Cell-autonomous retinoic acid receptor signaling alters enteric nervous system development with stage-specific effects in mice

Purpose: Determine how blocking retinoic acid receptor (RAR) signaling within enteric neural crest-derived cells (ENCDC) from E11.5 stomach or E13.5 colon alters mRNA abundance. These ENCDC become the enteric nervous system. Methods: Enteric nervous system precursors were isolated from mouse E11.5 stomach or E13.5 colon using fluorescence activated cell sorting to purify TdTomato or EYFP expressing cells respectively from fetal bowel. The RaraDN allele encodes a potent RAR dominant negative protein that is expressed after CRE-mediated DNA recombination. Tamoxifen-activated CRE-ERT2 was expressed from the Ret locus for E13.5 colon studies and mice were tamoxifen treated at E10.5 prior to cell sorting. The Wnt1Cre allele was employed for E11.5 stomach studies. RNA-SEQ was performed in quadruplicate using Illumina HiSeq 4000. Sequence reads that passed quality filters were aligned to remove repeat sequences and ribosomal RNA reads and then processed using RNA-Seq unified mapper (RUM) package. RUM files were visualized in the TessLA browser for subsequent analyses. Results: Transcriptional profiling shows RaraDN differentially impacts gene expression in E11.5 stomach and E13.5 colon enteric neural crest-derived cells (ENCDC) that become the enteric nervous system. Conclusions: Blocking of RAR signaling in ENCDC causes dramatic changes in gene expression during early development in a stage-specific manner. Overall design: Gene expression profiles for ENCDC that expressed a dominant negative RAR protein were compared to ENCDC that did not express this dominant negative RAR. All ENCDC expressed a fluorescent reporter (EYFP for E13.5 colon and TdTomato for E11.5 stomach ENCDC). Mice used for E13.5 colon studies had been tamoxifen-treated at E10.5.