14-3-3?? and epithelial differentiation in the eye and other tissues

描述（由申请人提供）：上皮细胞分化已被广泛研究，该过程可分为两个一般步骤：从常驻干细胞产生增殖祖细胞，以及随后这些祖细胞的捕获和分化。转录因子TCF3在激活因子和抑制因子之间的振荡是维持常驻干细胞群体和为这些干细胞产生祖细胞脉冲的关键。这些祖细胞增殖以填充组织场，然后Notch通路触发它们的细胞周期阻滞和分化。因此，当Notch1发生突变时，角膜、睑板腺和毛囊中的祖细胞不能分化，它们的增殖超出组织范围，导致病理性功能丧失。增殖祖细胞中Notch1信号起始的时间对于确定细胞的精确数量和组织拓扑结构至关重要。关于Notch1信号在这个过程中是如何启动的，现在才刚刚出现。上皮组织场是通过细胞-细胞接触抑制在增殖祖细胞接触时建立的。细胞接触抑制信号的突变导致祖细胞生长超出正常组织范围（如Notch1突变），组织功能丧失，最终导致癌症。但是，Notch1信号的启动是如何与这种细胞间接触联系起来的呢？钙粘蛋白启动的连接复合物不仅介导上皮细胞的顶端极性，它们还启动被称为Hippo的激酶级联反应，使一组转录因子磷酸化，这些转录因子调节细胞周期和分化程序。这种磷酸化为14-3-3提供了一个结合位点，而14-3-3又以无活性的形式将因子隔离在细胞质中。最近的研究结果表明，细胞接触抑制激酶级联组件的突变导致Notch1活性的丧失，同样，我们发现该途径中14-3-3的突变也导致Notch1表达的丧失。综上所述，这些结果表明，当祖细胞在组织中扩展时，它们最终的细胞接触信号是14-3-3依赖性Notch1的表达，从而发出阻滞和分化的信号。为了支持这一假设，我们发现小鼠的14-3-3突变导致增殖祖细胞的积累，并且在角膜、毛囊和睑板腺等导管结构中出现与Notch 1难以区分的表型。14-3-3或Notch1的缺失会导致严重的角膜缺损，导管阻塞导致末端囊膜的缺失，毛囊异常导致脱发。重要的是，我们证明了激活Notch1的表达可以修复14-3-3突变上皮祖细胞的分化缺陷。在此，我们提出了一系列的研究，旨在进一步建立14-3-3 -3和Notch1通路在角膜、眉板腺和毛囊上皮分化中的联系，并建立小鼠模型系统，为未来的R01提案研究14-3-3 -3和Notch1调控上皮分化开始的途径的分子细节奠定基础。

DESCRIPTION (provided by applicant): Epithelial differentiation has been widely studied, and the process can be divided into two general steps: generation of proliferating progenitors from resident stem cells, and the subsequent arrest and differentiation of these progenitors. Oscillation of the transcription factor TCF3 between activator and repressor is key to both maintaining a resident stem cell population and generating pulses of progenitor cells for these stem cells. These progenitors proliferate to fill the tissue field, and then the Notch pathway triggers their cell cycle arrest and differentiation. Accordingly when Notch1 is mutated, progenitors in the cornea, meibomian gland and hair follicles fail to differentiate and they proliferate beyond the tissue fields leading to pathologic loss of function. The timing of Notch1 signal initiation in proliferating progenitors is critical to define the precise number of cells and thus tissue topology. Insight into to how Notch1 signaling is initiated in this process is only now emerging. Epithelial tissue fields are established by cell-cell contact inhibition as proliferating progenitors come in contact. Mutations in cell contact inhibition signaling lead to progenitor outgrowth beyond normal tissue fields (as with Notch1 mutation), loss of tissue function and ultimately cancer. But, how might onset of Notch1 signaling be linked to such cell-cell contact? Cadherin-initiated junctional complexes not only mediate apical polarity in epithelial cells, they also initiate a kinase cascade known as Hippo which phosphorylates a set of transcription factors that regulate both the cell cycle as well as differentiation programs. This phosphorylation provides a binding site for 14-3-3, which in turn sequesters the factors in an inactive form in the cytoplasm. Recent results demonstrate that mutations in components of the cell contact inhibition kinase cascade lead to loss of Notch 1 activity, and likewise we have found that mutation of 14-3-3s in this pathway also leads to loss of Notch1 expression. Taken together, such results imply that as progenitors expand in tissues, their eventual cell contact signals 14-3-3s -dependent expression of Notch1 thereby signaling arrest and differentiation. In support of this hypothesis, we have found that mutation of 14-3-3s in mice leads to accumulation of proliferating progenitor cells and a phenotype indistinguishable from that of Notch 1 in the cornea, hair follicle and ductal structures such as those in the meibomian gland. This loss of 14-3-3s or Notch1 causes severe corneal defects, ductal obstruction leading to loss of terminal ascini and abnormal hair follicles leading to hair loss. Importantly, we demonstrate that expression of activated Notch1 rescues the differentiation defect in 14-3-3s mutant epithelial progenitors in culture. Here, we propose a series of studies designed to further establish linkage between 14-3-3s and the Notch1 pathway in epithelial differentiation in the cornea, meibomian gland and hair follicles and to development mouse model systems which can be used as a basis for a future R01 proposal examining the molecular details of the pathway through which 14-3-3s and Notch1 regulate the onset of epithelial differentiation.