Dynamic Mechanisms of Fate Control during Epithelial Organ Renewal

 描述(申请人提供)：有效和安全的干细胞疗法必须建立在干细胞如何产生精确数量的分化细胞以满足人体需求的知识基础上。在成人器官更新中，每一次干细胞分裂都会在不对称、对称干细胞和对称末端命运之间触发一个关键的决定。为了维持干细胞和分化细胞的数量不变，这三种命运的结果必须共同平衡。相反，如果命运失去平衡，就会出现发育不良或退化。然而，与执行命运结果的经过充分研究的路径相比，决定命运结果的上游事件几乎是未知的。我们的长期目标是了解仲裁整个器官范围内分裂命运平衡的机制。为了实现这一目标，我们在这里通过将实时成像与果蝇的多种遗传工具相结合，在体内和实时探索对称和不对称命运决定的细胞基础。利用成年果蝇中肠，我们通过开发活体动物高细胞分辨率的上皮更新的长期成像，取得了一项开创性的创新。我们的方法使单个干细胞分裂能够 被捕捉到他们的原生环境和命运决定，以便实时可视化。我们将研究关于命运决定的细胞和分子性质的三个基本问题。在目标1中，我们询问命运是由分裂的母干细胞、等效子细胞还是两者的组合来决定的。利用实时成像，我们将直接测试定向细胞分裂和命运决定基因分割的母控机制，以及Notch介导的侧向抑制的子控机制。我们将评估不同的机制是否偏向不同的命运，并检查最初的命运决定是否可以被后来的作用机制推翻。目标2建立在令人兴奋的初步数据基础上，这些干细胞是可移动的，这引发了这样一个问题：移动性是否通过改变与空间定位信号的接近来影响命运。我们将确定运动如何影响命运的决定，探索运动和命运结果之间的相互作用，并确定刺激运动的细胞骨架调节因子。在目标3中，我们转向定义上皮结构的黏附连接，并询问这种多细胞黏附网络如何整合到命运决定中。我们将分别干扰干细胞、子代细胞和分化的相邻细胞上的基础、侧向和顶端黏附受体，并分析不同细胞表面上不同的受体如何影响 命运决定机制和结果。由于上皮干细胞的生物学和结构是广泛保守的，这里发现的命运决定机制可能会延伸到脊椎动物的上皮器官，包括人类。最终，了解决定分裂命运的基本机制将为对抗干细胞病理和促进器官再生开辟新的治疗途径。

 DESCRIPTION (provided by applicant): Effective and safe stem cell therapies must build upon knowledge of how stem cells generate precise numbers of differentiated cells to meet the body's needs. In adult organ renewal, each stem cell division triggers a pivotal decision between asymmetric, symmetric-stem, and symmetric-terminal fates. To sustain constant numbers of stem and differentiated cells, these three fate outcomes must be collectively balanced. Conversely, dysplasia or degeneration arises if fate balance is lost. Yet in contrast to the well-studied pathways that execute fate outcomes, the upstream events that decide between fate outcomes are virtually unknown. Our long-term goal is to understand the mechanisms that arbitrate the organ-wide balance of division fates. Toward this goal, here we probe the cellular basis of symmetric and asymmetric fate decisions-in vivo and in real time-by combining live imaging with the versatile genetic tools of Drosophila. Using the adult Drosophila midgut, we have made a path breaking innovation by developing long-term imaging of epithelial renewal at high cellular resolution in live animals. Our methodology enables individual stem cell divisions to be captured in their native context and fate decisions to be visualized in real time. We will investigate three fundamental questions about the cellular and molecular nature of fate decisions. In Aim 1, we ask whether fate decisions are made by the dividing mother stem cell, by equipotent daughter cells, or a combination. Using live imaging, we will directly test the mother-control mechanisms of oriented cell division and fate determinant partitioning, and the daughter-control mechanism of Notch-mediated lateral inhibition. We will evaluate whether different mechanisms bias toward different fates, and examine whether initial fate decisions can be overturned by later-acting mechanisms. Aim 2 builds upon exciting preliminary data those stem cells are motile, which provokes the question of whether motility influences fates by altering proximity to spatially localized signals. We will determine how motility impacts fate decisions, probe the interplay between motility and fate outcomes, and identify the cytoskeletal regulators that instigate motility. In Aim 3, we turn to the adhesion junctions that define epithelal architecture and ask how this multicellular adhesive network integrates into fate decisions. We will separately perturb basal, lateral, and apical adhesion receptors on stem, daughter, and differentiated neighbor cells and parse how distinct receptors on different cell surfaces influence fate decision mechanisms and outcomes. Because epithelial stem cell biology and architecture are broadly conserved, the fate decision mechanisms uncovered here will potentially extend to epithelial organs in vertebrates, including humans. Ultimately, understanding the basic mechanisms that decide between division fates will open new therapeutic avenues to combat stem cell pathologies and promote organ regeneration.