Neurotrophin-3 is required for the survival-differentiation of subsets of developing enteric neurons.

Neurotrophin-3 (NT-3) promotes enteric neuronal development in vitro; nevertheless, an enteric nervous system (ENS) is present in mice lacking NT-3 or TrkC. We thus analyzed the physiological significance of NT-3 in ENS development. Subsets of neurons developing in vitro in response to NT-3 became NT-3 dependent; NT-3 withdrawal led to apoptosis, selectively in TrkC-expressing neurons. Antibodies to NT-3, which blocked the developmental response of enteric crest-derived cells to exogenous NT-3, did not inhibit neuronal development in cultures of isolated crest-derived cells but did so in mixed cultures of crest- and non-neural crest-derived cells; therefore, the endogenous NT-3 that supports enteric neuronal development is probably obtained from noncrest-derived mesenchymal cells. In mature animals, retrograde transport of (125)I-NT-3, injected into the mucosa, labeled neurons in ganglia of the submucosal but not myenteric plexus; injections of (125)I-NT-3 into myenteric ganglia, the tertiary plexus, and muscle, labeled neurons in underlying submucosal and distant myenteric ganglia. The labeling pattern suggests that NT-3-dependent submucosal neurons may be intrinsic primary afferent and/or secretomotor, whereas NT-3-dependent myenteric neurons innervate other myenteric ganglia and/or the longitudinal muscle. Myenteric neurons were increased in number and size in transgenic mice that overexpress NT-3 directed to myenteric ganglia by the promoter for dopamine beta-hydroxylase. The numbers of neurons were regionally reduced in both plexuses in mice lacking NT-3 or TrkC. A neuropoietic cytokine (CNTF) interacted with NT-3 in vitro, and if applied sequentially, compensated for NT-3 withdrawal. These observations indicate that NT-3 is required for the normal development of the ENS.

Development of the interstitial cell of Cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand.

Kit is a marker for interstitial cells of Cajal (ICC). ICCs interact with enteric neurons and are essential for gastrointestinal motility. The roles of neural crest-derived cells, neurons, Kit, and Kit ligand (KL) in ICC development were analyzed. ICC development lagged behind that of neurons and smooth muscle. Although mRNA encoding Kit and KL was detected at E11, Kit-immunoreactive ICCs did not appear until E12 in foregut and E14 in terminal hindgut. Transcripts of Kit and KL and Kit-immunoreactive cells were found in aganglionic gut from ls/ls and c-ret -/- mice. ICCs also developed in crest-free cultures of ls/ls terminal colon. ICCs appeared in cultures of noncrest- but not those of crest-derived cells isolated from the fetal bowel by immunoselection with antibodies to p75(NTR). KL immunoreactivity was coincident in cells with neuronal or smooth muscle markers. The development of ICCs in cultures of mixed cells dissociated from the fetal gut was dependent on plating density. No ICCs appeared at /=200,000 cells/ml. Exogenous KL partially substituted for a high plating density. These data support the ideas that mammalian ICCs are neither derived from the neural crest nor developmentally dependent on neurons. ICC differentiation/survival requires KL, which can be provided by neurons or cells in a smooth muscle lineage. Neurons may be needed for development of myenteric ICCs and the mature ICC network.

Inhibition of in vitro enteric neuronal development by endothelin-3: mediation by endothelin B receptors.

