Transit-Amplifying Cells Coordinate Changes in Intestinal Epithelial Cell-Type Composition.

Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. How are complex milieus of microenvironmental signals interpreted to coordinate tissue-cell-type composition? Here, we investigate the responses of intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways. Using a high-throughput approach that combines enteroid monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways. Importantly, we found that modulation of transit-amplifying cell proliferation changes the ratio of differentiated secretory to absorptive cell types. These observations highlight an underappreciated role for transit-amplifying cells in the tuning of differentiated cell-type composition.

Generation and Quantitative Imaging of Enteroid Monolayers.

The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease. Recently, we developed an enteroid monolayer (2D) culture system that recapitulates important features of 3D organoids and the in vivo intestinal epithelium such as tissue renewal, representation of diverse epithelial cell types, self-organization, and apical-basolateral polarization. Enteroid monolayers are cultured in microtiter plates, enabling high-throughput experiments. Furthermore, their 2D nature makes it easier to distinguish individual cells by fluorescent microscopy, enabling quantitative analysis of single cell behaviors within the epithelial tissue.Here we describe experimental methods for generating enteroid monolayers and computational methods for analyzing immunofluorescence images of enteroid monolayers. We outline experimental methods for generating enteroid monolayers from freshly isolated intestinal crypts, frozen intestinal crypts, and 3D organoids. Fresh crypts are easily obtained from murine or human intestinal samples, and the ability to derive enteroid monolayers from both frozen crypts and 3D organoids enables genetic modification and/or biobanking of patient samples for future studies. We outline computational methods for identifying distinct epithelial cell types (goblet, stem, EdU+) in immunofluorescence images of enteroid monolayers and, importantly, individual nuclei, enabling truly single cell measurements of epithelial cell behaviors to be made. Taken together, these methods will enable detailed studies of epithelial homeostasis and intestinal disease.

Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization.

The intestinal epithelium maintains a remarkable balance between proliferation and differentiation despite rapid cellular turnover. A central challenge is to elucidate mechanisms required for robust control of tissue renewal. Opposing WNT and BMP signaling is essential in establishing epithelial homeostasis. However, it has been difficult to disentangle contributions from multiple sources of morphogen signals in the tissue. Here, to dissect epithelial-autonomous morphogenic signaling circuits, we developed an enteroid monolayer culture system that recapitulates four key properties of the intestinal epithelium, namely the ability to maintain proliferative and differentiated zones, self-renew, polarize, and generate major intestinal cell types. We systematically perturb intrinsic and extrinsic sources of WNT and BMP signals to reveal a core morphogenic circuit that controls proliferation, tissue organization, and cell fate. Our work demonstrates the ability of intestinal epithelium, even in the absence of 3D tissue architecture, to control its own growth and organization through morphogen-mediated feedback.