Eph/ephrin signaling in craniofacial development and disease

项目总结/摘要 候选 杰弗里·O布什是一名博士后，他获得了博士学位。在罗切斯特大学工作 在理解唇腭裂的自发发生的舞者突变小鼠。这项工作 确定转录因子Tbx 10的功能获得是唇腭裂的病因 Dancer小鼠中的表型，并确定了这种表型的极少数已知遗传原因之一， 小鼠作为Philippe Soriano实验室的博士后研究员，该候选人专注于 了解ephrin-B1在影响颅面发育的先天性疾病病因学中的功能， 颅额鼻综合征（CFNS）。这项工作表明，腭裂表型与 肝配蛋白-B1的功能丧失是由有缺陷的前腭架生长和增殖引起的， 前向信令丢失的后果。候选人的短期职业目标是发展这些研究 进入一个独立的研究项目，研究颅面发育中的Eph/ephrin信号， 长期职业目标是扩大这项研究计划，以研究其他颅面先天性缺陷，重点是 参与颅面发育和疾病的信号分子。 环境 拟议的工作将在菲利普·索里亚诺教授的实验室进行， 西奈山医学院的发育和再生生物学。实验室有一个世界- 在发育过程中信号功能的小鼠遗传学研究方面取得的成就， 对血小板衍生生长因子信号传导的理解做出了重大贡献， 包括颅面发育。发展与再生系 生物学包括14个全职教师，研究以发展为中心的各种问题， 再生和器官的模式化。实验室和部门内可用的资源将 在辅导阶段和向独立过渡期间为候选人提供重要支持。 研究 颅面畸形是非常常见的，在四分之三的先天性 出生时发现的异常。这些包括腭裂，一个失败的屋顶口加入在出生时 每1000个活产婴儿中约有1个发生这种情况。颅面疾病的治疗涉及多种 侵入性手术，并对受影响的个人的儿童健康和家庭产生巨大影响。一 这种遗传性疾病，颅额鼻综合征（CFNS）是由肝配蛋白-B1基因突变引起的。 这是一种X染色体连锁疾病，可导致许多颅面缺陷，包括间距过宽、鼻 沟，冠状颅缝早闭和唇腭裂。在小鼠中相同基因的突变导致 同样的缺陷，支持小鼠是研究这个和其他生物的良好模型生物的想法。 颅面疾病Ephrin-B1是通过激活EphB起作用的信号分子家族的成员 受体。该基因家族在多种发育和疾病中具有重要功能， contexts.我们有数据表明，一个额外的肝配蛋白基因，肝配蛋白-B2，可能发挥重要作用， 颅面发育和疾病。在本申请的第一个目的中，我提议研究肝配蛋白-B2的功能 通过研究小鼠的颅面发育， 在发展过程中。我将通过利用目前可用的等位基因来从腭中去除肝配蛋白-B2来做到这一点。 架上皮，在那里它是高度表达的。此外，我将采取组织特异性拯救策略来测试 在鳃弓形成过程中需要ephrin-B2。基于初步表达数据，EphB 4， 肝配蛋白-B2的一种重要受体在其形成期间在腭内高度表达。在第二 因此，为了达到这个目的，我建议通过产生一个EphB 4基因，来测试EphB 4在第二腭形成过程中的作用。 条件等位基因在神经嵴来源的EphB 4细胞中执行EphB 4功能的组织特异性破坏 间充质最后，我已经启动了研究，以确定下游组件的Eph/ephrin信号转导 网络通过开发基于质谱的蛋白质组学方法来识别磷酸化靶点， 腭中的EphB/ephrin-B信号传导。这种方法已经确定了大量的优秀 用于下游信号转导的候选分子。这些候选人的角色大多不为人知 因此，我在第三个目标中建议优先考虑并研究这些候选人， 其目的是阐明控制腭形成的肝配蛋白-B信号传导下游的信号传导途径。 这项研究将大大提高对腭裂的遗传原因的理解，通过确定 ephrin-B2和EphB 4在其发展中的重要性，并通过表征新的基因与以前的 在颅面发育和疾病中的重要性未知。

Project Summary/Abstract Candidate Jeffrey O. Bush is a postdoctoral fellow who received his Ph.D. at the University of Rochester for work in understanding cleft lip and palate in the spontaneously-occurring Dancer mutant mouse. This work identified gain of function of the transcription factor Tbx10 as being causative to the cleft lip and palate phenotype in the Dancer mice and identified one of a very few known genetic causes for this phenotype in mice. As a postdoctoral fellow in the laboratory of Philippe Soriano, the candidate has focused on understanding ephrin-B1 function in the etiology of a congenital disorder affecting craniofacial development, Craniofrontonasal syndrome (CFNS). This work has shown that the cleft palate phenotype associated with ephrin-B1 loss of function is caused by defective anterior palatal shelf outgrowth and proliferation, as a consequence of loss of forward signaling. The candidate's short-term career goal is to evolve these studies into an independent research program studying Eph/ephrin signaling in craniofacial development, with a long- term career goal to expand this research program to study other craniofacial congenital defects with a focus on signaling molecules involved in craniofacial development and disease. Environment The proposed work will take place in the laboratory of Professor Philippe Soriano in the Department of Developmental and Regenerative Biology at the Mount Sinai School of Medicine. The laboratory has a world- class record of accomplishment in mouse genetics studies of signaling function during development, and has made significant contribution into the understanding of Platelet-Derived Growth Factor signaling throughout development, including craniofacial development. The Department