Identification and characterization of new factors involved in feedback regulation of stem cell proliferation

干细胞是人体内最重要的细胞之一。它们是相对未分化的细胞，具有增殖和数量扩增的能力，同时它们也可以分化成更专门的细胞类型。从胚胎干细胞到过多的成体（组织特异性）干细胞，它们对我们组织和器官的生成和再生至关重要。因此，这些关键细胞如何发挥作用以及它们如何受到调节一直是深入研究的焦点。特别关键的是发现了干细胞小生境，它提供了一个空间限制的环境，在其中干细胞保持在其增殖的未分化状态。因此，小生境驻留干细胞具有独特的增殖和扩增能力，尽管它们也可以以非活性或静止状态存在于那里。干细胞是保持静止还是增殖是它们从各种来源获得的生长因子整合的结果。一种重要的生长因子是胰岛素/IGF-1信号，其全身分布以刺激营养摄取后大多数（如果不是全部）干细胞的增殖。然而，并不是所有的干细胞群体在动物的组织内和跨同样地响应于该信号，使得在任何一个时间：一些干细胞群体可以增殖，但其他干细胞群体可能不增殖。我们和其他人发现，干细胞增殖是由来自干细胞分化后代的稳态信号局部调节的证据。这种反馈信号是由组织中需要新分化的细胞来取代旧的或受损的细胞而产生的。因此，当给定组织中不需要新的分化细胞时，这种反馈信号局部拮抗胰岛素/IGF-1并阻断亲本干细胞群的增殖。组织对新分化细胞的需求如何超越系统性胰岛素/IGF-1信号以局部调节成体SC增殖速率是生物学中未解决的主要问题。通过一种新的筛选方法，我们已经开发了一个方便的，良好的无脊椎动物模型（C。elegans），我们已经分离了5个新的等位基因，其特异性地阻止生殖系干细胞增殖的稳态调节。我们发现其中一个突变影响了一种叫做Papilin的细胞外基质蛋白。我们的目的是通过以下三个具体目标来解决Papilin，细胞外基质和四个新的因子如何参与反馈干细胞调控：1）确定Papilin在GSC增殖的稳态调节中的作用。2)定义Papilin的PLAC结构域介导的相互作用网络在稳态GSC调节。3)克隆和表征涉及稳态GSC调节的四个新突变。根据NSERC的授权，拟议研究的广泛目标是扩大我们对调节干细胞活性的体内机制的基本知识。

Stem cells are amongst the most important cells in the human body. They are relatively undifferentiated cells that have the capacity to proliferate and expand in numbers, while they can also differentiate into more specialized cell types. From the embryonic stem cells to the plethora of adult (tissue-specific) stem cells, they are essential to the generation and regeneration of our tissues and organs. How these critical cells function and how they are regulated has therefore been the focus of intensive investigation. Particularly key was the discovery of the stem cell niche, which provides a spatially restricted environment in which stem cells are maintained in their proliferative, undifferentiated state. Niche-resident stem cells thus uniquely have the capacity to proliferate and expand in numbers, although they can also exist there in an inactive or quiescent state. Whether stem cells remain quiescent or proliferate is the result of the integration of the growth factors that they receive from various sources. One important such growth factor is an insulin/IGF-1 signal that is systemically distributed to stimulate the proliferation of most (if not all) stem cells following nutrient uptake. Yet, not all stem cell populations across and within an animal's tissues equally respond to this signal, such that at any one time: some stem cell populations may proliferate, but others may not. We, and others, found evidence that stem cell proliferation is adjusted locally by homeostatic signals originating from the stem cells' differentiated progeny. This feedback signal is generated by the need for newly differentiated cells in a tissue, to replace old or damaged cells. As such, when no new differentiated cells are needed in a given tissue, such feedback signals locally antagonize insulin/IGF-1 and block proliferation of the parental stem cell population. How a tissue's needs for new differentiated cells overrides the systemic insulin/IGF-1 signal to locally adjust adult SC proliferation rates is a major unsolved problem in biology. Through a new screening method that we have developed in a convenient, well-characterized invertebrate model (C. elegans), we have isolated 5 new alleles that specifically prevent homeostatic regulation of germline stem cell proliferation. We found that one of these mutations affects an extracellular matrix protein called Papilin. We aim to resolve how Papilin, the extracellular matrix and four new factors participate in feedback stem cell regulation through the following three specific objectives: 1) Establish the role of Papilin in homeostatic regulation of GSC proliferation. 2) Define Papilin's PLAC domain-mediated interaction network in homeostatic GSC regulation. 3) Clone and characterize four new mutations implicated in homeostatic GSC regulation. In line with NSERC's mandate, the broad goal of the proposed research is thus to expand our fundamental knowledge about the in vivo mechanisms that regulate stem cell activity.