CDNA Cloning And Developmental Expression Patterns Of Reproduction-related DEAD-box Family And Boule Genes From Chlamys Farreri

Ontogenesis of germ cells is an important problem in developmental biology. The outstanding studies in model animals are revealing the molecular mechanisms of germ cell origin and development, in which some functionally conserved genes through evolution have been discovered. These genes might be useful as markers for an extensive investigation on germ cells of non-model animals. In this thesis, cDNAs of reproduction-related genes including Cf-vasa, Cf-PL10 and Cf-p68 of DEAD-box family and Cf-boule of DAZ family were isolated from Zhikong scallop Chlamys farreri using a homologous cloning strategy, followed by analysis of characterization and spatio-temporal expression. Cf-vasa mRNA was used as a molecular marker to establish the early developmental pattern of scallop primordial germ cells (PGCs) including the model and timing of PGCs origin and the migration in early larvae.DEAD-box family genes encode ATP-depended RNA helicase proteins, which are divided into VASA, PL10 and p68 sub-family by phylogenetic analysis. The vasa gene is essential both for germ cell formation and gametogenesis. Some PL10-related genes, such as human DBY and Drosophila bel, are involved in the gametogenesis. Cf-vasa, Cf-PL10 and Cf-p68 cDNAs isolated from C. farreri testis are 5578 bp, 2494 bp and 2950 bp long. The open reading frames (ORFs) encode proteins of 801, 760 and 703 amino acids. Cf-VASA, Cf-PL10 and Cf-p68 proteins share nine conserved motifs and a GG repeat with other DEAD-box members. ARKF motifs only found in VASA- and PL10-related proteins are present in Cf-PL10 and Cf-VASA, which also contains three zinc-finger CCHC motifs conserved among VASA-related proteins. Phylogenetic analysis showed that Cf-VASA, Cf-PL10 and Cf-p68 appeared more closely related to other members of their sub-families, respectively, which indicates that these genes are highly conserved through evolution. RT-PCR analysis of spatial expression and in situ hybridization experiments on developmental cycles of gonads revealed a specific expression of Cf-vasa in all germ cells except mature spermatozoids, suggesting that Cf-vasa might be involved in C. farreri gametogenesis. Cf-vasa mRNAs were detected by RT-PCR in early developmental stages from fertilized oocyte to gastrula. Whole-mount in situ hybridization showed that Cf-vasa mRNAs were localized to the vegetal pole of fertilized oocyte and transmitted to a specific blastomere during cleavages. Two cells containing Cf-vasa mRNAs were observed in gastrula, and the number of these cells increased in following stages. Based on above results, it is postulated that the C. farreri PGCs are determined at early development by maternal germ plasm containing Cf-vasa mRNAs. The putative PGCs with Cf-vasa expression form in gastrula stage, and are located symmetrically at both sides of the stomodeum in trochophore. These cells subsequently migrate to the posterior pole of D-shaped larva and then migrate towards abdomen. Moreover, expression of Cf-PL10 and Cf-p68 in gonads and other adult tissues were detected by RT-PCR. Expression level of Cf-PL10 in proliferative and mature gonads was higher than that in mantle, aductor muscle and gill. Cf-p68 expression in ovary increased along with gonad development. These suggest that Cf-PL10 and Cf-p68 might be involved in C. farreri gametogenesis.boule, a member of DAZ gene family, encodes an RNA-binding protein, which play a functionally conserved role in regulating the meiosis entry. A full length Cf-boule cDNA of 1879 bp was isolated from C. farreri testis and encodes a protein of 278 amino acids. Cf-BOULE protein shares the RNP-1, RNP-2 motifs and a DAZ repeat with other DAZ members. Cf-boule expression was detected by RT-PCR in gonads, aductor muscle and mantle, with the most abundance in gonads, where expression level in growing stage was higher than that in proliferative stage. Experiments of in situ hybridization revealed an extensive expression of Cf-boule in all germ cells except mature spermatozoids, suggesting that Cf-boule might be involved in regulating meiosis during C. farreri spermatogenesis. Similar amount of Cf-boule mRNAs in early embryos from fertilized oocyte to blastula was detected by RT-PCR, and that in gastrula and trochophore increased obviously. Above results indicate that Cf-boule mRNAs in early embryos are maternally inherited, and zygotic expression occurs in gastrula and trochophore. Whole-mount in situ hybridization showed a non-specific localization of Cf-boule mRNAs in early development from fertilized oocyte to gastrula. The distributional difference between Cf-boule and Cf-vasa mRNAs suggests that Cf-boule mRNAs are not involved in assembling germ plasm and determining PGCs fate in C. farreri. A high level expression of Cf-boule was observed in the putative PGCs and several cells above velum in trochophore, and disappeared in following stages. The role of Cf-boule in embryonic development including the origin and differentiation of C. farreri germ cells remains unknown.