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Identification and analysis of factors that regulate the plasmatocyte lineage during Drosophila hematopoiesis
thesis
posted on 2013-04-18, 00:00 authored by Paul Timothy KroegerDrosophila have become an important model organism in the study of hematopoiesis due to conservation of the myeloid blood cell lineage with conserved transcription factors and signaling pathways used between organisms. In order to study hematopoiesis, we analyzed Eater, a transmembrane protein that mediates phagocytosis of bacterial pathogens in Drosophila. The gene was shown to be expressed solely in one of three types of Drosophila hemocytes, the plasmatocyte, and only after embryonic development. The transcriptional enhancer of eater was extensively analyzed and the gene was found to be regulated by GATA factor Serpent. The specificity of the eater transcriptional enhancer has allowed the generation and use of eaterGal4 to selectively knockdown genes in plasmatocytes via UAS-RNAi technology. Hemocyte samples obtained from these larvae are observed for cell number irregularities and other aberrations caused by targeted gene knockdown. This approach identified a critical gene, Eip78C, which caused a three-fold decrease in cell number when compared to control. This decreased cell number was rescued by overexpression of Drosophila inhibitor of apoptosis. Interestingly, it was noted that there was a significant increase in the number of crystal cells in embryonic, as well as larval samples. These data suggest Eip78C plays a role in plasmatocyte survival, but also repression of the crystal cell fate. The RNAi screen also identified components of the SCF complex, which targets proteins for degradation via the ubiquitin ligase pathway. To investigate the SCF complex, all known members were analyzed via RNAi knockdown or mutant analysis. lin19, SkpA, Roc1a, Nedd8 and Skp2 were identified as the components of this specific complex, as knockdown of each elicited an enlarged cell phenotype. Further investigation determined that knockdown of SCF members caused an accumulation of Cyclin E and Double-parked in the nucleus of large cells, which are also P1-positive, indicating these cells are plasmatocytes. Double-parked accumulation in the nucleus forced these enlarged cells to undergo re-replication causing polyploidy and centriole overduplication. Conversely, Geminin, the inhibitor of Double-parked, was removed from the nucleus in the enlarged cells. By overexpressing Geminin, we partially rescued the SCF knockdown phenotype, suggesting that Double-parked accumulation in the nucleus is responsible for these re-replication events. Together, these studies show the value of investigating Drosophila hematopoiesis during larval development, as the screen identified Eip78C and the SCF complex, which had not yet been implicated in fly plasmatocyte development. Understanding Drosophila blood cell development may elucidate genes and pathways that function in hematopoiesis of higher organisms, such as mammals.
History
Date Modified
2017-06-05Defense Date
2013-04-11Research Director(s)
Robert A. SchulzCommittee Members
Tracy Vargo-Gogola Rebecca Wingert Zach SchaferDegree
- Doctor of Philosophy
Degree Level
- Doctoral Dissertation
Language
- English
Alternate Identifier
etd-04182013-120244Publisher
University of Notre DameProgram Name
- Biological Sciences
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