The parallel actin filament bundling protein Fascin is a critical protein in both disease and development. Overexpression of Fascin is linked to increased aggressiveness in a number of cancer types, including breast and colon carcinomas. Importantly, Fascin is not normally expressed in adult epithelial cells from which many of these cancers arise. Therefore, Fascin is increasingly cited as both a potential biomarker and therapeutic target in many types of cancer. Fascin is most commonly associated with the formation of filopodia and invadapodia (parallel actin filament bundle structures) to drive migration and invasion. However, Fascin activity and regulation remain poorly understood. In order for Fascin to be an effective target for cancer therapeutics, a better understanding of the mechanisms regulating Fascin activities in the cell is necessary. Prostaglandins (PGs) are short-lived lipid signaling molecules that mediate a wide range of biological activities. PGs act through G protein-coupled receptors to initiate signaling cascades that affect downstream targets, including actin cytoskeletal remodeling. Importantly, the key enzymes in the synthesis of PGs, cyclooxygenase (COX) 1 and 2, are the targets of non-steroidal anti-inflammatory drugs like aspirin. Interestingly, like Fascin, PGs have been independently implicated in cancer development and metastasis and aspirin may reduce the risk of aggressive cancer. However, the exact mechanisms by which PGs mediate cancer development are unknown. The work presented in this thesis focused on novel PG-dependent regulation and activity of Fascin. The research presented here utilized Drosophila oogenesis as a model system to analyze PG-dependent Fascin activity. Drosophila oogenesis is an ideal model in which to study the activity and regulation of actin binding proteins like Fascin. Oogenesis consists of 14 morphologically defined stages, which are observable many times over within a single isolated pair of ovaries. A developing follicle consists of 16 germline cells – 15 nurse, or support cells, and a single oocyte. The nurse cells are of particular interest because they are the sites of dynamic actin remodeling during mid-late oogenesis. During stage 10B, an array of radially-aligned actin filament bundles form at the nurse cell membranes and extend inwards towards the nucleus. A network of cortical actin is also strengthened during this stage. These actin structures are essential for the completion of oogenesis, and ultimately female fertility. Importantly, PGs and Fascin are required for this actin remodeling; genetic loss of Fascin or the Drosophila COX-like enzyme Pxt (Peroxinectin-like) leads to disruption of cytoplasmic actin remodeling, and ultimately, female sterility. Using this model system, work presented here describes the discovery of Fascin as a downstream target of PGs to promote actin bundle formation, described in Chapter 2. Additionally, Fascin is required for strengthening of the cortical actin network downstream of PGs. This observation is one of the first to describe a role for Fascin in a branched actin network. Additionally, Fascin is regulated by a specific PG – PGF2α – during S10B to promote follicle development. Finally, Chapter 2 shows that PGs target specific actin binding proteins to promote cytoskeletal remodeling; Villin, another actin bundling protein, does not interact with PGs. Chapter 3 describes the novel observation that Fascin localizes to the nucleus and the nuclear periphery in Drosophila nurse cells. This finding is significant, as it is the first to describe Fascin in a context other than cytoplasmic. Fascin localization in and around the nucleus is specific and dynamic, and changes throughout late stage oogenesis, suggesting regulated functions at these sites. Fascin localization is regulated by PGs, and loss of Pxt leads to reduced nuclear Fascin localization and failure to localize to the nuclear periphery. Additionally, Fascin has novel potential functions in the nucleus and at the nuclear periphery. Loss of Fascin leads to disruption of nucleolar morphology in the nurse cell nuclei. Additionally, loss of PGs, which cause reduced nuclear Fascin levels, also causes abnormal nucleolar morphology. These data suggest that PGs regulate Fascin to control nucleolar organization. At the nuclear periphery, Fascin localization requires components of the protein complex that links the nucleoplasm to the cytoplasm, termed the LINC complex. Loss of an essential LINC complex protein, Koi, leads to a loss of nuclear periphery Fascin localization. These data suggest that Fascin may be a novel component of the LINC complex. Finally, Chapter 4 describes regulation of Fascin by phosphorylation at conserved serine residues. PGs affect Fascin phosphorylation, and loss of PGs leads to more heavily phosphorylated Fascin. Additionally, phosphorylation of Fascin alters localization to the nucleus and to the nuclear periphery. These data suggest that one mechanism by which PGs regulate Fascin is to control its phosphorylation status to affect subcellular distribution. In summary, the work presented in this thesis has demonstrated novel regulation and function of the actin bundling protein Fascin using Drosophila oogenesis as a model. Importantly, these functions and regulation of Fascin are likely conserved in mammals, and may have implications in human health and disease. Continued study of the activity and regulation of actin binding proteins like Fascin in Drosophila will likely have great effect on our understanding of many human diseases.

