Genome-Wide Characterization of the wnt Gene Family Reveals a wnt5b-Mediated Regulatory Mechanism of Testicular Development in Cynoglossus semilaevis

The wnt gene family encodes a group of highly conserved secreted glycoproteins that play essential roles in vertebrate development, including tissue patterning, cell differentiation, and gonadal regulation. However, the genomic organization, evolutionary dynamics, and functional roles of Wnt signaling components in flatfish remain poorly understood. In this study, we performed a comprehensive genome-wide identification, evolutionary characterization, expression profiling, and functional analysis of wnt genes in <i>Cynoglossus semilaevis</i>, a flatfish species exhibiting ZW/ZZ sex determination and temperature-induced sex reversal. A total of 20 wnt genes were identified and classified into 13 subfamilies, displaying conserved structural organization and phylogenetic relationships consistent with other teleosts. Chromosomal mapping revealed lineage-specific WNT clusters, including a unique wnt3-wnt7b-wnt5b-wnt16 block, as well as syntenic associations with reproduction-related genes (e.g., <i>adipor2</i>, <i>sema3a</i>, <i>nape-pld</i>, <i>erc2</i>, <i>lamb2</i>), suggesting coordinated genomic regulation. Tissue transcriptome analysis demonstrated strong sex- and tissue-biased expression patterns, with <i>wnt5a</i> predominantly expressed in ovaries and <i>wnt5b</i> specifically upregulated in pseudo-male testes. Functional assays revealed that knockdown of <i>wnt5a</i> or <i>wnt5b</i> induced testis-specific genes (<i>sox9b</i>, <i>tesk1</i>) and suppressed ovarian markers (<i>foxl2</i>, <i>cyp19a1a</i>), indicating antagonistic regulatory roles in gonadal fate determination. Promoter analysis identified <i>yy1a</i> as a selective repressor of <i>wnt5b</i>, but not <i>wnt5a</i>, providing a mechanistic basis for paralog divergence. Furthermore, pull-down combined with LC-MS/MS analysis showed that WNT5b interacts with proteins enriched in ribosome biogenesis and ubiquitin-mediated proteolysis, suggesting a role in translational regulation and protein turnover during spermatogenesis. Together, these findings establish WNT5 signaling-particularly <i>wnt5b</i>-as a key driver of testicular development in <i>C. semilaevis</i> and provide new insights into the molecular mechanisms underlying sex differentiation and sex reversal in flatfish.