The signals and cell mechanics driving stem cell niche morphogenesis

驱动干细胞生态位形态发生的信号和细胞机制

• 批准号: 9770537
• 负责人: Lauren M. Anllo
• 金额: $ 6.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9770537
• 关键词: Ablation; Actins; Adult; Affect; Aging; Anterior; Architecture; Area; Automobile Driving; Behavior; Biological Assay; Biological Models; Biological Process; Cell Adhesion Process; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Coupled; Cues; Data; Development; Drosophila genus; Embryo; Engineering; Erinaceidae; Exhibits; Fishes; Frequencies; Genes; Genetic; Germ Cells; Gonadal structure; Homeostasis; Image; Image Analysis; Integral Membrane Protein; Knowledge; Lasers; Letters; Lipids; Measurement; Mechanics; Mesoderm; Molecular; Molecular Cloning; Morphogenesis; Natural regeneration; Neoplasm Metastasis; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Positioning Attribute; Preparation; Process; RNA Interference; Resolution; Shapes; Signal Pathway; Signal Transduction; Source; Stem cells; Stratification; Structure; System; TNFRSF1A gene; Techniques; Temperature; Testing; Testis; Tissues; Visceral; cancer cell; cancer stem cell; cell behavior; daughter cell; design; driving force; experimental study; fly; germline stem cells; high resolution imaging; knock-down; male; mechanical force; monolayer; mutant; neurotrophic factor; notch protein; novel; prospective; recruit; response; stem cell biology; stem cell division; stem cell fate; stem cell niche; stem cell population

Abstract/Summary Stem cells are responsible for tissue homeostasis and for protective responses to aging or damage. This behavior requires contact with a well-designed niche: one positioned accurately, and with its constituent cells properly organized. It is therefore important to understand how a niche forms. Unfortunately, most niches are either not well defined, or not tractable at the necessary resolution. For this reason, I have chosen a model system to study niche development, the embryonic Drosophila male gonad. In adult testes, the niche is well-defined, and is a paradigm for studying stem cell-niche interactions. Until now, morphogenesis of this niche, which occurs in the embryo, has only been analyzed in fixed preparations. Our lab recently pioneered live-imaging of the entirety of niche development. This approach revealed key cell behaviors during morphogenesis: 1) Polarized assembly of prospective niche cells at the gonad anterior, and 2) Compaction of the niche into a stratified sphere. I propose to uncover signaling and cell mechanics that guide these processes. Aim 1 will define the source and identity of cues directing niche Assembly. My experiments have clearly implicated the visceral mesoderm (Vm) as the tissue guiding niche placement. I will genetically ablate sub-regions of the Vm to determine the portion required to guide niche assembly. I will then use molecular cloning techniques to generate a driver that specifically expresses in the relevant sub-region of the visceral mesoderm. To identify guidance cues, I will use the driver to drive RNAi against transmembrane and secreted factors that are expressed in the Vm. For candidate cues where there is no RNAi line directed against the cue, I will use CRISPR to engineer a GFP-tagged version of the gene, and then employ deGradFP knockdown. Candidates with a phenotype will be verified by misexpressing them and testing for changes in niche position. Aim 2 will identify the forces affecting Compaction and identify the signals that regulate those forces. I will use quantitative cell mechanics assays and post acquisition image analysis. I will utilize laser ablation to disrupt candidate pulling forces directing niche cell ingression, and test changes to ingression frequency. I will disrupt candidate signaling pathways, such as Hedgehog and Notch, and assess changes to niche area and circularity, and to cell bond tension along forming niche-stem cell interfaces. These experiments will define the physical forces and signaling required to form the niche structure required for proper communication with the stem cells.

摘要/摘要 干细胞负责组织稳态和对衰老的保护反应 或损坏。这种行为需要与精心设计的利基市场接触：一个定位 准确及其成分细胞正确组织。因此，重要的是 了解利基是如何形成的。不幸的是，大多数壁ches的定义不是很好，或者不是 可在必要的分辨率下进行处理。因此，我选择了一个模型系统来研究 利基发育，胚胎果蝇男性腺。 在成人睾丸中，利基市场定义明确，是研究干细胞细胞细胞的范式 互动。到目前为止，发生在胚胎中的这种利基的形态发生仅是 在固定准备工作中进行了分析。我们的实验室最近开创了整个利基的现场模仿 发展。这种方法揭示了形态发生过程中的关键细胞行为：1）极化 在性腺前的前瞻性小众细胞组装，2）将小众压实到一个 分层球。我建议发现指导这些过程的信号传导和细胞力学。 AIM 1将定义指导利基组装的提示的来源和身份。我的 实验清楚地将内脏中胚层（VM）视为指导小众的组织 放置。我将在遗传上消除VM的子区域，以确定所需的部分 指导利基组装。然后，我将使用分子克隆技术来生成一个驱动器 在内脏中胚层的相关子区域中特别表达。确定指导 提示，我将使用驾驶员对跨膜和分泌因素驱动RNAi 在VM中表达。对于没有针对提示的RNAi线的候选提示，我 将使用CRISPR来设计GFP标记的基因，然后使用DeGradFP 击倒。具有表型的候选者将通过对其进行验证并测试 利基位置的变化。 AIM 2将确定影响压实的力并确定调节的信号 那些力量。我将使用定量的细胞力学测定法和收购后图像 分析。我将利用激光消融来破坏候选候选力，指导小众细胞 摄入和测试变化对入口频率。我会破坏候选人的信号 途径，例如刺猬和缺口，并评估对利基区域和循环的变化，以及 沿形成小裂茎细胞界面的细胞键张力。这些实验将定义 形成适当需要的利基结构所需的物理力和信号传导 与干细胞通信。