Msx2在小眼球畸形中对晶状体视网膜发育及诱导的调控机制研究

Microphthalmia is a developmental failure of the anterior and posterior segment tissues of the eye and an important cause of severe visual impairment in infants and young children. Complex molecular interactions dictate developmental steps leading to a mature and functional lens and retina. Msx2 has recently been implicated as a potential gene critical for eye development. However, the role of Msx2 within the complex mechanisms of eye development remains elusive. Our previous study firstly observed the morphological changes in conventional Msx2 knockout mice. Msx2 knockout mice were found to demonstrate phenotype of microphthalmia seen in human. Loss of Msx2 down regulates FoxE3 expression in the lens and down regulates Sox2 expression in the retina. And Sox2 is one of most important regulator in eye development and almost 20% microphthalmia caused by Sox2. Our present study focus on the mechanism of retina development and retina-lens induction during development process according to the results we found. We investigated the relationship between Msx2, Sox2 and downstream pathway, such as Notch and Wnts using Msx2 conditional knockout mice and Sox2 transgenic mice by promoter analysis, ISH and immunofluorescein. We will provide the evidence that the Msx2 gene is critically involved in retina and lens development. The results will provide the first direct genetic evidence for the mechanism of microphthalmia and the distinct function of Msx2 in regulating the growth and development of eye.

肌节同源盒基因同系物2（Msx2）表达缺失引起眼组织发育障碍的分子机理是目前小眼球畸形发病机制的研究热点。申请人前期工作中利用Msx2基因敲除小鼠在国际上首次发现Msx2作为上游调控因子调控FoxE3基因在晶状体内表达及Sox2基因在视网膜内表达，对眼组织发育有重要的调控作用。而Sox2基因是非常重要的眼发育调控基因，在此基础上本项目从晶状体、视网膜发育及发育过程中的组织间诱导的调控机制入手，以研究Msx2调控Sox2基因及其下游Notch和Wnt信号通路的分子机制为核心，通过荧光素酶活性测定、原位分子杂交、免疫荧光等实验结合不同的Msx2条件敲除小鼠和Sox2转基因小鼠模型，在体及离体对Msx2与Sox2基因的调控关系进行从表征到分子机理的研究。进一步阐明Msx2在晶状体、视网膜的诱导和发育过程中的转录调控机制，为明确小眼球畸形的成因，实现小眼球畸形的产前诊断和基因治疗提供实验依据。

眼前节发育障碍（ASD）是一种严重的先天性发育性眼病。明确晶状体发育调控机制及发育过程中的组织间相互诱导调控机制是深入研究该疾病的基础。肌节同源盒基因同系物2（Msx2）是调控晶状体发育的重要转录因子之一，已发表的研究结果证实Msx2基因过表达或敲除都会引起眼发育障碍，分子机理是目前小眼球畸形发病机制研究的热点。本项目利用RNA-seq高通量测序、HE染色、增殖细胞和凋亡细胞检测、原位杂交、免疫组化、实时定量PCR等实验方法，得到以下实验结果：1）在表面外胚层条件敲除Msx2基因后，会引起晶状体发育障碍及先天性白内障等异常；2）Msx2基因条件敲除后，Gja8表达在晶状体上皮细胞内下降；Tgm2, Capn1及CAMK2B 的表达升高；3）晶状体细胞增殖、凋亡发生改变，晶状体细胞的增殖、凋亡异常出现在胚胎发育E12.5天之后； 4）通过对条件敲除Msx2基因小鼠的成鼠晶状体进行RNA-seq，发现有1911个差异表达基因，其中1586个差异基因为上调，其余为下调；5）电镜下观察到Msx2基因的条件敲除可以影响晶状体上皮间的缝隙连接。以上实验结果说明Msx2基因是眼部发育的重要调控基因，为进一步阐明Msx2在角膜及前眼部发育过程中的重要作用及揭示ASD等眼部发育性疾病的发病机制提供了实验基础，指明了未来的研究方向。