Prostaglandins (PGs) are small, lipid signaling molecules produced downstream of cyclooxygenase (COX) enzymes. PG signaling regulates many processes including pain, inflammation, fertility, cardiovascular function and disease, and cancer. One mechanism by which PG signaling exerts its function is by regulating the dynamics of the actin cytoskeleton; however, the exact mechanisms remain largely undefined. Drosophila oogenesis provides an ideal system to determine how PG signaling regulates the actin cytoskeleton. Drosophila follicles, or eggs, pass through 14 well- characterized, morphologically defined stages of development. Each developing follicle is comprised of 16 interconnected germline-derived cells (15 nurse cells and 1 oocyte) that are surrounded by a layer of somatically derived epithelial cells. During Stage 10B (S10B), the nurse cells form a cage-like network of parallel actin filament bundles that extend from the nurse cell membranes inward, toward the nurse cell nuclei. During Stage 11 (S11), the nurse cells rapidly transfer their cytoplasmic contents into the oocyte in an actomysoin-dependent contraction termed nurse cell dumping. Previous work uncovered that the Drosophila COX-like enzyme, Peroxinectin-like (Pxt), and thus PG signaling, is required to promote both actin filament bundle formation during S10B and subsequent nurse cell dumping. This finding established Drosophila oogenesis as a genetically tractable model in which to elucidate the conserved mechanisms underlying PG- dependent actin remodeling. The research presented in this dissertation focused on identifying actin-binding proteins that are regulated by PG signaling during Drosophila oogenesis. To identify these downstream effectors, we performed a dominant modifier screen to uncover factors that could suppress or enhance the ability of COX inhibitors to block nurse cell dumping in vitro. This screen revealed a number of actin-binding proteins that enhance the dumping defects caused by COX-inhibition, including the actin bundling protein, Fascin (Drosophila Singed, Sn); the actin filament elongation factor, Enabled (Ena); and the actin filament capper, Capping protein (Drosophila Capping protein alpha, Cpa, and beta, Cpb). Through a collaborative effort between Christopher Groen and myself, Fascin was shown to mediate PG-dependent cortical actin integrity and actin bundle formation during Drosophila ooogenesis. Ena and Capping protein regulate actin filament elongation through opposing actions: Ena promotes their elongation, while Capping protein binds to, or caps, the growing end of actin filaments to prevent their further elongation. However, genetic reduction of either Ena or Capping protein enhance the nurse cell dumping defects caused by COX inhibition. These findings suggest that Ena activity must be balanced to promote proper actin remodeling during S10B. Ena localization to the growing ends of actin filament bundles is reduced in pxt mutants during S10B, suggesting that PG signaling is required to promote Ena localization at this stage. Together, these data support a model in which PG signaling promotes actin remodeling during S10B, at least in part, by modulating Ena-dependent actin remodeling. While PG signaling promotes parallel actin filament bundle formation during S10B, PGs also restrict actin remodeling during Stage 9 (S9). Loss of Pxt results in early actin remodeling, including the formation of extensive actin filaments and actin aggregate structures within the posterior nurse cells of S9 follicles. Wild-type follicles exhibit similar structures at a low frequency. Ena preferentially localizes to the early actin structures observed in pxt mutants and reduced Ena levels strongly suppress early actin remodeling in pxt mutants. These data indicate that PG signaling temporally restricts actin remodeling during Drosophila oogenesis, at least in part, through negative regulation of Ena localization or activity during S9. The data presented here support a model in which PG signaling coordinates the concerted activity of a number of actin-binding proteins to regulate actin remodeling during Drosophila oogenesis. Specifically, PG signaling temporally restricts actin remodeling during S9 of Drosophila oogenesis, but promotes parallel actin filament bundle formation during S10B. PG signaling achieves this temporal regulation, at least in part, through differential regulation of Ena-dependent actin remodeling. Based on prior pharmacologic studies, we hypothesize that PGE2 is required to restrict Ena-dependent actin remodeling during S9, while PGF2Α; is required to promote Ena-dependent actin remodeling during S10B. Determining how these signaling cascades achieve differential regulation of Ena throughout Drosophila oogenesis is an important area for future investigation. As both the actin cytoskeletal machinery and PG signaling are conserved across species, the data presented here provide new and significant insights into the likely conserved mechanisms by which PG signaling regulates actin remodeling across species.

