Oogenesis in Drosophila melanogaster: a model system for studying cell differentiation and development

Drosophila oogenesis is a complex developmental process involving the coordinated differentiation of germ line and somatic cells. Correct execution and timing of cell fate specification and patterning events is achieved during this process by the integration of different cell-cell signalling pathways, eventually leading to the generation of positional information inside the oocyte, that is instrumental for the establishment of embryonic polarity. The large body of data accumulated at both cellular and molecular levels in the last decade clearly demonstrated how Drosophila oogenesis is a genetically tractable system particularly suited for the investigation of key developmental biology questions. Our recent contribution to the field relies on the characterisation of three different mutants named tegamino (teg), hold hup (hup) and tulipano (tip), identifying novel gene functions required during oogenesis. Specifically, teg is implicated in the morphogenesis of the follicular epithelium surrounding the germ line cells in the egg chamber, hup is involved in the establishment of egg chamber polarity and tip in the regulation of the dynamic germ cell chromatin organisation.     

Histone methylation is required for oogenesis in Drosophila

SET domain proteins are histone lysine methyltransferases (HMTs) that play essential roles in development. Here we show for the first time that histone methylation occurs in both the germ cells and somatic cells of the Drosophila ovary, and demonstrate in vivo that an HMT, the product of the eggless (egg) gene, is required for oogenesis. Egg is a SET domain protein that is similar to the human protein SETDB1 and its mouse ortholog ESET. These proteins are members of a small family of HMTs that contain bifurcated SET domains. Because depletion of SETDB1 in tissue culture cells is cell-lethal, and an ESET mutation causes very early periimplantation embryonic arrest, the role of SETDB1/ESET in development has proven difficult to address. We show that egg is required in the Drosophila ovary for trimethylation of histone H3 at its K9 residue. In females bearing an egg allele that deletes the SET domain, oogenesis arrests at early stages. This arrest is accompanied by reduced proliferation of somatic cells required for egg chamber formation, and by apoptosis in both germ and somatic cell populations. We propose that other closely related SET domain proteins may function similarly in gametogenesis in other species.     

Profile of a mammalian sperm receptor

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.     

Physical interactions of Dmnk with Orb: implications in the regulated localization of Orb by Dmnk during oogenesis and embryogenesis.

The Dmnk (Drosophila maternal nuclear kinase) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. We report here that Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression pattern of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

The RNA-binding proteins PUF-5, PUF-6, and PUF-7 reveal multiple systems for maternal mRNA regulation during C. elegans oogenesis

In metazoans, many mRNAs needed for embryogenesis are produced during oogenesis and must be tightly regulated during the complex events of oocyte development. In C. elegans, translation of the Notch receptor GLP-1 is repressed during oogenesis and is then activated specifically in anterior cells of the early embryo. The KH domain protein GLD-1 represses glp-1 translation during early stages of meiosis, but the factors that repress glp-1 during late oogenesis are not known. Here, we provide evidence that the PUF domain protein PUF-5 and two nearly identical PUF proteins PUF-6 and PUF-7 function during a specific period of oocyte differentiation to repress glp-1 and other maternal mRNAs. Depletion of PUF-5 and PUF-6/7 together caused defects in oocyte formation and early embryonic cell divisions. Loss of PUF-5 and PUF-6/7 also caused inappropriate expression of GLP-1 protein in oocytes, but GLP-1 remained repressed in meiotic germ cells. PUF-5 and PUF-6/7 function was required directly or indirectly for translational repression through elements of the glp-1 3' untranslated region. Oogenesis and embryonic defects could not be rescued by loss of GLP-1 activity, suggesting that PUF-5 and PUF-6/7 regulate other mRNAs in addition to glp-1. PUF-5 and PUF-6/7 depletion, however, did not perturb repression of the maternal factors GLD-1 and POS-1, suggesting that subsets of maternal gene products may be regulated by distinct pathways. Interestingly, PUF-5 protein was detected exclusively during mid to late oogenesis but became undetectable prior to completion of oocyte differentiation. These results reveal a previously unknown maternal mRNA control system that is specific to late stages of oogenesis and suggest new functions for PUF family proteins in post-mitotic differentiation. Multiple sets of RNA-binding complexes function in different domains of the C. elegans germ line to maintain silencing of Notch/glp-1 and other mRNAs.     

