Enteric Nervous System Progenitors Are Coordinately Controlled by the G Protein-Coupled Receptor EDNRB and the Receptor Tyrosine Kinase RET

AbstractThe enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals

Multiple Roles of Ret Signalling During Enteric Neurogenesis

The majority of the enteric nervous system is formed by vagal neural crest cells which enter the foregut and migrate rostrocaudally to colonise the entire length of the gastrointestinal tract. Absence of enteric ganglia from the distal colon are the hallmark of Hirschsprung disease, a congenital disorder characterised by severe intestinal dysmotility. Mutations in the receptor tyrosine kinase RET have been identified in approximately 50% of familial cases of Hirschsprung disease but the cellular processes misregulated in this condition remain unclear. By lineage tracing neural crest cells in mice homozygous for a knock-in allele of Ret (Ret51/51), we demonstrate that normal activity of this receptor is required in vivo for the migration of enteric nervous system progenitors throughout the gut. In mutant mice, progenitors of enteric neurons fail to colonise the distal colon, indicating that failure of colonisation of the distal intestine is a major contributing factor for the pathogenesis of Hirschsprung disease. Enteric nervous system progenitors in the ganglionic proximal guts of mutant mice are also characterised by reduced proliferation and differentiation. These findings suggest that the functional abnormalities in Hirschsprung disease result from a combination of colonic aganglionosis and deficits in neuronal circuitry of more proximal gut segments. The reduced neurogenesis in the gut of Ret51/51 mutants was reproduced in the multilineage enteric nervous system progenitors isolated from these animals. Correction of the molecular defects of such progenitors fully restored their neurogenic potential in culture. These observations enhance our understanding of the pathogenesis of Hirschsprung disease and highlight potential approaches for its treatment

Contribution of neural crest-derived cells in the embryonic and adult thymus

Abstract Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26eYfp reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.</jats:p

IDENTIFICATION AND CHARACTERIZATION OF THE H19 LOCUS WHICH IS UNDER THE CONTROL OF THE MURINE RAF AND RIF GENES

The mouse $\alpha$-fetoprotein (AFP) gene is under the control of two loci, raf and Rif, in the mouse liver. The raf locus is thought to be responsible for the repression of the AFP locus after birth, and the Rif gene is part of the mechanism that induces the expression of the AFP gene during liver regeneration. The experiments described in this thesis establish that raf and Rif are not specific for the AFP locus but they control at least another murine structural locus, H19. The H19 locus was identified by constructing a fetal liver cDNA library and examining fetal liver specific clones for the diagnostic levels in C3H/He, BALB/cJ and C57BL/6 inbred mouse strain. The H19 gene is expressed in visceral endoderm, fetal liver and fetal gut, tissues that express also high levels of AFP. In addition, the H19 gene is expressed in fetal and adult myocardium and skeletal muscle. These tissues do not express detectable levels AFP. The expression of the H19 gene in myocardium and skeletal muscle is not under the control of the raf locus. Northern blot analysis was used to examine the expression of the H19 gene in cells in culture. The structure and the sequences of the H19 gene, which is highly conserved among mammalian species, were determined and compared to those of the AFP gene. The results suggest that although the AFP and H19 genes do not share a common, evolutionary ancestor, they contain in their 5\sp\prime flanking regions homologous sequences which could potentially mediate the coordinated regulation of the two genes. In order to study in detail the regulatory sequences of the H19 gene, constructs that contain the entire gene along with various amounts of 5\sp\prime and 3\sp\prime flanking sequences were introduced into cells into culture and the transcriptional efficiency of the introduced sequences were analyzed by Northern blot or S1 protection analysis. These experiments strongly suggest that both 5\sp\prime and 3\sp\prime flanking sequences play an important role in the regulation of the expression of the H19 gene

The LIM homeodomain transcription factors Lhx6 and Lhx7 are key regulators of mammalian dentition

Genes encoding LIM homeodomain transcription factors are implicated in cell type specification and differentiation during embryogenesis. Two closely related members of this family, Lhx6 and Lhx7, are expressed in the ectomesenchyme of the maxillary and mandibular processes and have been suggested to control patterning of the first branchial arch (BA1) and odontogenesis. However, mice homozygous for single mutations either have no cranial defects (Lhx6) or show only cleft palate (Lhx7). To reveal the potential redundant activities of Lhx6 and Lhx7 in cranial morphogenesis, we generated mice with all combinations of wild-type and mutant alleles. Double homozygous mice have characteristic defects of the cranial skeleton and die shortly after birth, most likely because of cleft palate. In addition, Lhx6/7 deficient embryos lack molar teeth. The absence of molars in double mutants is not due to patterning defects of BA1 but results from failure of specification of the molar mesenchyme. Despite molar agenesis, Lhx6/7-deficient animals have normal incisors which, in the maxilla, are flanked by a supernumerary pair of incisor-like teeth. Our experiments demonstrate that the redundant activities of the LIM homeodomain proteins Lhx6 and Lhx7 are critical for craniofacial development and patterning of mammalian dentition