FIGURE 1

Generation and characterization of slc30a1a/slc30a1b double‐knockout zebrafish. (A) Strategy used to perform comparative genomic mining for neural crest (NC)‐related studies using human, mouse, and zebrafish data, resulting in five overlapping studies. (B) Two of the five studies identified in (A) were selected based on in vivo experiments and data involving multiple time points, revealing a total of 38 overlapping genes in these two databases. These genes were then ranked based on their expression levels. (C) Embryonic phenotype mining revealed 12 candidate genes. Nine of these genes have been characterized in vivo, while the other three genes have not been characterized at the functional level. The six genes in green are known to play a role in NC development (mycn, mthfd1l, sox10, polr1b, and mecom) and/or pharyngeal arch (PA) patterning (sox10 and prrx1b). (D) Real‐time quantitative polymerase chain reaction (RT‐qPCR) analysis of the indicated genes at 18–22 hpf. (E) in situ hybridization of wild‐type zebrafish embryos using antisense slc30a1a and slc30a1b probes. The white dashed outlines indicate the pharyngeal region, and the gut is indicated with an orange arrow in the 4 dpf images. (F) Real‐time qPCR analysis of slc30a1a and slc30a1b mRNA in sox10 ⁺ and sox10⁻ cells (n = 3 sets of 3 × 10⁴ cells/group). ***p < 0.001. (G) Representative images of slc30a1a/slc30a1b double‐knockout mutants and control embryos. The red arrows in the mutant embryos indicate a smaller head and loss of the lower jaw. (H–K) Representative images of head cartilage in control and mutant zebrafish embryos stained with Alcian blue (H and J) and the corresponding diagrams (I and K). Residual palatoquadrate cartilage in the mutant embryos is indicated with red arrows. Abbreviations for cartilage: M, Meckel's; Pq, palatoquadrate; Bh, basihyal; Ch, ceratohyal; Hs, hyosymplectic; Bb, basibranchial; Cb, ceratobranchial; Hb, hypobranchial; Ih, interhyal