Defining the mechanism of Clara cell dedifferentiation into basal stem cells

摘要 最近，对祖细胞和干细胞的定义越来越受到关注。 肺的种群，并解剖管理的分子机制 这些细胞在呼吸道再生和修复过程中的行为。最近的多项研究 不同的器官系统也证明了明确定义的分化细胞 因为祖细胞和干细胞群体比以前更具可塑性 被认可了。然而，损伤后的呼吸道上皮细胞的可塑性一直不佳。 有记录在案。 白喉毒素诱导的细胞死亡与遗传谱系追踪相结合已被 用来严格证明皮肤和皮肤中不同类型的上皮干细胞 肠道可以相互转换。同样的战略组合也证明了 分化的肠腔上皮细胞可“去分化”为肠干细胞 细胞。我们提供的初步数据表明，腔内的Clara细胞可以将 在损伤后进入呼吸道基础干细胞。 我们通过表达白喉(DTA)毒素在 基底层细胞，这会导致这些基底层细胞发生凋亡。作为对此的回应 基底干细胞耗尽，腔内Clara细胞前体细胞开始复制，然后 这些Clara细胞开始表达基础细胞特异性标记p63和细胞角蛋白5。 此外，经过分离纯化的Clara细胞和体外培养的细胞失去了它们的 特征性的Clara细胞标志物，开始表达YAP1转录共激活因子， 然后表达基础干细胞特有的标记。这些去分化的基底样细胞 然后，细胞开始复制并形成集落。我们假设光芒四射的克拉拉 当基础干细胞库被激活时，细胞前体细胞可以转化为基础干细胞 耗尽了。为了验证这一假设，我们将追溯Clara细胞对 DTA介导的气道基底干细胞消融后的基础干细胞库。到时候我们会的 检测去分化的Clara细胞是否具有干细胞功能。我们 也假设YAP1转录共激活子是Clara细胞所必需的 去分化。我们将使用小鼠遗传学来测试YAP1的表达是否 对Clara细胞的可塑性是充分的和/或必要的。

Summary Recently, increasing attention has been paid to defining the progenitor and stem cell populations of the lung and dissecting the molecular mechanisms that govern the behavior of these cells during airway regeneration and repair. Recent studies in multiple different organ systems have demonstrated that well-defined differentiated cells as well as progenitor and stem cell populations are much more plastic than previously recognized. However, the plasticity of airway epithelial cells after injury has not been well documented. Diphtheria toxin-induced cell death in combination with genetic lineage tracing has been used to stringently demonstrate that distinct types of epithelial stem cells in the skin and intestine can interconvert. The same pairing of strategies has also demonstrated that differentiated luminal intestinal epithelial cells can "dedifferentiate" into intestinal stem cells. We present preliminary data that suggests that a luminal Clara cell can convert into an airway basal stem cell following injury. We ablated airway basal stem cells by expressing diphtheria (DTA) toxin exclusively in basal cells, which causes those basal cells to undergo apoptosis. In response to this depletion of basal stem cells, luminal Clara cell progenitors begin replicating and then these Clara cells begin to express the basal cell-specific markers p63 and cytokeratin 5. Furthermore, Clara cells that are sorted to purity and cultured ex vivo lose their characteristic Clara cell markers, begin expressing the Yap1 transcriptional co-activator, and then express markers specific to basal stem cells. These dedifferentiated basal-like cells then start replicating and give rise to colonies. We hypothesize that luminal Clara cell progenitors can convert into basal stem cells when the basal stem cell pool is depleted. To test this hypothesis, we will lineage trace the contribution of Clara cells to the basal stem cell pool after DTA-mediated airway basal stem cell ablation. We will then test whether the dedifferentiated Clara cells possess functional stem cell capacity. We also hypothesize that the Yap1 transcriptional co-activator is necessary for Clara cell dedifferentiation. We will use mouse genetics to test whether Yap1 expression is sufficient and/or necessary for Clara cell plasticity.