Project Summary The clinical and biological consequences of telomere dysfunction manifest in telomere biology disorders like dyskeratosis congenita (DC). DC is an inherited multisystem disorder characterized by degenerative phenotypes in tissues with high cell turnover, which often includes bone marrow failure and gastrointestinal (GI) disease. GI pathologies in DC patients include enterocolitis, mucosal ulceration, and malabsorption—findings that indicate a link between telomere dysfunction, intestinal regeneration, and gut barrier integrity. Thus, we seek to elucidate how telomere dysfunction impacts the homeostatic turnover of the intestinal epithelium (IE) to inform future therapeutic approaches. In the IE, intestinal stem cells (ISCs) require canonical Wnt/β-catenin pathway activity to maintain the tissue’s rapid cell turnover. Our lab discovered that Wnt activity and telomere fidelity lie in a positive feedback loop—telomerase and telomere capping genes are Wnt pathway targets, and telomere fidelity is required to sustain Wnt target gene expression. Recently, a population of mesenchymal Foxl1+ telocytes in the lamina propria directly underlying the IE has been identified as the critical niche source of Wnt ligands required for ISC proliferative self-renewal. However, in the context of telomere dysfunction, ISCs exhibit a loss of self-renewal and a broad downregulation of Wnt target gene expression, corresponding to the downregulation of Wnt ligands now known to secreted from subepithelial telocytes. The goal of this proposal is therefore to understand the consequences of telomere dysfunction on ISC niche function. Our overarching hypothesis is that telomere failure suppresses telocyte-derived niche support for ISCs, contributing to the intestinal phenotypes associated with telomeropathies. To address this hypothesis, we will utilize a novel Foxl1CreERT2-2A-tdTomato reporter mouse line that will allow for direct in situ visualization of Foxl1+ telocytes, crossed with an mTerc-/- mouse line that features genetic deletion of the telomerase RNA component (mTerc), leading to loss of telomerase activity and progressive telomere dysfunction. We seek to determine the niche factor gene expression and telomere defects in Foxl1+ telocytes in mTerc-/-::Foxl1CreERT2-2A-tdTomato mice by a combination of in situ assays and single- cell RNAseq on isolated telocytes. We will also determine the functional consequences of telocyte-specific defects on ISC self-renewal by utilizing primary organotypic co-culture assays in which primary telocytes and crypt epithelia-derived organoids from mutant mTerc-/-::Foxl1CreERT2-2A-tdTomato and control reporter mice are isolated and co-cultured. Lastly, we will determine whether wildtype donor bone marrow-derived telocytes have the ability to engraft within the lamina propria of mTerc-/- mice and rescue ISC function by restoring niche-derived Wnt activators to the ISC compartment. The findings from this proposal will eluci...