Delta-like 1(Dll1) and Sox10 in hedgehog : Notch mediated enteric neural crest cell development

Abstract

During the development of enteric nervous system(ENS), enteric neural crest cells (NCCs) migrate along the gut, proliferate and differentiate to neurons and glia. Hedgehog (Hh) and Notch signaling pathways are two known pathways implicated in these processes. Aberrant Hh and Notch signaling have been shown to impair ENS development in mice. Recently, our group has demonstrated that Hh promotes proliferation and gliogenesis of enteric NCCs through activation of Notch pathway. In addition, Dll1, a Notch ligand, was found to be up-regulated by Hh, integrating Hh and Notch signaling pathways. Nevertheless, the molecular mechanisms underlying the Hh-induced Dll1 expression and the Hh-Notch mediated proliferation and gliogenesis are largely unknown. Therefore, the aims of my study are: i) to examine the transcriptional regulation of Dll1 expression by Hh; ii) to determine the candidate gene implicated in Hh-Notch mediated enteric NCCs proliferation and gliogenesis. Given that Gli is the major transcription factor of Hh signaling, we hypothesized that Hh may up-regulate Dll1expression through Gli. In silico sequence analysis revealed that there are multiple putative Glibinding motifs residing in the proximal promoter (4.2kb) of the mouse Dll1gene. To further identify the functional Gli-binding domain(s), a series of 5’deletion and site-directed mutation of the Dll1promoter were generated and tested with the luciferase promoter assays in a neuroblastoma cell line, SK-N-SH. We found that the proximal 0.7kb of Dll1promoter confers the full promoter activity. In addition, Gli2 but not Gli1 was found to significantly transactivate the Dll1 promoter. The Gli2 responsive region is mainly localized in the minimal promoter (the proximal 0.7kb).

In parallel, the question on how Hh and Notch mediate proliferation and gliogenesis of enteric NCCs has also been addressed in this project. Sox10 is a member of SRY-like HMG-box family transcription factor and is expressed only in undifferentiated enteric NCCs and glia. Importantly, loss functional Sox10 favors undifferentiated enteric NCCs and glia. Importantly, loss functional Sox10 favors neuronal differentiation of enteric NCCs and accompanied by loss of their progenitor state, implying that Sox10 is the key player in both proliferation and gliogenesis of enteric NCCs and may represent the target for the Hh-Notch pathway.

To address this question, enteric NCCs were isolated from Sox10 mutant embryos (Sox10dom/+), which carry a Sox10gene of a dominant negative mutation, and their growth and differentiation capacities were monitored in presence/absence of Hh agonist, purmorphamine.

Consistently, Sox10mutation significantly attenuated the Hh-Notch mediated proliferation and gliogenesis of enteric NCCs. Our previous study has shown that constitutive activation of Hh signaling in the enteric NCCs of the Ptch1mutants (Wnt1-cre; Ptch1f/f) leads to a reduced cell proliferation as a consequence of premature gliogenesis. With the Wnt1-cre; Ptch1f/f; Sox10N/+ double mutant, we found that loss of a copy of functional Sox10 allele can also rescue the proliferation defects of the Ptch1mutants. Therefore, both in vitro and in vivo data suggest that Sox10 is required for Hh-Notch mediated proliferation and gliogenesis. Taken together, Gli2 and Sox10 represent the two transcription factors implicated in Hh-Notch mediated ENS development.