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

## TL;DR

This study identifies wnt5b as a key regulator of testicular development in flatfish, revealing its role in sex differentiation and protein regulation during spermatogenesis.

## Contribution

The study discovers a wnt5b-mediated regulatory mechanism in testicular development and sex differentiation in flatfish, with insights into paralog divergence and protein interactions.

## Key findings

- wnt5b is highly expressed in pseudo-male testes and promotes testis-like gonad development.
- yy1a selectively represses wnt5b, explaining functional divergence between wnt5a and wnt5b.
- WNT5b interacts with proteins involved in ribosome biogenesis and protein turnover during spermatogenesis.

## Abstract

We identified and analyzed 20 wnt genes in the Chinese tongue sole, a flatfish with ZW/ZZ sex determination and temperature-induced sex reversal. These genes showed conserved structures but also lineage-specific genomic features, including a unique wnt gene cluster linked to several reproduction-related genes. Expression analyses revealed strong sex-biased patterns: wnt5a is mainly expressed in ovaries, while wnt5b is highly expressed in pseudo-male testes. Functional experiments showed that reducing wnt5a or wnt5b pushes gonads toward a testis-like state, activating male genes and suppressing female markers. We further found that the transcription factor yy1a specifically represses wnt5b, explaining functional divergence between the two paralogs. Protein interaction analysis suggested that WNT5b is involved in ribosome biogenesis and protein turnover during spermatogenesis. Overall, our findings highlight wnt5—especially wnt5b—as a key regulator of testis development and sex differentiation in flatfish.

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 Cynoglossus semilaevis, 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., adipor2, sema3a, nape-pld, erc2, lamb2), suggesting coordinated genomic regulation. Tissue transcriptome analysis demonstrated strong sex- and tissue-biased expression patterns, with wnt5a predominantly expressed in ovaries and wnt5b specifically upregulated in pseudo-male testes. Functional assays revealed that knockdown of wnt5a or wnt5b induced testis-specific genes (sox9b, tesk1) and suppressed ovarian markers (foxl2, cyp19a1a), indicating antagonistic regulatory roles in gonadal fate determination. Promoter analysis identified yy1a as a selective repressor of wnt5b, but not wnt5a, 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 wnt5b—as a key driver of testicular development in C. semilaevis and provide new insights into the molecular mechanisms underlying sex differentiation and sex reversal in flatfish.