Masukichi Okada

degenerative spermatocyte, a novel gene encoding a transmembrane protein required for the initiation of meiosis in Drosophila spermatogenesis

Abstract We have identified a novel Drosophila gene, degenerative spermatocyte (des), that may be required for the initiation of meiosis in spermatogenesis. In des mutant testes, the primary spermatocytes become mature in size but degenerate without initiating meiotic chromosome condensation in their nuclei. We have mapped the locus of the des gene at 26A on the left arm of the second chromosome, and identified two related, differentially spliced transcripts (des-1, des-2) from the locus. The genomic DNA fragment encoding both transcripts is able to rescue the des phenotype when introduced into mutant flies. The expression of the des-1 transcript is significantly suppressed in the des mutant testes, whereas expression of the des-2 transcript is undetectable in both wild-type and mutant testes, indicating that the des-1 transcript encodes the des function in spermatogenesis. The des-1 transcript is selectively expressed in primary spermatocytes during normal spermatogenesis. The nucleotide sequence of the des-1 transcript predicts that it encodes a novel transmembrane protein. These results raise the possibility that the des gene product may be required for interactions between primary spermatocytes and surrounding somatic cells.

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non‐hybridized probes

We report a simplified and reliable method for non‐radioactive in situ hybridization to whole Drosophila embryos. In the previous method (Tautz and Pfeifle, 1989) the post‐hybridization wash, or the procedure for washing non‐hybridized probe away from embryos depends simply on diffusion. We modified the method with application of electrophoresis to the wash. After hybridized with RNA probe, embryos were transferred to a small well where an electric charge was given to drive non‐hybridized probe away from the embryos. This procedure enables us to acquire a much higher signal‐to‐noise ratio than that obtained from a conventional method. Furthermore, this is a time‐saving method. We propose a term “electro‐wash” for this procedure.

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos.

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.

Accumulation and Spatial Distribution of Poly(A)+RNA in Oocytes and Early Embryos of Drosophila melanogaster

We describe the accumulation and distribution of poly (A)+RNA during oogenesis and early embryogenesis as revealed by in situ hybridization with a radio‐labeled poly (U) probe. The amount of poly (A)+RNA in nurse cell cytoplasm continuously increased untill mid‐vitellogenic stage (st. 10), then decreased with the rapid increase of poly (A)+RNA in the oocyte (st. 11). The localization of poly (A)+RNA at stage 10 was in the anterior region of the oocyte, where it is connected by cytoplasmic bridge to the nurse cells. These observations indicate that most of the poly (A)+RNA synthesized in the nurse cells is transferred to the oocyte through the cytoplasmic bridges at stage 10–11. During the remainder of oogenesis (st. 11–14) and during preblastodermal embryogenesis, poly (A)+RNA was evenly distributed over the cytoplasm of oocytes and embryos. At blastoderm stage, poly(A)+RNA became concentrated in the peripheral region of embryos. Though the somatic nuclei of the blastoderm contained a detectable amount of poly (A)+ RNA, the pole cell nuclei did not. The cytoplasmic RNA visualised by acridine orange staining and the poly (A)+RNA detected by hybridization with [3H]poly (U) exhibited identical distributions during oogenesis and early embryogenesis. These observations provide a basis to assess the unique distributions of specific RNA sequences involved in early development.

