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FIGURE 3
Mutation in tprb gene is responsible for mutantcas7 phenotypes. (A–I) Morpholino knocking down of tprb mutantcas7 phenocopies. WISH results of ae1-globin (A,B), alas2 (C,D), and band3 (E,F) expression in control and tprb morphants at 3 dpf. The percentages of indicated phenotype are listed at the bottom of each panel. Arrow in panel (E,F) indicates the alas2 staining in heart region. (G,H) Giemsa staining for erythrocytes at 4 dpf in control and tprb morphants. More immature erythrocytes in tprb morphants are shown. Scale bars represent 10 μm. (I) Statistical analysis of the nucleus-to-cytoplasm (N/C) ratio in control and tprb morphants. N = 100, error bars represent SEM. ****p < 0.0001. (J) Construction of the plasmid used in Tol2-transposease-mediated rescue assay. (K–M) WISH analysis of ae1-globin expression in sibling, tprbcas7 mutants, and rescued embryos at 3.5 dpf. After WISH and photographing, all embryos were extracted for genomic DNA and genotyped by sequencing, then the rescue percentage was evaluated. The percentage of fully rescued mutant embryos is about 77% (10 out of 13 mutant embryos), while the rest are rescued partially. (N,O) Generation of tprb– 3+1 mutant via CRISPR-Cas9 technique. The alignment of WT and mutated sequences is listed. The underlined sequence is gRNA target site. The sequencing result of tprb genomic DNA shows a 3-bp deletion and 1-bp addition at exon 7 (N), which caused a premature stop codon leading to the production of a truncated 246-amino-acid Tpr protein (O). (P,Q) WISH results of ae1-globin expression in sibling and tprb– 3+1 mutant at 3 dpf. After WISH and photographing, all embryos were extracted for genomic DNA and genotyped by sequencing, then the mutant percentage was evaluated. The number of embryos in tprb– 3+1 mutant incross clutch with the expression pattern as shown in the tprb– 3+1 column and corresponding percentages are listed inside each panel.