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T P Rothman

Publications and source records attributed to T P Rothman.

10 recordsLinked to original sources
PubMed1991

Time of origin of neurons in the murine enteric nervous system: sequence in relation to phenotype.

The hypothesis was tested that developing enteric neurons withdraw from the cell cycle in a sequence related to their phenotype. The birthdays of immunocytochemically identified myenteric and submucosal neurons were determined in the murine duodenum and jejunum. [3H]thymidine ([3H]TdR) was injected into timed pregnant mice or pups at 4-8 hour intervals over a 24 hour period. Pups were killed on postnatal day 30 (P30). [3H]TdR incorporation was detected by radioautography in enteric neurons, which were phenotypically identified by the simultaneous detection of the immunoreactivities of 5-hydroxytryptamine (5-HT), choline acetyl transferase (ChAT), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and vasoactive intestinal peptide (VIP). The dates of the earliest withdrawal from the cell cycle of neurons containing these markers were determined, as well as the length of time during which the identified neurons continued to be born, and the date on which their rate of birth was maximal. The birthdates of myenteric neurons that contained 5-HT (E8-E14, peak at E10) or ChAT (E8-E15, peak at E12) tended to be earlier than those that contained ENK (E10-E18, peak at E14), NPY (E10-E18, peak at E15), VIP (E10-P5, peak at E15), or CGRP (E10-P3, peak at E17). For any given immunocytochemically defined neuronal phenotype, submucosal neurons tended to be born later than their myenteric counterparts and submucosal neurons that contained neuropeptides were born later than those that contained only ChAT immunoreactivity. The day (E8) on which the first 5-HT- and ChAT-immunoreactive neurons became postmitotic is earlier than the day (E9) on which the colonization of the bowel by crest-derived cells has been detected. The population of neural precursors that colonizes the gut, therefore, is heterogeneous; many cells are proliferating, but a specific subset, which will ultimately give rise to serotoninergic or cholinergic neurons, is already postmitotic. Neurons continued to be born throughout fetal life and even after birth. Consequently, terminally differentiated neurons coexist in the developing enteric nervous system with dividing neural precursor cells. This observation is consistent with the idea that early developing neurons could affect the development of enteric neural precursors; moreover, they also demonstrate that it is possible to add neurons to the enteric plexuses even after the neural circuits on which the bowel depends have become functional.

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PubMed1991

Enteric glia.

The structure of the enteric nervous system (ENS) is different from that of extraenteric peripheral nerve. Collagen is excluded from the enteric plexuses and support for neuronal elements is provided by astrocyte-like enteric glial cells. Enteric glia differ from Schwann cells in that they do not form basal laminae and they ensheath axons, not individually, but in groups. Although enteric glia are rich in the S-100 and glial fibrillary acidic proteins, it has been difficult to find a single chemical marker that distinguishes enteric glia from non-myelinating Schwann cells. Nevertheless, two monoclonal antibodies have been obtained that recognize antigens that are expressed on Schwann cells (Ran-1 in rats and SMP in avians) but not enteric glia. Functional differences between enteric glia and non-myelinating Schwann cells, including responses to gliotoxins and in vitro proliferative rates, have also been observed. Developmentally, enteric glia, like Schwann cells, are derived from the neural crest. In both mammals and birds the precursors of the ENS appear to migrate to the bowel from sacral as well as vagal levels of the crest. These crest-derived emigrés give rise to both enteric glia and neurons; however, analyses of the ontogeny of the enteric innervation in a mutant mouse (the ls/ls), in which the original colonizing waves of crest-derived precursor cells are unable to invade the terminal colon, suggest that enteric glia can also arise from Schwann cells that enter the gut with the extrinsic innervation. When induced to leave back-transplanted segments of avian bowel, enteric crest-derived cells migrate into peripheral nerves and form Schwann cells. Enteric glia and Schwann cells thus appear to be different cell types, but ones that derive from lineages that diverge relatively late in ontogeny.

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PubMed1991

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.

