Examination of the role of the forkhead box L2 (Foxl2) in gonadal and embryonic development in the boring giant clam Tridacna crocea

Abstract

Tridacna crocea, the smallest and earliest sexually maturing giant clam species, harbours important ecological value in maintaining coral reefs and holds potential for development as an ornamental species. At present, the molecular mechanism of gonadal development of this species remains largely unclear, and this hinders its artificial breeding and subsequent resource proliferation. The transcription factor forkhead box L subfamily member 2 (FoxL2) is one of the most conserved genes involved in the process of ovarian differentiation and development in many vertebrates and invertebrates. In this study, the Foxl2 gene (Tc-Foxl2) was cloned and characterized from T. crocea for the first time. The ORF of Tc-Foxl2 cDNA encoded a putative protein of 384 amino acid residues with a deduced molecular mass of 43.39 kDa and a theoretical isoelectric point (pI) of 8.92. Phylogenetic analysis indicated that the amino acid sequence of Tc-Foxl2 contained a typical FH domain and clustered with molluscan Foxl2 homologues. Tc-Foxl2 was mainly expressed in the gonads of females and hermaphroditic individuals but was fairly low in the gonads of males and resting individuals. Quantitative RT–PCR revealed that the expression of Tc-Foxl2 mRNA was significantly upregulated during vitellogenesis, while in situ hybridization indicated that Tc-Foxl2 mRNA was mainly detectable in follicle cells in the ovary. Furthermore, RNAi-mediated Tc-Foxl2 knockdown impeded the further growth of oocytes on the 14th day postinjection in the process of early vitellogenesis. Whole-mount in situ hybridization and qRT–PCR analyses demonstrated that during embryonic development, Tc-Foxl2 mRNA had a conserved expression profile, which may be involved in the formation of primordial germ cells. These results collectively suggest that Tc-Foxl2 is a female-biased gene that is involved in the vitellogenesis and embryonic development in Tridacna crocea.