Determining the Role of Notch Ligands in Regulating Adult Satellite Cell Fate

 描述（由申请人提供）：肌肉干细胞，也称为卫星细胞（SC），具有维持组织稳态和再生骨骼肌的功能。在成年人中，SC通过内在机制和来自小生境的外在信号维持在静止状态。干细胞的静止性质使干细胞库免于过早耗尽。一个基本的问题是：在生物体的生命过程中，静止干细胞的身份是如何建立和维持的？以及利基如何有助于保持这种身份？notch信号通路是干细胞静止的主要调节器。在这项提议中，我们的目标是1）确定维持SC静止的细胞来源和特定Notch配体，以及2）Notch信号在活化的SC中如何变化。我们将删除Mindbomb 1，一种激活Notch配体的酶，特别是在肌纤维中，以确定Notch配体在稳态条件下的作用及其对SC在体内再生反应下发挥功能的能力的影响。我们还将删除肌纤维中的Notch配体Dll 4，因为初步数据表明，在出生后成熟期间，肌纤维中的Dll 4表达增加，这与静止SC的出现相关。初步数据还显示，完整肌纤维上的活化SC在其细胞质中积累Dll 4;其在不存在肌纤维的情况下丢失。这表明肌纤维是激活SC中Dll 4蛋白增加所必需的。我们将针对Notch靶点测定肌纤维上的激活SC，以观察Notch信号传导是否减少，并将对激活SC中的Dll 4进行功能丧失和获得，以观察Notch信号传导是否受到影响。该提议的具体目的是：1）确定Notch配体在肌纤维中维持SC静止的作用，2）确定Dll 4是否是Notch信号传导的关键介体， 3）确定Dll 4在活化的卫星细胞子细胞中的功能作用。该项目的成功完成将提供对小生境中Notch配体以非细胞自主方式作用的SC静止调节的分子见解，以及这种调节如何在激活的SC中过渡到细胞自主信号传导。

 DESCRIPTION (provided by applicant): Muscle stem cells, also called Satellite Cells (SC) function to maintain tissue homeostasis and regenerate skeletal muscle. In the adult, the SCs are maintained in a quiescent state both by intrinsic mechanisms and extrinsic signals from the niche. The quiescent nature of stem cells preserves the stem cell pool from premature exhaustion. One fundamental question is: how is the identity of the quiescent stem cells established and maintained during the life of an organism? And how the niche contributes to maintaining this identity? The notch signaling pathway is a major regulator of stem cell quiescence. In this proposal we aim to 1) identify the cell source and the specific Notch ligands that maintain SC quiescence and 2) how Notch signaling changes in the activated SCs. We will delete Mindbomb1, an enzyme that activates Notch ligands, specifically in the muscle fiber to determine the role of Notch ligands under homeostatic conditions and their impact on the ability of SCs to function under a regenerative response in vivo. We will also delete Dll4, a Notch ligand in the muscle fiber as preliminary data suggests that Dll4 expression increases in the muscle fiber during postnatal maturation which correlates with the appearance of quiescent SCs. Preliminary data also shows that activated SCs on an intact muscle fiber accumulate Dll4 in their cytoplasm; which is lost in the absence of muscle fibers. This suggests that muscle fiber is required for the increase in Dll4 protein in activated SC. We will assay the activated SC's on muscle fibers for Notch targets to see if there is a decrease in Notch signaling and will perform loss and gain-of-function for Dll4 in the activated SCs to see if Notch signaling is affected. The specific aims of this proposal are: 1) To determine the role of Notch ligands in the muscle fiber to maintain SC quiescence, 2) To determine if Dll4 is the critical mediator of Notch signaling that maintains SC quiescence and 3) To determine the functional role of Dll4 in activated satellite cell daughters. Successful completion of this project will provide molecular insights into the regulation of SC quiescence by the Notch ligands in the niche acting in a non-cell autonomous manner and how this transitions to cell-autonomous signaling in activated SCs.