Experiments were done to test the hypothesis that the avian gut is colonized by cells derived from both vagal and sacral regions of the neural crest. A fluorescent dye, diI (1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and a replication-deficient retrovirus (LZ10; Galileo et al. 1990) were employed as tracers. Since LZ10 was constructed with lacZ of E. coli as a reporter gene, infected cells were identified by demonstrating beta-galactosidase immunoreactivity. DiI and LZ10 were injected between the neural tube and surface ectoderm (before the migration of crest cells away from the injection sites) at vagal, truncal (diI only), or sacral axial levels. The bowel was examined 4 days later in order to allow crest-derived cells sufficient time to migrate to the gut. Following injections of either tracer into the vagal crest, labelled cells were found in the gizzard and duodenum. When diI or LZ10 was injected into the sacral crest, labelled cells were seen in the post-umbilical bowel and ganglion of Remak. In the hindgut, marked cells were concentrated in the mesenchyme, just internal to the serosa, and were never observed rostral to the umbilicus. No fluorescent cells were ever found in the bowel following truncal injections of diI, although such cells were observed in sympathetic ganglia. Labelled cells were always found in dorsal root ganglia, no matter which tracer or level of the crest was injected. In embryos injected with LZ10, infected cells in the gut and dorsal root ganglia displayed a neural crest marker (NC-1 immunoreactivity). These observations confirm that the gut is colonized by cells from the sacral as well as the vagal region of the neural crest and that the emigrés from the sacral crest are confined to the post-umbilical bowel.

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PubMed1990

Distribution of hyaluronic acid and chondroitin sulfate proteoglycans in the presumptive aganglionic terminal bowel of ls/ls fetal mice: an ultrastructural analysis.

The terminal colon of the ls/ls mouse is aganglionic because an intrinsic defect prevents its colonization by cells migrating from the neural crest. Previous studies showed that laminin, type IV collagen, and glycosaminoglycans accumulate in the region of the presumptive aganglionic ls/ls bowel through which crest-derived cells would be expected to migrate. It was suggested that crest-derived cells might fail to enter the abnormal bowel because they receive inappropriate signals from a defective extracellular matrix. This hypothesis was evaluated by analyzing the ultrastructure of the extracellular matrix in mutant and control gut. Tissue was fixed in the presence of ruthenium red before or after selective enzymatic digestion. Heparan sulfate proteoglycan (diameter approximately equal to 15 nm) and chondroitin sulfate proteoglycan (diameter approximately equal to 20-50 nm) granules were found in both control and presumptive aganglionic gut. The heparan sulfate proteoglycan granules were primarily located within formed basal laminae, while chondroitin sulfate proteoglycan granules decorated plasma membranes and 5 nm hyaluronic acid microfibrils that formed a network in the extracellular matrix. At day E11.5, the mutant gut differed from the control in the following: 1) Hyaluronic acid microfibrils were longer and more numerous. 2) There were larger numbers of chondroitin sulfate proteoglycan granules associated with cell membranes and with hyaluronic acid microfibrils. By day E13 the spaces between mesenchymal cells of the outer wall of the control bowel contained a regular lattice of hyaluronic acid microfibrils studded with chondroitin sulfate proteoglycan granules. Instead of this lattice, tangles of excessively long hyaluronic acid microfibrils, coated more heavily than in the control with chondroitin sulfate proteoglycan granules, were found in the presumptive aganglionic gut. These results confirm that the extracellular matrix is abnormal in the presumptive aganglionic bowel of the ls/ls mouse; moreover, they also indicate that the defect involves not one, but several components of the extracellular matrix, as well as their distribution. The defective extracellular matrix is apparent at a time when crest-derived cells would be expected to be migrating in the terminal bowel and is located in their path. The observations thus support the idea that a localized abnormality of the extracellular matrix interferes with the colonization of the terminal bowel by crest-derived cells in the ls/ls mouse.

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PubMed1990

Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos.