The terminal colon is aganglionic in mice lacking endothelin-3 or its receptor, endothelin B. To analyze the effects of endothelin-3/endothelin B on the differentiation of enteric neurons, E11-13 mouse gut was dissociated, and positive and negative immunoselection with antibodies to p75(NTR )were used to isolate neural crest- and non-crest-derived cells. mRNA encoding endothelin B was present in both the crest-and non-crest-derived cells, but that encoding preproendothelin-3 was detected only in the non-crest-derived population. The crest- and non-crest-derived cells were exposed in vitro to endothelin-3, IRL 1620 (an endothelin B agonist), and/or BQ 788 (an endothelin B antagonist). Neurons and glia developed only in cultures of crest-derived cells, and did so even when endothelin-3 was absent and BQ 788 was present. Endothelin-3 inhibited neuronal development, an effect that was mimicked by IRL 1620 and blocked by BQ 788. Endothelin-3 failed to stimulate the incorporation of [3H]thymidine or bromodeoxyuridine. Smooth muscle development in non-crest-derived cell cultures was promoted by endothelin-3 and inhibited by BQ 788. In contrast, transcription of laminin alpha1, a smooth muscle-derived promoter of neuronal development, was inhibited by endothelin-3, but promoted by BQ 788. Neurons did not develop in explants of the terminal bowel of E12 ls/ls (endothelin-3-deficient) mice, but could be induced to do so by endothelin-3 if a source of neural precursors was present. We suggest that endothelin-3/endothelin B normally prevents the premature differentiation of crest-derived precursors migrating to and within the fetal bowel, enabling the precursor population to persist long enough to finish colonizing the bowel.

Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception.

Studies of the guinea pig small intestine have suggested that serotonin (5-HT) may be a mucosal transmitter that stimulates sensory nerves and initiates peristaltic and secretory reflexes. We tested the hypothesis that guinea pig villus epithelial cells are able to inactivate 5-HT because they express the same 5-HT transporter as serotonergic neurons. A full-length cDNA, encoding a 630-amino acid protein (89.2% and 90% identical, respectively, to the rat and human 5-HT transporters) was cloned from the guinea pig intestinal mucosa. Evidence demonstrating that this cDNA encodes the guinea pig 5-HT transporter included 1) hybridization with a single species of mRNA ( approximately 3.7 kb) in Northern blots of the guinea pig brain stem and mucosa and 2) uptake of [3H]5-HT by transfected HeLa cells via a saturable, high-affinity (Michaelis constant 618 nM, maximum velocity 2.4 x 10(-17) mol . cell-1 . min-1), Na+-dependent mechanism that was inhibited by chlorimipramine > imipramine > fluoxetine > desipramine > zimelidine. Expression of the 5-HT transporter in guinea pig raphe and enteric neurons and the epithelium of the entire crypt-villus axis was demonstrated by in situ hybridization and immunocytochemistry. Inhibition of mucosal 5-HT uptake potentiates responses of submucosal neurons to mucosal stimulation. The epithelial reuptake of 5-HT thus appears to be responsible for terminating mucosal actions of 5-HT.

Promotion of the development of enteric neurons and glia by neuropoietic cytokines: interactions with neurotrophin-3.

Neurotrophin-3 (NT-3) is known to promote enteric neuronal and glial development. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) were investigated to test the hypothesis that the development of subsets of enteric neurons and/or glia is also affected by a neuropoietic cytokine, by itself, or together with NT-3. Crest-derived cells, immunoselected from the fetal rat gut (E14) with antibodies to p75NTR, were found by RT-PCR and immunocytochemistry (after culture) to express both alpha (CNTER alpha) and beta components (gp130 and LIFR beta) of the tripartite CNTF receptor. In situ, myenteric ganglia below the esophagus were CNTFR alpha-immunoreactive by E16-E18. In vitro, CNTF and LIF induced in crest-derived cells nuclear translocation of STAT3 (signal transducer and activator of transcription 3), a concentration-dependent increase in expression of neuronal or glial markers, and a decrease in expression of the precursor marker, nestin. LIFR beta was expressed by more cells than CNTFR alpha; therefore, although the factors were equipotent, the maximal effect of LIF > CNTF. The cytokines and NT-3 were additive in promoting neuronal but not glial development. Specifically, the development of neurons expressing NADPH-diaphorase activity (an early marker found in inhibitory motor neurons) was promoted by CNTF and NT-3. These observations support the idea that a ligand for the tripartite CNTF receptor complex plays a role in ENS development.

Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of GFRalpha-1 in vitro and in vivo.

