The forebrain is the brain region which has undergone the most dramatic changes through vertebrate evolution. Analyses conducted in lampreys are essential to gain insight into the broad ancestral characteristics of the forebrain at the dawn of vertebrates, and to understand the molecular basis for the diversifications that have taken place in cyclostomes and gnathostomes following their splitting. Here, we report the embryonic expression patterns of 43 lamprey genes, coding for transcription factors or signaling molecules known to be involved in cell proliferation, stemcellness, neurogenesis, patterning and regionalization in the developing forebrain. Systematic expression patterns comparisons with model organisms highlight conservations likely to reflect shared features present in the vertebrate ancestors. They also point to changes in signaling systems –pathways which control the growth and patterning of the neuroepithelium-, which may have been crucial in the evolution of forebrain anatomy at the origin of vertebrates.

The Wnt/β-catenin pathway is crucial for ovarian differentiation in mammals, and WNT4 is an important protein that regulates this process. While the role of Wnt4 in gonadal differentiation is relatively well characterized in mammals, little is known regarding its role in teleost fish. Therefore, we investigated the potential activity of wnt4 in gonadal differentiation in rainbow trout (Oncorhynchus mykiss), focusing on the teleost and salmonid gene duplications. Phylogenetic and synteny analyses demonstrated that teleost fish possess two wnt4 genes, wnt4a and wnt4b, as a consequence of the teleost-specific whole-genome duplication (3R). In rainbow trout, we also identified an additional wnt4 gene, which is a wnt4a paralog that likely resulted from the salmonid-specific whole-genome duplication (4R). These two Wnt4a proteins (Wnt4a1 and Wnt4a2) share a high identity (>80%) with other vertebrate Wnt4 proteins, whereas Wnt4b is clearly more divergent (60% identity). During embryogenesis and adulthood, the wnt4a1/2 transcripts were expressed in various tissues, including the ovaries and testes. In contrast, wnt4b expression was restricted to the nervous system, suggesting a sub- or a neo-functionalization of this divergent paralog. During early gonadal differentiation in both males and females, the wnt4a1/2 transcripts were detected in the somatic cells surrounding the germ cells, with a slight sexual dimorphism in favor of males. These results demonstrate that, unlike mammals, rainbow trout do not display an ovary-predominant wnt4 expression profile during early gonadal differentiation.

Tbx1 is a member of the T-box transcription factor gene family involved in embryogenesis and organogenesis. Recently, within a pan-genomic screen using rainbow trout (Oncorhynchus mykiss) cDNA microarrays, we identified a tbx1 homolog with testicular over-expression during sex differentiation. Here, we characterized two very similar rainbow trout tbx1 paralogs, tbx1a and tbx1b. In adult tissues, tbx1a expression is restricted to the gonads, with high expression in the testis, while tbx1b is more widely expressed in gonads, gills, brains, muscle, and skin. During gonadal differentiation, both genes are differentially expressed in favor of testis formation shortly after hatching. These genes are expressed in somatic cells surrounding germ cells of the differentiating testis, while no or only weak expression was observed in the differentiating ovary. tbx1a and tbx1b were also both down-regulated in the differentiating testis during feminization with estrogens and up-regulated in the differentiating ovary during masculinization with an aromatase inhibitor. These results suggest that tbx1a and tbx1b are probably involved in the regulation of testicular differentiation in rainbow trout. Since Tbx1 is known to interact with the retinoic acid (RA) signaling pathway, we also examined the effect of RA on the rainbow trout tbx1 expression pattern. Expression of tbx1a and tbx1b was down-regulated in RA-treated male gonads, suggesting that tbx1 interacts with the RA signaling pathway and thus could be involved in the control of rainbow trout gonadal differentiation.

In mammals, follistatin (FST) plays an important role in early ovarian differentiation, acting downstream of the Wnt pathway. In teleost fish, fst is implicated in folliculogenesis and oocyte maturation, and an early and specific expression during ovarian differentiation has been described in rainbow trout, Oncorhynchus mykiss. By in situ hybridization, we demonstrated that during rainbow trout gonadal differentiation, fst shares a similar expression pattern with cyp19a1a, the gene encoding ovarian aromatase, a key steroidogenic enzyme needed for ovarian differentiation in fish. Expression of fst and cyp19a1a was first detected in a few scattered cells in the embryonic ovary several days before hatching. Then, after histological differentiation, fst and cyp19a1a expression was localized in clusters of cells lining the future ovarian lamellae. As FST expression is known to be induced by the Wnt/β-catenin pathway in mammals, the Wnt pathway was inhibited in vivo with the IWR-1 molecule, and we analyzed by qPCR the effects of this treatment on fst expression. We found that IWR-1 decreased fst expression in female gonads, consistent with a regulation of fst expression by the Wnt pathway in rainbow trout. Furthermore, expression of cyp19a1a was also downregulated, suggesting an implication of the Wnt pathway in ovarian differentiation.