Modeling pattern formation: counting to two in the Drosophila egg

The EGF receptor pathway patterns the Drosophila egg and specifies the position of its dorsal appendages. A new mathematical analysis of this patterning network has highlighted its crucial features and provided novel insights into the spatial and temporal kinetics controlling patterning.     

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation. This work was supported by the National Science Foundation (grant MCB-0234245). O'Keith Dellafosse was supported by the Louisiana Alliance for Minority Participation (LAMP).     

Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster

Mutations in gurken and torpedo cause a ventralization in the follicle cell epithelium during Drosophila oogenesis and in the pattern of the embryo that develops in the resultant egg. Both genes lie midway in an epistatic series between fs(1)K10 and dorsal; the mutations block the dorsalization normally observed in K10 eggs but have no effect on the phenotype of embryos derived from dorsal mothers. Analysis of germ-line mosaics demonstrates that both ovarian and embryonic phenotypes will be produced when either the gurken+ gene is removed from the germ line or torpedo+ is removed from the soma. This shows that the dorsoventral pattern of the Drosophila egg chamber depends on the transfer of spatial information from the germ line to the somatic follicle cells, and from somatic cells to the oocyte.     

Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation

Egg activation is the process that modifies mature, arrested oocytes so that embryo development can proceed. One key aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. wispy (wisp) maternal-effect mutations in Drosophila block development during the egg-to-embryo transition. We show here that the wisp gene encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). The WISP protein is required for poly(A) tail elongation of bicoid, Toll, and torso mRNAs upon egg activation. In Drosophila, WISP and Smaug (SMG) have previously been reported to be required to trigger the destabilization of maternal mRNAs during egg activation. SMG is the major regulator of this activity. We report here that SMG is still translated in activated eggs from wisp mutant mothers, indicating that WISP does not regulate mRNA stability by controlling the translation of smg mRNA. We have also analyzed in detail the very early developmental arrest associated with wisp mutations. Pronuclear migration does not occur in activated eggs laid by wisp mutant females. Finally, we find that WISP function is also needed during oogenesis to regulate the poly(A) tail length of dmos during oocyte maturation and to maintain a high level of active (phospho-) mitogen-activated protein kinases (MAPKs).     

RNA synthesised during oogenesis and early embryogenesis in an insect egg (Euscelis plebejus)

Summary RNA labelled during oogenesis or early embryogenesis was isolated from eggs of the leaf hopperEuscelis plebejus. The polyadenylated RNA fraction deposited during early oogenesis accounted for approximately 2.7% of the total RNA content of the newly laid egg. This fraction differed significantly in molecular weight (15–32 S) from poly(A)-containing RNA synthesised between early cleavage and early germ anlage stages (4–20S). Locally injected³H-uridine spread through the egg within approximately 3 h. A considerable fraction (25–35%) of label injected as³H-uridine during early cleavage was recovered in DNA at subsequent stages (10–20 h later); labelled RNA was not found prior to the cellular blastoderm stage. When the yolk-endoplasm was separated from the blastoderm cells, only the latter contained demonstrable amounts of RNA synthesised by the embryo. Of the precursor incorporated into embryonic RNA, approximately 10% was found in the polyadenylated fraction at the early blastoderm stage, but only 3% at the early germ anlage stage. No differences in size distribution of polyadenylated RNA were evident between anterior and posterior halves of the early germ anlage stage.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Shining light on Drosophila oogenesis: live imaging of egg development

Drosophila oogenesis is a powerful model for the study of numerous questions in cell and developmental biology. In addition to its longstanding value as a genetically tractable model of organogenesis, recently it has emerged as an excellent system in which to combine genetics and live imaging. Rapidly improving ex vivo culture conditions, new fluorescent biosensors and photo-manipulation tools, and advances in microscopy have allowed direct observation in real time of processes such as stem cell self-renewal, collective cell migration, and polarized mRNA and protein transport. In addition, entirely new phenomena have been discovered, including revolution of the follicle within the basement membrane and oscillating assembly and disassembly of myosin on a polarized actin network, both of which contribute to elongating this tissue. This review focuses on recent advances in live-cell imaging techniques and the biological insights gleaned from live imaging of egg chamber development.     

