DUSP5 Characterization and ETSRP Target Identification in Development

描述（由申请人提供）：我们的目标是了解胚胎发生过程中来自中胚层的造血干细胞（HSC）和成血管细胞谱系规范的潜在机制。作为实现这一目标的切入点，我们选择研究在斑马鱼血管发育早期阶段表达的两个分子因子，ETSRP和DUSP5。本研究的主要目标是通过采用新开发的染色质免疫沉淀测序（ChlP-Seq）方法，确定在血管母细胞中表达的一种新型转录因子ETSRP的分子靶点，其中转录因子结合的染色质区域通过直接测序进行免疫沉淀、扩增和定量。读取值高于预定阈值的染色质区域随后被计算映射到它们在基因组中的位置，以识别目标。这种方法适用于斑马鱼，因为它的基因组序列最近可以通过公共数据库获得。斑马鱼也是这种应用的一个极好的系统，因为通过随后的生物信息学、表达和功能研究，潜在的靶标可以在体内得到验证，并置于发育遗传网络的背景下。作为验证过程的一个例子，我们将检查ETSRP遗传下游的一个遗传实体，其表达模式是血管特异性的，双特异性磷酸酶，DUSP5。功能的损失和获得方法都将用于此目的，如果发现它与循环发育相关，则将检查其功能相关性。这些项目对公共卫生具有重大意义，因为各种临床疾病，如癌症、类风湿性关节炎和缺血，要么依赖于循环形成，要么是由循环问题引起的。缓解这些疾病的医学方法依赖于仔细和系统的研究，而基础科学研究（如本文提出的工作）的重点是为未来的治疗发展提供有关循环生物学的宝贵信息

DESCRIPTION (provided by applicant): Our goal is to understand the underlying mechanisms regulating lineage specification of hematopoietic stem cells (HSC) and vascular angioblast cells from mesoderm during embryogenesis. As an entry point to achieve this we have chosen to study two molecular factors that are expressed during the early stages of blood and vessel development in zebrafish, ETSRP and DUSP5. The main goal of this proposal is to identify the molecular targets of a novel transcription factor expressed in hemangioblasts, ETSRP, by adopting the newly developed Chromatin Immunoprecipitation-Sequencing (ChlP-Seq) approach, whereby the chromatin regions bound by a transcription factor are immunoprecipitated, amplified and quantified by direct sequencing. Chromatin regions with reads above a predetermined threshold are then computationally mapped to their locations in the genome to identify the targets. This approach is applicable to zebrafish as its genome sequence has recently become available through public databases. Zebrafish is also a superb system for such an application because through subsequent bioinformatics, expression, and functional studies, the potential targets can be validated in vivo and placed in the context of developmental genetic networks. As an example of this validation process, we will examine one genetic entity that is genetically downstream of ETSRP and whose expression pattern is blood and vessel specific, Dual Specific Phosphatase, DUSP5, Both loss and gain of function approaches will be used for this purpose, and it's functional relevance will be examined if it is found to be relevant to circulatory development. These projects bear significant relevance to public health because various clinical disorders such as cancer, rhematoid arthritis, and ischemias either rely on circulatory formation or are caused by problems with circulation. Medicinal approaches to alleviate such disorders depend on careful and methodical research, and basic scientific research such as the work proposed here is focused on providing future therapeutic development with valuable information on the biology of circulation