Effects of UV-irradiation at Various Wavelengths on Sterilizing Drosophila Embryos: (Drosophila embryo/UV-effects/pole cells/polar plasm/nucleic acids).

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. Sterility, expressed either as the frequency of pole-cell-deficient embryos, or as the frequency of agametic adults, was found to be dependent on the wavelength of UV light. UV-irradiation at 280 nm was found moot effective in causing sterility on Drosophila embryos. The minimum dose of radiation to give a 100% sterility was 200 J/m2 at 280 nm, and 400 J/m2 at 254 nm. In contrast, mortality showed no dependency on the wavelength. The possibility that nucleic acids in the posterior region is a target of 280 nm radiation is discussed.

Mutations Affecting Embryonic F-Actin Reorganization also Affect Separation of Nuclei from their Sisters and from the Cortex in Drosophila Cleavage Embryos: (Drosophila/nuclear migration, F-actin, mutants).

We showed in Drosophila that nuclear migration was reduced all through cleavage stages in embryos with any one of the maternal-effect mutations, gs(1)N441 and gs(1)N26, in which F-actin reorganization in cleavage embryos is disordered. Moreover, we determined nuclear positions in embryos at cycle 1 and 2 in the wild type and two mutants, gs(1)N441 and gs(1)N26, in order to test if the nuclear migration is regulated within a nuclear cycle. At cycle 1, there was no difference in nuclear position among the strains that we observed. At cycle 2 the two sister nuclei had already migrated posteriorly in the wild type. However, migration was not detectable at cycle 2 in the mutants. Besides, the two sister nuclei were less-separated from each other, and orientation of the two nuclei with regard to the anteroposterior axis was random, different from the wild type. These results support the hypothesis that F-actin is involved in the regulation to separate cleavage nuclei from each other and from the egg cortex. This regulation is apparently required for posteriorward nuclear migration, and for synchronous nuclear arrival in the whole egg cortex.

A Mutation in the Drosophila Profilin Homolog Gene Affects Gametogenesis and Bristle Development in Both Sexes: (Drosophila mutant/profilin/gametogenesis/bristle).

We have isolated a Drosophila melanogaster mutant, allelic to the profilin gene reported as chickadee. We named the allele chickadeebin , in which the oogenesis and the spermatogenesis are disrupted, and the bristles are malformed. In the mutant nurse cells, cytoplasmic actin filaments fail to polymerize, and nuclei are displaced. The flow of cytoplasm from nurse cells to the oocyte is abortive. These ovarian phenotypes are principally the same as those reported in chickadeewc57 and WF57 (2). In addition, the egg chamber of chickadeebin contains a reduced number of cystocytes that are binucleafed. In some egg chambers, the oocyte fails to differentiate. All cystocytes in such an egg chamber are morphologically similar to nurse cells with polyploid nuclei. Mutant male flies have defective testes in which the spermatocyst is deficient or reduced in number. Mutant adults have shortened and forked bristles. We discuss the function of profilin in the gametogenesis and bristle development.

Differences in Fine Structures between Normal and RNA-induced Drosophila Pole Cells: (Drosophila pole cell/fine structure/polar granule/nuclear body/cytoplasmic determinant).

Fine structures were compared between normal pole cells and those induced in embryos that had been uv-irradiated and then injected with intact polar plasm or with poly(A)+ RNA extracted from cleavage embryos. Nuclei in nomal pole cells were spherical. In contrast, those in the induced pole cells were deformed to variable extents depending on materials injected with. Polar granules were smaller in pole cells induced by injection of poly(A)+ RNA than in normal pole cells. The size of polar granules in polar-plasm-induced pole cells was intermediate between those in poly(A)+ RNA-induced and normal pole cells. Small polar granules were observed in posterior cells of embryos uv-irradiated, nevertheless those cells were columnar and with identical morphology to somatic cells. Nuclear bodies showed a similar tendency in size differences as observed in polar granules in three types of pole cells observed.

Activation of Heat-Shock Genes by Digitonin is Selectively Repressed in Preheated Drosophila Salivary Glands: (Drosophila/heat-shock puff/digitonin/gene expression).

