Periodic Reporting for period 2 - GHOSTINTHESHELL (Impact of yolk-cellular blastoderm-egg shell interactions on the evolution of animal gastrulation)
Periodo di rendicontazione: 2022-07-01 al 2023-12-31
inner surfaces of egg shells that surround the embryos impact morphogenetic processes during early development. This ERC projects builds on the discovery that integrins, a class of proteins responsible for linking intracellular skeleton of the cell with extracellular matrices, are also involved in attaching the embryo to the rigid external structures of the eggs, at least in case of insects. The aim is to understand the molecular and physical mechanisms of those attachments and how the forces originating from them interact with forces generated within the living tissue to shape the embryo. Besides this mechanistic aspect, the project has two exploratory dimensions. One concerns the impact of another atypical interaction occurring within animal embryos, namely that between cells and the yolk component of the egg. Second, we aim to generalize the lessons learned about the embryo-eggshell-yolk ensemble in insects to other animal species undergoing early developmental phases within confined spaces of rigid egg structures. Early development is surprisingly diverse, despite common descent and the unavoidable constraints of starting multicellular life from a single cell. It remains however unclear, how early development evolves, especially in the context of species where embryos are heavily shielded from the interactions with outside environment. Our work will shed life on this remaining mystery of multicellular life.
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.
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.
We are well on the way to understand the function of embryo shell attachment in stabilizing gastrulation morphogenesis and suppressing cell intrinsic asymmetries in animal embryos. We hope to further substantiate the notion that internal mechanical conflict is one of the drivers of the evolution of developmental mechanisms. Finally, we will move on to generalize the findings beyond insects.