During morphogenesis, epithelial tissues undergo dramatic changes in shape, transitioning from flat sheets to three-dimensional folded structures. This remarkable transformation relies on dynamic changes in mechanical tension at both their apical and basal surfaces. While it is well-established that the generation of mechanical tension at the apical side is driven by the actomyosin network, research on this process has often overlooked the generation of mechanical tension at the basal surface. Moreover, the mechanical response to stress, encompassing both elastic (spring-like) and viscous (fluid-like) properties, is important for epithelial transformations, yet this mechanical response is poorly understood for the basal cell surface. In this thesis, we investigated how basal tension is influenced by the basement membrane - an extracellular matrix layer which has been widely regarded as a passive scaffold for cells. We probed the material mechanical response of the basement membrane and directly measured and analyzed basal tension in the wing imaginal disc epithelium of Drosophila. To study the mechanical response, I used long-term confocal imaging and fluorescence recovery after photobleaching (FRAP) to analyze the turnover and mobility of Collagen IV, a component of the basement membrane. The low Collagen IV mobility and turnover (≈ 40 hours) suggest a solid-like behavior of the basement membrane at the time scale of hours. Moreover, Atomic Force Microscopy (AFM) force-indentation curves reveal low hysteresis and an elastic solid-like response. To measure basal mechanical tension, I probed the basement membrane with an AFM. Interpreting the results of AFM shallow indentations on the basal side of explanted wing discs as indenting into a fixed, elastic, stretched thin film, I investigated in control conditions and after molecular perturbations basal mechanical tension. Mechanical tension was ≈ 0.4 mN/m. The removal of collagen IV by collagenase significantly reduced basal tension while increasing basal cell surface area. In addition, inhibition of actomyosin activity through different reagents reduces basal tension while decreasing basal cell surface area. These results indicate that basal tension depends on both the ECM and actomyosin activity. They also indicate that the basement membrane is under expansile stress. Finally, to further investigate the mechanisms underlying the generation of stretch in the basement membrane, I analyzed the influence of hydrostatic pressure and actomyosin contractility along the lateral cell surfaces. These mechanisms exert mechanical forces that increase basal cell area, inducing a stretch in the basement membrane. Mild hypo- or hyperosmotic shocks resulted in increased or decreased basal cell area and basal tension, respectively. Moreover, optogenetic activation of actomyosin at lateral cell surfaces resulted in an increase in both basal cell area and basal tension. In summary, our research quantifies basal tension and unveils that the basement membrane is an elastic material (at time scale of hours). Furthermore, our data suggest that the basement membrane is under elastic stretch generated by hydrostatic pressure and actomyosin contractility. Thus, rather than being a passive scaffold for cells, the elastic properties of the basement membrane contribute to basal tension and thereby the shaping of cells and tissues.

The purpose of this thesis is to develop a vertex model framework that can be used to perform computational experiments related to the dynamics of epithelial tissues in developmental systems. We focus on three example systems: the Drosophila wing imaginal disc, the Drosophila epidermis and the visceral endoderm of the mouse embryo. Within these systems, key questions pertaining to size-control mechanisms and coordination of cell migration remain unanswered and are amenable to computational testing. The vertex model presented here builds upon existing frameworks in three key ways. Firstly, we include novel force terms, representing, for example, the reaction of a cell to being compressed and its shape becoming distorted during a highly dynamic process such as cell migration. Secondly, we incorporate a model of diffusing morphogenetic growth factors within the vertex framework, using an arbitrary Lagrangian-Eulerian formulation of the diffusion equation and solving with the finite-element method (FEM). Finally, we implement the vertex model on the surface of an ellipsoid, in order to simulate cell migration in the mouse embryo. Throughout this thesis, we validate our model by running simple simulations. We demonstrate convergence properties of the FEM scheme and discuss how the time taken to solve the system scales with tissue size. The model is applied to biological systems and its utility demonstrated in several contexts. We show that when growth is dependent on morphogen concentration in the Drosophila wing disc, proliferation occurs preferentially in regions of high concentration. In the Drosophila epidermis, we show that a recently proposed mechanism of compartment size-control, in which a growth-factor is released in limited amounts, is viable. Finally, we examine the phenomenon of rosettes in the mouse embryo, which occur when five or more cells meet at a common vertex. We show, by running simulations both with and without rosettes, that they are crucial facilitators of ordered migration, and are thus critical in the patterning of the early embryo.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

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