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a multi-zinc finger DNA binding factor required for the gypsy insulator function. At the gypsy element, Su(Hw) recruits partners Centrosomal Protein of 190 kD (CP190) and Modifier of mdg4 67.2 kD isoform (Mod67.2), which facilitate the enhancer blocking and barrier functions of the insulator. Our genome-wide studies have identified thousands of endogenous non-gypsy Su(Hw) binding sites (SBSs) in Drosophila genome, constitutively occupied throughout development. Yet, only a third of SBSs associate with CP190 and Mod67.2, suggesting that the endogenous function of Su(Hw) may not necessarily involve formation of a gypsy-like chromatin insulator. To understand the function of endogenous SBSs, we investigated the requirement for Su(Hw) during female germline development. To this end, we performed genome-wide transcriptional analyses in su(Hw) mutant ovaries coupled with the genome-wide definition of ovary SBSs. We identified 49 direct targets of Su(Hw) regulation in the ovary, with 80% of these genes showing increased RNA accumulation when Su(Hw) is lost. Derepressed Su(Hw) targets are normally highly expressed in central nervous system, suggesting that Su(Hw) has a critical role in silencing neural genes in the non-neural tissues. We find that a single upregulated target gene is largely responsible for the female sterility of the su(Hw) mutants. This gene encodes an elav family factor RNA binding protein 9 (Rbp9), and Su(Hw)-mediated repression of Rbp9 is required at a specific stage of germline development. Decreasing the levels of Rbp9 restores female fertility of su(Hw) null mutants. Further, we demonstrate that Su(Hw) is required for transcriptional activation of pointed, gene involved in eggshell patterning during late embryogenesis. Importantly, both CP190 and Mod67.2 are dispensable for Rbp9 regulation, indicating that mechanism of Rbp9 repression is independent of the insulator function of Su(Hw). Our studies extend the known transcriptional activities of Su(Hw), indicating that it can function as an insulator, activator and repressor, the latter function being essential for oogenesis. These findings highlight that insulator proteins are versatile transcriptional regulators, suggesting that tissue specific contributions to transcription result from direct regulation of individual genes.

Abstract not available

Understanding the genetic control of gametogenesis is a central goal of developmental biology and is important for treating infertility in humans. An approach to identifying critical genes in mammals is to search for and study homologues of genes known to play key roles in other organisms. In the fly, Drosophila melanogaster, GUS protein is a component of nuage, an electron-dense aggregation in early germ cells, and is required for oocyte development. GUS physically interacts with VASA, an RNA helicase thought to regulate mRNA metabolism. I identified two murine genes, SSB-1 and SSB-4, that are similar to and likely homologues of gus. SSB-1, SSB-4 and GUS each contain two conserved regions, termed the SPRY domain and the SOCS box, respectively. SSB-1 and SSB-4 share about 75% sequence identity and about 70% identity with GUS. Both SSB-1 and SSB-4 RNA and protein were found to be express in mouse ovarian granulosa cells of all stages of folliculogenesis. These cells support oocyte development and also produce steroids. Unexpectedly, SSB-1 and SSB-4 were only weakly or not detectable in oocytes, that contrasts with the expression of GUS in Drosophila oocytes. However, SSB-1 mRNA and protein were expressed in male germ cells; specifically in spermatocytes and spermatids. SSB-1 in spermatids was localized in a specialized structure known as the chromatoid body. Although the function of this structure is not quite clear, it has been compared to nuage, and one of its components is MVH, the murine homologue of VASA. Finally, using RNAi technology, SSB-1 was transiently depleted SSB-1 from a granulosa cell line. These cells showed a transient decrease in expression of the gene encoding P450scc, the rate-limiting enzyme in steroid synthesis. Preliminary results also indicated a decrease in progesterone synthesis. Taken together, these results establish the expression pattern of murine homologues of Drosophila GUS in mouse ovary and testis, reveal it might play function in translation regulation in male spermatogenesis, and identify a potential role in steroidogenesis by ovarian granulosa cells.

