Location of stem cells for the enteric nervous system.

Hirschsprung disease is the result of aganglionosis of a variable length of the terminal bowel, which arises from the incomplete colonisation of the embryonic gut by vagal neural crest-derived cells (NCC) that migrate caudally from the pharyngeal gut to the rectum. We have previously shown that a very small group of NCC, at the leading edge of this wave of migration, can proliferate and differentiate to innervate the entire distal gut. It remains unknown if this capability is unique to those cells at the leading edge of NCC migration. The hypothesis tested was that NCC capable of acting as stem cells are found throughout the developing enteric nervous system (ENS). Gut was taken from mice at embryonic day 11.5 as the leading edge of NCC migration enters the colon. Terminal colon was separated as aganglionic recipient gut and its rostral end juxtaposed to the caudal end of the small intestine or caecum. The explants were cultured on nitrocellulose filters for up to 120 h, after which time the apposed segments had fused. The gut was then fixed and examined by immunohistochemistry to detect the neuronal markers PGP9.5 and nitric-oxide synthase (NOS) to assess development of enteric ganglia. NCC migrated from the proximal gut into the terminal colon, colonising it along its entire length. The pattern of NCC colonisation and differentiation of NOS-positive neurons was the same, regardless of whether the NCC were derived from the leading edge of migration in the caecum or from more proximal regions of the small intestine. Vagal NCC have the capacity to migrate into separated aganglionic terminal colon and differentiate into neurons. NCC at the leading edge of migration and those located more proximally within the gut demonstrate equivalent ability to migrate to and differentiate in the terminal rectum. Further studies are required to confirm which of these migrating NCC have the properties of ENS stem cells.

Assessment of protein gene product 9.5 as a marker of neural crest-derived precursor cells in the developing enteric nervous system.

The neurons and glial cells of the enteric nervous system (ENS) are derived from the neural crest. To study the developmental events involved in congenital abnormalities of the ENS, it is essential to identify all neural-crest cells (NCC) in the prenatal gut. The low-affinity neurotrophin receptor p75 is currently considered to be a gold-standard marker, but because it is a membrane protein, it is lost during procedures that permeabilise cells that are necessary to identify intracellular components and in apoptosis and cell-proliferation assays. We have therefore assessed the potential of the intracellular neuronal marker protein gene product (PGP) 9.5 as a label for neural-crest-derived precursor cells during gut development. Gut was taken from mouse embryos at 11.5 days post-coitum, at which time NCC had reached the proximal colon. Cellular p75 and PGP9.5 expression was determined by double-labelling immunofluorescence. The leading edge of neural-crest migration was defined as the 10 most distal p75-labelled cells. The neuronal marker PGP9.5 labelled NCC as they migrated along the gut at day 11.5. At the leading edge of migration, over 95% of p75-positive cells also expressed PGP9.5, and all PGP9.5-positive cells were also labelled for p75. PGP9.5 is expressed by at least 95% of neural-crest-derived precursor cells at the leading edge of migration along the gut. Thus, it can be used as a robust marker for developing NCC in the gut.

Reduced endothelin-3 expression in sporadic Hirschsprung disease.

BACKGROUND: Enteric aganglionosis in Hirschsprung disease has been linked to genes coding for endothelin-3 (EDN3) and the endothelin B receptor (EDNRB), but there is no such linkage in most patients with sporadic Hirschsprung disease. However, the similarity between the distal colonic aganglionosis in Hirschsprung disease and that due to EDN3 or EDNRB mutations led to the hypothesis that levels of expression of these genes might be affected in the absence of mutation, thus causing the Hirschsprung disease phenotype. The aim of this study was to determine EDN3 and EDNRB messenger RNA (mRNA) levels in tissue samples from patients with sporadic Hirschsprung disease. METHODS: RNA and DNA were isolated from the ganglionic and aganglionic colonic segments of ten children with sporadic Hirschsprung disease, and from the colon of ten age-matched controls. The DNA was analysed for mutations in the genes coding for endothelin-3 (ET-3) and endothelin B receptor (ET-B) proteins. Relative levels of EDN3 and EDNRB mRNA were determined by semi-quantitative transcriptase-polymerase chain reaction. RESULTS: Three children had sequence variants in EDN3 and EDNRB. In the remaining seven patients, EDN3 mRNA levels were reduced in both the ganglionic and aganglionic colon compared with levels in controls; there was no difference in expression of EDNRB between groups. CONCLUSION: In the absence of mutation, EDN3 is downregulated in short-segment Hirschsprung disease, suggesting that this may be a common step leading to aganglionosis.

Expression of endothelin 3 by mesenchymal cells of embryonic mouse caecum.

