| Abstract |
Gastrulation involves a complex series of morphogenetic processes that unfold in precise coordination alongside tissue genetic patterning. During the gastrulation of Drosophila melanogaster, a monolayer of cells known as the blastoderm undergoes a sequence of tissue rearrangements that reveal axis determination and cell fates. The Drosophila model provides a comprehensive toolkit for studying embryogenesis, offering insights into its streamlined developmental program, which efficiently produces the final organism while maintaining robustness. This prompts an exploration of the mechanisms that coordinate individual morphogenetic events. Active forces, generated by cytoskeletal-driven cell shape changes, exert themselves on the viscoelastic blastoderm tissue, resulting in passive transmission. Traditional analysis involved studying these events at molecular, cellular, and tissue scales. However, the emergence of single-plane illumination microscopy, enabling whole volume (in toto) imaging of developing embryos with high temporal and spatial resolution, has reshaped this approach. This advancement in microscopy allows us to investigate how morphogenetic events synergise or hinder one another and how the embryo integrates this information to coordinate gastrulation. A recent development in gastrulation research involves identifying regions of heightened friction between the cells and the eggshell within the embryo. Integrins expressed near the midgut primordium have been found to mediate this interaction, challenging the notion that cell movement is the sole driving force of morphogenesis. Instead, static regions emerge as vital contributors to mechanical stability and the preservation of left-right symmetry during germ band extension. This role of integrins is not confined to Drosophila alone, as similar findings have been observed in the beetle Tribolium castaneum, emphasizing their role in orchestrating tissue flows and raising questions about their conservation. Consequently, my focus centered in understanding the physical forces underlying gastrulation, encompassing their interplay, balance, and conflicts in the developing embryo. I considered not only the active forces generated by cell shape changes, but also the passive forces transmitted in the surrounding tissue and the friction generated by the interaction with the physical constraint of the eggshell. In the initial chapter of this thesis, I delved into a lesser-explored morphogenetic event: the formation of the cephalic furrow. This transient tissue fold delineates the head from the trunk but differs from other invaginations in that it does not give rise to further differentiated structures. Instead, it unfolds later in development without leaving a trace of its origin. By employing in toto imaging alongside genetic and photo-manipulation techniques, I investigated the influence of the active forces driving the furrow formation on the head-trunk boundary tissue, and the consequences upon their absence. I found that the invagination is driven by active forces that propagate in the surrounding tissue. By genetic inhibition of the furrow, the presence of ectopic buckling was confirmed in the head-trunk boundary, appearing in between mitotic domains in a rather stochastic fashion. Next, I assessed the impact of further morphogenetic events in the head-trunk boundary tissue using whole embryo imaging and quantitative strain analysis. From my observations, I was able to conclude that the cephalic furrow primes tissue folding to dissipate forces coming from two sources, the local mitotic domains' expansion and the remote germ band compression. These discoveries suggest that the cephalic furrow and its genetic patterning may have evolved in response to the coalition of compressive forces in the head-trunk boundary tissue. The second chapter focused on understanding the ensemble of tissue flows while considering the newfound importance of static regions, mediated by the integrin subunit scab. The possibility to visualise the whole embryo upon mechanical photo-manipulation and integrin mutation, allowed me to uncover how enhanced friction's localised forces contribute to the directionality of tissue movements during the posterior midgut invagination. I was able to verify that cell-to-shell attachment helps maintaining the speed and direction of germ band extension, a highly mechanically unstable process. At the same time, I explored the effects of two milder expression sites of scab not addressed in the past on the mid-dorsal and ventral-anterior region of the embryo. I determined that the first contributes to the shift of the cephalic furrow posteriorly, which is required to allow mitotic domains divisions in the dorsal side of the head. Upon integrin depletion, ectopic buckling in between mitotic domains was observed, resembling our results in the first chapter. Lastly, thanks to whole volumetric imaging, the newly found expression site in the ventral-anterior region was determined to stabilise the head in face of torque generation of the deviating germ band. These results indicate a potential early safety mechanisms against symmetry breaking in the unstable stages of germ band extension, before posterior midgut invagination. In the concluding chapter, I explored the origins of left-right organismal symmetry instability during germ band extension. Analysing cartographic projections of the whole blastoderm surface, I discovered an inherent chirality identified in symmetric embryos at both the cellular and local tissue levels. By quantification of geometric features, along with tissue strain and curl rates, I found significant and consistent differences in all specimens in a specific region of the embryo in the lateral posterior side. These finding raises questions about the potential role of early chiral determinants in embryogenesis, that could translate local asymmetries into organismal twisting. In summary, this thesis underscores the significance of a multi-scale and interdisciplinary approach to embryonic development. The view of genetic patterning setting up the canvas for morphogenesis is taken to the next level, considering this canvas as an active material that needs fine coordination of single cellular events and the forces generated by them. These forces, in turn, shape molecular components and gene expression, culminating in a dynamic picture of a somewhat unstable, though robust, equilibrium of embryonic development. |