Gonad Differentiation in Zebrafish Is Regulated by the Canonical Wnt Signaling Pathway1

Abstract

Zebrafish males undergo a "juvenile ovary-to-testis" gonadal transformation process. Several genes, including nuclear receptor subfamily 5, group A (nr5a) and anti-Müllerian hormone (amh), and pathways such as Tp53-mediated germ-cell apoptosis have been implicated in zebrafish testis formation. However, our knowledge of the regulation of this complex process is incomplete, and much remains to be investigated about the molecular pathways and network of genes that control it. Using a microarray-based analysis of transforming zebrafish male gonads, we demonstrated that their transcriptomes undergo transition from an ovary-like pattern to an ovotestis to a testis-like profile. Microarray results also validated the previous histological and immunohistochemical observation that there is high variation in the duration and extent of commitment to the juvenile ovary phase among individuals. Interestingly, global gene expression profiling of diverging zebrafish juvenile ovaries and transforming ovotestes revealed that some members of the canonical Wnt/beta-catenin signaling pathway were differentially expressed between these two phases. To investigate whether Wnt/beta-catenin signaling plays a role in zebrafish gonad differentiation, we used the Tg (hsp70l:dkk1b-GFP)w32 line to inhibit Wnt/beta-catenin signaling during gonad differentiation. Activation of dkk1b-GFP expression by heat shock resulted in an increased proportion of males and corresponding decrease in gonadal aromatase gene (cyp19a1a) expression. The Wnt target gene, lymphocyte enhancer binding factor 1 (lef1), was also down-regulated in the process. Together, these results provide the first functional evidence that, similarly to mammals, Wnt/beta-catenin signaling is a "pro-female" pathway that regulates gonad differentiation in zebrafish.

Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes

“…In fish, masculinization by elevated temperature usually involves down-regulation of profemale genes, such as cyp19a1a and foxl2, and up-regulation of promale genes, such as dmrt1 or amh (8)(9)(10)(11)(12)(13)(14). In accordance with this general pattern, the FHT2 females with a male-like gonadal transcriptome exhibited down-regulation of both key profemale genes such as cyp19a1a and vtg5 as well as downregulation of profemale pathways such as oocyte meiosis, progesterone-mediated oocyte maturation, canonical Wnt signaling (28,53), and Fanconi anemia (26). Another profemale pathway, the NF-κB pathway, involved in blocking apoptosis through an inflammatory process during ovarian development (27), was not affected, although some of its genes (e.g., bcl2, nfkb, tradd) were down-regulated.…”

“…In contrast, ovarian differentiation continues in the future females (25), with an increase in cytoplasm volume of oocytes at around 60 dpf. Transcriptomic analysis of zebrafish sex differentiation has shown that apoptosis (24), activated by the p53 signaling pathway (26), is required for testis differentiation, whereas up-regulation of the canonical Wnt and NF-κB signaling pathways is needed for ovarian differentiation (27,28).…”

R-spondin1, WNT4, and the CTNNB1 signaling pathway: strict control over ovarian differentiation

“…Indeed, other wnt genes have been shown to display a sex-specific expression at different stages of development in the rainbow trout (Nicol & Guiguen 2011). Moreover, transgenic inhibition of wnt signaling results in male-biased sex ratios, suggesting that Wnt signaling is a conserved key pathway during gonadal differentiation in zebrafish (Sreenivasan et al 2014). In medaka (Oryzias latipes), Rspo1 and Rspo2 are prominently expressed in both germ cells and somatic cells (Zhou et al 2012).…”

Transcriptome Survey of a Marine Food Fish: Asian Seabass (Lates calcarifer)

“…Gene ontology terms of these differentially expressed genes included biological processes such as reproduction (GO:0000003), sexual reproduction (GO:0019953), the reproductive process (GO:0022414), the reproductive developmental process (GO:0003006), gamete generation (GO:0007276), fertilization (GO:0009566) and the reproductive cellular process (GO:0048610). Twenty-one of these transcripts were also shown to have sex-related roles or differential expression in the gonads in previous studies (Table 4) [8,[30][31][32][33][34]. This finding showed that the assembled transcriptome could be reliably used as a reference for differential expression analyses.…”