Engineering Morphogenesis Through Calcium Signaling and Cellular Mechanical Mediators

Cells communicate with each other to coordinate cellular processes across tissues during cell differentiation and tissue morphogenesis. This coded communication, facilitated by calcium as a second messenger, controls cell mechanics to shape the growth of organs and defines the “Rules of Life.” There are still many unresolved questions about the complex morphogenetic process due to challenges in precisely perturbing and measuring cell mechanics and signaling dynamics. How an organ’s size and shape are determined is a crucial question for developmental biology, with implications for regenerative medicine and cancer research. While many individual molecular regulators have been identified, how the collective action of multiple modular subsystems precisely controls organ size and coordinates uniform growth termination remains unclear. This knowledge gap limits our understanding of developmental disorders and prevents fundamental advances in regenerative medicine and cancer therapies. Thus, the overarching goal of this dissertation is to program calcium and its downstream mechanical modulators to elucidate the multi-layered steps of morphogenesis and elucidate growth control. Optogenetic and mechanosensitive tools were employed to define relationships between signal transduction and downstream cellular responses to bridge the gap of knowledge in growth control. These tools control intracellular calcium levels at the single-cell or whole-tissue scale to direct subsequent cellular processes and define organogenesis. Mechanochemical models of morphogenesis were developed next to capture the complexities of the process accurately. The overall aim was to uncover the general design principles that spatiotemporally govern morphogenesis. To program calcium dynamics and cellular mechanics for mapping organ development, the primordium wing imaginal disc and the terminal wing of Drosophila melanogaster were used as models to study tissue-level perturbations determining the eventual effect on the final size and shape of the organ. One specific goal of this dissertation was to identify the optimum level, or “Goldilocks Zone” of calcium, as defined in previous literature, specifically to promote cell, tissue, and organ growth and discover the roles of downstream cell-level processes and signaling pathways using optogenetics. Next, Piezo-mediated calcium dynamics and its role in ensuring the precise development of organs, homeostasis, and regulation of cell mechanics through experimental and computational endeavors in Drosophila were also discovered and outlined. Finally, multiscale computational models of epithelial morphogenesis and subcellular mechanisms during Drosophila wing disc development were developed. Specifically, tissue bending was investigated through cytoskeletal protein and gene expressions and the effect of morphogens and cellular processes downstream of calcium. A collaborative initiative with mathematical biologists resulted in the construction of these comprehensive computational frameworks simulating cell populations with varying mechanical properties organized into multilayer structures. The predictions made by the models were validated by performing multifactorial experiments involving perturbation of the extracellular matrix and actomyosin complex and defining the role of calcium in this vastly complicated chemical network. This will enable us to study how signaling regulators and mechanical forces synergistically determine the final size and shape of an organ. Cumulatively, these findings provide insights into practical applications for improving the development of tissues and organs in various pathological disorders.