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. Overall design: 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.