Shedding light on dynamic processes in the intestinal epithelium

The intestine is the primary site for the absorption of nutrients, minerals, and water from the food that we eat. This vital function is performed by the intestinal epithelium—a single layer of specialized cells lining the inner surface of the intestine. In addition to facilitating absorption, the intestinal epithelium acts as a protective barrier that protects the underlying tissue from the potentially harmful contents of the intestinal lumen. In essence, this thesis is about two things: the intestinal epithelium and time. Specifically, it investigates how the intestinal epithelium preserves its structural and functional integrity over time and it focuses on the dynamic processes that enable the epithelium to sustain its barrier function. Chapter 1 provides an overview of the structure, cell types, and core functions of the intestinal epithelium, along with the state-of-the-art methodologies used to study it. This chapter also introduces intestinal organoids, which are small, in vitro models of the intestinal epithelium, and serve as the central experimental system throughout this thesis.

Part I presents novel experimental insights into the dynamic mechanisms that regulate epithelial homeostasis and barrier function. A key feature of the intestinal epithelium is its ability to maintain constant cell density and integrity through continuous stem cell proliferation and extrusion of cells. In Chapter 2, we propose a new model for epithelial cell extrusion. Our findings reveal that epithelial cells engage in a constant mechanical “tug-of-war”, exerting dynamic pulling forces on their neighbors. This mechanical competition allows cells to assess the tension they generate relative to adjacent cells. Those that produce insufficient tension are more likely to be extruded. This mechanism selectively retains mechanically robust cells, thereby enhancing epithelial integrity and preserving barrier function. Chapter 3 explores how differentiation is regulated within the intestinal epithelium. We demonstrate that once stem cells lose access to Wnt ligands, they initiate a differentiation timer. IfWnt signaling is restored before the timer elapses, cells can reset the process and retain their stem cell identity. If not, they commit to a differentiated fate. These findings suggest that differentiation is a spatially dependent, reversible process rather than a strictly linear one. In Chapter 4, we investigate the differentiation of the rare and only recently identified BEST4/CA7+ cells. While absent in mouse intestinal epithelium, these cells were originally discovered in humans and have since been found in other animals. Using human intestinal organoids and transcriptional fluorescent reporters, we tracked the differentiation of this cell type. Our data show that BEST4/CA7+ cells and mucus producing (goblet) cells are mutually dependent: Goblet cells direct the differentiation of neighboring cells toward the BEST4/CA7+ lineage. In turn, BEST4/CA7+ cells are critical for the long-term survival of goblet cells.

Part II introduces new tools and protocols for studying dynamic processes in the intestinal epithelium. Chapter 5 presents a machine learning-based algorithm for the in silico labeling of nuclei and membranes in 3D organoids. This approach conserves fluorescent channels for other reporters and enables high-resolution, single cell tracking with minimal phototoxicity. Finally, Chapter 6 details protocols for long-term live-cell imaging combined with in-situ fixation. These methods allow for the real-time tracking of organoid development with experimental perturbations and identification of cell types at the endpoint.