Date of Award
Doctor of Philosophy (PhD)
The process of epiboly, or the thinning and spreading of a tissue, is a well-conserved morphogenetic process. As one of four conserved gastrulation cell movements, epiboly is important to help organize the overall body plan. Epiboly in zebrafish involves the thinning and spreading of the blastoderm originating from the animal pole to completely enclose the yolk. It is driven by a multitude of physical processes that involve the three cell types comprising the embryo: the yolk syncytial layer (YSL), enveloping layer (EVL), and deep cells. These physical aspects can be broadly described as involving cell-cell interactions through adhesion proteins, actomyosin contractility for cell shape changes and exerting forces on the cell layers, and the yolk microtubule skeleton responsible for exerting pulling forces on the YSL, from which it emerges. Through attachments of the YSL to the EVL and the EVL to deep cells, these pulling forces are a significant contributor to epiboly progression. My thesis work focuses on understanding the roles for the three atypical cadherins, Dachsous 1a (Dchs1a), Dchs1b, and Dchs2, in regulating microtubules, particularly those within the yolk during epiboly. Previous work from our lab established multiple roles for Dchs1b during early embryogenesis and epiboly. Maternal-zygotic (MZ) dchs1b mutants exhibit defects due to aberrant actomyosin and microtubule networks including insufficient yolk cytoplasmic streaming and improper dorsal determinant transport, respectively. Our collaborative work on dchs1b established a novel mechanism for dchs1b-dependent regulation of microtubule dynamics during early cell cleavages. Briefly, we show Dchs1b promotes microtubule dynamics by recruiting Tetratricopeptide repeat-containing protein (Ttc28) to the membrane and away from the cytoplasm and microtubule organizing centers (MTOCs), which limits the inhibitory effect of Ttc28 on microtubule dynamics. This Dchs1b-Ttc28 interaction somehow exerts effects on microtubules via Aurora B kinase (AurkB), which is known for regulating a multitude of microtubule associated proteins (MAPs). I also showed that correct levels of dchs1b expression were required for proper AurkB localization to mitotic spindles, which may be responsible for altering AurkB activity. Through studies of epiboly, I showed the partial redundant functions for the three dchs genes in this process. Additionally, we showed endogenous expression of Dchs1b-6xsfGFP at cell membranes during zebrafish epiboly for the first time. Yolk microtubules during epiboly in triple dchs mutants are bundled, and I used high-speed time lapse confocal microscopy to show that microtubule polymerization is longer in duration and plus end displacement, suggesting fewer catastrophe events and more stable plus ends. At the minus end, dchs triple mutants yolk microtubules exhibit increased association with CAMSAP1, indicating increased stability at the minus end as well. Together, my studies support roles for dchs in promoting microtubule dynamics at both ends of microtubules. Lastly, we developed a workflow to manipulate AurkB activity in yolk microtubules to be used in the future to better understand roles for AurkB in regulating microtubules outside of cell division and whether Dchs regulates microtubules in epiboly using a similar mechanism to that in early cell cleavages.
Chair and Committee
James Skeath, Gregory Longmore, Mohammed Mahjoub, Helen McNeill,
Castelvecchi, Gina Danielle, "Regulation of Yolk Microtubule Dynamics by Dachsous Cadherins" (2020). Arts & Sciences Electronic Theses and Dissertations. 2306.