vasa基因研究进展

DEAD-box家族基因编码一类ATP依赖的RNA解旋酶。经系统进化分析可将该家族蛋白分为VASA、PL10和p68三个亚家族。其中,vasa基因最先在果蝇(Drosophila melanogaster)中被发现,在许多动物中都已经克隆得到其同源基因,研究显示,vasa基因在生殖细胞系中特异性表达,在许多生物中为生殖细胞形成和配子发生所需。有趣的是在果蝇中VASA蛋白是生殖质的组成部分,而在斑马鱼(Danio rerio)中vasa mRNA才是生殖质的组成部分。本文主要综述了vasa基因及其蛋白的结构、功能、表达和作为原生殖细胞分子标记物的应用等方面的内容,并展望了其研究前景。     

中华鳖vasa基因克隆及在卵母细胞中的表达分析

研究利用中华鳖为研究模型进行爬行类生殖细胞发育分化成熟等生物学研究,克隆了中华鳖vasa基因的cDNA序列,全长3865 bp,包括5'端非编码区90 bp,3'端非编码区1699 bp,开放阅读框长2076 bp,共编码691个氨基酸。中华鳖Vasa氨基酸序列包含DEAD-box家族蛋白8个保守保守功能域,在N末端有4个RGG重复序列和2个GG富集区,与小鼠Vasa蛋白的同源性较高（72%）。荧光定量PCR的结果表明,中华鳖vasa mRNA主要精巢和卵巢中表达,其他体组织中均难检测到表达。卵巢冰冻切片原位杂交结果显示:中华鳖vasa mRNA在生殖细胞中特异表达;在卵子发生过程中的不同发育期卵母细胞中呈现动态的变化。即vasa mRNA在初级卵母细胞及生长期卵母细胞中表达最强,且均匀分布在细胞质中,随着卵母细胞的逐渐增大,信号逐渐减弱,直至在成熟的卵母细胞中几乎检测不到表达信号,说明vasa可能在中华鳖早期卵母细胞发育中起重要作用。同时,vasa基因可作为中华鳖生殖细胞分子标记物,根据其mRNA的表达水平来鉴别不同发育时期的卵母细胞。研究结果为进一步开展中华鳖胚胎生殖细胞发育及配子生成,特别是研究中华鳖,乃至爬行类原始生殖细胞（Primordial Germ Cells,PGCs）的起源、迁移、分化等研究奠定了基础。     

Germ line development in the grasshopper Schistocerca gregaria: vasa as a marker

Vasa is a widely conserved germline marker, both in vertebrates and invertebrates. We identify a vasa orthologue, Sgvasa, and use it to study germline development in the grasshopper Schistocerca gregaria, a species in which no germ plasm has been identified. In adults, Sgvasa is specifically expressed in the ovary and testis. It is expressed at high levels during early oogenesis, but no detectable vasa RNA and little Vasa protein are present in mature unlaid eggs. None appears to be localized to any defined region of the egg cortex, suggesting that germline specification may not depend on maternal germ plasm expressing vasa. Vasa protein is expressed in most cleavage energids as they reach the egg surface and persists at high levels in most cells aggregating to form the embryonic primordium. However, after gastrulation, Vasa protein persists only in extraembryonic membranes and in cells at the outer margin of the late heart-stage embryo. In the embryo, it then become restricted to cells at the dorsal margin of the forming abdomen. In older embryos, these Vasa-positive cells move toward the midline; Vasa protein accumulates asymmetrically in their cytoplasm, a pattern closely resembling that of germ cells in late embryonic gonads. Thus, we suggest that the Vasa-stained cells in the abdominal margin are germ cells, as proposed by Nelson (1934), and not cardioblasts, as has been proposed by others.     

vasa-related genes and their expression in stem cells of colonial parasitic rhizocephalan barnacle Polyascus polygenea (Arthropoda: Crustacea: Cirripedia: Rhizocephala)

vasa (vas)-related genes are members of the DEAD-box protein family and are expressed in the germ cells of many Metazoa. We cloned vasa-related genes (PpVLG, CpVLG) and other DEAD-box family related genes (PpDRH1, PpDRH2, CpDRH, AtDRHr) from the colonial parasitic rhizocephalan barnacle Polyascus polygenea, the non-colonial Clistosaccus paguri (Crustacea: Cirripedia: Rhizocephala), and the parasitic isopodan Athelgis takanoshimensis (Crustacea: Isopoda). The colonial Polyascus polygenea, a parasite of the coastal crabs Hemigrapsus sanguineus and Hemigrapsus longitarsis was used as a model object for further detailed investigations. Phylogenetic analysis suggested that PpVLG and CpVLG are closely related to vasa-like genes of other Arthropoda. The rest of the studied genes form their own separate branch on the phylogenetic tree and have a common ancestry with the p68 and PL10 subfamilies. We suppose this group may be a new subfamily of the DEAD-box RNA helicases that is specific for parasitic Crustacea. We found PpVLG and PpDRH1 expression products in stem cells from stolons and buds of internae, during asexual reproduction of colonial P. polygenea, and in germ cells from sexually reproducing externae, including male spermatogenic cells and female oogenic cells.     

