DUSP5 characterization and ETSRP target identification in zebrafish development

描述（由申请人提供）：我们的目标是了解胚胎发生过程中调控造血干细胞（HSC）和中胚层血管成血管细胞谱系特化的潜在机制。作为实现这一目标的切入点，我们选择研究在斑马鱼血液和血管发育的早期阶段表达的两种分子因子，ETSRP和DUSP 5。该提案的主要目标是通过采用新开发的染色质免疫沉淀测序（ChIP-Seq）方法来鉴定成血管细胞中表达的新型转录因子ETSRP的分子靶点，从而通过直接测序对转录因子结合的染色质区域进行免疫沉淀、扩增和定量。然后将具有高于预定阈值的读数的染色质区域计算映射到它们在基因组中的位置以鉴定靶标。这种方法适用于斑马鱼，因为其基因组序列最近已通过公共数据库获得。斑马鱼也是这种应用的极好系统，因为通过随后的生物信息学，表达和功能研究，可以在体内验证潜在的靶点，并将其置于发育遗传网络的背景下。作为该验证过程的一个例子，我们将检查一个遗传实体，该遗传实体是ETSRP的遗传下游，并且其表达模式是血液和血管特异性的，双特异性磷酸酶，DUSP 5，功能丧失和获得方法都将用于此目的，并且如果发现其与循环发育相关，则将检查其功能相关性。这些项目与公共卫生具有重要的相关性，因为各种临床疾病，如癌症、类风湿性关节炎和缺血，要么依赖于循环系统的形成，要么由循环系统的问题引起。缓解这些疾病的医学方法依赖于仔细和系统的研究，而基础科研（如本文所提出的工作）的重点是为未来的治疗发展提供有关循环生物学的有价值的信息

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