Identification of the genetic pathways that give rise to the chicken rod-free zone and human fovea using epigenomic profiling

 描述（由申请人提供）：这个指导临床科学家研究职业发展奖是一个培训计划，旨在帮助候选人实现他的长期职业目标，有一个专注于视网膜的学术生涯。对于目前的培训和未来的职业生涯，布赖恩哈夫勒打算进行研究，重点是了解人类中央凹和视网膜疾病的翻译应用程序的发展的分子机制。中央凹负责高敏锐度的中央视觉，但建立这一区域的基因是未知的。这项研究计划将侧重于确定使用鸡作为模型系统产生该区域的分子途径，因为它有一个中心无杆点，很像人类的中央凹。在这项研究计划中，Hafler博士将使用ATAC-seq等新技术对发育中的无鸡条区的细胞进行开放染色质分析。ATAC-seq数据将与RNA-seq数据进行比较，RNA-seq数据正在Cepko实验室中生成，以优先考虑感兴趣的候选基因。将进行免疫组织化学和原位杂交，以评估与开放染色质区域相关的转录物是否特异性定位于无视杆细胞的中央区域。 鸡胚胎视网膜中的区域。将位于开放染色质区域的候选顺式调节模块与含有中央凹的物种进行比对和评估以进行保守性。将最有希望的序列克隆到报告构建体中，并使用电穿孔测定法测试其在发育中的鸡视网膜中的活性。将使用电泳迁移率变动分析来鉴定与顺式调节模块结合的转录因子。免疫组织化学和原位杂交胚胎人视网膜将被用来检查，如果感兴趣的转录因子在人类视网膜发育过程中是保守的。这项工作的完成不仅可以深入了解中央凹在人类发育过程中是如何产生的，还将有助于为年龄相关性黄斑变性患者开发细胞替代疗法。候选人有一个优秀的导师，博士康斯坦斯Cepko，谁是在该领域的领导者，并致力于科学发展和项目的执行。她是哈佛医学院遗传学和眼科学的布拉德教授，也是霍华德休斯医学研究所的研究员。哈佛医学院的环境有一个强大的和建立的研究计划与视网膜发育。他们将提供必要的设施，以促进职业发展，成为一名独立的调查员。有许多课程提供，研讨会，期刊俱乐部和演示文稿与其他研究科学家的智力互动的机会。通过研究计划和职业发展活动获得的经验，知识和技能将使候选人成为独立的临床科学家。

 DESCRIPTION (provided by applicant): This mentored clinical scientist research career development award is a training program designed to help the candidate achieve his long-term career goal to have an academic career with a focus on the retina. For the current training and future career, Brian Hafler intends to perform research focused on understanding the molecular mechanisms underlying development of the human fovea and translational applications to retinal disease. The fovea is responsible for high acuity central vision, yet the genes that establish this region are unknown. This research proposal will focus on identifying the molecular pathways that generate this region using the chicken as a model system, as it has a central rod-free spot, much like the human fovea. In this research proposal, Dr. Hafler will use new technologies like ATAC-seq to perform open chromatin profiling of cells in the developing chicken rod-free zone. ATAC-seq data will be compared with RNA-seq data, which is in the process of being generated in the Cepko lab to prioritize candidate genes of interest. Immunohistochemistry and in situ hybridization will be performed to assess whether the transcripts associated with regions of open chromatin specifically localize to the rod-free central zone in the chicken embryonic retina. Candidate cis- regulatory modules located in regions of open chromatin will be aligned and assessed for conservation with species that contain a fovea. The most promising sequences will be cloned into reporter constructs and tested for activity in the developing chicken retina using electroporation assays. Transcription factors that bind to the cis-regulatory modules will be identified using electrophoretic mobility shift assays. Immunohistochemistry and in situ hybridization on embryonic human retinas will be used to examine if the transcription factors of interest are conserved during human retinal development. Completion of this work would not only give insight into how the fovea is generated during human development, it will also help with the development of cell replacement therapies for patients with age-related macular degeneration. The candidate has an excellent mentor, Dr. Constance Cepko, who is a leader in the field and is committed to the scientific development and execution of the project. She is the Bullard Professor of Genetics and Ophthalmology at Harvard Medical School and a Howard Hughes Medical Institute Investigator. The environment at Harvard Medical School has a strong and established research program related to retinal development. They will provide necessary facilities for career enhancement to become an independent investigator. There are numerous courses offered, seminars, journal clubs, and presentations with opportunities for intellectual interactions with other research scientists. The experience, knowledge, and skills gained through the research plan and career development activities will carry the candidate forward towards a career as an independent clinician-scientist.