Zebrafish tgfbr3 null mutants survive to adulthood. A, Exon 2 sequence from wild type and loss of function Tgfbr3 mutant alleles (WT, ∆7, ∆7.2, ∆ + 11). Highlighted are the translation start codon (blue), Cas9 target sequence (green), deletion/insertion sequence in mutant alleles (red) and novel in frame stop codon (underlined, *). B, Scheme comparing the structural domains encoded in the tgfbr3 WT allele vs the predicted tgfbr3 mutant alleles. C, Representative images of defective crispant embryos obtained by the injection of 100 pg of sgRNA at 96 hpf and 200 pg at 24 hpf. D, Representative image of a polyacrylamide gel showing embryos genotyping. Exon 2 amplicons derived from the DNA to be genotyped DNA were mixed with exon 2 probing amplicons of known sequence, + for WT, ∆7 for mutant allele, − for control test without probing amplicon. The presence of migration-retarded heteroduplex complexes (doublet near loading well) is observed only when a discordant WT or ∆7 probing DNA is added to the annealing mix. The heterozygous samples yield these duplexes even in the absence of added probes (−). E, Tgfbr3 immunohistochemistry of WT (tgfbr3 +/+) or null mutant (tgfbr3 ∆7/∆7) 72 hpf embryos obtained from heterozygous parents (WT/∆7) intercrosses. Genotyping (lower panel) of these specimens corroborated their allele identity. F, Endogenous Tgfbr3 was revealed by crosslinking to Tgfb2-I125 in the AFLIP experiment done with protein extracts from tgfbr3 +/+ (WT) embryos but not with protein extracts from tgfbr3 ∆7/∆7 (Mut) embryos. G, Quantitation of malformed embryos from mutant homozygous parents for the 3 different alleles, alone or combined, showing no defects in embryos at 72 hpf. H, Image of 6-month-old WT (tgfbr3 +/+) and null (tgfbr3 ∆7/∆7) specimens did not reveal any major morphological defects. I, Relative expression of tgfbr3 mRNA by RT-qPCR in WT and mutant (tgfbr3 ∆7/∆7) total RNA purified from 7 dpf embryos
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