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster

SummaryTwo temperature-sensitive sex-linkedgrandchildless (gs)-like mutations (gs(1)N26 andgs(1)N441) were induced by ethylmethane sulphonate inDrosophila melanogaster. They complemented each other and mapped at two different loci (1−33.8±0.7 forgs(1)N26 and 1−39.6±1.7 forgs(1)N441), which were not identical to those of any of thegs-like mutants reported in earlier work.Homozygous females of the newly isolated mutants produced eggs that were unable to form pole cells and developed into agametic adults. Competence of the embryos to form pole cells was not restored by wild-type sperm in either mutant; that is, the sterility caused by these mutations is controlled by a maternal effect.Fecundity and fertility ofgs(1)N26 females were low, and their male offspring showed a higher mortality than that of female offspring, causing an abnormal sex ratio. The frequency of agametic progeny was 93.1% and 55.8%, when the female parents were reared at 25° C and 18° C, respectively. In eggs produced by thegs(1)N26 females reared at 25° C, the migration of nuclei to the posterior pole was abnormal, and almost no pole cell formation occurred in these egg. Furthermore, half of these eggs failed to cellularize at the posterior pole. When the females were reared at 18° C, almost all of the eggs underwent complete blastoderm formation, and in half of these blastoderm embryos normal pole cells were formed.In the other mutant,gs(1)N441, the fecundity and fertility of the females were normal. The agametic frequency in the progeny was 70.8% and 18.6% when the female parents were reared at 25° C and 18° C, respectively. In the eggs laid by females reared either at 25° C or at 18° C, the migration of nuclei to the periphery and cellularization proceeded normally; nevertheless, in the majority of the embryos no pole cell formation occured at the stage when nuclei penetrated into the periplasm. When the females were reared at 18° C, some of the embryos from these females formed some round blastoderm cells with cytologically recognizable polar granules and nuclear bodies, which are attributes of pole cells. The temperature sensitive period ofgs(1)N441 was estimated to extend from stage 9 to 13 of Kings stages of oogenesis.

Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm☆

Abstract A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.

Monoclonal antibodies against Drosophila ovaries: their reaction with ovarian and embryonic antigens

A library of monoclonal antibodies (MAbs) against Drosophila ovarian antigens was established. Each of the MAbs was characterized by its immunohistochemical binding pattern to sections from egg chambers at various stages of oogenesis. Sixteen of the 18 MAbs were found to bind to antigens in mature oocytes. Among the 16 antigens, two were also located in cytoplasm of cell types in the egg chamber other than the oocyte, at all stages of oogenesis. Four made their appearance in nurse cell cytoplasm at mid-vitellogenic stages and shifted to oocyte cytoplasm at a later stage, and ten appeared at the vitellogenic stage and confined their distribution to oocyte cytoplasm. All these antigens were distributed evenly in cytoplasm of mature oocytes. However, some of these antigens were noticed to change their distribution during early embryogenesis as to be localized in a specific region of embryos.

Maternal messenger RNA as a determinant of pole cell formation in Drosophila embryos

Some polar plasm components are UV‐sensitive. Messenger RNA extracted from oocytes or cleavage embryos can to induce pole cells in embryos that have been deprived of ability to form pole cells by UV‐irradiation. This article reviews studies on the role of this mRNA in the developmental pathway leading to germ cell formation.

Effects of UV-irradiation at Various Wavelengths on Sterilizing Drosophila Embryos

Embryos of Drosophila melanogaster at the early intravitelline nuclear multiplication stage were irradiated with UV light at the posterior pole. The sterility and mortality of these embryos were examined in relation to the dose and wavelength of the UV light.

Nonradioactive In Situ Hybridization Methods for Drosophila Embryos Detecting Signals by Immunogold-Silver or Immunoperoxidase Method for Electron Microscopy

We present details of in situ hybridization methods for electron microscopy applicable for Drosophila embryos. Improvements upon the foregoing methods were made at 1) hybridization and visualization of signals were carried out with whole embryos that were then processed for electron microscopy, and 2) digoxigenin‐labeled probes were detected by the immunogold silver enhancement method or by the immunoperoxidase method. Using these methods. we demonstrated the localization of fushi tarazu transcripts in the apical region of blastodermal cells. We also showed that mitochondrial large ribosomal RNA is associated with polar granules in pole plasm of cleavage embryos. These methods will make a useful tool to determine the precise subcellular distribution of specific transcripts in Drosophila embryos.