Dynamic Mechanisms of Fate Control during Epithelial Organ Renewal

 描述（由申请人提供）：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身体需要的知识基础上。在成体器官更新中，每次干细胞分裂都会触发不对称、干细胞-干细胞和干细胞-终末命运之间的关键决定。为了维持干细胞和分化细胞的恒定数量，这三种命运结果必须共同平衡。相反，如果失去命运平衡，就会出现发育不良或退化。然而，与执行命运结果的已被充分研究的途径相反，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器官范围内分裂命运平衡的机制。为了实现这一目标，在这里，我们探测对称和不对称的命运决定的细胞基础-在体内和真实的时间-通过结合活体成像与果蝇的通用遗传工具。利用成年果蝇中肠，我们通过在活体动物中以高细胞分辨率开发上皮更新的长期成像，做出了开创性的创新。我们的方法使单个干细胞分裂， 在他们的自然环境中被捕获，命运决定被真实的时间可视化。 我们将探讨三个基本问题的细胞和分子的性质的命运决定。在目标1中，我们问命运决定是由分裂的母干细胞，由等能的子细胞，或组合。使用实时成像，我们将直接测试定向细胞分裂和命运决定子分配的母控机制，以及Notch介导的侧抑制的子控机制。我们将评估不同的机制是否偏向于不同的命运，并研究是否最初的命运决定可以推翻后作用机制。目标2建立在令人兴奋的初步数据基础上，这些干细胞是能动的，这引发了一个问题，即能动性是否通过改变与空间定位信号的接近程度来影响命运。我们将确定运动如何影响命运的决定，探测运动和命运的结果之间的相互作用，并确定细胞骨架调节剂，煽动运动。在目标3中，我们转向定义上皮结构的粘附连接，并询问这种多细胞粘附网络如何整合到命运决定中。我们将分别干扰干细胞、子细胞和分化的相邻细胞上的基底、侧向和顶端粘附受体，并分析不同细胞表面上的不同受体如何影响 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将有可能扩展到脊椎动物，包括人类的上皮器官。最终，了解决定分裂命运的基本机制将开辟新的治疗途径，以对抗干细胞病理学和促进器官再生。

 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.