Dynamic expression patterns of <Emphasis Type="Italic">vasa</Emphasis> during embryogenesis in the cricket <Emphasis Type="Italic">Gryllus bimaculatus</Emphasis>

The specification of germ cells during embryogenesis is an important issue in the development of metazoans. In insects, the mode of germ cell specification appears to be highly variable among species and molecular data are not sufficient to provide an evolutionary perspective to this issue. Expression of vasa can be used as a germ line marker. Here, we report the isolation of a vasa-like gene in a hemimetabolous insect, the cricket Gryllus bimaculatus (Gb'vas), and its expression patterns during oogenesis and embryogenesis. Gb'vas is preferentially expressed in the germarium and the expression of Gb'vas is detectable throughout vitellogenesis including mature eggs subjected to oviposition, suggesting that Gb'vas is maternally contributed to the cricket eggs. The zygotic expression of Gb'vas appears to start at the mid blastoderm stage in the posterior region of the egg, expanding in a developing germ anlage. In early germbands, an intense expression of Gb'vas is restricted to the posterior end. In later embryos, Gb'vas expression extends over the whole body and then distinctly localized to the embryonic gonad at the stage immediately before hatching. These results suggest that, in the cricket, germ cells are specified early in development at the posterior end of an early germband, as proposed by Heymons (1895) based on cytological criteria.     

Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila.

The steroid hormone ecdysone regulates larval development and metamorphosis in Drosophila melanogaster through a complex genetic hierarchy that begins with a small set of early response genes. Here, we present data indicating that the ecdysone response hierarchy also mediates egg chamber maturation during mid-oogenesis. E75, E74 and BR-C are expressed in a stage-specific manner while EcR expression is ubiquitous throughout oogenesis. Decreasing or increasing the ovarian ecdysone titer using a temperature-sensitive mutation or exogenous ecdysone results in corresponding changes in early gene expression. The stage 10 follicle cell expression of E75 in wild-type, K10 and EGF receptor (Egfr) mutant egg chambers reveals regulation of E75 by both the Egfr and ecdysone signaling pathways. Genetic analysis indicates a germline requirement for ecdysone-responsive gene expression. Germline clones of E75 mutations arrest and degenerate during mid-oogenesis and EcR germline clones exhibit a similar phenotype, demonstrating a functional requirement for ecdysone responsiveness during the vitellogenic phase of oogenesis. Finally, the expression of Drosophila Adrenodoxin Reductase increases during mid-oogenesis and clonal analysis confirms that this steroidogenic enzyme is required in the germline for egg chamber development. Together these data suggest that the temporal expression profile of E75, E74 and BR-C may be a functional reflection of ecdysone levels and that ecdysone provides temporal signals regulating the progression of oogenesis and proper specification of dorsal follicle cell fates.     

The phylogenetic origin of oskar coincided with the origin of maternally provisioned germ plasm and pole cells at the base of the Holometabola

The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program.     

DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes

DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups-eutherians, marsupials, and monotremes.     

mRNA localization patterns in zebrafish oocytes

In both invertebrate and vertebrate systems, the localization of maternal mRNAs is a common mechanism used to influence developmental processes, including the establishment of the dorsal/ventral axis, anterior/posterior axis, and the germ line (for review, see Bashirullah et al., 1998. Annu. Rev. Biochem. 67, 335-394). While the existence of localized maternal mRNAs has been reported in the zebrafish, Danio rerio, the precise localization patterns of these molecules during oogenesis has not been determined. In this study, in situ hybridization experiments were performed on zebrafish ovaries and activated eggs to examine different mRNA localization patterns. The results establish that while some maternal mRNAs remain ubiquitously distributed throughout the oocyte, other mRNAs follow specific localization patterns, including localization to the animal pole, localization to the vegetal pole, and cortical localization. The animal/vegetal axis is first apparent in stage II oocytes when the earliest mRNA localization is seen. Unique patterns of localization are seen in mature eggs as well. Some mRNAs maintain their oocyte localization patterns, while others localize upon egg activation (fertilization).