Endothelin-3 regulates neural crest cell proliferation and differentiation in the hindgut enteric nervous system

Department of Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School, Warren 1153, Boston, MA 02114, USA.
Developmental Biology (Impact Factor: 3.55). 06/2006; 293(1):203-17. DOI: 10.1016/j.ydbio.2006.01.032
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Neural crest cells (NCC) migrate, proliferate, and differentiate within the wall of the gastrointestinal tract to give rise to the neurons and glial cells of the enteric nervous system (ENS). The intestinal microenvironment is critical in this process and endothelin-3 (ET3) is known to have an essential role. Mutations of this gene cause distal intestinal aganglionosis in rodents, but its mechanism of action is poorly understood. We find that inhibition of ET3 signaling in cultured avian intestine also leads to hindgut aganglionosis. The aim of this study was to determine the role of ET3 during formation of the avian hindgut ENS. To answer this question, we created chick-quail intestinal chimeras by transplanting preganglionic quail hindguts into the coelomic cavity of chick embryos. The quail grafts develop two ganglionated plexuses of differentiated neurons and glial cells originating entirely from the host neural crest. The presence of excess ET3 in the grafts results in a significant increase in ganglion cell number, while inhibition of endothelin receptor-B (EDNRB) leads to severe hypoganglionosis. The ET3-induced hyperganglionosis is associated with an increase in enteric crest cell proliferation. Using hindgut explants cultured in collagen gel, we find that ET3 also inhibits neuronal differentiation in the ENS. Finally, ET3, which is strongly expressed in the ceca, inhibits the chemoattraction of NCC to glial-derived neurotrophic factor (GDNF). Our results demonstrate multiple roles for ET3 signaling during ENS development in the avian hindgut, where it influences NCC proliferation, differentiation, and migration.

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Available from: Allan M Goldstein, Oct 09, 2015
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    • "Between the caecum and the cloaca, the HNK1 antibody labeled all NCCs within the myenteric and submucosal plexuses as well as the avian-specific nerve of Remak on the dorsal side of the cloaca, derived from sacral NCCs (Fig. 1A) (Doyle et al., 2004; Nagy et al., 2012). Both plexuses also strongly expressed Ednrb mRNA (Fig. 1C,E) (Nataf et al., 1996; Nagy and Goldstein, 2006). Our strategy of pharmacological inhibition of ECE1 by phosphoramidon was expected to reduce the generation of mature endothelin peptides in the chick embryo, and consequently silence both EDNRA and EDNRB, which are required to build a normal craniofacial skeleton and establish a complete enteric nervous system, respectively. "
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    ABSTRACT: The enteric nervous system originates from neural crest cells that migrate in chains as they colonize the embryonic gut, eventually forming the myenteric and submucosal plexus. Failure of the neural crest cells to colonize the gut leads to aganglionosis in the terminal gut, a pathological condition called Hirschsprung disease (HSCR) in humans, also known as congenital megacolon or intestinal aganglionosis. One of the characteristics of the human HSCR is its variable penetrance, which may be attributable to the interaction between genetic factors, such as the endothelin-3/endothelin receptor B pathway, and non-genetic modulators, although the role of the latter has not well been established. We have created a novel HSCR model in the chick embryo allowing to test the ability of non-genetic modifiers to alter the HSCR phenotype. Chick embryos treated by phosphoramidon, which blocks the generation of endothelin-3, failed to develop enteric ganglia in the very distal bowel, characteristic of an HSCR-like phenotype. Administration of dexamethasone influenced the phenotype, suggesting that glucocorticoids may be environmental modulators of the penetrance of the aganglionosis in HSCR disease. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 04/2015; 4(5). DOI:10.1242/bio.201410454 · 2.42 Impact Factor
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    • "Consistent with previous reports [15,85], we found that EDN3 promoted the differentiation of ENS precursors into myofibroblasts as evidenced by their morphology and by smooth muscle actin immunoreactivity. Interestingly, the addition of RA supported the formation of neuronal and glial cells, in addition to myofibroblasts. "
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    ABSTRACT: Signaling through the endothelin receptor B (EDNRB) is critical for the development of the enteric nervous system (ENS) and mutations in endothelin system genes cause Hirschsprung's aganglionosis in humans. Penetrance of the disease is modulated by other genetic factors. Mutations affecting retinoic acid (RA) signaling also produce aganglionosis in mice. Thus, we hypothesized that RA and endothelin signaling pathways may interact in controlling development of the ENS. Rat immunoselected ENS precursor cells were cultured with the EDNRB ligand endothelin-3, an EDNRB-selective antagonist (BQ-788), and/or RA for 3 or 14 days. mRNA levels of genes related to ENS development, RA- and EDNRB-signaling were measured at 3 days. Proliferating cells and cells expressing neuronal, glial, and myofibroblast markers were quantified. Culture of isolated ENS precursors for 3 days with RA decreases expression of the endothelin-3 gene and that of its activation enzyme. These changes are associated with glial proliferation, a higher percentage of glia, and a lower percentage of neurons compared to cultures without RA. These changes are independent of EDNRB signaling. Conversely, EDNRB activation in these cultures decreases expression of RA receptors β and γ mRNA and affects the expression of the RA synthetic and degradative enzymes. These gene expression changes are associated with reduced glial proliferation and a lower percentage of glia in the culture. Over 14 days in the absence of EDNRB signaling, RA induces the formation of a heterocellular plexus replete with ganglia, glia and myofibroblasts. A complex endothelin-RA interaction exists that coordinately regulates the development of rat ENS precursors in vitro. These results suggest that environmental RA may modulate the expression of aganglionosis in individuals with endothelin mutations.
    PLoS ONE 09/2013; 8(9):e74311. DOI:10.1371/journal.pone.0074311 · 3.23 Impact Factor
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    • "The biologically active 21 amino acid long EDN3 is produced by proteolytic cleavage of preproendothelin-3 by endopeptidases to yield proendothelin-3, which is finally cleaved by endothelin converting enzyme-1 to produce the mature active EDN3 [32,33]. Studies have shown that EDN3/EDNRB interaction is required for the proper development of neural crest derived melanocytes and enteric neurons [34-36]. Mouse models for homozygous mutations in the EDNRB or EDN3 genes show pigmentation anomaly, aganglionic megacolon and cochlear disorder [31,37]. "
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    ABSTRACT: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.
    Molecular vision 07/2012; 18:2022-32. · 1.99 Impact Factor
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