Fig. 1

Characterization of Zebrafish Heat Shock Factor 5 and Generation of hsf5 Mutants Using CRISPR/Cas9-Based Strategy

(A) Schematic representation of four members of zebrafish heat shock factor (Hsf) protein family for comparing the relative location of specific domains. For Hsf5, two transcript variants (hsf5_tv1 and hsf5_tv2) are shown. HR, heptad repeat. (B) Phylogenetic analysis of vertebrate heat shock factors showed that all orthologs of zebrafish Hsf5 are located on a separate branch (in bold rectangle) clearly apart from other heat shock factor family members. The tree was constructed with the neighbor-joining method based on the amino acid sequences; we performed a 1000× bootstrapping for a robust comparison. hs, Homo sapiens; mm, Mus musculus; gg, Gallus; xt, Xenopus tropicalis; om, Oncorhynchus mykiss; and dr, Danio rerio (GenBank IDs shown in Table S2). (C) Schematic representation of three mutant lines generated by CRISPR/Cas9 method: Hsf5^sg40 has a deletion of five bases, and it introduces a stop codon after 121 amino acids (AA). Hsf5^sg41 has a deletion of 7 bases, whereas Hsf5^sg42 has an addition of 25 bases, introducing a stop codon after 114 AA and 131 AA, respectively. All three mutations are frameshift type resulting in a truncated protein. (D) Western blot confirmed loss of the C-terminal regions in the mutants. Adult testes from Hsf5^sg40 and Hsf5^sg41 and WT were used. Actin was used as an internal control; n = 2 biological replicates in each group. (E) Blue Native PAGE analysis using total testicular lysates showed a band of 242 kDa (arrow), suggesting that Hsf5 is present either in oligomeric state or as complexed with other proteins; n = 4 biological replicates. See also Figures S2 and S3 and Tables S2 and S3.