Project Summary: “Differential Cell Size in Epithelial Packing, Homeostasis, and Disease” A striking increase in cellular size heterogeneity is a salient feature of both organ development and cancer pro- gression. Our goal is to discover the principles that underpin how cells with divergent sizes pack into epithelial tissue and how alterations in cellular packing influence tissue homeostasis. The shape and size of individual components dictate the morphology and integrity of any structure. Indeed, the regulation of cell shape through modulation of cellular mechanical properties is a key factor in tissue morphogenesis. However, in contrast to shape, the role of cell size in determining the form of epithelial tissues is largely unknown. This study will test the hypothesis that cell size is a causal, rather than merely a consequential factor in epithelial homeostasis. To determine the role of cell size in tissue packing, we will take advantage of the two distinct domains of the mouse small intestine as a model to ask the following fundamental questions: 1) How do cells of diverse sizes pack and maintain stereotypical organization in healthy epithelial tissues, and 2) What are the implications of packing cells with ectopic sizes in the context of epithelial diseases. These overarching questions will be addressed with the following Specific Aims. In Aim 1, we will determine how the large Paneth cells and small Lgr5+ stem cells at the base of the intestinal crypt domain maintain a checkerboard-like order. In Aim 2, we will interrogate how aberrant changes in nuclear and cellular size are accommodated in the intestinal villi. Both aims will use sophisticated genetics with quantitative biophysical approaches to determine how a shift in cell size is physically accommo- dated in epithelial tissue. First, we will alter cellular size in a spatially and temporally controlled manner by ma- nipulating gene expression using cell-type drivers. Subsequently, we will integrate confocal imaging with bio- physical measurements to quantitatively distinguish the influence of cell mechanics, shape, and size in tissue packing. By integrating highly interdisciplinary methodologies, we anticipate that our study will extend the under- standing of how cells self-assemble into tissues with stereotypical forms and reveal how functional tissue archi- tecture is perturbed in disease. Ultimately, the findings from this work have the potential to contribute to the identification of novel molecular targets critical for the control of epithelial cell size, tissue packing, and disease progression.