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 RarαDN 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 RarαDN 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. 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.

研究目的：明确阻断胚胎第11.5天（E11.5）胃组织或胚胎第13.5天（E13.5）结肠组织来源的肠神经嵴来源细胞（enteric neural crest-derived cells, ENCDC）内的视黄酸受体（retinoic acid receptor, RAR）信号通路后，其mRNA丰度的变化情况。此类肠神经嵴来源细胞最终将发育为肠神经系统。实验方法：分别从小鼠胚胎第11.5天的胃组织与胚胎第13.5天的结肠组织中分离肠神经系统前体细胞：通过荧光激活细胞分选（fluorescence activated cell sorting）分别纯化胎肠中表达TdTomato与EYFP的细胞。RarαDN等位基因可编码一种强效的显性负效视黄酸受体蛋白，该蛋白的表达依赖于CRE介导的DNA重组。针对胚胎第13.5天结肠组织的实验，采用由Ret基因座驱动表达的他莫昔芬激活型CRE-ERT2，且在细胞分选前于胚胎第10.5天（E10.5）对小鼠进行他莫昔芬处理；针对胚胎第11.5天胃组织的实验，则使用Wnt1Cre等位基因。本研究采用Illumina HiSeq 4000测序平台进行4次生物学重复的RNA测序（RNA-Seq）。通过质量过滤的测序读数首先进行比对，以去除重复序列与核糖体RNA读数，随后使用RNA测序统一比对工具（RNA-Seq unified mapper, RUM）进行数据分析。RUM分析结果文件通过TessLA浏览器进行可视化，用于后续分析。实验结果：转录组谱分析显示，RarαDN对将发育为肠神经系统的胚胎第11.5天胃组织来源与胚胎第13.5天结肠组织来源的肠神经嵴来源细胞的基因表达具有差异性调控作用。研究结论：在肠神经嵴来源细胞中阻断RAR信号通路，会以阶段特异性的方式在发育早期对基因表达产生显著改变。本研究将表达显性负效RAR蛋白的肠神经嵴来源细胞的基因表达谱，与未表达该蛋白的对照组细胞进行了对比。所有受试肠神经嵴来源细胞均表达荧光报告基因：胚胎第13.5天结肠来源的细胞表达EYFP，胚胎第11.5天胃来源的细胞表达TdTomato。用于胚胎第13.5天结肠组织实验的小鼠，均在胚胎第10.5天接受了他莫昔芬处理。