Reverse Engineering Epithelial Morphogenesis: A Systems Biology Approach

Organogenesis is a complex process that relies on the integration of a diverse set of inputs to control multiple downstream cellular processes forming a bow-tie network. Second messengers, such as Ca²⁺, play a central role in this network and are essential for mediating input signals to the desired output. While much is known about how different dynamical trends of these second messengers regulate single cell processes, how they come together in a multicellular perspective is still unclear. Recent discoveries across multiple biological systems, such as zebrafish, plants and fruit flies, have revealed the existence of specific dynamical patterns of these second messengers, often observed to be directly correlated with organ growth, which raises significant questions about their overall impact on the regulation of morphogenesis. Furthermore, research has shown a Ca²⁺ response upon wounding of epithelial tissue, but the functional role of how Ca²⁺ regulates the wound healing response remains unclear. To investigate the role of Ca²⁺ signaling in regulation of epithelial morphogenesis, we conducted a systematic screening of genes regulating Ca²⁺ signaling using Drosophila melanogaster, an established model system for epithelial morphogenesis. The loss-of function screening combined with a machine learning driven framework for phenotypic quantification identified neuropeptide receptors as novel regulators of epithelial morphogenesis (Chapter 1, 2). Our screening also highlighted Piezo, recently discovered mechanosensitive ion channels, as regulators of epithelial morphogenesis. Piezo gets activated upon mechanical stretch or pressure and allows the entry of Ca²⁺ into the cytosol. While much work has been done to understand how extrinsic activation of Piezo regulates cell-level processes, the intrinsic role of Piezo in organ development is still unclear. Through model simulations and experiments, we propose and test the key hypothesis that Piezo protein expression regulates the activation tension of these channels. We further describe that Piezo coordinated regulation in cell proliferation, apoptosis, and cell tension is required for maintaining the epithelial topology and hexagonality in packing. This outlines novel implications of Piezo as being a feedback controller to "fine-tune" morphogenesis (Chapter 3). Ultimately, our findings contribute to a better understanding of how second messengers like Ca²⁺ regulate morphogenesis and organ development.

Elucidating the molecular principles governing cellular dynamics during tissue morphogenesis is challenging due to the system complexity of many molecular components and reactions. Multicellular systems exhibit a high degree of nonlinear mechanical and cell signaling interactions between multiple cell types and tissue layers, which are patterned in space and time by morphogenetic programs. Further, the mechanical-biochemical signaling crosstalk that directs multiple cellular processes such as the regulation of cell-cell and cell-ECM adhesion, cytoskeletal organization, metabolic growth and proliferation is pervasive. Computational models provide critical insights into morphogenesis. However, most models are limited in their predictive ability since most simulate biological processes at a single length scale. Further, the ability to test predictions through controlled manipulations and dynamic imaging at the whole organ scale is often very limited. New realistic multi-scale, mechano-chemical models of morphogenesis are critically needed to infer general design principles of morphogenesis that operate across multiple length and time scales.

Model simulations have been combined with an advanced model-based, statistics-guided design of experiments and machine-learning driven image analysis to generate and test specific hypotheses of molecular mechanisms regarding how molecular signaling activity within cells regulate internal forces and how these activities are coordinated with external forces applied on cells by adjacent cell layers and the extracellular matrix (Chapter 4-6). This project focuses on testing the overall guiding hypothesis that epithelial cell shape regulation depends on the interplay between two key morphogen pathways (BMP and Wingless/WNT) to pattern actomyosin contractility through multiple Rho GTPases. This hypothesis also posits a functional role of the cellular interaction with the extracellular matrix as a morphogenic ‘memory’ system of earlier developmental stages. This work expands our understanding that morphogen signaling pathways not only regulate cell differentiation and growth during developmental processes but also modulate cell mechanics under faster time scales.