Treatment of Drosophila salivary glands with a mild detergent, digitonin, activates puffing at 35 chromosome loci. These digitonin-activated puffs include all of the nine heat-shock puffs known in D. melanogaster. Here we show that the activation of heat-shock genes, but not of other digitoninstimulated puffs, is repressed in salivary glands which have been subjected to and have recovered from heat shock before being treated with digitonin. The findings indicate that, (a) the activation of heat-shock genes by digitonin, as that by temperature elevation, is self-regulated by the heat-shock proteins (HSPs). (b) the gene repressive activity of HSPs is heat-shock-gene specific, and (c) the repression mechanism of heat-shock genes by HSPs is resistant to digitonin, in contrast to that the suppression of heat-shock genes is prevented by the detergent in non-heat-shocked salivary glands. The selective repression of heat-shock genes in preheated salivary glands suggests that the heat-shock genes and other digitonin-activated genes may be controlled by a different mechanism(s).

Maternal Messenger RNA as a Determinant of Pole Cell Formation in Drosophila Embryos: (polar plasm/mRNA/cytoplasmic determinant/pole cells/germ cells).

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.

Regional expression of the transcript encoding sterol carrier protein x-related thiolase and its regulation by homeotic genes in the midgut of Drosophila embryos.

We have isolated a gene designated terminal rings in the midgut (trim), which encodes a transcript expressed in the embryonic midgut in a region-specific manner. The 1.4 kb trim transcript is expressed in two discrete regions of the midgut epithelium during late embryogenesis. The anterior domain of trim expression is found between the proventriculus and the first midgut constriction. The posterior domain locates in the region posterior to the third midgut constriction. Furthermore, we show that normal function of two homeotic genes, Antp and abd-A, which express in the visceral mesoderm covering the midgut epithelium, is required for the regional expression of trim. The trim gene is thus reasoned to be activated through inductive interaction between two midgut layers. The 1.4kb mRNA encodes a sterol carrier protein x-related thiolase. As thiolase activity is required for lipid metabolism, the expression of the 1.4 kb trim transcript can represent a state of differentiation and can be used as an appropriate marker for analyzing regional differentiation of the midgut in terms of physiologically distinct domains.

Digitonin Activates Different Sets of Puff Loci Depending on Developmental Stages in Drosophila melanogaster Salivary Glands: (salivary gland chromosomes/detergent/gene regulation/ecdysone-inducible puffs/Drosophila melanogaster).

We showed previously that treatment of Drosophila melanogaster salivary glands with a mild detergent, digitonin, induces heat shock puffs and many developmentally regulated puffs. To find if the mechanism underlying the puff induction by digitonin is related to the temporal control of gene expression in salivary glands, we examined effects of digitonin on salivary glands at various puff stages from late third instar larva to white prepupa. The results indicate that (a) all the heat shock puffs are induced by digitonin irrespective of the developmental stage of the treated glands, (b) intermolt and early puff loci are always irresponsive to digitonin, and (c) late puff loci respond to digitonin to form puffs only before the stage of their developmentally programmed puffing. Based on the stage at which the locus becomes digitonin responsive, the digitonin-responsive late puff loci were divided into two groups: group A loci, responsive to digitonin continuously from PS1 until programmed puffing begins, and group B loci, responsive to digitonin only in a short period of time immediately before the programmed puffing. The results suggest that a digitonin-sensitive suppression mechanism(s) is involved in the temporal control of gene expression in Drosophila salivary glands.

A simplified and efficient method for in situ hybridization to whole Drosophila embryos, using electrophoresis for removing non-hybridized probes: (Drosophila/whole mount-in situ hybridization).

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.

Developmentally Regulated Splicing of the Third Intron of P Element in Somatic Tissues in Drosophila Embryos: (transposon/P element/splicing/Drosophila embryo).

