Investigating the role of telomere failure on intestinal stem cell niche function

项目摘要 端粒功能障碍的临床和生物学后果表现为端粒生物学疾病，如 先天性角化不良(DC)。DC是一种以退行性表型为特征的遗传性多系统疾病 在细胞周转率高的组织中，通常包括骨髓衰竭和胃肠道(GI)疾病。美国退伍军人协会 DC患者的病理包括小肠结肠炎、粘膜溃疡和吸收不良--这些发现表明 端粒功能障碍、肠道再生和肠道屏障完整性之间的联系。因此，我们试图澄清 端粒功能障碍如何影响肠上皮(IE)的动态平衡周转 治疗方法。在IE中，肠干细胞(ISCs)需要规范的Wnt/β-Catenin途径活性 以维持组织细胞的快速周转。我们的实验室发现，Wnt活性和端粒保真度存在于 正反馈环-端粒酶和端粒覆盖基因是Wnt途径的靶点，并且端粒的保真度 是维持Wnt靶基因表达所必需的。最近，一群间充质Foxl1+端细胞在 直接位于IE下方的固有层被认为是Wnt配体的关键生态位来源 ISC增殖性自我更新所必需的。然而，在端粒功能障碍的背景下，ISCs表现出一种损失 自我更新和Wnt靶基因表达的广泛下调，与下调相对应 现在已知的Wnt配体是由上皮下端粒细胞分泌的。因此，这项提议的目标是 了解端粒功能障碍对ISC小生境功能的影响。我们最重要的假设是 端粒衰竭抑制了端粒细胞对ISCs的支持，从而导致肠道表型 与端粒病有关。为了解决这一假设，我们将利用一个新的Foxl1CreERT2-2A-tdTomato报告程序 可以直接原位观察Foxl1+端粒细胞的小鼠品系，与mTerc-/-小鼠品系杂交 其特征是端粒酶RNA组分(MTerc)的基因缺失，导致端粒酶活性丧失 和进行性端粒功能障碍。我们试图确定利基因子基因的表达和端粒 用原位检测和单细胞培养相结合的方法检测mTerc-/-：：Foxl1CreERT2-2a-td番茄小鼠Foxl1+端粒细胞的缺陷。 分离的端粒细胞上的细胞RNAseq。我们还将确定特定于Telcell的功能后果 利用原代器官型共培养试验研究间质干细胞自我更新的缺陷 突变型mTerc-/-：：Foxl1CreERT2-2a-td番茄和对照报告小鼠的隐窝上皮细胞衍生的有机物 隔离和共培养。最后，我们将确定野生型供者骨髓来源的端粒细胞是否有 MTerc-/-小鼠移植到固有层的能力和通过修复生态位衍生的ISC功能的能力 将WNT激活剂送到ISC隔间。这项研究的发现将阐明端粒功能障碍 改变ISC利基，从而为未来旨在减轻端粒的治疗方法提供信息 先天性角化不良等由功能障碍引起的疾病。

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 elucidate how telomere dysfunction alters the ISC niche, and thus inform future therapeutic approaches aimed toward alleviating telomere dysfunction-driven disorders like dyskeratosis congenita.