Dokument: Functional analysis of a new dominant-negative allele of miranda during CNS development in Drosophila
|Titel:||Functional analysis of a new dominant-negative allele of miranda during CNS development in Drosophila|
|URL für Lesezeichen:||http://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=3450|
|Dokumententyp:||Wissenschaftliche Abschlussarbeiten » Dissertation|
|Autor:|| Takizawa, Chieko [Autor]|
|Beitragende:||Prof. Dr. Wodarz, Andreas [Gutachter]|
Prof. Dr. Knust, Elisabeth [Gutachter]
|Stichwörter:||asymmetric cell division, Drosophila, neuroblast|
|Dewey Dezimal-Klassifikation:||500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie|
|Beschreibung:||Multicellular organisms generate a variety of different cell types during development. Cell diversity can be achieved by asymmetric cell division, which leads to the generation of two distinct cell types from one progenitor cell. The central nervous system (CNS) of Drosophila has served as an excellent model system to investigate asymmetric cell division. In the Drosophila CNS, neural stem cells, called neuroblasts (NBs), divide asymmetrically along the apical-basal axis, giving rise to two distinct daughter cells that differ in size and mitotic potential. The larger apical daughter cell remains as a NB that retains its stem cell characteristics and will continue to divide asymmetrically. The smaller basal daughter cell, called a ganglion mother cell (GMC), undergoes one terminal division to produce two post mitotic neurons or glial cells. Asymmetric segregation of cell fate determinants is essential for the cell fate choice between NB and GMC.|
I performed two genetic screens to search for novel genes involved in the asymmetric cell division of Drosophila NBs. In the second screen, I isolated a novel mutant allele of miranda (mira) named miraE326. Mira is an adaptor protein for the cell fate determinant Prospero (Pros), which is localized at the basal cortex of NBs and exclusively segregates into GMCs. In mira mutants, Pros does not segregate correctly into the GMC, which leads to defects in cell fate determination in the developing CNS
The mutant Mira protein produced in miraE326 had an in-frame deletion and a point mutation in the N-terminal region. Since all of the mutant mira alleles that have been isolated so far produce C-terminally truncated Mira proteins, MiraE326 has unique characteristics. Consistent with previous studies proposing that the N-terminal region of the Mira protein is responsible for its basal localization, MiraE326 failed to be localized at the basal cortex. Instead it was distributed in the cytoplasm. However, MiraE326 appeared to bind to Pros, since the C-terminal region of MiraE326, which is responsible for binding Pros, was intact. Consequently, the MiraE326 protein recruits Pros into the cytoplasm in miraE326 mutant NBs, which leads to mislocalization of the cell fate determinant Pros in the cytoplasm.
Taking advantage of the unique behavior of the MiraE326 protein, I examined the effect of Pros mislocalization in miraE326 mutant NBs on animal development and neuronal differentiation in CNS development. Firstly, transheterozygotes for miraE326 and an amorphic pros allele do not survive to adulthood. A possible explanation is that the amount of Pros segregated into GMCs in the transheterozygote for miraE326 and the amorphic pros allele is one-quarter, in comparison with that in the wild type, and this amount is insufficient for viability. Secondly, I examined cell fate determination in the CNS in transheterozygotes for miraE326 and the amorphic pros allele. Fifty percent of the RP2 neurons did not express the neural marker Even-skipped (Eve), suggesting that RP2 neurons failed to be properly differentiated.
I also examined the protein-protein interactions between MiraE326 and its interaction partners Inscuteable (Insc) and Myosin VI (Jaguar, Jar), to investigate which protein is responsible for the cortical localization of Mira. The results of the yeast two-hybrid assay revealed that the N-terminal part of MiraE326 lost its binding ability with Insc, whereas it retained its interaction with Jar. This suggests that Insc might be involved in the cortical localization of Mira in NBs.
|Fachbereich / Einrichtung:||Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie|
|Dokument erstellt am:||07.07.2006|
|Dateien geändert am:||12.02.2007|
|Datum der Promotion:||22.05.2006|