Fig. 1

Genome editing generates likely null alleles of zebrafish smyhc1. (A) Schematic of smyhc1 gene, mRNA sequences and proteins showing nature of smyhc1^kg179 and smyhc1^kg180 mutant alleles. Underline indicates the guide RNA sequence, red and blue bases were deleted and inserted, respectively. Most of the S1 (cyan) and all the S2 (magenta) and LMM domains (purple) of the protein are lost in the mutants, ablating most highly-conserved regions (black). Location of other smyhc1^mb16-18 and smyhc1^stl583 truncation alleles are marked (green). (B) In situ RNA hybridisation for smyhc1 mRNA in smyhc1 mutant and their siblings from smyhc1^kg179/+ and smyhc1^kg180/+ in-crosses reveal nonsense-mediated decay of mutant smyhc1^kg179 and smyhc1^kg180 mRNA at 24 hpf. In a randomly-selected sample from a smyhc1^kg179/+ incross, 4/21 had reduced expression and were shown to be mutant; among 17 ‘normal’ expressors 11/20 were heterozygotes and 6/20 were wild type upon sequence genotyping. From the smyhc1^kg180/+ incross, 6/29 embryos had low expression and were shown to be mutant; among 23 normal expressors 15/23 were heterozygous and 8/23 were wild type upon sequence genotyping. (C) Quantitative RT-PCR for smyhc1, smyhc2 and smyhc3 on pools of ten 2 dpf embryos from a smyhc1^kg179/+ incross that had been sorted into immotile mutant and motile siblings at 1 dpf. Each symbol represents a separate reverse transcription with symbol shapes distinguishing biological replicate lays from separate parents. The dashed line represents the motile sibling level (D). Maximum intensity projections of Slow MyHC-stained 24 hpf embryos showing region centred on somite 17 (S17). Wild-type siblings (left) show slow fibre staining and smyhc1^kg179/kg179 and smyhc1^kg180/kg180.mutant siblings show no slow fibre stain (right).