Generation of zebrafish polr2d mutant zebrafish. (a) Multiple amino acid sequence alignment of Polr2d/Rpb4 protein. Black and grey colors respectively indicate completely and highly conserved residues among eukaryotes. Black boxes H1–H6 mark the region of helix domain 1–6. The position of frame-shift mutation in zebrafish is indicated by a red arrow in H1. Following NCBI data were used: human (H. sapines) NP_004796; rhesus monkey (Macaca mulatta) NP_001254716; mouse (Mus musculus) NP_081278; chicken (Gallus gallus) NP_001264338; african clawed frog (Xenopus laevis) NP_001087315; zebrafish (Danio rerio) NP_001002317; fruit fly (Drosophila melanogaster) NP_001014633; fission yeast (Schizosaccharomyces pombe) NP_595415; budding yeast (Saccharomyces cerevisiae) NP_012395. (b) A genomic deletion and a consequent frame-shift mutation in zebrafish polr2d. The 8-bp deletion was generated in the spacer sequence of the CRISPR. The position of forward and reverse primers for genotyping are indicated. (c) Genotyping PCR. The size of wild-type and mutant bands were 48 and 40 bp, respectively. (d, e) Spatial and temporal expression of polr2d. Whole-mount in situ hybridization of polr2d in zebrafish embryos at 24 hpf (d) and 48 hpf (e). Magnified views of the boxed region indicate polr2d expression in the spinal cord. (f–i) Images of polr2d mutants. The polr2d mutants showed developmental defects at 24 hpf (h) and 48 hpf (i) compared to wild-type (f, g). Note that small eye and cardiac edema were evident in mutants at 48 hpf.
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