The technique of back-transplantation was used to investigate the developmental potential of neural crest-derived cells that have migrated to and colonized the avian bowel. Segments of quail bowel (removed at E4) were grafted between the somites and neural tube of younger (E2) chick host embryos. Grafts were placed at a truncal level, adjacent to somites 14-24. Initial experiments, done in vitro, confirmed that crest-derived cells are capable of migrating out of segments of foregut explanted at E4. The foregut, which at E4 has been colonized by cells derived from the vagal crest, served as the donor tissue. Comparative observations were made following grafts of control tissues, which included hindgut, lung primordia, mesonephros and limb bud. Additional experiments were done with chimeric bowel in which only the crest-derived cells were of quail origin. Targets in the host embryos colonized by crest-derived cells from the foregut grafts included the neural tube, spinal roots and ganglia, peripheral nerves, sympathetic ganglia and the adrenals, but not the gut. Donor cells in these target organs were immunostained by the monoclonal antibody, NC-1, indicating that they were crest-derived and developing along neural or glial lineages. Some of the crest-derived cells (NC-1-immunoreactive) that left the bowel and reached sympathetic ganglia, but not peripheral nerves or dorsal root ganglia, co-expressed tyrosine hydroxylase immunoreactivity, a neural characteristic never expressed by crest-derived cells in the avian gut. None of the cells leaving enteric back-grafts produced pigment. Cells of mesodermal origin were also found to leave donor explants and aggregate in dermis and feather germs near the grafts. These observations indicate that crest-derived cells, having previously migrated to the bowel, retain the ability to migrate to distant sites in a younger embryo. The routes taken by these cells appear to reflect, not their previous migratory experience, but the level of the host embryo into which the graft is placed. Some of the population of crest-derived cells that leave the back-transplanted gut remain capable of expressing phenotypes that they do not express within the bowel in situ, but which are appropriate for the site in the host embryo to which they migrate.

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PubMed1989

Mitogenic effect of muscle on the neuroepithelium of the developing spinal cord.

A previous study revealed that segments of bowel grafted between the neural tube and somites of a younger chick host embryo would induce a unilateral increase in cellularity of the host's neural tube. The current experiments were done to test the hypotheses that muscle tissue in the wall of the gut is responsible for this growth-promoting effect and that the spinal cord enlargement is the result of a mitogenic action on the neuroepithelium. Fragments of skeletal (E8-15) or cardiac muscle (E4-14) were removed from quail embryos and grafted between the neural tube and somites of chick host embryos (E2). Both skeletal and cardiac muscle grafts mimicked the effect of bowel and induced an increase in cell number as well as a unilateral enlargement of the region of the host's neural tube immediately adjacent to the grafts. The growth-promoting effect of muscle-containing grafts was restricted to the neural tube itself and was not seen in proximate dorsal root or sympathetic ganglia. The action of the grafts of muscle was neither species- nor class-specific, since enlargement of the neural tube was observed following implantation of fetal mouse skeletal muscle into quail hosts. Grafts of skeletal muscle or gut increased the number of cells taking up [3H]thymidine in the host's neuroepithelium as early as 9 h following implantation of a graft. The increase in the number of cells entering the S phase of the cell cycle preceded the increase in cell number. These observations demonstrate that muscle-containing tissues can increase the rate of proliferation of neuroepithelial cells when these tissues are experimentally placed together.

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PubMed1988

Neural and glial phenotypic expression by neural crest cells in culture: effects of control and presumptive aganglionic bowel from ls/ls mice.

The enteric nervous system is formed by cells that migrate to the bowel from the neural crest. Previous experiments have established that avian crest cells in vitro will colonize explants of murine bowel and there give rise to neurons. It has been proposed that phenotypic expression by the crest-derived precursors of enteric neurons and glia is critically influenced by the microenvironment these cells encounter within the gut. To test this hypothesis, quail crest cells were cocultured with explants of control or presumptive aganglionic bowel from the ls/ls mutant mouse, and the effects of the enteric tissue on five phenotypic markers of crest cell development were followed. Aganglionosis develops in the terminal region of the colon of the ls/ls mouse because viable crest-derived neural and glial precursors fail to colonize this tissue. Expression of the phenotypic markers in the cocultures was compared with that in cultures of crest alone, crest plus neural tube, and gut grown alone. The markers examined were melanogenesis and immunostaining with antisera to 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) and the monoclonal antibodies, NC-1 and GlN1. Explants of control, but not presumptive aganglionic ls/ls gut were found to increase the incidence of the expression of 5-HT and NC-1 immunoreactivities; moreover, especially near the gut, the assumption of a neuronal morphology by 5-HT-, NC-1-, and GlN1-immunoreactive cells was also increased. Coincidence of expression of 5-HT with NC-1 and GlN1 immunoreactivities was observed. The effect of the bowel was selective in that the expression of TH immunoreactivity, which is not a marker of mature enteric neurons, was reduced rather than enhanced. The effect of enteric explants on crest cell development was specific in that it was not mimicked by explants of metanephros, which inhibited expression of 5-HT immunoreactivity and the acquisition of a neuritic form by NC-1-immunoreactive cells. It is concluded that the enteric microenvironment affects the phenotypic expression of subsets of crest cells and that this action of the bowel is manifested in vitro. The inability of presumptive aganglionic gut from ls/ls mice to influence neural phenotypic expression may be due to the failure of this tissue to produce putative factor(s) required for the effect or to the inability of the crest-derived precursor cells to migrate into the abnormal enteric tissue.

