Grant: Discovery Grants Program - Individual

The epithelial lining of our intestine rapidly turns over, completely replaced every week, and fueled by adult intestinal stem cells. Intestinal stem cells divide daily and give rise to all intestinal epithelial lineages including absorptive enterocytes, mucus-producing goblet cells, and antimicrobial-secreting Paneth cells. In contrast, during the fetal stage, there is no such hierarchy, and virtually any cell can be the clonal origin for large patches of tissue. Postnatally, a switch occurs from this fetal non-hierarchal system to the adult stem-cell based system, via an unknown mechanism. Organoids are 3-dimensional organ-like structures cultured in petri dishes that mimic their in vivo counterparts. They have eased the study of epithelium ex vivo and are important in reducing animal use in research. Although this is encouraging, the analysis of organoids and their cellular composition remains challenging due to the size of these structures and the variability within the culture system. Therefore, unbiased analysis tools are necessary. For example, brightfield microscopy could be used to define organoid state (e.g., spheroid vs. budding), and techniques to determine cellular composition such as confocal microscopy and flow cytometry are necessary. However, these analysis techniques are not commonly established research tools, especially in labs with limited funds. Epigenetic modifiers are enzymes regulating persistent gene expression changes through the modification of histones. Thus, they are hypothesized to contribute to the switch from a fetal-to-adult intestinal epithelium. Indeed, we have recently discovered that Lysine-specific demethylase 1 (LSD1/KDM1A) is required for the early-life change in intestinal epithelial differentiation. However, it is not defined how LSD1 is mediating this, and how this relates to known mediators of the early-life transition, such as the WNT pathway. The long-term goal of my research program is to determine how early-life intestinal maturation is regulated. To achieve this goal within the next five years, our focus will be on testing the hypothesis that epigenetic modifiers, in concert with other transcriptional regulators, are the main epithelial-intrinsic drivers of this early-life process. We have 2 short-term objectives: Objective 1: Establishment and implementation of an organoid screening platform to discover regulators of intestinal epithelial biology. We will develop an organoid analysis toolbox and use this to screen a library of inhibitors targeting epigenetic modifiers to determine how and which epigenetic modifiers are involved in early-life intestinal epithelial maturation. Objective 2: Define the molecular mechanism for LSD1-controlled intestinal epithelial maturation. We will use organoid analysis tools, as well as biochemical and (epi)genomic assays, to define how and through what pathways LSD1 orchestrates intestinal epithelial biology.