A Novel Bidirectional Interaction between endothelin-3 and Retinoic Acid in Rat Enteric Nervous System Precursors

Abstract

BackgroundSignaling 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.MethodsRat 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.ResultsCulture 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.ConclusionsA 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.

A novel enteric neuron–glia coculture system reveals the role of glia in neuronal development

“…; Sato & Heuckeroth, ; Gisser et al . ). A procedure of myenteric explant culture of ganglia in which EGCs were eliminated through a combination of antibody complement‐mediated cytolysis and antimitotic agent resulted after 20 DIV in a neuron‐enriched culture (Bannerman et al .…”

Isogenic enteric neural progenitor cells can replace missing neurons and glia in mice with Hirschsprung disease

“…Although Kruger et al 35 demonstrated that Ednrb deficiency did not alter the potency of multilineage differentiation of embryonic gut NCSCs, including glial differentiation, a more recent study showed that Et3 decreased glial proliferation presumably by downregulating Sox10 expression, resulting in decreased glial cell numbers in cultured p75+ rat enteric neural crest cells. 49 In addition, increased numbers and early appearance of S-100-expressing glial cells have been observed in developing peripheral nerves of Ednrb-null rats. 50 Our results are consistent with these observations and implicate a possible role for Ednrb signaling in balancing neuronal and glial cell numbers in the postnatal ENS.…”

Ion Channel Expression in the Developing Enteric Nervous System

“…In contrast, addition of drugs that block the activities of a range of Ca 2+ , Cl - and K + channels at concentrations that did not cause cell death did not perturb the migration of ENCCs in gut explants. The lack of effect of the channel-blocking drugs on ENCC migration is unlikely to be due to poor penetration of the drugs as intact E11.5 gut explants are permeable to antibodies and large (250 kDa) toxins [ 47 , 66 ], and often similar concentrations of drugs are used to affect ENCCs in intact embryonic gut explants to those used in dissociated ENCCs [ 64 , 67 ]. We had previously shown that tetrodotoxin, which blocks voltage-gated sodium channels, and clotimazole, which blocks the intermediate conductance Ca 2+ -dependent K + channel (K Ca 3.1), also do not affect ENCC migration [ 50 ].…”