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.