Vasa protein expression is restricted to the small micromeres of the sea urchin, but is inducible in other lineages early in development

Vasa is a DEAD-box RNA helicase that functions in translational regulation of specific mRNAs. In many animals it is essential for germ line development and may have a more general stem cell role. Here we identify vasa in two sea urchin species and analyze the regulation of its expression. We find that vasa protein accumulates in only a subset of cells containing vasa mRNA. In contrast to vasa mRNA, which is present uniformly throughout all cells of the early embryo, vasa protein accumulates selectively in the 16-cell stage micromeres, and then is restricted to the small micromeres through gastrulation to larval development. Manipulating early embryonic fate specification by blastomere separations, exposure to lithium, and dominant-negative cadherin each suggest that, although vasa protein accumulation in the small micromeres is fixed, accumulation in other cells of the embryo is inducible. Indeed, we find that embryos in which micromeres are removed respond by significant up-regulation of vasa protein translation, followed by spatial restriction of the protein late in gastrulation. Overall, these results support the contention that sea urchins do not have obligate primordial germ cells determined in early development, that vasa may function in an early stem cell population of the embryo, and that vasa expression in this embryo is restricted early by translational regulation to the small micromere lineage.     

Expression of vasa (vas)-related genes in germ cells and specific interference with gene functions by double-stranded RNA in the monogenean, Neobenedenia girellae

Neobenedenia girellae, a monogenean, is an important pathogen in marine cultured fish such as yellowtail and amberjack. An effective control method is required but none has yet been established. Aiming to establish a new control method by interfering with the gametogenesis of N. girellae, we focused on vasa (vas)-related genes that are expressed exclusively in the germline granules in Drosophila, Caenorhabditis elegans and other animals. Three vas-related genes (N. girellae vasa-like gene, Ngvlg1, Ngvlg2 and Ngvlg3) were isolated by PCR and Ngvlg1 and Ngvlg2 were shown to be expressed only in germ cells. We demonstrated that introduction of double-stranded Ngvlg1 or Ngvlg2 RNA by soaking resulted in partial or complete loss of germ cells. Moreover, the hatching rate of eggs from animals showing partial loss of germ cells decreased significantly. These results suggest that Ngvlg1 and Ngvlg2 are essential genes for germ cell quantity and quality. The possibility that a new control method can be developed by controlling gametogenesis of N. girellae was proven, because sterilised N. girellae could be produced.     

Exploring embryonic germ line development in the water flea, Daphnia magna, by zinc-finger-containing VASA as a marker

VASA is an ATP-dependent RNA helicase belonging to the DEAD-box family that, in many organisms, is specifically expressed in germ line cells throughout the life cycle, making it a powerful molecular marker to study germ line development. To obtain further information on germ line development in crustaceans, we cloned VASA cDNAs from three branchiopod species: water fleas Daphnia magna and Moina macrocopa, and brine shrimp Artemia franciscana. RNA helicase domains in branchiopod VASA were highly conserved among arthropod classes. However, N-terminal RNA-binding domains in branchiopod VASA were highly diverged and, unlike other arthropod VASA reported so far, possessed repeats of retroviral-type zinc finger (CCHC) motifs. Raising specific antibodies against Daphnia VASA revealed that the primordial germ cells (PGCs) in this organism segregate at a very early cleavage stage of embryogenesis in parthenogenetic and sexual eggs. Clusters of PGCs then start to migrate inside the embryo and finally settle at both sides of the intestine, the site of future gonad development. RNA analyses suggested that maternally supplied vasa mRNA was responsible for early VASA expression, while zygotic expression started during blastodermal stage of development.     

Vasa expression in a colonial ascidian, Botrylloides violaceus

Evolution of solitary or colonial life histories in tunicates is accompanied by dramatic developmental changes that affect morphology and reproduction. We compared vasa expression in a solitary ascidian and a closely related colonial ascidian, in an effort to uncover developmental mechanisms important during the evolution of these contrasting life histories, including the ability to reproduce by budding. In this study, we explored the origin of germ cells in new buds developing by asexual reproduction in a colonial ascidian, Botrylloides violaceus and compared it to the source of germ cells in a solitary ascidian Boltenia villosa. We studied expression by in situ hybridization of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans. In B. villosa, bv-vasa mRNA was expressed in putative germ cells and oocytes of adult gonads, and was sequestered into a posterior lineage during embryogenesis. In mature colonies of the ascidian B. violaceus, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and in some circulating cells in the zooids and differentiating buds. We propose that expression of vasa in cells other than gonadal germ cells of zooids in a colonial ascidian may serve as a source of germ-line stem cells in the colony.