Fig. 2

sema3fb mutants have cardiac edema. A Overview of the sema3fb locus targeted by CRISPR/Cas9 mutagenesis to exon 2 (guide RNA: B1) and primers used to identify the mutation. sema3fb^ca305 fish have a 19 bp deletion (dots) and sema3fb^ca306 fish have a 10 bp insertion (red sequence). Schematic representation of WT and mutant proteins. In the sema3fb^ca305 allele, exon 1 is spliced in frame with exon 3, with loss of 62 amino acids (aa) at the beginning of a protein that would be translated, including the signal sequence (ss) and the first 14 aa of the SEMA domain. In the sema3fb^ca306 allele, the 10 bp insertion introduces a frame shift and a premature stop codon to generate a predicted truncated 58 aa protein. F, forward primer; R, reverse primer. B RT-PCR indicates that exon 2 is skipped in the sema3fb^ca305 allele; no PCR product is generated with exon 2-exon 3 primers, but product is generated with exon 3-exon 4 primers. Exon 2-exon 3 primers reveal exon 2 is present in the sema3fb^ca306 allele. C Brightfield images of sema3fb^+/+, sema3fb^ca305 and sema3fb^ca306 72 hpf embryos. Dotted line outlines the pericardial sac. D The percent of embryos that present with cardiac edema at 48 hpf (N = 3, n = 60 for each genotype) and 72 hpf (N = 3, n = 60 for each genotype). E The severe mutant phenotype displaying major cardiac edema and failure to thrive (E), and the abnormal linear arrangement of the heart (E’, inset in E). F Quantitation of the severe edema phenotype at 72 hpf (N = 3, n = 180 per genotype). G Quantitation of the area of the pericardial sac (N = 2; WT n = 27; sema3fb^ca305 n = 26, p < 0.0001). H The average ventricle wall width (µm) in 72 hpf sema3fb^ca305 (n = 6) embryos is significantly thinner (p = 0.024) than that of WT (n = 3). Scale bar in C and E is 1 mm