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This research was designed to gain a better understanding of the oogenesis of the autogenous black-fly, Simulium vittatum and to compare it with studies on other Diptera. The autogenous character in S. vittatum showed only for the first gonadotropic cycle. Since the cytoblasts are first produced in the late larval period, the late larvae, early pupae, late pupae and adults of various ages were prepared separately for histological and histochemical examination. The adult female flies were fed only sugar and water. The changes in size, histology, and histochemistry of the oocyte, follicular cells and nurse-cells during oogenesis were described and the possible physiological role of these cells in oogenesis suggested. The amount and composition of larval fat-body remaining in females was compared with the stages of the oocyte development and the age of the flies. / Thesis / Master of Science (MS)

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Master of Science

The JAK-STAT pathway is an intracellular signaling pathway that is found to have crucial roles in hematopoiesis, immune response and the development of many other tissues in mammals. The pathway is conserved in Drosophila melanogaster, and is much simpler: there is only one Drosophila JAK (Hopscotch, Hop) and STAT (STAT92E) respectively, while there are at least 4 JAKs and 7 STATs in mammals. The pathway has been intensively studied in Drosophila, and has been implicated in many tissue development and cellular processes. In this work, I present several roles of JAK signaling in oogenesis.First, JAK signaling is required for cell differentiation within a specific lineage of follicle cells – stalk cells and polar cells. Unpaired (upd), which encodes the known ligand for the pathway, is expressed specifically in the polar cells in the developing egg. Reduced function of Upd or Hop results in fusions of egg chambers, which is primarily caused by improper formation of stalk cells, while general activation of the pathway in the egg chamber produces an extra number of stalk cells and sometimes eliminates polarfollicle cells. Based on the known function of the Notch pathway in oogenesis, we propose a model that Notch signaling determines a pool of precursors for the polar and stalk cells while JAK activity determines their specific fates within that pool.Second, JAK signaling is also involved in epithelial follicle cell differentiation. Consistent with the expression pattern of upd in the ovary, there is a gradient of JAK activity expanding from the poles, and this JAK activation gradient is both required and sufficient to suppress the main body follicle cell fate. Also, different levels of JAK activity are required and sufficient to determine both anterior and posterior terminal follicle cell fates. Consistent with these data is a model that a gradient of JAK activity triggered by Upd from the poles pre-patterns the epithelium into three domains and pre-determines sub-populations of terminal follicle cell fates prior to the EGFR activation, and cooperates with EGFR activity later to define posterior terminal follicle cell fates. This provides the first evidence for a morphogenic function of the JAK-STAT pathway in any organism.

Cell death is a key component in development and for the continued renewal of tissues. Phagoptosis is a process in which phagocytes directly lead to the death of other cells. This process of cell death is significantly less characterized when compared to other mechanisms of cell death, such as apoptosis. In the Drosophila ovary, phagoptosis appears to play a key role in the developmental process of oogenesis. Recent studies have shown that genes associated with phagocytosis are required for the programmed death of nurse cells in the Drosophila ovary. Ovaries are made up of 15 nurse cells, a single oocyte, and a layer of follicle cells bordering them. During the process of egg chamber development, all of the nurse cells undergo programmed cell death. During late oogenesis, each nurse cell is surrounded by a group of follicle cells referred to as stretch follicle cells. These stretch follicle cells have recently been implicated as a main promoter of nurse cell phagoptosis. However, an exact mechanism to explain how these stretch follicle cells induce nurse cell death is not fully characterized. To achieve a more detailed understanding of this mechanism, we are examining the function of the cytoskeleton in this process via live imaging. We hypothesize that the follicle cell cytoskeleton plays a significant role in nurse death due to the importance of actin during phagocytosis. Further, we intend to use these live imaging studies to investigate the role of calcium before, during, and after clearance of the nurse cells. Previous studies have shown that calcium bursts within the cell are associated with the initiation of phagocytosis in macrophages, as well as other phagocytic cell types. Studies in this thesis were done by utilizing live imaging and have shown dynamic changes in follicle cell actin before and during the death of nurse cells. These confocal microscopy real time videos have revealed that follicle cell actin polymerizes towards the nurse cell immediately before acidification. Following acidification of the nurse cells, the follicle cell actin changes direction, moving towards the phagocytic follicle cell. Additionally, through live imaging we have observed calcium bursts in the follicle cells immediately before nurse cell death. Overall, this work has provided a more detailed understanding of nurse cell death.

