Cloning Of Genes Wnt4, β-catenin, Dax1, And Their Correlation To The Gonadal Development In Scallop Chlamys Farreri

Gonadal development is one of the major research fields in the developmentalbiology. It has been cognized to some extent on genes of gonadal development invertebrates especially in mammals. While, limited information about the field isavailable in invertebrates especially in marine bivalves. In the present study, wecloned full-length cDNA sequences of the genes Wnt4, β-catenin and Dax1from theimportant commercial bivalves in china-scallop Chlamys farreri by the method ofrapid-amplification of cDNA ends (RACE). Furthermore, tissue distributions of thethree target genes were detected by semi-quantitative RT-PCR technique (sqRT-PCR),and expressions of the three genes in gonads during different developmental stageswere analysized by quantitative real-time PCR (qRT-PCR) technique.Immunohistochemistry technique was employed to determine the cytologicallocations of the three genes and their functions were analysized during thegametogenesis. Two repressors, DKK-1(an inhibitor of canonical Wnt signalmolecule) and quercetin (an inhibitor of β-catenin) were used to treat C. farreritestis cells cultivated in vitro to probe into their roles of the three genes during thegonadal development and the gametogenesis. In addition, we identified the location ofthe gene Dax1in chromosomes by fluorescence in situ hybridization (FISH)technique.Wnt4(Wingless-type MMTV integration site family, member4), a growth factor, ismember of Wnt family. The full-length cDNA sequence of Wnt4from C. farreri ovarywas obtained which was1,239bp including an open reading frame (ORF) of1,068bpwhich encoded355amino acids. The deduced amino acid sequence of Wnt4had theconserved sequence of Wnt family, and identity of over60%with most species Wnt4s. Semi-quantative RT-PCR result showed that C. farreri Wnt4expressed with lowexpression level in most detected tissues including testes, ovaries, adductor muscle,hepatopancreas, gill and mantle, except kidney, implying that Wnt4could involve in avarious of biological processes and played roles as a signal molecule in C. farreri.qRT-PCR results indicated that C. farreri Wnt4mRNA expression was the highestlevel in testis and ovary at mature stage, and higher in testis than in ovay at the samestage during the whole reproductive cycle. We inferred that Wnt4contributed to thedevelopmental processes of gonad and germ cell maturation.Armadillo (ARM) family is composed of highly conserved proteins, containingthe ARM motif consisted of about50amino aids and playing roles in variousbiological processes, such as the transduction of cell signal and the regulation ofcytoskeleton. β-catenin is a member of ARM family and as a key signal transductionprotein wildly involved in the development process of animals. The whole cDNAsequence of C. farreri β-catenin was3,353bp in length including an ORF of2,511bpwhich encoded836amino acids. The deduced amino acid sequence of β-catenin washighly conserved and had the repeated sequences of ARM family, and the typicalN-terminal and C-terminal region. Semi-quantative RT-PCR result revealed that C.farreri β-catenin was also expressed in multiple tissues. qRT-PCR result showed thatthe expression of β-catenin in gonad increased gradually as the gonad developmentand mature, and reached the highest level at mature stage, and then it decreasedsharply to the lowest level at the resting stage. Meanwhile, the expression of β-cateninin gonad presented a sexually dimorphic pattern, that was with the significant higherexpression in ovaries than in testis at the same stage (P<0.05). Immunohistochemistryresult showed that β-catenin protein was mainly located in germ cells, such asspermatogonia and spermatocytes in testis, oogonia and oocytes in ovary, indicatingthat this gene maybe participate in the gametogenesis in C. farreri.DAX1[dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC)critical region on the X chromosome gene1], a member of nuclear receptorsuperfamily, is known to involve in sex determination and gonadal development inseveral vertebrate species. The cDNA sequence of C. farreri Dax1is2,093bp in full-length including1,404bp ORF encoding a predicted467-amino acid. Unlike invertebrates, no conserved LXXLL-related motif existed in putative DNA bindingregion of C. farreri DAX1. FISH result showed that C. farreri Dax1was located onthe short arm of a pair of subtelocentric chromosome. Semi-quantitative RT-PCRrevealed that C. farreri Dax1mRNA expressed wildly in adult scallop tissues. Duringthe reproductive cycle (proliferative stage, growing stage and mature stage) of C.farreri, the Dax1also assumed a sex-dimorphic expression pattern, whereas with asignificant higher expression in testis than in ovay at the same stage (p<0.05). Inaccordance with the cytolocalization of β-catenin, C. farreri DAX1was also mainlylocated in spermatogonia and spermatocytes of testis, in oogonia and oocytes of ovary,implying that DAX1may involved in gametogenesis in C. farreri.When DKK-1with concentrations of0.1μg/ml and0.2μg/ml were added to themedium for48h, the expression of β-catenin in the C. farreri testis cells in vitrowas significantly down-regulated (p<0.05) in treated groups compared with thecontrol group detected by qRT-PCR, which indicated the canonical Wnt pathwayexisted in C. farreri testis. Furthermore，when, quercetin with concentrations of50μmol/L and100μmol/L were added to the medium for48h respectively, also theexpression level of Dax1significantly decreased compared with the control, implyingDax1acts as the downstream gene of β-catenin to regulate the gonadal developmentand gametogenesis.