Eph signaling through RhoGTPase in cell segregation and craniofacial disease

通过 RhoGTPase 的 Eph 信号传导在细胞分离和颅面疾病中的作用

• 批准号: 8830858
• 负责人: Audrey Kathleen O'Neill
• 金额: $ 5.68万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830858
• 关键词: Affect; Alleles; Back; Behavior; Binding; Biological Assay; Cell Culture System; Cell Culture Techniques; Cell Separation; Cells; Child; Cleft Lip; Cleft Palate; Congenital Abnormality; Craniofacial Abnormalities; Craniosynostosis; Data; Defect; Deformity; Development; Disease; E-Cadherin; Embryo; Eph Family Receptors; EphB2 Receptor; Ephrin B Receptor; Ephrin-B1; Ephrins; Face; Family; Female; Genes; Genetic; Guanosine Triphosphate Phosphohydrolases; Head; Heterozygote; Human; Investigation; Lead; Ligand Binding; Link; Mammalian Cell; Mediating; Metalloproteases; Methods; Modeling; Molecular; Morphogenesis; Mus; Mutate; Mutation; Orbital separation excessive; Palate; Pathogenesis; Pathway interactions; Patients; Pharmacogenetics; Phenotype; Phosphotransferases; Play; Population; Process; Protein Tyrosine Kinase; Publishing; Receptor Protein-Tyrosine Kinases; Role; Roller Bottle; Severe dysplasia; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Sorting - Cell Movement; System; Testing; Work; X Inactivation; base; craniofacial; craniofrontonasal syndrome; embryo culture; embryonic cell culture; functional loss; human disease; in vivo; male; mutant; public health relevance; receptor; rho; segregation

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.

描述（由申请方提供）：人EFNB 1基因突变导致一种称为颅额鼻综合征（CFNS）的疾病，该疾病涉及多种颅面异常，包括颅缝早闭、额鼻发育不良、重度距离过宽和腭裂。在小鼠中突变efnB 1会导致类似的缺陷，使这些小鼠成为人类疾病的良好模型。EFNB 1在小鼠和人类中都是X连锁的;因此，半合子突变雄性没有功能性肝配蛋白-B1，而杂合突变雌性由于随机X失活而嵌合能够表达肝配蛋白-B1的细胞。然而，杂合子雌性比半合子无效雄性或纯合子无效雌性具有更严重的表型。这种现象被认为与肝配蛋白-B1介导的细胞分离成大的肝配蛋白-B1阳性和阴性斑块有关。在这个建议中，我的目的是探索这种隔离的机制。 Ephrin-B1是一种细胞结合的信号分子，其部分通过在称为正向信号传导的过程中激活EphB受体酪氨酸激酶来起作用。EphB受体可以刺激通过Rho家族GTP酶和Ras/ERK途径的信号传导，并且还促进ADAM家族金属蛋白酶对E-钙粘蛋白的切割。所有这三种机制已被提出来介导细胞分离在各种发展的背景下，但是，目前还不清楚是哪种机制在CFNS的工作。除了正向信号传导外，肝配蛋白- B1作为一种跨膜分子，还可以将“反向信号”传回其自身细胞;已经观察到在某些情况下细胞分离需要反向信号传导。我的初步数据表明，CFNS的情况并非如此。我的数据还表明，RhoA和Rho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开始。 在第一个目标中，我建议确定反向信号是否是必要的，并足以导致隔离使用反向信号死亡等位基因efnB 1。我还将调查由肝配蛋白-B1的受体EphB 2和EphB 3的前向信号的贡献，以隔离测试是否遗传损失的前向信号能力可以拯救分选表型在杂合子efnB 1 +/-小鼠。在第二个目标中，我将在HEK 293细胞培养中使用药物遗传学方法鉴定参与细胞分选的下游信号通路。基于我的初步数据，我将专注于Rho家族GTdR信号通路的组成部分。然后，我将检查分离培养和胚胎中的细胞，以激活Rho和EphB-Rho信号通路的其他潜在组分。最后，我将验证这些研究结果在滚瓶胚胎培养测定系统，我已经建立，其中胚胎分离开始前分离和培养滚瓶，直到一个点，在该点分离通常是显而易见的。 这项研究的结果将阐明一个不太了解的先天性疾病，并有助于更好地了解基本的Eph/ephrin信号转导机制。