I have investigated the mechanisms by which developmental asymmetry arises, using oocyte determination in Drosophila melanogaster as a model system. The Bicaudal-D (Bic-D) gene is required early in oogenesis for the asymmetric localization of specific mRNAs and proteins and for the differentiation of an oocyte from one of a cluster of 16 interconnected germarial cells. To better understand how Bic-D functions in creating this asymmetry, I took two approaches. First, I examined the role of Bic-D in the asymmetric localization of mRNA and other cellular components during later oogenesis. Second, I molecularly and genetically characterized a gene that interacts with Bic-D in oocyte determination. To determine the role of Bic-D in later oogenesis, I used an inducible source of Bic-D activity to selectively rescue the block at oocyte determination in Bic-D null mutants. Using this system, I find that Bic-D is indeed required in the later stages of oogenesis for the localization of specific mRNAs at both the anterior and posterior of the oocyte. Bic-D is also required for oocyte growth and nuclear positioning, processes which also depend on microtubules. / In the second part of this thesis, I describe the characterization of a Bic-D interacting gene which I have identified as the Drosophila homologue of the human Lissencephaly-1 (Lis-1) gene, DLis-1. Human Lis-1 is the causative gene for Miller-Dieker Syndrome and is required for neuronal migration in the developing brain, while fungal homologues have been implicated in dynein dependent nuclear migration. Like Bic-D , DLis-1 is essential for oocyte determination and for intracellular localization throughout oogenesis. DLis-1 is required for correct positioning of the oocyte nucleus, and appears to function upstream of dynein in this process. Immunolocalization studies suggest that DLis-1 functions as part of a cortical anchor that links microtubules and the oocyte nucleus, via dynein and microtubules, to the cell cortex. DLis-1 and Bic-D are also required for nuclear positioning during neural development in Drosophila, supporting a model in which the neuronal migration defects in Miller-Dieker Syndrome are due to a disruption of dynein/Lis-1 dependent nuclear migration.

loki was identified in our lab in a screen for novel serine-threonine protein kinases that are specifically expressed in the ovary. loki transcripts are expressed in early embryos and adult ovaries. In the ovaries, loki transcripts accumulate in the oocyte during early oogenesis, and by stage 8, are found to localize anteriorly to the oocyte-nurse cell interface. In order to understand the role of loki in Drosophila development, I created a loki mutant through excision of a P-element that had inserted 700bp upstream of loki. loki mutants display no apparent phenotype. / I was also able to identify the transcription unit responsible for valois (vls) function since it is located downstream from loki in the 38B region. vls is a member of the posterior group gene family and vls mutant mother produce embryos that lack pole cells and show abdomen patterning defects. Further characterization of vls indicates that it is a fairly direct activator of Vasa protein: vls function is required between Oskar protein and Vasa in the posterior patterning pathway since Vasa fails to localize to the posterior pole of vls mutant eggchambers. I also find that Vasa is differentially modified in vls ovaries and embryos, even though it is not yet known whether this modification is required for localization, proper function or both processes.

The Janus kinase (JAK) pathway is an integral part of signaling through a variety of ligands and receptors in mammals. The extensive reutilization and pleiotropy of this pathway in vertebrate development is conserved in other animals as well. In Drosophila melanogaster, JAK signaling is involved in embryonic pattern formation, sex determination, larval blood cell development, wing venation, planar polarity in the eye, and formation of other adult structures. Here we describe several roles for JAK signaling in Drosophila oogenesis. The gene for a JAK pathway ligand, unpaired, is expressed specifically in the polar follicle cells, two pairs of somatic cells at the anterior and posterior poles of the developing egg chamber. A primary defect of chambers with reduced JAK activity is fusion of successive chambers. These chambers exhibit an expansion of the polar cell population and concomitant loss of interfollicular stalk cells. Mosaic analysis of both JAK pathway transducers, hopscotch and stat92E, reveals that JAK signaling is specifically required in the somatic follicle cells. Another role of JAK signaling is in oocyte localization. In chambers mosaic for loss of hop activity, oocyte mislocalization results. Proper localization occurs only when the posterior follicle cells are wild type for hop.

The goal of the work presented here was to investigate the hypothesis that cytoplasmic SLBP is required for translation of somatic H1 mRNA, and their translational repression is due to a lack of SLBP in the cytoplasm of oocytes, and early cleavage-stage embryos. To this end, the expression of SLBP in murine oocytes and pre-implantation embryos was characterized by RT-PCR, Western blotting, and immunocytochemical. techniques. / mRNA encoding SLBP was detected throughout oogenesis and pre-implantation development, from small growing oocytes to the late blastocyst stage. SLBP protein was found in the nucleus and cytoplasm of growing and fully-gown prophase I-arrested oocytes. SLBP accumulated to extremely high levels during meiotic maturation in a process requiring translation. The protein remained abundant both in the nucleus and cytoplasm throughout the 1- and 2-cell stages. SLBP was depleted in 4-cell embryos in a process independent of DNA replication, and was not detected again until the late 8-cell stage. From the late 8-cell stage to the early blastocyst stage, SLBP was detected exclusively in the cytoplasm. Interestingly, in late blastocysts, SLBP was translocated to the nucleus. (Abstract shortened by UMI.)

At fertilization, the remodelling of the sperm nucleus into the male pronucleus is critical for normal development. Morphological and functional changes to the nucleus are underpinned by biochemical changes in the chromatin composition, most notably the removal of sperm specific protamines and assembly of histones onto the paternal DNA. This exchange is controlled by oocyte factors, as exemplified in Xenopus by nucleoplasmin. Though mammalian factors remain unidentified, a functional assay based on antibodies recognizing core histones has been developed to test the activity in oocytes that transfers histones onto sperm DNA, named histone transfer activity (HTA). The assay was applied to growing and maturing murine oocytes to determine when during oogenesis HTA develops, and to probe potential regulatory mechanisms. Fully-grown oocytes develop HTA upon maturation, in a protein-synthesis dependent manner. Large, growing oocytes also develop HTA upon entry into M-phase. Small growing meiotically incompetent oocytes, ones that do not spontaneously enter M-phase, do not develop HTA, though this can be overcome by culture of oocytes to meiotic competence, or by treatment with strontium to induce intracellular calcium oscillations. Taken together these findings form a model of how HTA develops throughout oogenesis. Finally, an attempt is made to identify a potential mammalian HTA factor. Transcripts for two remodelling factors, mNAP and Npm3, are identified in the murine oocyte, and injection of anti-sense oligonucleotides reveals that Npm3 plays a significant role in the deposition of histories and the remodelling of sperm chromatin at fertilization. Combined with the findings of the HTA assay, the data forms a testable model of how Npm3 may be regulated throughout oogenesis.

Thesis (Ph.D.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR29321

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on June 19, 2009) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Illinois State University, 1978. / Title from title page screen, viewed Jan. 24, 2005. Dissertation Committee: Fritz Schwalm (chair), Herman Brockman, Harry Huizinga, Mathew Nadakavukaren, Arlan Richardson. Includes bibliographical references (leaves 96-105) and abstract. Also available in print.

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