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PubMed1988

Accumulation of components of basal laminae: association with the failure of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice.

Aganglionosis occurs in the terminal colon of the ls/ls mouse because an intrinsic defect of the presumptive aganglionic tissue prevents the entry and colonization of this portion of the bowel by migrating neural crest cells. The current study was undertaken to determine if abnormalities of the extracellular matrix could be identified in this segment that might account for migratory failure. Since basal laminae of the muscularis mucosa are overproduced in the aganglionic segment of adult ls/ls mice, we examined components of basal laminae in fetal gut from Day E 11 to Day E 16 of gestation. This period spans the time of enteric ganglion formation. Laminin and collagen type IV were studied by immunocytochemistry and proteoglycans by staining glycosaminoglycans with Alcian blue. Abnormalities of each of these components occur during development of the presumptive aganglionic bowel in the ls/ls mouse and could be detected as early as Day E 11. These defects consist mainly of an overabundance of these materials, both in defined basal laminae and throughout the extracellular space of the mesenchyme. Electron microscopic observations in the presumptive aganglionic ls/ls colon revealed a thickening of basal laminae and exceptionally wide intercellular spaces between smooth muscle myoblasts that contained an irregular fibrillar material, consisting of 4.5- to 6.0-nm filaments associated with 14- to 20-nm granules. Fibrillar and flocculant material was continuous with formed basal laminae, and was concentrated in the same areas found to have an overabundance of laminin immunoreactivity. These observations indicate that there is an accumulation of extracellular matrix material, including components of basal laminae, that (i) precedes the formation of enteric ganglia, (ii) is in the path through which enteric neural precursors from the crest would have to migrate, and (iii) is limited to the aganglionic and hypoganglionic ls/ls bowel. These data are consistent with the hypothesis that components of basal laminae contribute to the inability of crest cells to colonize the terminal bowel of ls/ls mice.

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PubMed1976

Separately developing axonal uptake of 5-hydroxytryptamine and norepinephrine in the fetal ileum of the rabbit.

Uptake of 5-hydroxytryptamine (5-HT) by adult and fetal rabbit's ileum was studied. The adult myenteric plexus accumulated tritium when incubated with tritiated 5-HT. However, in addition to labeled 5-HT, tritiated 5-hydroxyindole acetic acid and, when monoamine oxidase (MAO) was inhibited, 5-HT-o-glucuronide were found in the tissue. Two uptake processes differing in affinity could be defined. Only the high affinity process was saturable. Fetal ileum took up tritiated 5-HT but glucuronidation did not occur when MAO was inhibited. The uptake of tritiated 5-HT by the fetal ileum was due to a single, saturable, temperature sensitive (Q10 at 27-37 degress C = 2.4) process inhibited by ouabain. It was identical to the high affinity uptake found in adult tissue. This specific high affinity uptake could be found as early as the 16th day of gestation, 5-8 days before uptake of norepinephrine (NE) begins. Light and electron microscope radioautography revealed that the uptake of 5-HT was primarily into axons and a characteristic structure called the expanded process, both in the myenteric plexus. Both contained dense-cored vesicles. Axons were not labeled by tritiated NE until after 24 days and the expanded process was never labeled by tritiated NE. This study shows that uptake of 5-HT is a property of distinct system of axons in the mammalian myenteric plexus which develops prior to adrenergic axons during ontogeny.

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