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

研究目的：明确阻断E11.5天小鼠胃组织或E13.5天小鼠结肠组织内的肠神经嵴来源细胞（enteric neural crest-derived cells, ENCDC）中的视黄酸受体（retinoic acid receptor, RAR）信号通路后，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：本研究分别从小鼠E11.5天胃组织及E13.5天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术（fluorescence activated cell sorting, FACS）纯化胎肠中分别表达TdTomato与EYFP的细胞。RarαDN等位基因编码一种强效的RAR显性负性蛋白，该蛋白的表达依赖于CRE介导的DNA重组。针对E13.5天结肠的实验，本研究采用了由Ret基因座驱动表达的他莫昔芬激活型CRE-ERT2，并在细胞分选前于E10.5天对小鼠施以他莫昔芬处理；针对E11.5天胃组织的实验，则采用了Wnt1Cre等位基因。采用Illumina HiSeq 4000平台完成四次生物学重复的RNA测序（RNA-Seq）。对通过质量过滤的序列读数进行比对，以去除重复序列及核糖体RNA读数，随后使用RNA测序统一映射器（RNA-Seq Unified Mapper, RUM）工具包进行数据分析。利用TessLA浏览器可视化RUM输出文件，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E11.5天胃组织及E13.5天结肠来源的肠神经嵴来源细胞的基因表达具有差异化调控作用。
实验结论：在肠神经嵴来源细胞中阻断RAR信号通路，会以发育阶段特异性的方式，在早期发育过程中引发基因表达的显著改变。
实验整体设计：将表达显性负性RAR蛋白的肠神经嵴来源细胞的基因表达谱，与未表达该显性负性RAR蛋白的肠神经嵴来源细胞进行对比。所有肠神经嵴来源细胞均表达荧光报告基因：E13.5天结肠来源的细胞表达EYFP，E11.5天胃组织来源的细胞表达TdTomato。用于E13.5天结肠实验的小鼠，均在E10.5天接受了他莫昔芬处理。