Expression analysis of Hoxb5 in enteric neurons and generation of tamoxifen inducible Cre mice for neuronal Hoxb5 signaling perturbation

Abstract

Vagal NCC migrate and colonize the intestine rostro-caudally, differentiate into Enteric Nervous System (ENS) ganglia. Expression analysis in human has suggested distinct functions of Hoxb5 at different ENS development stages. Perturbation of Hoxb5 signaling in vagal NCC at the early stage of ENS development impaired NCC migration. However, functions of Hoxb5 in enteric neurons at the late stage of ENS development remains unanswered. In this study, we (i) examine the Hoxb5 expression patterns in ENS in mice; and (ii) generate transgenic mice that express a tamoxifen inducible Cre (MerCreMer) in neurons driven by neuron specific enolase gene (NSE) promoter. In mice, migratory vagal NCC express high level of Hoxb5, and Hoxb5 expression is down-regulated in NCC when these cells undergo differentiation. Later in enteric ganglia, Hoxb5 is re-expressed and the expression persists into adulthood. Co-immunofluorescence analysis revealed Hoxb5 expression in various neuron subtypes, suggesting that Hoxb5 may be involved in the development of different neuron subtypes. Transgenic construct (NSE-MerCreMer) is microinjected to generate founder mice. Four transgenic lines are generated and Cre expressions in neural tissues in these mice are confirmed by RT-PCR, Western blotting, and immuno-fluorescence. Transgenic mice are crossed to reporter lines ROSA26R and Z/AP to evaluate the efficiency and the specificity of tamoxifen inducible cre-recombination. Our NSE-MerCreMer mice shall be an important tool for the investigation of specific roles of Hoxb5 in the development of neurons of ENS.