In the fruit fly, Drosophila melanogaster, accumulation of osk mRNA at the posterior pole of the oocyte and local translation initiate assembly of the pole plasm, which is required for germ cell formation and posterior patterning of the embryo. I have used fluorescence in situ hybridization (FISH) in combination with immunofluorescence and laser scanning confocal microscopy to examine the spatial and temporal control of osk transcript localization and translation. Drosophila oocytes develop within cysts of 16 interconnected cells. One cell in each cyst differentiates to form the oocyte while the remaining cells form nurse cells that produce RNAs and proteins that are transported to the oocyte. osk mRNA is produced by the nurse cells and accumulates in the oocyte throughout oogenesis, but is only specifically localized to the posterior pole and translated during mid to late oogenesis. My studies help define distinct steps in the osk mRNA localization process. An early step in posterior localization is removal of osk mRNA from most of the cortex, leading to accumulation in the oocyte interior. This process requires microtubules, the microtubule motor protein Kinesin I, the actin binding protein Tropomyosin, and the RNA binding protein Staufen. Transcript then moves from the oocyte interior to the posterior pole through a microtubule independent process. The genes cappuccino, chickadee, spire, armitage, maelstrom, par-1 and gurken are all required for this next step in osk mRNA localization. The final capturing or tethering osk mRNA at the cortex requires an intact actin filament system, but additional components of this anchoring system remain to be identified. I also find that osk mRNA first begins to accumulate at the posterior pole during oogenesis stage 8, but protein is not detectable until stage 9. In addition, grk and par-1 mutations that block osk mRNA localization to the posterior pole and lead to transcript accumulation in the interior do not prevent translation; again, Osk protein production is only observed during stage 9 and later. These observations indicate that posterior localization is neither sufficient nor necessary to trigger osk mRNA translation, which appears to be under tight temporal control.

Suppressor of Hairy-wing [Su(Hw)] is a twelve zinc-finger (ZF), DNA binding transcription factor. Su(Hw) has been well characterized as critical component of the gypsy insulator complex, required for the enhancer blocking and the barrier activity of the insulator. In addition to gypsy, Su(Hw) localizes to ~3,000 binding sites in the Drosophila genome, with association to a subset of sites required for female germline development. Loss of Su(Hw) results in activation of a developmental checkpoint and apoptosis at mid- oogenesis, with a critical role during oogenesis in down-regulation of neural genes. Studies of Su(Hw) function have identified transcriptional activator, repressor and insulator roles at distinct binding sites. Current investigations aim to understand the factors that dictate the regulatory output of Su(Hw) at individual sites in the Drosophila genome, with a focus on the ZF domain. A genetic screen was completed to generate novel mutations in su(Hw). After screening more than 8,000 mutagenized chromosomes, we identified four new su(Hw) alleles, including two deletion mutations and two amino acid substitutions disrupting individual ZFs (ZF4 and ZF8). Studies of the ZF4 mutant, Su(Hw)M4M393, revealed that Su(Hw) requires this ZF for female fertility, but notgypsy insulator function. To achieve a comprehensive understanding of the Su(Hw) ZF domain, we generated Su(Hw) mutant proteins carrying disruptions in individual ZFs. Analyses utilizing these proteins have defined the requirement for each ZF in DNA association in vitro. To complement extant ZF alleles, Su(Hw) ZF mutants were expressed in vivo. Analyses of these mutants established how each ZF contributes to SBS occupancy, gypsy insulator function and female fertility. Gene expression and ChIP analyses suggest that some Su(Hw) ZFs may execute roles apart from direct DNA recognition. Genome-wide binding analyses of Su(Hw)M4M393, combined with previous studies, found that the SBS binding motif contains three DNA sequence cores (termed upstream, central and downstream). Analyses of these sequence cores in combination with Su(Hw) ZF mutants have outlined which ZFs associate with each core. Interestingly, the class containing all three sequence cores represents high occupancy SBSs that are enriched for protein factors from functional classes including transcriptional repression, nucleosome remodeling and DNA replication. The class containing the upstream and central core correlates with insulator function, while the class containing the central and downstream cores correlates with activation or repression of Su(Hw) target genes. Finally, in vitro studies of Su(Hw) ZF mutants revealed a DNA bound conformation distinct from wild type Su(Hw). Su(Hw) is a versatile transcription factor able to act as an insulator, activator and repressor. Analyses of SBSs with these functions suggest that DNA sequence, ZF usage, protein partnership and Su(Hw) conformation, combine to dictate regulatory output. Together, these studies provide insight into how discrete ZFs contribute to the roles of a multifunctional, polydactyl transcription factor.