A Microfluidics-Driven Study into the Biomechanics of Epithelial Organogenesis and Homeostasis

Epithelial tissues function to maintain physical barriers and protect organs against pathogens, microbes, and toxins. Tissue-wide coordination of mechanical stiffness requires second messengers such as calcium ions (Ca²⁺). Dysregulation of calcium signaling is associated with a broad range of epithelial diseases ranging from poor wound healing to carcinogenesis and metastasis. However, there remain significant knowledge gaps regarding the connections between calcium signaling dynamics and cell mechanics. In particular, how the coordination of calcium signaling across cell populations leads to robustness in tissue morphogenesis and homeostasis remains largely a mystery. Discerning how the spatiotemporal dynamics of calcium signaling encode the regulation of cell mechanics requires innovative research tools that enable defined chemical and mechanical perturbations and 3D live-imaging.

In this dissertation, Drosophila is used as a model organism to study calcium-driven dynamic mechanics of epithelial growth. Chapter 1 introduces the complexities of epithelial tissues and discusses the tools available for studying fragile epithelial tissues. In particular, precise microenvironment control is required to study endogenous cell signaling in epithelial tissues. In Chapter 2, we developed a novel microfluidic platform to chemically and mechanically perturb cells and micro-organs. This platform was used to create a pneumatic micro-mechanical device to quantify the mechanical properties of a wing imaginal disc, a prototypical micro-organ. Using the aforementioned device, we identified that tissue strain under the same applied force increased with age for our model micro-organ. This method was further developed and cataloged as a general microfluidic technique to demonstrate how to fabricate both educational and live-culture microfluidic devices (Appendix A).

The next chapter focuses on the cytoskeletal components responsible for tissue bending during development to further research the biomechanics of changing mechanical properties with age (Chapter 3). We took a combined experimental and computational approach to study the formation and maintenance of the stereotypical dome-shaped pouch of a Drosophila wing disc. Studies of Drosophila disc development are crucial to understanding the complex developmental processes in humans. Interestingly, we found separable contributions from ECM prestrain and actomyosin tension during epithelial organogenesis and homeostasis. Actomyosin has an active role in bending the disc to shape. Once bent, the tissue was then held in place by the ECM. The ECM had a minimal force-generating role during the active process.

As a key component that converts mechanical stretching into calcium signals, we addressed the homeostatic role of the mechanosensitive ion channel, Piezo, in organ size control (Chapter 4). Piezo has multiple roles in promoting both proliferation and cell extrusion in epithelial tissues. We identified upregulation of both integrin and phosphorylated myosin when Piezo was overexpressed. We confirmed that short-term pharmacological activation of Piezo stimulates mitosis in wing discs. It also acts as a homeostatic regulator by promoting cell death in overcrowded tissues. These results provide support for the hypothesis that Piezo functions through a homeostatic size-control feedback loop: overcrowding from proliferating cells leads to increased cell death to maintaining tissue size. In the absence of this Piezo-mediated feedback loop, the robustness of organ size control is significantly impacted, resulting in bilateral asymmetry in limb development.

Next, a preliminary study addresses the dynamic regulation of actomyosin-driven contraction (Chapter 5). First, an assay was developed to study the distinct configurations and resultant force profiles of actomyosin during wound healing. Second, we identified a potential function of the endoplasmic reticulum calcium pump, SERCA, for regulating actomyosin-driven contractility. Pharmacological inhibition of SERCA led to increased contraction as measured through tissue morphology and cell bond tension. Overall, these biomechanical studies introduce hypotheses for the relationship between cellular calcium and tissue-wide forces.

Future directions are proposed to expand the use of microfluidics in traditional and remote teaching environments (Chapter 6). Recent advances in biomicrofluidics have the potential to revolutionize lab-style courses within the biomolecular engineering curriculum. Microfluidic devices show great promise in fulfilling the need for remote educational experiences because they are small, transportable, cheap, and commercially safe volumes of reagents to complete experiments.

In sum, this work establishes key features of calcium signaling–actomyosin axis of cell mechanic regulation during Drosophila wing disc development and homeostasis. Microfluidic devices and accompanying fabrication methods were developed for culture and imaging of cultured micro-organs. The future of microfluidics includes uses for researching other dynamic biomechanical systems and for advancing STEM education.