|Other Abstract||In most animals, primordial germ cells（PGCs）are set aside from somatic cells in early stage of embryo development and migrate to the gonads where they differentiate into gametes for transmitting genetic information from one generation to another. Understanding the mechanism of germline development is very important for genetic manipulation of germ cells. Piwi and vasa were studied among different organisms. This work was aimed at making use of the medaka as a model to study the expression pattern and function of piwi and vasa, two highly conserved germ cell markers in the germline development.
Piwi was first isolated from Drosophila which is important for germ line stem cell self renewal. Piwi encodes an RNA binding protein and has been shown to be highly conserved in structure and germ cell specific. At present, medaka piwi has not been reported. Here we show that medaka piwi (opiwi) is 2.9kb for a putative protein of 857 aa with conserved PAZ domain at its N-terminus and PIWI domain at its C-terminus respectively. A phylogenetic tree shows that opiwi belongs to the piwi sub-family and has a closest identity with that of zebrafish piwi. Opiwi has the same genome structure and chromosome location as the human piwi gene. During embryogenesis, opiwi RNA is maternally provided and its expression persists throughout embryogenesis. During early stages of embryo development, opiwi RNA and protein evenly distribute in every blastomere. During the somitegenesis stage, opiwi RNA expression is high in PGC and weak in somatic cells such as the eye, brain and tail. In PGCs oPiwi protein distributes in peri-muclear particles. In spermatogonia and oogonia, oPiwi protein localizes to the same structure. In adult tissues, except for their preferential expression in gonads, opiwi RNA and protein are also weakly expressed in the eye and brain. In the gonads, both opiwi RNA and protein expression are restricted to germ cells. In the ovary, opiwi RNA and protein are high in oogonia and early stages of oocytes, declines in growing oocytes, in the late and mature stage oocytes, opiwi either distributes into dot-like structure in the yolk or forms a thin layer around the cortex of oocytes. In addition, opiwi RNA and protein localize to Balbiani body with mitochondria in early stage of oocytes. In the testis, opiwi is high in spermatogonia and spermertocyte, opiwi RNA is absent from spermatids and sperm, in contrast, oPiwi is persisting in spermatids where it localizes to chromatoid body also with mitochondria. Furthermore, we find that gfp fused to opiwi3’UTR (untranslated region, 3’UTR) can identify first PGCs as early as later blastula stage (9hpf), compared to the early gastrula stage (13hpf) when PGCs become distinguished by nanos3’UTR... Opiwi function was investigated by increasing or decreasing its expression, PGCs are distinguished at the mid gastrula by gfp expression in double transgenic medaka driven by nanos promoter and vasa promoter (NgVg). Injection of opiwi morpholino (MOpw) gives rise to decreased PGC number and somatic development defect during organgenesis. Opiwi depleted embryos in cell culture shows that opiwi depletion affected PGCs and somatic cells proliferation and survival. By injection of splicing MO and antibody, we find that its maternal expression affects germ cells development while the zygotic expression affects somatic development. Injection of opiwi RNA and dominant negative RNA without PAZ domain also decreases PGC number and defects in early stages of embryo development.
Vasa was identified in a genetic screening as one of the several maternal-effect genes for the embryonic polarity and germ cell formation in Drosophila. It is best known for its expression and role in the germline in different organisms. So far, Medaka vasa RNA is restricted to germ cells and its promoter activity is germ cells specific. However, Vasa protein expression and function still remains unclear. Here we report that vasa RNA and protein have the same expression pattern of that of opiwi , Vasa RNA is maternally provided with high expression till early gastrula stage, its expression persists throughout the embryogenesis. During early stages of embryo development, vasa is evenly distributed in every blastomere. During organgenesis stage, we find that vasa RNA is weak in somatic cells such as brain, trunk and tail except for its preferential expression in PGCs. In adult tissues, vasa RNA also is weak in somatic tissues although it is high in the gonads. In the gonads, vasa RNA and protein are restricted to germ cells. In the ovary, vasa expression is throughout the whole stage of oogenesis and co-localized with opiwi in Balbiani body. In the testis, vasa is co-localized with oPiwi in chromatoid body in spermatids. In spermatogonia , Vasa distributes in peri-nuclear particles with oPiwi. Vasa and oPiwi have the same expression pattern in oogonia and PGCs. In madaka, vasa has dual roles in PGC migration and somatic development. Loss of function by injection of MOvas led to many ectopic PGCs. MOvas embryos subjected to cell culture show that vasa depletion does not affect PGC motility, proliferation and survival. Surprisingly, even PGCs at ectopic sites are able to maintain the PGC identity and capable of proliferation, mobility and survival. By chimeric analysis we show that vasa depleted PGCs can move but can not migrate properly in the normal host environment. Surprisingly, upon vasa knockdown, embryonic lethality is compromised during organogenesis, and massive somatic cell death takes place in living embryos and their cell cultures. Therefore, medaka vasa has dual roles. In germline development vasa specifically controls PGC migration cell-autonomously. In somatic development vasa controls embryonic viability and cell survival. Accordingly, the medaka vasa has the conserved role in the germline and previously unidentified roles in somatic development, suggesting that vasa has a broader role than previously thought.
In conclusion, both opiwi and vasa expression and function are mainly in germ cells; however we find that opiwi and vasa have a new role in the maintenance of somatic cells development.
Key words: germ cells, opiwi, vasa, spermatogenesis, chromatoid body, oognesis，balbiani body, medaka|