Dynamic polarity mechanisms controlling stem cell asymmetry during tissue development

项目总结/摘要 细胞极性是组织形成所必需的，也是干细胞产生细胞多样性所必需的 在发育和体内平衡过程中。因此，极性缺陷可以驱动发育障碍， 疾病，包括癌症。以前的研究已经阐明了产生极性和 打破单细胞的对称性。这些机制如何整合以产生复杂的组织仍然存在 不太了解。拟南芥叶表皮气孔谱系是一个理想的定量研究系统 确定内在细胞极性如何传递到子细胞不对称性，由于其良好的特征， 发育轨迹、光学可及性和遗传易处理性。在这个建议中，我将利用气孔 谱系1）测试一种新的微管依赖性抑制环如何利用保守逻辑产生极性 在干细胞和2）描绘极性依赖机制，驱动子细胞不对称。完成 提出的目标，我已经开发了新的体内工具，以操纵极性的气孔谱系与高 时间和亚细胞精度。在目标1中，我将使用靶向消除，分子遗传学和高分辨率 成像来测试微管和皮质极性蛋白之间的相互抑制如何产生一个单一的 多能干细胞的极性轴。此外，我将使用定量体内成像实验测试 将抑制电路与并行的依赖于MAPK的正反馈回路集成如何增加极性 鲁棒性在目标2中，我将利用气孔谱系的优势来确定干细胞极性 调节不同的子细胞大小。通过敏锐地操纵极性和跟踪子细胞的大小， 生长和命运的决定，使用长期的时间推移显微镜在体内，我将直接测试极性如何调节 下游不对称此外，通过鉴定气孔谱系中的新极性调节因子，我将 鉴定将极性与不同子代生长联系起来的分子途径。完成拟议目标 将在多细胞发育过程中测试极性模型，并将确定将极性与茎连接起来的机制。 细胞不对称阐明极性机制在一个进化分歧的物种有着广泛的意义 极性模型，并可以定义合成策略，以纠正人类疾病状态中的极性缺陷。

PROJECT SUMMARY/ABSTRACT Cell polarity is necessary to pattern tissues and is essential in stem cells to generate cellular diversity during development and homeostasis. Accordingly, polarity defects can drive developmental disorders and diseases, including cancer. Previous studies have elucidated molecular mechanisms that generate polarity and break symmetry in single cells. How these mechanisms are integrated to generate complex tissues remains poorly understood. The stomatal lineage in the Arabidopsis leaf epidermis is an ideal system to quantitatively determine how intrinsic cell polarity is transmitted to daughter cell asymmetry due to its well-characterized developmental trajectory, optical accessibility, and genetic tractability. In this proposal, I will utilize the stomatal lineage to 1) test how a novel microtubule-dependent inhibitory loop utilizes conserved logic to generate polarity in stem cells and 2) delineate polarity-dependent mechanisms that drive daughter cell asymmetry. To accomplish the proposed aims, I have developed novel in vivo tools to manipulate polarity in the stomatal lineage with high temporal and subcellular precision. In Aim 1, I will use targeted depletion, molecular genetics, and high-resolution imaging to test how mutual inhibition between microtubules and cortical polarity proteins generate a single polarity axis in multipotent stem cells. Additionally, I will use quantitative in vivo imaging to experimentally test how integrating an inhibitory circuit with a parallel MAPK-dependent positive feedback loop increases polarity robustness. In Aim 2, I will leverage the strengths of the stomatal lineage to determine how stem cell polarity regulates differential daughter cell size. By acutely manipulating polarity and tracking resulting daughter cell size, growth, and fate decisions using long-term time-lapse microscopy in vivo, I will directly test how polarity regulates downstream asymmetry. Furthermore, by identifying novel polarity regulators in the stomatal lineage, I will identify molecular pathways that link polarity to differential daughter growth. Completion of the proposed aims will test polarity models during multicellular development and will identify the mechanisms linking polarity to stem cell asymmetry. Elucidation of polarity mechanisms in an evolutionarily divergent species has broad implications for polarity models and may define synthetic strategies to correct polarity defects in human disease states.