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/总结 干细胞负责组织稳态和对衰老的保护性反应 或损坏。这种行为需要接触一个精心设计的利基市场：一个定位的 准确地，并且其组成细胞正确组织。因此，重要的是 了解利基市场如何形成。不幸的是，大多数利基要么没有明确定义，要么没有 在必要的分辨率下易于处理。为此，我选择了一个模型系统来研究 生态位发育，胚胎果蝇雄性性腺。 在成年睾丸中，生态位定义明确，是研究干细胞生态位的范例 互动。到目前为止，这种发生在胚胎中的生态位的形态发生仅被了解 在固定制剂中进行分析。我们的实验室最近开创了整个利基市场的实时成像 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 性腺前部预期生态位细胞的组装，以及 2) 将生态位压缩成 分层球体。我建议揭示指导这些过程的信号传导和细胞机制。 目标 1 将定义指导利基组装的线索的来源和身份。我的 实验清楚地表明内脏中胚层 (Vm) 作为组织引导生态位 放置。我将从基因上消融 Vm 的子区域，以确定所需的部分 引导利基组装。然后我将使用分子克隆技术生成一个驱动程序 在内脏中胚层的相关亚区域中特异性表达。确定指导 提示，我将使用驱动程序来驱动针对跨膜和分泌因子的 RNAi 以Vm表示。对于没有针对该线索的 RNAi 线的候选线索，我 将使用 CRISPR 设计该基因的 GFP 标记版本，然后使用 deGradFP 击倒。具有表型的候选人将通过错误表达并进行测试来验证 利基位置的变化。 目标 2 将识别影响压实的力并识别调节的信号 那些势力。我将使用定量细胞力学测定和采集后图像 分析。我将利用激光烧蚀来破坏引导利基细胞的候选拉力 进入，并测试进入频率的变化。我会扰乱候选人信号 途径，例如 Hedgehog 和 Notch，并评估利基面积和循环度的变化，以及 沿着形成生态位-干细胞界面的细胞键张力。这些实验将定义 形成适当的利基结构所需的物理力和信号 与干细胞的通讯。