BACKGROUND: Mutations in endothelin 3 (EDN3) and endothelin B receptor (EDNRB) genes cause terminal colonic aganglionosis in mice, and mutations in these genes have also been linked to the terminal aganglionosis seen in human Hirschsprung's disease. However, details of EDN3 expression during embryogenesis are lacking, and consequently the cellular mechanism by which EDN3 regulates innervation of the terminal gut is unclear. AIMS: To localise the expression of EDN3 and EDNRB in the embryonic mouse gut. METHODS: Expression of EDN3 and EDNRB mRNA was analysed by reverse transcription polymerase chain reaction and in situ hybridisation. RESULTS: High levels of EDN3 mRNA expression were restricted to mesenchymal cells of the caecum before and after the arrival of neural crest cells. In contrast, EDNRB expression along the gut displayed a time dependent pattern similar to those of the protein tyrosine kinase ret and the neural crest cell marker PGP9.5. CONCLUSIONS: Mesenchymal cells of the caecum express high levels of EDN3 mRNA during embryogenesis and hence the production of EDN3 at the caecum is likely to act on neural crest cells as a paracrine factor necessary for subsequent innervation of the terminal gut.

Evaluation of the long-acting somatostatin analogue octreotide in the management of insulinoma in three dogs.

The response of dogs with insulinoma to surgical and medical management is variable, with the majority developing intractable hypoglycaemia. A long-acting somatostatin analogue, Octreotide (SMS 201-995; Sandostatin) has been useful in the management of hypoglycaemia in humans with insulinoma, and preliminary reports suggest a beneficial clinical response in dogs with insulinoma. The present study objectively evaluated Octreotide in the management of three dogs with immunohistochemically confirmed insulinoma. Octreotide had no benefit over placebo, and little effect on circulating glucose and insulin concentrations despite clearly detectable plasma concentrations of Octreotide. No clinical improvement was apparent in two dogs given Octreotide over a period of two and three weeks. These results contrast with the positive clinical responses noted previously and indicate that further placebo controlled, objective studies are necessary before clear statements on the treatment of insulinoma with Octreotide are made.

The abdominal air sac ostium of the domestic fowl: a sphincter regulated by neuro-epithelial cells?

A microscopic study of the ostium of the abdominal air sac of the domestic fowl has shown that the ostium has a sphincter-like ring of well innervated smooth muscle. Three types of neuro-epithelial cell characterised by their content of numerous large granular vesicles are found in the wall of the ostium. Type I cells are present within the submucosal nerve plexus and appear to be morphologically similar to SIF cells. Type II cells occur in the lamina propria, in clusters or cords, are often associated with fenestrated capillaries, and have synaptic contact with axonal terminals containing small agranular vesicles. The cells of Types I and II are not intra-epithelial and therefore differ from the cells which have been found elsewhere in the respiratory tract of the domestic fowl and other vertebrates. Type III cells are intra-epithelial, and some of those in the basal region of the epithelium are associated with axon terminals. Type III cells are similar in ultrastructure and location to neuro-epithelial cells found elsewhere in the major airways of the domestic fowl. They also resemble cells in neuro-epithelial bodies in amphibian, reptilian and mammalian lungs, although neuro-epithelial bodies have not been found in the lung of this species of bird. The morphology of the ostium suggests that it may have a sphincter-like function, possibly regulated by the neuro-epithelial cells. The presence of a mucociliary epithelium and defensive tissue in the lamina propria indicates that the ostium is the site of defence mechanisms.

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract.

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.

Pancreatic polypeptide-like material in nerves and endocrine cells of the rat.

A region-specific antiserum (AbS11) raised against the carboxyl-terminal hexapeptide of pancreatic polypeptide has been employed to measure rat pancreatic polypeptide specifically and to demonstrate apparent immunoreactivity in nerves and in endocrine cells outside the pancreas. The concentration of pancreatic polypeptide in the head of the rat pancreas measured with AbS11 (176 +/- 47 pmol/g) was 750 fold higher than that measured with a conventional anti-bPP antiserum (0.23 +/- 0.08 pmol/g). Column chromatographs of rat pancreatic extracts demonstrated two peaks of immunoreactivity both eluting after the porcine pancreatic polypeptide standard. AbS11 also detected specific immunoreactivity in rat brain (470 fmol/g) which went undetected in convention assays. Although immunohistochemical studies with AbS11 and human pancreatic polypeptide antiserum demonstrated immunoreactivity in the same population of pancreatic endocrine cells, immunoreactive nerve fibres and enteroglucagon cells were only demonstrable with AbS11. These studies demonstrate that the carboxyl terminus of rat pancreatic polypeptide is immunochemically similar to that of higher mammals. Furthermore, neural and extrapancreatic endocrine variants of this peptide share an immunochemical determinant contained within the carboxyl-terminal hexapeptide.