Hoxb5 trans-activates Sox9 and regulates trunk neural crest cell development

Abstract

Background and objectives: We have previously shown that Hoxb5 regulates the development of vagal neural crest cells (NCC) through Ret. In the mutant b3-IIIa-Cre/enb5, in which the engrailed-Hoxb5 (enb5) protein was specifically expressed in vagal NCC under Cre induction, retarded migration of vagal NCC and defective enteric nervous system (ENS) development ranging from aganglionosis to hypoganglionosis were observed. The enb5 protein is a chimeric protein consisting of the Drosophila engrailed repressor domain and the mouse Hoxb5 DNA binding domain. Hence, enb5 protein functions as a dominant repressor of Hoxb5 by competing for the same binding site and normal Hoxb5 signaling would then be perturbed. Hoxb5 is also strongly expressed in NCC originated from trunk region of the neural tube, suggesting Hoxb5 may also regulate trunk NCC development. This study aims to investigate the function of Hoxb5 in trunk NCC.

Methods: We crossed enb5 mice with Wnt1-Cre mice to induce enb5 expression in NCC along the entire AP levels of the neural tube, investigated the NCC developmental abnormalities in Wnt1-Cre/enb5 mice and compared with those of Wnt1-Cre/Sox9flox/flox mice. Expression of Sox9 in Wnt1-Cre/enb5 mice was evaluated by in situ hybridization and immuno-histochemistry. In silico analysis, electro-mobility shift assay (EMSA), luciferase reporter assay and chromatin immuno-precipitation (ChIP) assay were performed to investigate the binding and trans-activation of Hoxb5 from SOX9 promoter.

Results: Wnt1-Cre/enb5 mice displayed neurocristopathies of hypopigmentation, hypoplastic dorsal root ganglion and ENS defects, which were similar to the neurocristopathies in Wnt1-Cre/Sox9flox/flox mice. In Wnt1-Cre/enb5 embryos, expression of Sox9 in NCC was down-regulated, and apoptosis of NCC was observed. Hox binding sites were predicted in SOX9 promoter by bioinformatics software, suggesting that SOX9 could be a downstream target of Hoxb5. Physical interaction between Hoxb5 proteins with SOX9 promoter was shown by EMSA. Trans-activation of Hoxb5 from SOX9 promoter in human neuroblastoma cell line, HTB-11, was confirmed by using luciferase reporter assay. Moreover, binding of Hoxb5 proteins onto the Sox9 promoter was further consolidated by ChIP assay in the central nervous system of mouse embryos.

Conclusions: Hoxb5 regulates the development of trunk neural crest cell by trans-activating Sox9.