Expression and Function of Mouse Pelota Gene

Expression and function of the pelota gene has originally been characterized in Drosophila. A loss-of-function mutation in Drosophila pelota was found to result in male infertility. Spermatogonia and spermatocytes are produced in mutant flies but the cell cycle of the first meiotic division is arrested during the G2/M transition.In the present work, the expression pattern and physiological function of the mouse pelota gene was studied. The expression of the pelota gene in different mouse tissues was analysed by Northern blot. A 1.6 kb pelota transcript was detected in the RNA of all studied tissues. An additional 2.1 kb pelota transcript was observed in RNA of the spleen. To investigate the expression of the pelota gene during prenatal development, RNA was extracted from the posterior part of 9.5 to 17.5 dpc. embryos and analysed for the presence of pelota transcript by RT-PCR. Pelota transcripts were detected in all studied embryos. To evaluate the expression of the pelota gene during testicular development, Northern blot with RNA extracted from testes of 5- to 60 day old mice was performed. The results of Northern blot hybridisation revealed that the pelota gene is expressed throughout postnatal testis development. Further Northern blot analyses with testicular RNA isolated from mutant mice indicate that the expression of the pelota gene is not restricted to spermatogenic cells of the testis.To elucidate the potential role of the mammalian pelota gene, the gene was deleted in mice through homologous recombination. Both male and female mice heterozygous for pelota deletion were phenotypically normal and fertile. Heterozygous animals were intercrossed and the offspring were genotyped by PCR analysis. No pelota-/- animals were detected in the F2 generation. These results indicate that pelota deficiency results in embryonic death. To assess the consequence of the pelota mutation for embryonic development, embryos were collected from heterozygous intercrosses at different days of prenatal development. PCR analysis of postimplantation embryos dissected from the deciduas at embryonic days (E) 8.5, 9.5 and 10.5 showed that none of studied embryos was pelota-/-, while analysis of preimplantation embryos at E2.5 and 3.5 revealed the presence of pelota-/- embryos in the expected ratio as defined by Mendelian distribution. These results demonstrate that pelota-/- embryos die between E3.5 and E8.5.To determine the cause and time of embryonic lethality, histological analysis of deciduas at embryonic days 6.5, 7.5, 8.5 and 9.5 were performed. The results of these studies revealed that the development of the pelota deficient embryos up to E6.5 appears normal but slows down at the E7.5. After E9.5, no viable pelota-/- were observed. The fact that differentiation of the three germ layers is initiated in the pelota-/- embryos suggests that the developmental arrest is not due to lack of differentiation, but rather results from a proliferation defect. To directly determine the growth capability of mutant pelota-/- embryos, E3.5 blastocyts from heterozygous intercrosses were collected and cultured individually for 7 days and then genotyped by PCR. During the first 4 days in culture, the inner cell mass (ICM) of pelota-/- blastocysts was indistinguishable from that of pelota+/+ and pelota+/-. However, while the ICM cells of pelota+/+ and pelota+/- embryos continued to expand throughout the 7-day, pelota-/- ICM cells failed to expand subsequent to day 4. In contrast, pelota-/- trophoblast gaint cells (TGC) continued to grow in size through 7 days of culture. The survival of mitotically inactive pelota-/- trophoblast gaint cells and the death of mitotically active inner cell mass of pelota-/- blastocysts support our hypothesis that pelota is required during mitosis.To address the question whether the mitotic cell cycle is arrested in the pelota-/- embryos, DNA contents in embryonic cells of E7.5 was determined with CAS image system. Results of these studies suggest that the deficiency of pelota results in a cell cycle arrest at the G2/M transition.To determine the subcellular localization of pelota protein, embryonic stem cells (ES) were transfected with green fluorescent protein (GFP)-targeted pelota and expressing clones were selected. Microscopic examination of the transfected ES cells showed that GFP-fluorescence signal was localized at the centrosomes. Immunostaining of the GFP-pelota transfected ES cells with monoclonal anti g-tubulin demonstrated that the g-tubulin, a marker for centrosomes, colocalized exactly with the GFP-fluorescence signal in interphase, prophase and metaphase. These results indicate that pelota protein is associated with the centrosome.