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

 描述(申请人提供)：肌肉干细胞，也被称为卫星细胞(SC)，功能是维持组织内稳态和再生骨骼肌。在成体中，干细胞通过内在机制和来自壁龛的外在信号维持在静止状态。干细胞的静止特性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，静止的干细胞的身份是如何建立和保持的？以及利基市场如何有助于保持这种身份？Noch信号通路是干细胞静止的主要调节因子。在这个方案中，我们的目标是1)确定维持SC静止的细胞来源和特定的Notch配体，以及2)Notch信号在激活的SC中如何变化。我们将删除Mindomb1，这是一种激活Notch配体的酶，特别是在肌肉纤维中，以确定Notch配体在体内稳态条件下的作用及其对SCs在体内再生反应下功能的影响。我们还将删除肌肉纤维中的Notch配体DLL4，因为初步数据表明，在出生后成熟的过程中，DLL4在肌肉纤维中的表达增加，这与静止SCs的出现相关。初步数据还显示，在完整的肌肉纤维上激活的干细胞在其细胞质中积累了DLL4；如果没有肌肉纤维，DLL4就会丢失。这表明，在激活的SC中，DLL4蛋白的增加需要肌肉纤维。我们将检测肌肉纤维上激活的SC的Notch靶标，以确定Notch信号是否减少，并将对激活的SCs中的DLL4进行功能丧失和功能获得，以了解Notch信号是否受到影响。这个提议的具体目的是：1)确定Notch配体在肌肉纤维中维持SC静止的作用，2)确定DLL4是否是Notch信号转导的关键介质 维持SC的静止；3)确定DLL4在激活的卫星细胞子体中的功能作用。该项目的成功完成将为了解Notch配体以非细胞自主方式作用于干细胞静默的调节以及在激活的SCs中这一过程如何转变为细胞自主信号提供分子洞察力。

 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.