of Developmental and Regenerative Biology includes fourteen full-time faculty that study various questions centered on the development, regeneration, and patterning of organs. The resources available within the laboratory and the department will provide significant support to the candidate during the mentored phase and transition to independence. Research Craniofacial malformations are extremely common, identified in three quarters of all congenital abnormalities identified at birth. These include cleft palate, a failure of the roof of the mouth to join at birth which occurs in approximately 1 in 1000 live births. The treatment of craniofacial conditions involves multiple invasive surgeries, and has a dramatic impact on an affected individual's childhood health and family. One such genetic disorder, Craniofrontonasal syndrome (CFNS) is caused by mutations in the ephrin-B1 gene. This is an X-linked disorder that causes a number of craniofacial defects including, hypertelorism, nasal grooves, coronal craniosynostosis and cleft lip and palate. Mutations in the same gene in mice cause the same defects, supporting the idea that the mouse is a good model organism for studying this and other craniofacial diseases. Ephrin-B1 is a member of a family of signaling molecules that act by activating EphB receptors. This family of genes have important functions in a wide variety of developmental and disease contexts. We have data indicating that an additional ephrin gene, ephrin-B2, may play important roles in craniofacial development and disease. In the first aim of this application, I propose to study ephrin-B2 function in craniofacial development by studying mice in which its function is removed from specific craniofacial tissues during development. I will do this by utilizing currently available alleles to remove ephrin-B2 from the palatal shelf epithelium, where it is highly expressed. Additionally, I will take a tissue-specific rescue strategy to test for requirement for ephrin-B2 during branchial arch formation. Based on preliminary expression data, EphB4, an important receptor for ephrin-B2 is highly expressed within the palate during its formation. In the second aim, I therefore propose to test for a role of EphB4 during formation of the secondary palate by generating a conditional allele to perform tissue-specific disruption of EphB4 function in the neural crest-derived mesenchyme. Finally, I have initiated studies to identify downstream components of the Eph/ephrin signaling network by developing a mass-spectrometry based proteomic approach to identify phosphorylation targets of EphB/ephrin-B signaling in the palate. This approach has already identified a large number of excellent candidate molecules for transduction of downstream signaling. These candidates have mostly unknown roles in craniofacial development, and I therefore propose in the third aim to prioritize and study these candidates, with the goal of elaborating signaling pathways downstream of ephrin-B signaling that control palate formation. This study will greatly enhance understanding of the genetic causes of cleft palate by identifying the importance of ephrin-B2 and EphB4 in its development, and by characterizing new genes with previously unknown importance in craniofacial development and disease.