角膜缘干细胞(LSC)分化异常可导致角膜丧失透明性，是世界主要的致盲眼病。然而其分化的调控机制并未阐明。我们前期研究揭示Wnt7a可通过激活pax6表达调控LSC分化。但另有研究显示，WNT通路关键效应因子β-catenin具有抑制pax6表达的作用。因此，LSC中β-catenin是否及如何参与Wnt7a激活分化作用，是阐明WNT通路如何调控分化的关键。研究表明，LSC中Notch1抑制β-catenin激活，且notch1敲除与pax6敲低使LSC分化的角膜上皮细胞出现相似的异常表型。由此我们推测，Notch1是β-catenin未被Wnt7a激活的关键，其通过抑制β-catenin激活间接维持了pax6的表达。本项目将在体外培养的人LSC中敲除notch1或持续激活β-catenin，通过观察相关蛋白的表达，结合notch1条件性敲除小鼠的表型，在体外和体内层面对上述推测进行验证。

Abnormality in limbal stem cell(LSC) differentiation causes opaque corneal epithelium, which leads to blindness of millions of people worldwide. However, the mechanism that governs LCS differentiation is not clarified yet. Our previous studies have shown that Wnt7a can regulate LSC differentiation by activating pax6. Nevertheless, studies on WNT pathways indicated that its key downstream effector β-catenin could inhibit pax6 expression. Thus, the role of β-catenin in Wnt7a regulated LSC differentiation serves as the key to clarify how WNT pathways affect LSC differentiation. In addition, previous works have shown that Notch1 represses β-catenin activation in LSC. Moreover, notch1 knock out LSC showed similar but abnormal phenotype as pax6 knock down LSC after cells’ differentiation. Therefore, we propose that Notch1 indirectly maintains pax6 expression through inhibiting β-catenin activation by Wnt7a. In this project, to investigate the above hypothesis, CRISPR/Cas9 will be used to generate notch1 knock out or β-catenin gain-of-function cells in primarily cultured human LSC. The expression of related proteins in these cells will be examined, combining with the results of phenotype of notch1 conditional knock out mouse.