Three strains of zebrafish fgfr4 knockout mutants generated using CRISPR/Cas9.

(A) Schematic of generation of fgfr4 knockout zebrafish. Single cell wildtype zebrafish embryos were injected with fgfr4 guide RNA and Cas9 protein to generate mutant founders. Potential founders were outcrossed to isolate CRISPR/Cas9-mediated mutations, and putative crispant F1s were subjected to high resolution melt analysis to identify mutation sequences. (B) High resolution melt analysis (HRMA) of potential F1 mutants revealed three distinct clusters of potential fgfr4 crispants. (C) Schematic of wildtype Fgfr4 and predicted knockout strain protein lengths. Premature stop codons produced truncated Fgfr4 proteins with predicted length of 223, 228, and 215 amino acids respectively. Solid blocks indicate regions of wildtype Fgfr4 amino acid sequence alignment. The protein sequence aligned to the wildtype Fgfr4 protein ends at the same amino acid for all three alleles, with various additions of amino acids that do not align to the wildtype protein afterwards. Hatching indicates these additional amino acids.

Zebrafish fgfr4 knockout validated by real time quantitative polymerase chain reaction (RT-qPCR).

(A) Schematic of wildtype zebrafish fgfr4 mature mRNA transcript and regions targeted for RT-qPCR. Blue triangle indicates approximate fgfr4 crispant mutation site for all strains relative to primers. Orange box indicates region 5’ of mutation site targeted by RT-qPCR primers, and green box indicates region 3’ of mutation site targeted by RT-qPCR primers. Relative expression of fgfr4 mRNA as measured by RT-qPCR targeting the regions 5’ (B) and 3’ (C) of the sequence mutation. Expression is normalized to gapdh and rpl13a. Each point represents a pool of n = 12, 24 hours post-fertilization (hpf) zebrafish embryos derived from a maternal zygotic. Multiple points represent biological replicates, while three technical replicates were used to generate each biological replicate. Error bar is the mean ± standard deviation. P values were calculated using a one-way Brown-Forsythe and Welch ANOVA, correcting for multiple comparisons with a Dunnett T3 test.

Homozygous fgfr4 knockout (KO) zebrafish have no embryonic phenotype but are significantly smaller than wildtype zebrafish at three months post-fertilization (mpf).

(A) Representative images from a phenotypic analysis of embryonic zebrafish. Homozygous fgfr4 mutant zebrafish do not display an early embryonic phenotype. Each point represents an individual fish standard length, and the bar represents the mean. An unpaired two-tailed t-test with Welch’s correction was used to calculate the p value. Scale bar is 560.1 μm in 24 hpf images and 700.4 μm in 48 hpf images. Standard length quantification was performed with n = 15 embryos per group. (B) Phenotypic analysis of adult zebrafish. Homozygous knockout fgfr4 zebrafish are significantly smaller than wildtype zebrafish at three months post fertilization. Standard length quantification was performed with n = 27 WT fish and n = 34 fgfr4 KO adult fish. Each point represents an individual fish standard length, and the bar represents the mean. An unpaired two-tailed t-test with Welch’s correction was used to calculate p value. Scale bar is 1 cm. (C) Representative hematoxylin and eosin (H&E) staining of a sagittal section from female and male adult fgfr4 knockout zebrafish. Internal anatomy in mutant zebrafish is normal in both male and female adult fgfr4 knockout zebrafish. This was assessed in 5 female and 3 male fgfr4 knockout fish total. Scale bar is 1mm.