Fig. 2

tubgcp6 is the causative gene of the smu1347 mutation.
A Genetic mapping localized the mutation site to a region between two SSLP markers, zH66D24 and zC11I22, on chromosome 25. This region harbored ten candidate genes. B Sequencing of candidate gene cDNAs revealed a C-to-A transversion in exon 12 of tubgcp6, resulting in a premature stop codon and producing a truncated Tubgcp6 protein. C A novel tubgcp6 mutant was generated using CRISPR/Cas9 technology. An additional 161-bp insertion in tubgcp6 exon 4 was identified, causing a frameshift mutation and protein truncation. WISH of cmyb showed that cmyb⁺ HSPCs were severely decreased in 4 dpf tubgcp6^+161/+161 mutants (D) and bi-allelic tubgcp6^smu1347/+161 mutants (E). The ratio in the upper right corner indicated the proportion of embryos with a representative expression pattern for each genotype. Red arrows indicate relevant signals in the region. Scale bars: 200 μm. F Quantification of cmyb⁺ cell numbers in the CHT region at corresponding stages of tubgcp6^+161/+161 mutants, tubgcp6^smu1347/+161 mutants, and their siblings (mean ± SD; n > 10; Student’s t test, ****p < 0.0001). Data were pooled from three independent experiments.