A new peroxinectin-like gene preferentially expressed during oogenesis and early embryogenesis in Drosophila melanogaster

Several peroxidase isozymes have been described in Drosophila melanogaster. We describe a peroxinectin-like gene (Dpxt) in D. melanogaster. Peroxinectin is a cell-adhesive hemoperoxidase which binds superoxide dismutase and mediates blood cells attachment and spreading in the crayfish Pacifastacus leniusculus. The Dpxt predicted protein has a putative RGD-integrin binding tripeptide. The Dpxt mRNA is present in high amounts in late oogenesis and in early embryogenesis until the cellular blastoderm stage. It is virtually absent at other stages of the Drosophila life cycle, suggesting that Dpxt function is restricted to the early stages of fly development.     

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.     

Bicaudal-D is essential for egg chamber formation and cytoskeletal organization in drosophila oogenesis

Bicaudal-D (Bic-D) is required for the transport of determinant mRNAs and proteins to the presumptive oocyte, an essential step in the differentiation of the oocyte. Bic-D protein contains four well-defined heptad repeat domains characteristic of intermediate filament proteins. We characterized the ovarian phenotypes of females expressing mutant Bic-D proteins (Bic-D(H)) deleted for each of the heptad repeat domains. The altered migration of follicle cells we observe in mutant ovaries suggests that Bic-D functions in the germline and directs the inward migration of somatic follicle cells. In the germarium Bic-D is required for the organization of the egg chamber and the structural integrity of the oocyte and nurse cells. Examination of the polarized microtubule network in Bic-D(H) ovaries shows that Bic-D function is required for both the establishment of the polarized microtubule network and its maintenance throughout oogenesis. To explain the multiple functions suggested by the pleiotropic Bic-D phenotype, we propose that Bic-D protein could form itself a filamentous structure and represent an integral, essential part of the cytoskeleton.     

Scanning electron microscopy of Drosophila embryogenesis: 1. The structure of the egg envelopes and the formation of the cellular blastoderm

As part of a series of detailed observations on embryogenesis in Drosophila, the protective coverings of the egg and surface changes in the embryo prior to gastrulation have been studied with the SEM. Four specializations of the chorion are described: the plastron, micropylar cone, operculum, and the posterior thickening. After removal of the protective coverings the surface changes during development can be observed. During the first eight synchronous nuclear divisions a dense array of thin microprojections covers the whole embryo. After the ninth division between 373 and 408 nuclei reach the surface and become located in cytoplasmic projections. From counts of the number of surface bulges during the syncytial blastema stages, it was established that 13 synchronous divisions take place producing between 5600 and 6500 surface nuclei. During formation of the cellular blastoderm, the location of the prospective cells becomes obscured by a dense pattern of microprojections from each cell. However, with the completion of the blastoderm, the surfaces of the cells become smooth and the cell outlines distinct. The usefulness of the SEM in developmental studies is discussed.     

The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.

The pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Here we report on a phenotypic analysis using pum(ovarette) mutations, which reveals multiple functions of pum in primordial germ cell proliferation, larval ovary formation, GSC division, and subsequent oogenic processes, as well as in oviposition. Specifically, by inducing pum(-) GSC clones at the onset of oogenesis, we show that pum is directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, we show that pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pum(ovarette) mutations, pum(1688) specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum(2003) and pum(4277) specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum(1688) and pum(2003) mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, we present molecular evidence suggesting that the two PUM isoforms share some of their primary structures.     

Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis

Two isoforms of a single nuclear lamin, distinguishable on one-dimensional SDS-polyacrylamide gels, have previously been identified in Drosophila nuclei during interphase. A third species, designated lamin Dmmit, has now been identified as soluble in extracts of Drosophila tissue culture cells blocked in mitosis by drugs. An apparently identical form is the only lamin species detectable in late-stage egg chambers and early embryos. Phosphoamino acid analyses suggest that the conversion of lamins Dm1 and Dm2 to lamin Dmmit is brought about by a specific rearrangement of phosphate groups rather than by dramatic net changes in the levels of lamin phosphorylation. The residues involved in these phosphorylation/dephosphorylation reactions have been tentatively mapped to a 17.8-kD cyanogen bromide fragment containing amino acids 385-547. This represents a potential "hinge" domain in the lamin structure between the end of coil 2 and the globular COOH terminus. These results have implications for understanding the regulation of nuclear envelope breakdown during mitosis and karyoskeletal dynamics during oogenesis and early embryogenesis.     

Maternal-effect loci involved in Drosophila oogenesis and embryogenesis: P element-induced mutations on the third chromosome

A collection of 1609 recessive P-lethal mutations on the third chromosome was tested in germline clones for effects on egg differentiation and embryonic development. In 164 lines, normal differentiation of the egg chamber is prevented and in 841 lines, embryos develop abnormally. This latter group of maternal-effect mutations was subdivided into 23 classes based on the cuticular phenotypes. Our collection comprises new alleles of previously characterized genes (e.g. kayak, punt, string, tramtrack). For some of the genes identified in this screen, a maternal contribution to embryonic development has not been described previously (e.g. extramacrochaete, Trithorax-like, single minded, couch potato, canoe). The genes classified in our study with a dual function during oogenesis and embryogenesis not only substantially extends the existing collection of maternal-effect genes but will also aid further understanding of how patterning of the Drosophila embryo is controlled by the maternal genome.     

Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. Here, we show that during early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. Our results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis.     

The role of the otu gene in Drosophila oogenesis

The ovarian tumor (otu) gene behaves as if it encodes a product (OGP) which is required during several early steps in the transformation of oogonia into functional oocytes. The ovarian phenotypes produced by various EMS-induced mutations can be explained as graded responses by individual mutant germ cells to the different levels of functionally active OGP they themselves synthesize. In addition, genetic evidence suggests that otu also encodes a second product that is utilized late in oogenesis. Molecular studies of the otugene demonstrate that it does transcribe at least two ovaryspecific RNAs. The possible function of the otu product in the cellular divisions that give rise to the oocyte and its sister nurse cells is discussed.     

The ontogeny of germ plasm during oogenesis in Drosophila

Primordial germ cells can be induced at both the anterior and ventral region of the Drosophila egg by transplanted posterior polar plasm. Two questions arise from these results: (1) Is fertilization required for germ plasm to be functional, and (2) at what stage during oogenesis does the posterior polar plasm become established as a germ-cell determinant?Polar plasm from unfertilized eggs and from oocytes at stage 10 to 14 of Drosophila melanogaster was implanted into the anterior region of cleavage embryos. Some injected embryos were analyzed at the ultrastructural level during blastoderm formation. Polar plasm from unfertilized eggs and from oocytes of stages 13 and 14 was found to be integrated into several anterior cells that resembled morphologically normal pole cells. The formation of such cells, however, could not be detected in embryos injected with polar plasm from oogenetic stages 10 to 12. Experimentally induced pole cells proved to be capable of differentiating into functional germ cells when cycled through the germ line of genetically different host embryos. About 5% of the flies developing from these embryos produced progeny that originated from the induced pole cells. Germ-line mosaicism in those flies also could be detected histochemically in their gonads. No germ cells were recovered with polar plasm transplants from oogenetic stages 10 to 12.The results show that posterior polar plasm of the unfertilized egg is functional in germ-cell determination, and that prior to egg maturation this cytoplasm has already acquired its determinative ability. This is the first demonstration that specific developmental information stored in the cytoplasm can be traced back to a particular region of the oocyte.