No enteric neurons or glia develop in the gut below the rostral foregut in mice lacking glial cell line-derived neurotrophic factor (GDNF) or Ret. We analyzed the nature and age dependence of the effects of GDNF and, for comparison, those of NT-3, on the in vitro development of the precursors of enteric neurons and glia. Positive and negative immunoselection with antibodies to p75(NTR) were used to isolate crest-derived and crest-depleted populations of cells from the fetal rat bowel at E12, 14, and 16. Cells were typed immunocytochemically. GDNF stimulated the proliferation of nestin-expressing precursor cells isolated at E12, but not at E14-16. GDNF promoted the development of peripherin-expressing neurons (E12 >> E14-16) and expression of TrkC. GDNF inhibited expression of S-100-expressing glia at E14-16. NT-3 did not affect cells isolated at E12, never stimulated precursors to proliferate, and promoted glial as well as neuronal development at E14-16. GFRalpha-1 was expressed both by crest- and non-crest-derived cells, although only crest-derived cells anchored GFRalpha-1 and GFRalpha-2 (GFRalpha-1 >> GFRalpha-2). GDNF increased the number of neurons anchoring GFRalpha-1. GFRalpha-1 is immunocytochemically detectable in neurons of the E13 intestine and persists in adult neurons of both plexuses. We suggest that GDNF stimulates the proliferation of an early (E12) NT-3-insensitive precursor common to enteric neurons and glia; by E14, this common precursor is replaced by specified NT-3-responsive neuronal and glial progenitors. GDNF exerts a neurotrophic, but not a mitogenic, effect on the neuronal progenitor. The glial progenitor is not maintained by GDNF.

The alpha1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut.

A plasmalemmal protein, LBP110, which binds to the alpha1 chain of laminin-1, is acquired by the neural crest-derived precursors of enteric neurons after they colonize the gut. We tested the hypothesis that laminin-1 interacts with LBP110 to promote enteric neuronal development. The effects of laminin-1 on neuronal development were studied in cultures of cells immunoselected from fetal mouse gut (E14-15) with antibodies to LBP110 or p75NTR, a marker for enteric crest-derived cells. No matter which antibody was used, the development of cells expressing neuronal markers was increased three- to fourfold by culturing the cells on a laminin-1-containing substrate. To determine whether this effect of laminin-1 is due to the selective adherence of a neurocompetent subset of precursors, immunoselected cells were permitted to preadhere to poly-D-lysine. Addition of soluble laminin-1 24 h later promoted neuronal but not glial development. The laminin-1-induced increment in neuronal development was abolished both by a peptide containing the sequence of the LBP110-binding domain, IKVAV, and by antibodies to laminin alpha1 that recognize the IKVAV domain. Neither reagent affected the total number of cells. In contrast, the response to laminin-1 was not affected by control peptides, preimmune sera, or antibodies to laminin beta1. Laminin-1 transiently induced the expression of nuclear Fos immunoreactivity; this action was blocked specifically by the IKVAV peptide. These data are consistent with the hypothesis that LBP110 interacts with the IKVAV domain of laminin alpha1 to promote the differentiation of neurons from enteric crest-derived precursors.

Increased expression of laminin-1 and collagen (IV) subunits in the aganglionic bowel of ls/ls, but not c-ret -/- mice.

