Wnt transfer in the intestinal stem cell niche

肠上皮的持续再生对人类健康至关重要，也是理解成体干细胞在其利基中的调节的一个模型。WNT信号调节肠道干细胞的增殖和分化。过度的Wnt信号导致干细胞分裂增加，导致腺瘤和癌症，而Wnts的缺乏会导致肠道萎缩。因此，Wnt信号对于严格调控ISCs的活动和静止是至关重要的。最近，几个研究小组独立地证明，控制肠道干细胞生态位的WNT是由称为端细胞的特殊基质细胞组成的。然而，尽管WNTs在干细胞调控中很重要，但人们对这些关键调控因子如何从端粒细胞传递到ISCs知之甚少。有人认为，端细胞的长细胞延伸可能是Wnt转移所必需的，因为它们可以使WNTs定向和精确地传递到ISCs。相关的结构，称为信号丝状伪足或细胞素，参与Wnt在其他组织中的运输。然而，目前尚不清楚肠道中端粒细胞延伸的形成、调节和运动的确切机制。本研究项目旨在对WNTs从端细胞向ISCs传递的机制提供更深入的了解。这项工作将在新加坡杜克-国立大学医学院的David Virshup教授的实验室进行，他是健康和疾病中Wnt途径调控ISC领域的国际公认专家。在我两年的博士后研究期间，我将首先通过高分辨率光片和电子显微镜可视化端细胞的WNT传递装置，使3D重建能够深入了解细胞延伸的本质。其次，我将分离Wnt分泌基质细胞，通过下一代测序鉴定对Wnt分泌装置可能重要的端粒细胞丰富的基因。在选择了特定的有希望的筛选命中后，将通过原位杂交和免疫荧光显微镜来验证表达。最后，我将在已建立的测量WNTS从端细胞向ISCs传递的功能分析中对选定的候选基因进行表征。综上所述，该项目将阐明WNTT从隐窝基质细胞运输到肠道干细胞以调节增殖、分化和自我更新的机制。由于Wnt信号在多种组织中是至关重要的，而且该系统的功能障碍经常伴随着癌症，这一知识将对其他器官产生重要影响，并可能有助于开发新的治疗方法来预防或治疗人类结直肠癌和其他Wnt成瘾疾病。

The ongoing regeneration of the intestinal epithelium is critical for human health and a model for understanding the regulation of adult stem cells in their niche. Wnt signaling regulates the proliferation and differentiation of intestinal stem cells (ISCs). Excessive Wnt signaling induces increased stem cell division leading to adenomas and cancer, while the lack of Wnts causes intestinal atrophy. Consequently, Wnt signals are of the utmost importance for the strict regulation of activity and quiescence of ISCs. Recently, several groups independently demonstrated that the Wnts that control the intestinal stem cell niche are made in specialized stromal cells called telocytes. However, despite the importance of Wnts in stem cell regulation, little is known about how these pivotal regulators are delivered from telocytes to the ISCs. It was proposed that long cellular extensions of the telocytes might be essential for Wnt transfer, since they could enable a directed and precise transmission of Wnts to the ISCs. Related structures, called signaling filopodia or cytonemes, are involved in Wnt transport in other tissues. However, the exact mechanisms underlying formation, regulation and movement of telocyte extensions in the intestine are currently unknown. This research project aims to provide deeper insights into the mechanisms involved in the transmission of Wnts from telocytes to ISCs. This work will be performed in the laboratory of Prof. David Virshup at the Duke-NUS Medical School in Singapore, who is an internationally recognized expert in the field of ISC regulation by the Wnt pathway in health and disease. During my two-year postdoctoral fellowship, I will first visualize the Wnt transmitting apparatus of telocytes by high resolution light sheet and electron microscopy enabling 3D reconstruction to gain insight into the nature of the cellular extensions. Second, I will isolate Wnt secreting stromal cells to identify telocyte-enriched genes potentially important for the Wnt secreting apparatus by next generation sequencing. After selection of particular promising screening hits, expression will be validated by in situ hybridization and immunofluorescence microscopy. Finally, I will characterize selected candidate genes in established functional assays measuring transmission of Wnts from telocytes to ISCs. In summary, this project will illuminate the mechanisms by which Wnts are transported from pericryptal, stromal cells to the stem cells in the intestine to regulate proliferation, differentiation and self-renewal. As Wnt signaling is vital in diverse tissues and malfunctions of this system are often accompanied by cancer, this knowledge will have important implications for other organs and might help to develop novel therapeutic approaches to prevent or treat colorectal cancer and other Wnt-addicted diseases in humans.