors of noncatecholaminergic enteric neurons. , 1990, Developmental biology.