Bulk RNA-seq of zebrafish 5dpf embryos comparing Klf9 knockout with matched controls at 3 timepoints flanking zeitgeber (lights on)

Krüppel-like factor 9 (Klf9) is a ubiquitously expressed transcription factor that is a feedforward regulator of multiple stress-responsive and endocrine signaling pathways. We previously described how loss of Klf9 function affects the transcriptome of zebrafish larvae sampled at a single time point 5 days post-fertilization (dpf). However, klf9 expression oscillates diurnally, and the time point that was sampled corresponded to its expression nadir. To determine if klf9-/- function varies with time of day, we performed bulk RNA-seq on 5 dpf zebrafish embryos sampled at three timepoints encompassing the predawn peak and midmorning nadir of klf9 expression. We found that while the major effects of the klf9-/- mutation that we reported previously are robust to time of day, the mutation has additional effects that manifest only at the predawn time point. We used a published single-cell atlas of zebrafish development to map the effects of the klf9-/- mutation onto different cell types and found that the mutation increased representation of genes associated with digestive organs (liver, pancreas, and intestine) and decreased representation of genes associated with differentiating neurons, blood, and somites. Measurements from confocally-imaged larvae suggest that overrepresentation of liver genes in klf9-/- mutants is due to development of enlarged livers. Comparative gene expression profiling analysis of RNA-seq data for zebrafish klf9-/- mutant larvae vs wild-type at three times of day. Klf9 -/- knockouts were generated through CRISPR, creating a 2 bp deletion in the first exon that causes a frameshift and premature stop codon, as previously described in Gans et al, Scientific Reports 10, 11415 (2020). https://doi.org/10.1038/s41598-020-68040-z