A study of the roles of microfilaments and calcium transients during epiboly in zebrafish embryos

The basic body plan of teleost embryos emerges during the gastrula period when a series of extensive cell movements and rearrangements---epiboly, involution, convergence and extension---lead to the formation of the three germ layers as well as the dorsoventral and anterioposterior body axes. Starting towards the end of the blastula period, epiboly consists of the thinning and spreading of both the blastoderm and the yolk syncytial layer (YSL) over the large (∼600 mum diameter) yolk cell toward the vegetal pole until, by the end of the gastrula period, the yolk is completed encompassed. The characterisation of microfilaments in zebrafish embryos from the earliest stages of development to around 50% epiboly has been previously described, thus the goal of my project was to complete this study up until the end of epiboly. Here in my thesis, I described four striking new actin-based structures: a punctate actin band in the external yolk syncytial layer (E-YSL); continuous rings of actin at the leading margins of both the enveloping layer (EVL) cells and deep cells (DCs); as well as a dense actin mat located at the vegetal pole (VP). Of these four structures, only one, the vegetal actin mat, is present throughout epiboly. The other three, the punctate actin band and the two marginal actin rings, only appear after the equator of the yolk cell has been crossed by the spreading blastoderm layers. It is suggested, therefore, that these structures are specific for the second half of epiboly and appear in response to the changing geometry of the embryo with respect to the spreading cell layers.; With regards to function, the punctate actin band appears to be associated with a region of macropinocytosis that acts to internalise the yolk cell membrane (YCM) in front of the advancing blastoderm margins. It is proposed that this actin-based contractile activity also generates force that helps to draw the E-YSL and attached EVL toward the VP. It is suggested that the EVL and DC marginal actin rings act as contractile purse-strings and thus help to bring together the margins of the EVL and DCs as they approach the VP. Finally, it is proposed that the vegetal actin mat acts as major component of the yolk cell cytoskeleton, maintaining structural integrity during epiboly.; I also present evidence to show that Ca2+ may play a key role in the formation and function of these actin-based structures. Treatment with the Ca2+ chelator dibromo-BAPTA (DBB) results in the disruption of the various actin-based structures and leads to the slowing or immediate arrest of epiboly, resulting in a failure of yolk cell occlusion followed by the eventual lysis of the embryo through the vegetal pole region. I also present data whereby embryos were loaded with f-aequorin (a Ca2+-sensitive bioluminescent reporter) at the single cell stage and then treated with either cytochalasin B or DBB during epiboly and the resulting Ca2+ signalling patterns visualised using a Photon Imaging Microscope system.; The possible roles of, and interactions between, microfilaments and Ca 2+ signalling during late epiboly will be discussed.