Gli3R-mediated inhibition of Hedgehog signaling alters the embryonic transcriptome in zebrafish

Hedgehog signaling is a key developmental pathway required for normal patterning of the embryonic body plan. In zebrafish, disruptions to hedgehog signaling cause well-characterized defects in specific cell types including neurons and glia derived from the ventral spinal cord. We inhibited hedgehog signaling by overexpressing the Gli3 repressor (Gli3R) ubiquitously and performed bulk RNA-seq of 30 hour post-fertilization zebrafish embryos. Consistent with known roles of hedgehog signaling, we observed reduced expression of genes marking floor plate, motor neurons, Kolmer-Agduhr cells, dopaminergic neurons, slow muscle cells, and anterior pituitary. Gene set enrichment analysis using marker genes derived from the Daniocell atlas also revealed downregulation of genes expressed in ionocytes, which are located in the embryonic skin and are responsible for osmotic homeostasis. Reduced expression of ionocyte-specific transporter genes and the transcription factor foxi3a suggests that Gli activity may play a role in the development of this cell type. RNA-sequencing of whole Tg(ubb:zGli3R) larve at 30 hpf. A mosaic F0 male was crossed to a wildtype female, and embryos from one clutch were collected at 30 hours post-fertilization (hpf). Transgenic embryos and wildtype control siblings were sorted based on the myl7:GFP transgenesis marker. Four dechorionated embryos were pooled for each biological replicate, and four biological replicates were collected per genotype.

刺猬信号通路（Hedgehog signaling）是胚胎体模式正常构建所必需的关键发育通路。在斑马鱼中，刺猬信号通路的功能紊乱会导致已被充分表征的特定细胞类型缺陷，包括腹侧脊髓来源的神经元与神经胶质细胞。本研究通过泛在过表达Gli3阻遏蛋白（Gli3 repressor, Gli3R）抑制刺猬信号通路，并对受精后30小时的斑马鱼胚胎开展批量RNA测序（bulk RNA-seq）。与刺猬信号通路的已知功能一致，我们观察到标记底板、运动神经元、Kolmer-Agduhr细胞、多巴胺能神经元、慢肌细胞以及垂体前叶的基因表达出现下调。利用源自Daniocell图谱的标记基因进行基因集富集分析（gene set enrichment analysis）还显示，位于胚胎皮肤中并负责渗透压稳态的离子细胞（ionocytes）相关基因表达下调。离子细胞特异性转运蛋白基因与转录因子foxi3a的表达水平降低，提示Gli活性可能在该细胞类型的发育过程中发挥作用。本研究对受精后30小时（hpf）的全株Tg(ubb:zGli3R)幼虫进行RNA测序：将嵌合F0雄鱼与野生型雌鱼杂交，收集单批受精后30小时（hpf）的胚胎；基于myl7:GFP转基因标记筛选转基因胚胎与同窝野生型对照胚胎；每个生物学重复混合4枚去卵膜的胚胎，每个基因型设置4个生物学重复。