Progestagenic compounds are emerging contaminants found in surface and ground water around the world. Information on the effects and potency of progestagens is needed in order to understand the environmental risks posed by these compounds. Using the Xenopus (Silurana) tropicalis test system, developmental and reproductive toxicity after exposure to selected progestagens were determined. Larval exposure to levonorgestrel (LNG) severely impaired oviduct and ovary development causing sterility. No effects on testicular development, spermcount or male fertility were observed. Hepatic mRNA expression of the androgen receptor was increased in the females indicating that the receptor is involved in LNG-induced developmental reproductive toxicity. Exposure of adult females to LNG, norethindrone (NET) or progesterone (P) increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes compared with the controls, indicating an inhibited vitellogenesis. The effects on oocyte development were ascertained at environmentally relevant concentrations of LNG, NET and P (1.3, 1 and 10 ng/L respectively). Since unintentional co-exposure of progestagens and ethinylestradiol (EE2) occurs in wildlife and also in human infants, data on mixture effects of combined exposures to these hormones during development are needed. Co-exposure during development showed antagonistic effects of EE2 and LNG. EE2 caused a female biased sex ratio which showed a tendency to be antagonized by LNG. Moreover, the hepatic AR induction by LNG was counteracted by co-exposure to EE2. In conclusion, the results show that female amphibians are susceptible to reproductive toxicity of progestagens after developmental exposure as well as after adult exposure during the breeding period. The differentiating Müllerianduct and ovary, and the egg development are sensitive targets for progestagens. Finally, the findings reported in this thesis show that environmental progestagens impairs reproductive function in amphibians and may present a threat to reproduction in wild populations.

In most animals, oogenesis involves a syncytial “cyst” stage. Cysts are produced by incomplete mitotic divisions of gonial precursor cells, leaving the resulting cystocytes interconnected by cytoplasmic bridges. The bridges subsequently break down, liberating the developing gametes. In some animals (e.g. meroistic insects) cysts are “polarized”, such that certain cystocytes differentiate as supportive nurse cells, rather than oocytes. The variability of cysts in animal oogenesis contrasts with the relative universality of spermatogenic cysts, making the functional importance of cysts in oogenesis unclear. I have studied oogenesis in a polychaete worm, Ophryotrocha labronica (Annelida: Dorvilleidae). These worms produce polarized, two-celled oogenic cysts with one nurse cell and one oocyte. Such cysts resemble their better-characterized counterparts in meroistic insects. However, using a variety of light- and electron-microscopic techniques, I show here that the resemblance between O. labronica and meroistic insects is largely superficial. Rather, the roles of nurse cells and the mechanisms underlying cystocyte differentiation are quite distinct in both groups. Therefore, similarities between these polychaetes and insects are probably examples of convergent evolution rather than homology. These observations underscore the plasticity of oogenesis among animals. Mechanisms by which germ cells become distinct from somatic cells in animals are also a subject of considerable research activity. Two general modes of germ-cell specification have been described in animals: deterministic specification, which is typical of established model species (e.g., Drosophila melanogaster and Caenorhabditis elegans) and inductive specification, which, though it is the more-common mode among animals, has not been well studied. As an annelid worm, O. labronica likely specifies its germ cells inductively, and therefore has potential to serve as a model species for studies of inductive germ cell specification. Realizing this potential, however, will require the development of genetic resources for this species. I describe the beginnings of such work here: the isolation and characterization of a vasa/PL10-like gene whose expression is largely restricted to germ cells, the construction of a cDNA library, and the refinement of methods for in situ hybridization and immunostaining to visualize gene expression in whole worms.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the cell cycle proceeds. The Chk2 family of kinases plays a central role in mediating responses to DNA damage or DNA replication blocks in various organisms. My functional analysis of the Drosophila serine/threonine kinase Loki/Chk2 shows that fly chk2 monitors double-strand breaks caused by irradiation during S and G2 phases and induces cell cycle arrest in embryonic cells around cellularization. / loki is also required for the normal number of germ line cells to form in the embryo, and for normal modification of Vasa, a crucial factor in germ cell formation. However, during normal oogenesis loki expression is suppressed by orb. Another group described the involvement of Drosophila loki/chk2 in the meiotic pachytene checkpoint. Using our loki·null mutant, I obtained the opposite result: loki/chk2 does not have an essential function in this process. / The second part of my thesis deals with the question of how cells are instructed about their identity in a developing organism. (Abstract shortened by UMI.)