In Drosophila, it has been suggested that the third intron of the P element is spliced out only in germ-line cells. However, we found that the splicing took place in some somatic tissues during embryogenesis, as well as in pole cells, progenitor cells of the germ line. To detect the splicing activity in vivo, we designed an assay system to visualize the cells where the third intron was spliced out by the histochemical staining for ß-galactosidase activity. In this paper, we describe the somatic tissues where the third intron is spliced out during embryogenesis, with reference to the stages when the splicing occurs. Based on these findings we propose a modification of the previous documentation that the complete processing of transcripts from P element takes place only in germ-line cells.

Accumulation and Spatial Distribution of Poly(A)+ RNA in Oocytes and Early Embryos of Drosophila melanogaster: (Poly(A)+ RNA/in situ hybridization/oocytes and embryos/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 [3 H]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.

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster.

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.

BEHAVIOR OF INTERPHASE EMBRYONIC NUCLEI TRANSPLANTED IN NUCLEAR MULTIPLICATION STAGE EMBRYOS OF DROSOPHILA MELANOGASTER.

Interphase nuclei were transplanted from syncytial blastoderm into early cleavage embryos of Drosophila melanogaster. The transplanted nuclei, when exposed to host cytoplasm, were initiated to mitosis. During the period from 10 to 50 min after transplantation, the implanted nuclei and host nuclei were found not synchronous in their mitotic cycles. Synchrony was restored usually by the blastoderm stage. About 5% of eggs with transplanted nuclei developed significantly faster than control eggs, resulting in premature blastoderm formation. This finding is discussed in relation to chimera formation and to embryonic development of grandchildless mutants.

Changes in subcellular localization of mtlrRNA outside mitochondria in oogenesis and early embryogenesis of Drosophila melanogaster.

Mitochondrial large ribosomal RNA (mtlrRNA) has been identified as a cytoplasmic factor inducing pole cells in ultraviolet (UV)-sterilized Drosophila embryos. In situ hybridization studies have revealed that mtlrRNA is present outside mitochondria localized on the surface of polar granules during the cleavage stage. In the present study, we describe the developmental changes in extramitochondrial mtlrRNA distribution through early embryogenesis using in situ hybridization at the light and electron microscopic level. No mtlrRNA signal was discernible on polar granules in the mature oocyte, unless the oocyte was activated for development. mtlrRNA was localized on the surface of polar granules during a limited period of stages from oocyte activation to pole bud formation and disappeared as soon as being detached from polar granules without entering pole cells. These changes in the temporal and spatial distribution of mtlrRNA outside mitochondria are compatible with the idea that mtlrRNA is required for pole cell formation but not for the differentiation of pole cells as functional germ cells.

Nonradioactive In Situ Hybridization Methods for Drosophila Embryos Detecting Signals by Immunogold-Silver or Immunoperoxidase Method for Electron Microscopy: (electron microscopy/pre-embedding/transcripts/Drosophila embryo).

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.

Cytoplasmic factors determining anteroposterior polarity in Drosophila embryos.

Cytoplasm removal/transplant techniques applied to Drosophila cleavage-stage embryos induced changes in anteroposterior polarity. Removal of anterior cytoplasm or anterior transplantation of posterior cytoplasm caused the anterior formation of posterior (telson) structures, and the replacement of anterior cytoplasm with posterior cytoplasm induced double-abdomen embryos, as reported by Frohnhöfer et al. [J Embryol Exp Morphol 97 (suppl):169-179 (1986)]. Changing the conditions of anterior cytoplasm removal we showed that greater volumes, earlier stages, and removal from the periphery were efficient. In addition we found that double-cephalon embryos are induced by replacing posterior cytoplasm with anterior cytoplasm, while removal of posterior cytoplasm or the posterior transplantation of anterior cytoplasm was without effect. However, introduction of anterior cytoplasm into the posterior of nanos embryos, which are mutants not developing abdominal segments, caused the formation of double-cephalon embryos. Similarly, double-abdomen embryos are produced by introducing posterior cytoplasm into the anterior of bicoid embryos, which are mutants not forming cephalic and thoracic structures. These results are compatible with the initial involvement of separate anterior, posterior and terminal cytoplasmic factors deduced from mutant analysis (Nüsslein-Volhard and Roth 1989).

Isolation and characterization ofgrandchildless-like mutants inDrosophila melanogaster.

Two 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 King's stages of oogenesis.