Extracellular matrix molecules, including laminin, affect the development of enteric neurons and accumulate in the aganglionic colon of ls/ls mice. Quantitative Northern analysis revealed that mRNAs encoding the beta 1 and gamma 1 subunits of laminin and collagens alpha 1(IV) and alpha 2(IV) are increased in the colons of ls/ls mice. Transcripts of laminin alpha 1 were evaluated quantitatively with reverse transcription and the competitive polymerase chain reaction (RT-cPCR). The abundance of laminin alpha 1 transcripts was developmentally regulated, but greater in the ls/ls than the wild-type colon at each age examined. In situ hybridization revealed that transcripts in the colon encoding laminin alpha 1 and beta 1 and collagen alpha 2(IV) were initially expressed in the endoderm, but by E15, expression shifted to cells of the colonic mesenchyme (ls/ls > wild type) where crest-derived cells migrate. The expression of laminin alpha 1 was examined in the totally aganglionic intestine of E15 and newborn c-ret -/- mice, to determine whether an increase occurs when neurogenesis fails independently of the ls/ls defect. RT-cPCR revealed no difference from control in mRNA encoding laminin alpha 1 in the c-ret -/- colon in either E15 or newborn animals. The accumulation of immunohistochemically demonstrable laminin that is prominent in the newborn ls/ls colon could not be detected in that of c-ret -/- animals. These observations suggest that transcripts encoding laminin-1 and collagen (IV) are increased in the colon and surrounding pelvic mesenchyme of ls/ls mice because of an intrinsic lesion, rather than a secondary consequence of aganglionosis. The data are compatible with the hypothesis that the increased expression of laminin-1 contributes to the failure of crest-derived cells to complete their colonization of the ls/ls colon.

Neurotrophin-3 induces neural crest-derived cells from fetal rat gut to develop in vitro as neurons or glia.

The precursor cells that form the enteric nervous system (ENS) are multipotent when they arrive in the gut from the neural crest. Their differentiation thus depends on signals from the enteric microenvironment. Crest-derived cells were isolated from the fetal rat bowel by immunoselection at E14 with NC-1/HNK-1 antibodies and secondary antibodies coupled to magnetic beads. NC-1/HNK-1-immunoreactive cells were enriched approximately 36-fold. The NC-1/HNK-1-selected population and the residual population were plated at equal cell density and maintained in a defined medium for 6-7 d. The total number of cells found in the cultures of the residual cells was three- to fourfold that in cultures of immunoselected cells. Neurotrophin-3 (NT-3), but not nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-4/5 (NT-4/5), was found to increase the proportion of neurons (neurofilament-immunoreactive or neuron-specific enolase-immunoreactive) or glia (S-100-immunoreactive) (from 6.6 +/- 0.9% to 15.2 +/- 1.4%; p < 0.001). This effect was concentration dependent (from 1 to 40 ng/ml) and observed only in the cultures of immunoselected cells. NT-3 also enhanced neurite outgrowth. NT-3 increased neither cell number nor bromodeoxyuridine incorporation and thus was not mitogenic. Exposure of immunoselected cells to NT-3 rapidly and transiently induced the appearance of nuclear Fos immunoreactivity. Transcripts coding for TrkC, the transducing receptor for NT-3, were identified in the fetal rat gut (E14-E16) and in the immunoselected population of cells using reverse transcriptase and the polymerase chain reaction. It is concluded that NT-3 specifically promotes the differentiation of enteric crest-derived cells as neurons or glia and may thus play a role in the development and/or maintenance of the ENS.

Inhibition of migration of neural crest-derived cells by the abnormal mesenchyme of the presumptive aganglionic bowel of ls/ls mice: analysis with aggregation and interspecies chimeras.

The terminal bowel is congenitally aganglionic in ls/ls mice. The condition has been associated with an overabundance of laminin and other matrix molecules. Aggregation ls/ls<==>C3H chimeric mice and interspecies mouse<==>quail chimeras were constructed to test the hypothesis that the aganglionosis arises because the ls/ls gut and not the neural crest is abnormal. Demonstration of beta-glucuronidase activity permitted genotypically ls/ls and C3H cells to be distinguished in the ls/ls<==>C3H chimeras. Aganglionosis did not occur in the ls/ls<==>C3H mice and ls/ls neurons were observed in the terminal bowel. Following bactransplantation of control segments of mouse gut into quail host embryos, mouse cells migrated to host targets normally colonized by cells from the neural crest; moreover, quail crest-derived cells entered the mouse gut. In contrast, cells did not migrate to these targets from presumptive aganglionic ls/ls bowel and quail crest-derived cells neither entered the ls/ls gut nor migrated through it. Laminin immunoreactivity was present in the backgrafts of murine colon and was far more abundant and widespread in those from ls/ls than in those from control animals. These data suggest that the presumptive aganglionic ls/ls bowel does not contain crest-derived cells because these cells, which are normal in ls/ls mice, do not enter it. This failure of colonization may be related to the premature formation of neurons outside the abnormal gut, a response that may be promoted by the excessive secretion of laminin by the ls/ls enteric mesenchyme.

Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin.

The neural crest-derived cells that colonize the bowel are different from their predecessors in the premigratory crest. A procedure, which utilized the immunoselection of cells with a magnet, was thus devised to obtain crest-derived precursors from developing gut. Primary antibodies against cell surface antigens, NC-1 in chick, quail, and rat, or antibodies to a 110-kDa laminin binding protein (alpha-110) in mouse, were used in conjunction with secondary antibodies coupled to magnetic beads. Immediately after immunoselection with NC-1, almost all of the selected cells were NC-1-immunoreactive. Neurons and glia, identified immunocytochemically with antibodies to specific markers, developed preferentially in cultures of immunoselected cells. Some of the phenotypes expressed by neurons arising in vitro were appropriate for the bowel (serotonin- and vasoactive intestinal peptide-immunoreactive); however, catecholaminergic neurons, which are not present in the enteric nervous system, also differentiated in the cultures. Neuronal development, as well as neurite outgrowth, were promoted by laminin. Cells selected with alpha-110 from the fetal murine bowel preferentially gave rise in vitro to neurons and glia. These data suggest that the population of crest-derived cells that colonizes the gut is multipotent, that development of catecholaminergic neurons in situ is prevented by the intact enteric microenvironment, that laminin is important in the formation of enteric ganglia, and that the 110-kDa laminin binding protein is expressed on the surfaces of the immediate precursors of enteric neurons and glia.

Colonization of the bowel by neural crest-derived cells re-migrating from foregut backtransplanted to vagal or sacral regions of host embryos.

The enteric nervous system (ENS) in avian embryos is formed by cells that migrate to the bowel from vagal and sacral regions of the neural crest. Experiments were carried out to evaluate the developmental potential of crest-derived cells at the time they colonize the gut. Backtransplantation of E4 quail foregut (or control aneuronal hindgut) was used to determine whether crest-derived cells that have previously colonized the bowel are capable of following defined neural crest migration pathways in host embryos. Vagal and sacral, but not truncal, backgrafts provided donor cells for the host's bowel. These cells were immunostained by the neural crest marker, NC-1, restricted to the ENS, and appeared only when foregut was backgrafted; therefore, they were crest-derived. In order for cells to migrate to the host's bowel, backgrafts evidently had to be located in the vicinity of the neuraxis at the time crest-derived cells exited from them. When vagal grafts moved away from the neuraxis, crest-derived donor cells colonized cephalic ganglia and the vagus nerves near the grafts; however, such cells did not migrate down the vagi to the host's gut. Sacral backgrafts provided crest-derived cells for the bowel only if the donor gut was transplanted prior to the formation of somite 28, at the level of the disappearing primitive streak. Cells from vagal backgrafts were capable of reaching the host's cloaca, but backgrafts placed at a sacral level colonized only the post-umbilical bowel. In addition, donor cells proliferated extensively within the host's gut. Whenever the host's gut was colonized, donor crest-derived cells were also found in non-enteric targets including nerves, cephalic (vagal backgrafts), or sympathetic (sacral backgrafts) ganglia; however, donor cells did not form ectomesenchyme or melanocytes. These data suggest that (i) crest-derived cells that have colonized the bowel remain capable of re-migrating and following defined neural crest migration pathways in host embryos; (ii) remigrating cells must enter these pathways at their start; (iii) the gut stimulates the proliferation of enteric crest-derived cells; (iv) vagal crest-derived cells can follow sacral pathways to reach enteric, Remak's, or sympathetic ganglia; and (v) the migration of crest-derived cells within the gut is determined more by the route they follow to reach the bowel than by their level of origin in the neural crest.

From neural crest to bowel: development of the enteric nervous system.

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in ls/ls mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nerve-specific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue.

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.

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.

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.

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.

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.

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.

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.