Eph signaling through RhoGTPase in cell segregation and craniofacial disease

描述（由申请人提供）：人类EFNB1基因突变导致一种称为颅额鼻综合征（CFNS）的疾病，该疾病涉及许多颅面异常，包括颅缝紧闭、额鼻发育不良、严重远端肥大和腭裂。在小鼠中突变efnB1会导致类似的缺陷，使这些小鼠成为人类疾病的良好模型。EFNB1在小鼠和人类中都是x连锁的；因此，半合子突变体雄性没有功能性ephrin-B1，而杂合子突变体雌性由于随机x失活而嵌合能够表达ephrin-B1的细胞。然而，杂合子雌性比半合子零雄性或纯合子零雌性具有更严重的表型。这种现象被认为与ephrin- b1介导的细胞分离成大的ephrin- b1阳性和阴性斑块有关。在本提案中，我旨在探讨这种隔离的机制。Ephrin-B1是一种细胞结合信号分子，其作用部分是通过激活EphB受体酪氨酸激酶，这一过程被称为正向信号传导。EphB受体可以通过Rho家族gtpase和Ras/ERK途径刺激信号传导，也可以通过ADAM家族金属蛋白酶促进E-cadherin的裂解。所有这三种机制都被提出在各种发育背景下介导细胞分离；然而，目前尚不清楚哪种机制在CFNS中起作用。除了正向信号外，ephrin- B1作为一种跨膜分子，还可以“反向信号”返回自身细胞；在某些情况下，已经观察到细胞分离需要反向信号。我的初步数据表明，在CFNS中情况并非如此。我的数据还表明，RhoA和RhoA依赖性激酶ROCK的激活对分离至关重要，并且分离在发育早期就开始了。在第一个目标中，我建议确定反向信号是否必要和充分使用反向信号的efnB1死等位基因来引起分离。我还将研究ephrin-B1受体EphB2和EphB3的正向信号传导对分离的贡献，通过测试基因正向信号传导能力的丧失是否可以挽救杂合型efnB1+/-小鼠的分选表型。在第二个目标中，我将在HEK293细胞培养中使用药物遗传学方法确定参与细胞分选的下游信号通路。基于我的初步数据，我将重点关注Rho家族GTPase信号通路的组成部分。然后，我将检查培养和胚胎中的分离细胞，以激活Rho和EphB-Rho信号通路的其他潜在成分。最后，我将在我建立的滚轮瓶胚胎培养试验系统中验证这些发现，在分离开始之前，胚胎被分离出来，并在滚轮瓶中培养，直到分离通常很明显的一点。这项研究的结果将阐明一种鲜为人知的先天性疾病，并有助于更好地了解基本的Eph/ephrin信号传导机制。

DESCRIPTION (provided by applicant): Mutations in the human EFNB1 gene lead to a disease known as craniofrontonasal syndrome (CFNS), which involves a number of craniofacial anomalies including craniosynostosis, frontonasal dysplasia, severe hypertelorism, and cleft palate. Mutating efnB1 in mice causes similar defects, making these mice a good model for the human disease. EFNB1 is X-linked in both mice and humans; consequently, hemizygous mutant males have no functional ephrin-B1, while heterozygous mutant females are mosaic for cells capable of expressing ephrin-B1 due to random X-inactivation. However, heterozygous females have much more severe phenotypes than either hemizygous null males or homozygous null females. This phenomenon is thought to be related to ephrin-B1-mediated segregation of cells into large ephrin-B1-positive and -negative patches. In this proposal, I aim to explore the mechanisms underlying this segregation. Ephrin-B1 is a cell-bound signaling molecule that acts in part by activating EphB receptor tyrosine kinases in a process known as forward signaling. EphB receptors can stimulate signaling through Rho family GTPases and the Ras/ERK pathway and also prompt cleavage of E-cadherin by ADAM family metalloproteases. All three of these mechanisms have been proposed to mediate cellular segregation in various developmental contexts; however, it is unclear which mechanism is at work in CFNS. In addition to forward signaling, ephrin- B1, as a transmembrane molecule, can also "reverse signal" back into its own cells; reverse signaling has been observed to be required for cell segregation in some contexts. My preliminary data suggest that this is not the case in CFNS. My data also indicate that activation of RhoA and the Rho-dependent kinase ROCK are critical for segregation and that segregation begins early in development. In the first aim, I propose to determine whether reverse signaling is necessary and sufficient to cause segregation using a reverse-signaling dead allele of efnB1. I will also investigate the contribution of forward signaling by ephrin-B1's receptors EphB2 and EphB3 to segregation by testing whether genetic loss of forward signaling capacity can rescue the sorting phenotype in heterozygous efnB1+/- mice. In the second aim, I will identify the downstream signaling pathways involved in cell sorting using a pharmacogenetic approach in HEK293 cell culture. Based on my preliminary data, I will focus on components of Rho family GTPase signaling pathways. I will then examine segregating cells in culture and embryos for activation of Rho and other potential components of the EphB-Rho signaling pathway. Finally, I will validate these findings in a roller bottle embryo culture assay system tha I have established, in which embryos are isolated before segregation begins and cultured in roller bottles until a point at which segregation is normally readily apparent. The results of thi study will illuminate a poorly understood congenital condition and contribute to a better understanding of fundamental Eph/ephrin signaling mechanisms.