The purpose of this study was to begin a study characterizing the diapause state in Sarcophaga crassipalpis. Recent evidence suggests the diapause syndrome is a unique developmental state with its own set of genes expressed. Two cDNA libraries from the pupal brains of diapausing and nondiapausing S. crassipalpis were generated. These libraries will be the basis for future work determining the expressed and repressed genes during the diapause syndrome. The genetic regulation of diapause in S. crassipalpis is also of interest. This species enters diapause during metamorphosis, which is under regulation of the steroid hormone 20-hydroxyecdysone (20-HE). Because the metamorphic system is suspended for diapause in this species, whether or not the EcR is expressed in the diapause state is of interest. To address this issue, a partial EcR was cloned from S. crassipalpis by RT-PCR. This partial EcR was found in all developmental stages tested.

Systemic steroid hormone and intracellular signaling pathways are known to act cooperatively during the development of vertebrate and invertebrate epithelia. However, the mechanism of this interaction is poorly understood. Morphogenesis of Drosophila leg imaginal disc epithelia is regulated both by the steroid hormone 20-hydroxyecdysone (ecdysone) and the RhoA GTPase signaling pathway. Recent evidence suggests that these pathways act cooperatively to control imaginal disc morphogenesis. Thus, leg imaginal disc morphogenesis is an excellent system in which to study the interaction of steroid hormone and intracellular signaling pathways. We have identified mutations in three genes, 12-5, 18-5, and 31-6, with roles in the morphogenesis of leg epithelia. Of particular interest, these mutations interact genetically with each other, mutations in the RhoA signaling pathway, and the ecdysone regulated Sb-sbd (Stubble) transmembrane serine protease. This suggests that the 12-5, 18-5, and 31-6 gene products may link hormone and RhoA signaling responses. The goal of this research was to identify and characterize the 18-5 and 12-5 genes in order to discern the mechanistic relationship between the RhoA pathway and ecdysone hierarchy.18-5 and 12-5 were precisely mapped to molecular locations within the Drosophila genome utilizing a P-element recombination mapping technique. This work narrowed the location of the 18-5 locus to within an interval of 112 kb within the Drosophila genome sequence. This interval contains 17 known and predicted genes. I also mapped the location of the 12-5 locus to a 2.6 Mb interval of the 2nd chromosome. Based on phenotypic analyses and the site of the molecularly mapped interval, a candidate gene for the 18-5 mutation was identified. Sequence analysis of the candidate gene was inconclusive and requires further analysis. Genetic interaction assays indicate that the 18-5 gene product acts upstream or at the level of Rho kinase in the RhoA signaling pathway.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24912号 / 農博第2575号 / 新制||農||1102(附属図書館) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 大門 高明, 教授 松浦 健二, 准教授 小野 肇 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

The steroid hormone ecdysone plays vital roles during Drosophila development. Pulses of 20E during Drosophila life cycle function as temporal cues, signaling the onset of metamorphic processes, including the stage specific programmed cell death of larval tissues. Ecdysone is the critical developmental cue orchestrating the metamorphic reformation of CNS, resulting in the formation of adult-specific neural circuitry. Ecdysone signaling is transduced by a heterodimeric receptor complex formed between two nuclear receptors: EcR and Ultraspiracle (USP). There are 18 nuclear receptors known in Drosophila and EcR is the only receptor whose functions in neuronal PCD have been well recognized. Therefore, the current study is aimed to define the role of nuclear receptors in neuronal cell death mechanisms in Drosophila. Here, I examine the function of nuclear receptors in PCD of two groups of peptidergic neurons: vCrz and CCAP. EcR and USP receptor complex on activation results in the coordinated transcriptional regulation of a host of transcription factors regulating genes essential for PCD. USP plays a dual role in ecdysone response, as its function is necessary for both activation and repression of ecdysone primary response genes. I have developed a possible dominant-negative mutant USP (usp3), and expressed it in flies using the GAL4-UAS system to illustrate the role of USP in ecdysone mediated PCD of vCrz neurons. Targeted expression of usp3 in corazonin neurons results in a complete blockage of PCD pathway. Another interacting partner of USP, Drosophila Hormone Receptor 38, however shows no involvement in PCD of vCrz neurons. I have also designed an ecdysone sensor to monitor the developmental timing of EcR activation in vCrz neurons. Further, I investigate the survival factors required for preventing the untimely PCD of these two groups of neurons. The study reveals that DIAP1 is required for the survival of larval vCrz and CCAP neurons. Also, the nuclear receptor E75 is shown to be critical for preventing premature PCD of CCAP neurons.

Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 130-148). Also available on the Internet.

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Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 130-148). Also available on the Internet.