In most animals, oogenesis involves a syncytial “cyst” stage. Cysts are produced by incomplete mitotic divisions of gonial precursor cells, leaving the resulting cystocytes interconnected by cytoplasmic bridges. The bridges subsequently break down, liberating the developing gametes. In some animals (e.g. meroistic insects) cysts are “polarized”, such that certain cystocytes differentiate as supportive nurse cells, rather than oocytes. The variability of cysts in animal oogenesis contrasts with the relative universality of spermatogenic cysts, making the functional importance of cysts in oogenesis unclear. I have studied oogenesis in a polychaete worm, Ophryotrocha labronica (Annelida: Dorvilleidae). These worms produce polarized, two-celled oogenic cysts with one nurse cell and one oocyte. Such cysts resemble their better-characterized counterparts in meroistic insects. However, using a variety of light- and electron-microscopic techniques, I show here that the resemblance between O. labronica and meroistic insects is largely superficial. Rather, the roles of nurse cells and the mechanisms underlying cystocyte differentiation are quite distinct in both groups. Therefore, similarities between these polychaetes and insects are probably examples of convergent evolution rather than homology. These observations underscore the plasticity of oogenesis among animals. Mechanisms by which germ cells become distinct from somatic cells in animals are also a subject of considerable research activity. Two general modes of germ-cell specification have been described in animals: deterministic specification, which is typical of established model species (e.g., Drosophila melanogaster and Caenorhabditis elegans) and inductive specification, which, though it is the more-common mode among animals, has not been well studied. As an annelid worm, O. labronica likely specifies its germ cells inductively, and therefore has potential to serve as a model species for studies of inductive germ cell specification. Realizing this potential, however, will require the development of genetic resources for this species. I describe the beginnings of such work here: the isolation and characterization of a vasa/PL10-like gene whose expression is largely restricted to germ cells, the construction of a cDNA library, and the refinement of methods for in situ hybridization and immunostaining to visualize gene expression in whole worms.

We report the cloning and characterization of valois (vls), a posterior group gene of Drosophila melanogaster , which was initially identified in a screen for female steriles. Three EMS alleles of vls contain premature stop codons in the open reading frame. Sequence analyses show the presence of WD domains in Vls and find significant similarity with the human MEP50 protein which is involved in the assembly of the splicing machinery. We did not find evidence that this function is conserved in flies yet. / We created a null mutant for vls, which shows a maternal effect lethal phenotype accompanied by posterior polarity defects in the embryos. Hemizygous vlsEMS females show a weaker, partially maternal-effect lethal and a fully penetrant grandchildless phenotype. The posterior localization of Vasa is disrupted in vlsnull ovaries, but the initial distribution of Oskar protein and mRNA appear normal. However, levels of the Short Oskar isoform responsible for pole plasm assembly are greatly reduced and Vasa appears to be differently modified post-translationally. Furthermore, a Vls::GFP fusion protein is detected all throughout oogenesis in the nurse cell and oocyte cytoplasm. Taken together, these data suggest that Vls is a cytoplasmic protein involved in the transport or activation of Vasa at the posterior of the oocyte essential for the accumulation of Short Osk.