Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut.

Cells of the enteric nervous system are derived from the neural crest. Probes to a number of molecules identify neural crest-derived cells within the gastrointestinal tract of embryonic mice prior to their differentiation into neurons and glial cells. However, it is unclear whether the different markers are identifying all neural crest-derived cells. In this study the distribution of p75(NTR)-immunoreactivity was compared with that of Ret-, Phox2a-, Phox2b-, and tyrosine hydroxylase (TH) in undifferentiated neural crest-derived cells in the E10.5-E13.5 mouse intestine. Neural crest-derived cells colonise the embryonic mouse gut in a rostral-to-caudal wave between E9.5-E14, and differentiation into enteric neurons also occurs in a rostral-to-caudal wave. Thus, the most caudal neural crest-derived cells within the gut are undifferentiated. These most caudal neural crest-derived cells co-expressed p75(NTR)-, Phox2b- and Ret-immunoreactivity; at E10.5 a sub-population was also TH-positive. The most caudal cells did not show Phox2a-immunoreactivity at any stage. However, a sub-population of cells, which was rostral to the undifferentiated neural crest-derived cells, was Phox2a-positive, and these are likely to be cells beginning to differentiate along a neuronal lineage. The expression of Ret-, Phox2a-, Phox2b- and p75(NTR)-immunoreactivity by two classes of enteric neurons that differentiate prior to birth was also examined. Nitric oxide synthase (NOS) neurons showed Phox2b and Ret immunoreactivity at all ages, and Phox2a and p75(NTR) immunoreactivity only transiently. Calcitonin gene-related peptide (CGRP) neurons showed Phox2b and Ret-immunoreactivity, but not Phox2a immunoreactivity. It is concluded that all undifferentiated neural crest-derived cells initially express Phox2b, Ret, and p75(NTR); a sub-population of these cells also expresses TH transiently. Those cells that are beginning to differentiate along a neuronal lineage maintain their expression of Phox2b and Ret, and they start to express Phox2a, but down-regulate p75(NTR); those cells that differentiate along a glial lineage down-regulate Ret and maintain their expression of p75(NTR). Dev Dyn 1999;216:137-152.

Innervation of the esophagus in mice that lack MASH1.

The striated muscle of the esophagus differs from other striated muscle, because it develops by the transdifferentiation of smooth muscle, and the motor end plates receive a dual innervation from vagal (cholinergic) motor neurons and nitric oxide synthase (NOS)-containing enteric neurons. Mash1-/- mice have no enteric neurons in their esophagus and die within 48 hours of birth without milk in their stomachs (Guillemot et al. [1993] Cell 75:463-476). In this study, the innervation of the esophagus of newborn Mash1-/-, Mash1+/- and wild type mice was examined. There was no difference between Mash1-/-, Mash1+/-, and wild type mice in the transdifferentiation of the muscle and the development of nicotinic receptor clusters. However, there were significantly more cholinergic nerve terminals per motor end plate in Mash1-/- mice than Mash1+/- or wild type mice. Each of the Mash1-/- mice had fewer than 50 NOS neurons per esophagus, compared with approximately 3,000 in wild type mice. Newborn Mash1+/- mice also contained significantly fewer NOS neurons than wild type mice. In Mash1-/- mice, NOS nerve fibers were virtually absent from the external muscle but were present at the myenteric plexus. Unlike that of newborn wild type mice, the lower esophageal sphincter of Mash 1-/- mice lacked NOS nerve fibers; this may explain the absence of milk in the stomach. We conclude that 1) the transdifferentiation of the esophageal muscle and the development of the extrinsic innervation do not require enteric neurons or MASH1, 2) extrinsic NOS neurons only innervate the myenteric plexus.

Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation.

The embryonic gastrointestinal tract develops from a simple tube into a coiled, flexed, and regionalized structure. The changes in gut morphology coincide with the differentiation of multiple cell types in concentric layers, and include colonization by migratory neuron precursors, and the development of gastrointestinal motility. We describe a reliable method for growing embryonic mouse intestine in vitro by the attachment of segments of intestinal tract by their cut ends, with the intervening region suspended in the culture medium. These are termed "catenary cultures." E11-E11.5 mouse midgut, hindgut, or mid- plus hindgut segments were grown in catenary culture for up to 10 days and their growth, morphology, cell differentiation, ability to support neural precursor migration, and contractile activity were assessed. The increase in size of the cultured explants was not large, but morphogenesis proceeded, best exemplified by elongation of the caecum. Cell differentiation also proceeded. In the mucosa, goblet cells differentiated. Muscle layers, characterized by desmin expression, and kit-positive interstitial cells of Cajal differentiated in the correct positions. Where segments initially included neural precursors in a small sub-region, these migrated and proliferated to form uniform neuronal networks throughout the entire explant, and the cells expressed the neuron markers nitric oxide synthase and neuron specific enolase. Gut motility was attained 5-6 days into the culture period, and both contractile- and mixing-type movements were observed. Thus, cell types representative of all three germ layer contributions developed, and in addition, the gut, being mainly free, was able to elongate and bend (unlike on solid support cultures), while retaining its rostrocaudal identity.

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.

Identification of neurons that express stem cell factor in the mouse small intestine.

