The Role of MAP 3 kinase 1 in Ocular Surface Morphogenesis

描述(申请人提供)：组织形态发生是一个高度组织化和进化保守的过程，由信号转导通路的时间和组织特异性激活控制。脊椎动物的眼皮形态发生经历了一个常见的短暂的眼皮闭合过程，导致闭合的眼皮包裹着一个富含粘蛋白的结膜囊，保护角膜的成熟和发育。在小鼠中，眼皮关闭发生在胚胎第16天(E16)，重新打开发生在出生后12-14天；因此，胚胎眼皮关闭缺陷总是导致出生时睁眼(EOB)表型，很容易在出生时追踪到。因此，显示EOB的遗传突变小鼠构成了独特的模型，以研究眼睑形态发生的分子因素和复杂的调控机制。MAP3K1(MAP3K1，又称MEKK1)是一种“眼皮闭合因子”，负责将形态发生蛋白B信号传递给Jun N-末端激酶(JNKs)。JNK反过来使转录因子c-jun磷酸化，以调节与眼皮闭合有关的基因表达事件。而激活素B激活MAP3K1-JNK/Jun通路导致上皮细胞迁移，在激活素2B-/-、MAP3K1-/-和皮肤特异性c-jun-/-小鼠中阻断这一途径，或在Map3k1Jnk1-/-和Map3k1Jnk1Jnk2复合突变小鼠中通过该途径降低信号传递效率，都会导致上皮细胞迁移缺陷和EOB表型。尽管JNK/Jun途径有明显的贡献，但MAP3K1的形态发生作用很大程度上依赖于JNK非依赖的途径以及与其他“眼皮闭合因子”的串扰。根据MAP3K1在发育中的眼睑上皮细胞中特异表达的研究结果，我们推测MAP3K1是一个主控基因，协调多条信号通路调控眼睑上皮的形态发生和闭合。拟议的研究将使用遗传和分子方法结合系统生物学来理解MAP3K1如何在眼表形态发生中协调复杂的信号网络。我们提出了三个特定的目标：(1)阐明转录因子招募和表观遗传修饰作为MAP3K1启动子激活的机制，从而将生理/环境因素与眼皮闭合联系起来；(2)描述负责MAP3K1介导的基因表达的下游途径；以及(3)了解MAP3K1如何与Smad途径在协调眼皮闭合的转录程序中发生相互作用。人类的眼皮经历了惊人的相似的形态发生过程；然而，与小鼠不同的是，人类的眼皮关闭和重新打开完全是在子宫中完成的，因此无法在临床上诊断眼皮关闭缺陷。因此，识别与小鼠眼睑闭合缺陷相关的遗传和表观遗传变化将为人类相关先天性眼病的预后提供有用的工具。从这项工作中获得的信息将为进一步研究遗传和环境在眼表发育中的相互作用和识别先天性角膜异常的病因铺平道路。公共卫生相关性了解人类发育性疾病的机制和病因学是一个重大挑战。这项工作的结果将为识别人类先天性角膜畸形的遗传和表观遗传原因提供基本的理解和有用的预后工具。此外，我们的工作可能导致对MAP3K1参与的许多生理病理过程的机械性见解，如伤口愈合、血管生成和肿瘤转移。

DESCRIPTION (provided by applicant): Tissue morphogenesis is a highly organized and evolutionarily conserved process controlled by the temporal and tissue-specific activation of signal transduction pathways. Vertebrate eyelid morphogenesis undergoes a common process of transient eyelid closure, resulting in closed eyelids that enclose a mucin-enriched conjunctiva sac protecting corneal maturation and development. In mice, eyelid closure takes place at embryonic day 16 (E16) and its re-opening occurs at postnatal day (P)12-14; thus, embryonic eyelid closure defects always result in an eye-open at birth (EOB) phenotype that is easily traceable at birth. Genetic mutant mice displaying EOB therefore constitute unique models to study the molecular factors and complex regulatory mechanisms underlying eyelid morphogenesis. The MAP3 kinase 1 (MAP3K1, also known as MEKK1), an "eyelid closure factor", is responsible for transmission morphogenetic activin B signals to Jun N-terminal kinases (JNKs). JNK in turn phosphorylates transcription factor c-Jun to regulate gene expression events involved in eyelid closure. While activation of the MAP3K1-JNK/Jun pathway by activin B leads to epithelial cell migration, blocking this pathway in the Activin 2B-/-, Map3k1-/- and skin-specific-c-Jun-/- mice, or reduction of the signal transmission efficiency by this pathway in Map3k1Jnk1-/- and Map3k1Jnk1Jnk2 compound mutant mice, all result in defective epithelial cell migration and EOB phenotypes. Despite of the clear contribution of the JNK/Jun pathway, the morphogenetic effects of MAP3K1 depend largely on JNK- independent pathways and crosstalks with other "eyelid closure factors". Based on the findings that MAP3K1 is specifically expressed in the developing eyelid tip epithelial cells, we hypothesize that MAP3K1 is a master gene that coordinates multiple signaling pathways for eyelid epithelial morphogenesis and closure. The proposed studies will use genetic and molecular approaches in combination with systems biology to understand how MAP3K1 orchestrates complex signaling networks in ocular surface morphogenesis. We propose 3 specific aims: (1) to elucidate transcription factor recruitment and epigenetic modifications as mechanisms for Map3k1 promoter activation thereby connecting physiological/environmental factors to eyelid closure; (2) to delineate the downstream pathways responsible for MAP3K1-mediated gene expression; and (3) to understand how MAP3K1 crosstalk with the Smad pathway in coordinating transcription programs for eyelid closure. Human eyelid undergoes strikingly similar morphogenetic processes; however, unlike in mice, eyelid closure and re-opening in humans is entirely accomplished in utero, making it impossible to clinically diagnose eyelid closure defects. Identifying genetic and epigenetic changes associated with eyelid closure defect in mice therefore will offer useful prognostic tool for related congenital ocular diseases in human. Information derived from this work will pave the way for further investigation of genetic and environmental interactions in ocular surface development and identification of the etiology of congenital corneal anomalies. PUBLIC HEALTH RELEVANCE It is a major challenge to understand the mechanism and etiology of human developmental diseases. Results from the proposed work will provide basic understanding and useful prognostic tool for identifying the genetic and epigenetic causes of congenital corneal anomalies in human. Furthermore, our work may lead to mechanistic insights into numerous physiopathological processes in which MAP3K1 participates, such as wound healing, angiogenesis and tumor metastasis.