In the first phase of the project, we focused on understanding the molecular mechanism of embryo attachment to the egg shell. In the process, we discovered that in contrast to our prior beliefs, there are multiple attachment points and their dynamic interplay appears to be necessary to suppress mechanical instabilities during massive tissue movements of gastrulation. We have investigated the impact on these putative attachment sites on tissue flow. We found that the dorsal-anterior attachment is together with a local expansion due to a mitotic domain, responsible for the posterior shift of the cephalic furrow. The functional significance of this process is at the moment unclear. The anterior ventral attachment on the other hand appears to be necessary to stabilize the head portion of the blastoderm experiencing torque forces originating on the other end of the embryo during germ-band extension process. This attachment is transient and appears to be released by the appearance of the mitotic domain. This conclusion about the function of the ventral attachment is based on our discovery of the tendency of the embryo to twist. We believe that the twist originates from molecular inherent asymmetries in the machinery that underlies cell shape changes.
The phenotype of the lack of dorsal attachment prompted us to realise that concurrent occurrence of several independent cellular movements has the potential to generate instabilities in early embryos. Our data suggest that embryos evolved active mechanisms to suppress such instabilities. These active mechanisms are controlled by the genome, however, our comparative analysis of the processes across the phylogeny points to an interesting possibility that the driving force of the evolution of those mechanisms may have been the emergence of the said mechanical conflict. We supported this hypothesis by both experiment and theory. More recently, we developed an exciting collaboration with the group of Eric Glowacki at CEITEC in Brno, Czechia, that established unique technology to generate local hypoxia. Since we serendipitously observed that the process that we study in this context is extraordinarily sensitive to hypoxia, we aim to use the electrochemical spatial hypoxia (named Faraday scalpel by Eric’s lab) to abolish the structure specifically in the otherwise normally developing Drosophila embryo. This will allow us to assess the potential fitness cost of the mechanical conflict between different processes in the embryo. Taken together, these experiments will provide a novel and fundamental insight into how developmental regulation evolves.
To facilitate the analysis of embryo-eggshell-yolk ensembles, we have adapted expansion microscopy and histological staining to reveal the ultrastructure of interfaces at near electron microscopy (EM) resolution without the need to engage in complex and costly EM studies outlined in the original proposal. We also developed and characterized a fluorescent reporter of scab protein (the integrin mediating the attachment). We are currently using the line to study the subcellular localization of the integrin in expanded embryos.