BACKGROUND & AIMS: Enteric neurons in the murine intestine express stem cell factor (SCF), which may provide an important signal in the development of the interstitial cells of Cajal (ICC). Our aim was to identify the subpopulation(s) of myenteric neurons that express SCF. METHODS: Myenteric plexus preparations from postnatal SCF-lacZ mice were processed for beta-galactosidase histochemistry followed by immunohistochemistry. RESULTS: Approximately 60% of the nitric oxide synthase-immunoreactive neurons, which projected to myenteric ganglia and to circular muscle, expressed SCF, and more than 80% of the calbindin-immunoreactive neurons, which projected exclusively to myenteric ganglia, expressed SCF. A small subpopulation of calretinin-immunoreactive neurons expressed SCF transiently. Many of the remainder of SCF-expressing neurons were choline acetyltransferase immunoreactive, but their projections are unknown. CONCLUSIONS: SCF-expressing neurons that project within the myenteric plexus may be an important source of SCF for the development of Kit-expressing ICC at this level. The only possible neuronal source of SCF for the ICC of the deep muscular plexus is a subpopulation of nitric oxide synthase-immunoreactive neurons.

Transient expression of neuronal nitric oxide synthase by neurons of the submucous plexus of the mouse small intestine.

Although neurons containing neuronal nitric oxide synthase (NOS) are abundant in the myenteric plexus of the small intestine of all mammalian species examined to date, NOS-containing neurons are sparse in the submucous plexus, and there does not appear to be an innervation of the mucosa by nerve fibres containing NOS. In this study, we used immunohistochemical techniques to examine the presence of neuronal NOS in the mouse intestine during development. At embryonic day 18 and postnatal day 0 (P0), about 50% of the neurons in the submucous plexus of the small intestine showed strong immunoreactivity to NOS, and NOS-immunoreactive nerve fibres were present in the mucosa. By P7, there was a gradation in the intensity of NOS immunostaining exhibited by submucosal neurons, varying from intense to extremely weak. During subsequent development, the proportion of submucous neurons showing NOS immunoreactivity decreased, and immunoreactive nerve fibres were no longer observed in the mucosa. In adult mice, NOS neurons comprised only 3% of neurons in the submucous plexus, which is significantly less than at P0. In contrast to the submucous plexus, the percentage of neurons that showed NOS immunoreactivity in the myenteric plexus did not change significantly during development.

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues.

The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes-neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Inhibitory transmission to the longitudinal muscle of the mouse caecum is mediated largely by nitric oxide acting via soluble guanylyl cyclase.

In this work we describe a region of mouse intestine, the caecum, in which inhibitory transmission to the longitudinal muscle is predominantly due to nitric oxide. In the presence of muscarinic receptor blockade, electrical stimulation of intramural nerves in the longitudinal muscle of the mouse caecum evoked a relaxation. The relaxation was reduced to about 25% of the control amplitude by the nitric oxide synthase inhibitor L-NMMA (NG-methyl-L-arginine), but was unaffected by D-NMMA. In the presence of the nitric oxide scavenger oxyhaemoglobin, the relaxation was reduced to less than 10% of the control amplitude. In the circular muscle of the caecum and the longitudinal muscle of the ileum, colon and rectum, electrical field stimulation either evoked only small relaxations, or relaxations that were unaffected by L-NMMA. Nitric oxide synthase-containing neurons in the caecum were localized immunohistochemically using an antibody to neuronal nitric oxide synthase or with NADPH diaphorase histochemistry. Reactive nerve cell bodies were observed in the myenteric plexus, and varicose nerve fibres were present in the longitudinal and circular muscle layers of the caecum. The transduction mechanism of the nitric oxide-mediated relaxation in the longitudinal muscle of the caecum was examined using ODQ (1 H-[1,2,4]oxadiazolo[4,3,-alpha-]quinoxalin-1-one), a selective inhibitor of soluble guanylyl cyclase. ODQ abolished the relaxations induced by applied sodium nitroprusside (0.01-1 mM) and reduced the relaxation induced by electrical stimulation to about 40% of control values. However, ODQ reduced the relaxations induced by electrical stimulation to a lesser extent than L-NMMA. Hence, although the relaxation in this tissue mediated by NO (or an NO-related substance) is largely via soluble guanylyl cyclase, an action of NO on other targets cannot be ruled out.

Origin of interstitial cells of Cajal in the mouse intestine.

The interstitial cells of Cajal (ICC) are found in a number of different locations in the gastrointestinal tract, where they form close associations with both muscle cells and nerve terminals. In this study we examined the embryological origin of ICC in the mouse intestine to determine whether they arise from the neural crest or from the intestinal wall. Segments of intestine were removed from embryonic mice either before or after the arrival of neural crest cells (the precursors of enteric neurons and glial cells) and transplanted under the renal capsule of host (adult) mice and allowed to develop for 18-41 days. In the mouse intestine, antibodies to c-kit protein selectively label ICC at a variety of locations, and antibodies to the NK1 receptor (the receptor for substance P) labels ICC at the level of the deep muscular plexus in the small intestine and a subpopulation of enteric neurons in the large intestine. The presence of neurons in the explants was examined using antisera to neuron-specific enolase, substance P, and calretinin. In segments of small and large intestine explanted after the arrival of neural crest cells, immunoreactive neurons and c-kit- and NK1-immunoreactive ICC were present with a distribution similar to that seen in control tissue at a similar developmental age. In segments of large intestine explanted before the arrival of neural crest cells, neurons were not present; however, c-kit-immunoreactive ICC were present in these aneuronal explants, indicating that ICC do not arise from the neural crest. The source of ICC in mammals is therefore likely to be the mesenchyme of the gut.