Interrogating Three-Dimensional Structure and Response to Exogenous Stimuli in Multicellular Systems

Organ development requires precise coordination of extrinsic and intrinsic stimuli at a cellular level. However, the underlying principles of how cell signaling is coordinated within the organ context is poorly understood. Many components of the chemical signaling pathways that regulate organ development have been identified through the genetic manipulation of model organisms. However, defining how stimuli are integrated at the scale of multicellular systems has proven challenging due to the difficulties with simultaneous stimulation and visualization of delicate tissues. This thesis describes several milestones that advance the state of the art for analyzing multicellular systems, including insect micro-organs, tissue biopsies, and thromboses (blood clots). These advances include a detailed analysis of how cell geometries impact the propagation of intercellular calcium transients (Chapter 2). This study implicated the distribution of mechanical stress in a tissue as an important input to calcium propagation. This prompted the development of a microfluidic device to allow for more detailed exogenous stimulation at the organ scale (Chapter 3). The device lead to the discovery that it is the release, and not the application, of mechanical loading that stimulates an intercellular calcium response – a distinction that is inaccessible by previously developed methods. The device is generalizable to many model systems and is a multi-modal tool for analyzing how exogenous stimuli impact cell behavior and organ development. Further, this device led to the creation of an automated platform for staining and imaging tissue biopsy samples in 3D (Chapter 4). This advance provides an avenue for diagnosticians to more accurately analyze patient biopsy samples. Finally, the optical clearing strategies employed in Chapter 4 were modified to optically clear human blood clots and enable deep 3D imaging (Chapter 5). This revealed a distinct difference in the fibrin structure between contracted and non-contracted clots and will lead to new insights into pathological thrombosis. Opportunities for further advancing each discovery are discussed in the concluding chapter (Chapter 6).