Fate Specification of CFTR High Expresser Cells in the Intestine

Yale University, New Haven CT

Investigators

Abstract

Project Summary Rare cell types have been traditionally difficult to study due to their low abundance and lack of markers to facilitate isolation and enrichment. We lack foundational knowledge about how rare cell types contribute to tissue function and how their unique functional roles are established through differentiation from progenitors. Recently, a rare and distinct population of cells has been identified in human small intestine and colon, marked by Bestrophin 4 (Best4). The small intestinal population of Best4+ cells is striking, as these cells are highly enriched for CFTR (cystic fibrosis transmembrane conductance regulator), a feature that Best4+ colon cells lack. This CFTR High Expresser (CHE) cell population may serve an important role in the localized coordination of pH regulation in the small intestine, and is also a compelling target cell type for the small intestinal phenotypes of cystic fibrosis, for which there is currently no effective treatment. Despite mounting need to understand how CHE cells contribute to intestinal homeostasis and disease, we do not know how CHE cells are made (mechanisms controlling their differentiation) or what they do (their contribution to intestinal physiology). The goal of this proposal is to elucidate the fate specification of CFTR High Expresser (CHE) cells in the intestine, with the long- term goal of understanding the lineage origin of CHE cells as they arise from stem cells, and their role in homeostasis and disease. The overall objectives are (i) to define the transcriptional network driving intestinal CHE cell fate specification (Aim 1) and (ii) to define the role of Notch signaling in CHE cell fate specification (Aim 2). As CHEs are found only in rats and humans and genetic tools are limited in rats, I have developed a novel rat intestinal organoid model that allows for genetic perturbation. Aim 1 will determine the role of key candidate transcription factors identified by single-cell RNA sequencing in promoting CHE cell fate in rat intestinal organoids. Then, chromatin immunoprecipitation sequencing (ChIP-seq) will be performed to identify direct interactions between candidate transcription factors and CHE-enriched genes. Additionally, Notch signaling is critical for determining the initial cell fate decision of whether an intestinal progenitor cell becomes absorptive, "Notch on", or secretory, "Notch off." Surprisingly, our single cell RNA-seq data suggests that active Notch signaling may also be involved in specifying later cell fate decisions within the secretory lineage, particularly CHE cell fate. I will investigate this in Aim 2 using genetic manipulation of Notch in the rat intestinal organoid model. Lastly, I will use our ability to modulate CHE cell numbers by regulating Notch signaling to correlate CHE abundance to the physiological function of CFTR-mediated fluid secretion. Completion of this project will identify factors required for specifying CHE cells in the small intestine, informing insights into the specification of CHEs and other rare secretory intestinal cells, building a foundation to observe how CHEs arise from stem cells, and illuminating a potential novel target cell type in pathological conditions associated with CFTR dysregulation.