Single-Cell Analysis of Proxy Reporter Allele-Marked Epithelial Cells Establishes Intestinal Stem Cell Hierarchy

• Proxy intestinal stem cell reporter alleles often mark heterogeneous populations
• Discrepancies exist between proxy reporter activity and endogenous transcripts
• Reserve and active intestinal stem cells are molecularly distinct
• Reserve intestinal stem cells give rise to active stem cells during homeostasis

SummaryThe recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.

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