Novel technology for analysis of cardiac & BMP-specific gene expression profiling

描述(申请人提供)：先天性心脏畸形的发生率约为人类活产的1%和死产的8%。虽然心血管发育的几个关键组成部分已经被阐明，但支配这些过程的基因表达谱和信号机制在很大程度上还没有确定。其中一个挑战是在特定的发育阶段提纯特定组织和谱系的样本，以评估mRNA表达或翻译的细微变化。为了应对这一挑战，我们开发了一种新技术，通过从特定的细胞谱系(在这种情况下是心脏和BMP反应谱系)中纯化多聚体来捕获斑马鱼的基因表达谱，以确定在发育的特定时间点正在翻译的mRNA。为了实现这一点，我们建立了一个双组分系统，其中生物素连接酶识别肽(BLRP)表位连接到两个独立的核糖体蛋白(Rpl18a和Rpl23)的可及区，第二组分以谱系或细胞信号响应的方式驱动Bira的表达。BIRA是一种将生物素共价添加到BLRP标记的构建体中的酶。利用稳定的转基因斑马鱼品系，目标是普遍驱动BLRP-RPL，并以组织特异性转录启动子以细胞谱系受限的方式驱动Bira 这些元件(例如，特定于心肌细胞，或受热休克启动子的时间调节，或对特定细胞信号的响应)。因此，只有已定义的细胞谱系才会同时拥有这两种细胞 BRLP标记的核糖体和Bira酶将具有标记了生物素的核糖体。这些生物素标记的核糖体的免疫纯化(IP)将使我们能够纯化主动翻译的mRNAs。作为原理的证据，我们测试了这样的假设，即我们可以使用Bira来生物素化鱼类中特定的RPL，以及我们可以使用心脏特异性(Cmlc2)启动子驱动的Bira表达来标记心脏谱系中的IP多聚体。我们提出的第一个目标是在关键的早期发育时间点使用RNA-Seq研究IP‘ed多聚体的心脏特异性表达谱。此外，众所周知，转化生长因子超家族的成员，如骨形态发生蛋白(BMPs)，控制着不同的细胞过程，如细胞生长和分化，以调节发育。因此，我们的第二个目标是捕捉通过BMP途径主动传递信号的细胞的基因表达谱，并与其他细胞信号传递途径进行比较分析。该项目将产生一种新的方法来发现整个心脏和特定心脏谱系中的基因表达谱和翻译控制。培训环境一流，拥有心血管研究、生物信息学和全基因组分析方面的专业知识。这项研究的目标是产生翻译图谱，识别心脏发育和细胞信号依赖的形态发生中的候选基因和新途径。这些结果将对基础研究产生重大影响，为斑马鱼和心血管研究社区提供工具，并为临床治疗的潜在靶点提供新的途径。

DESCRIPTION (provided by applicant): Congenital heart malformations occur at a rate of approximately 1% of human live births and 8% of stillbirths. While several critical components of cardiovascular development have been elucidated, the gene expression profiles and signaling mechanisms that govern these processes is largely undetermined. One of the challenges is purifying tissue and lineage-specific samples during specific developmental stages to assess subtle changes in mRNA expression or translation. To address this challenge, we generated a new technology that captures gene expression profiles in zebrafish by purifying polysomes from specific cell lineages (in this case cardiac and BMP-responding lineages) in order to identify the mRNAs that are being translated during specific timepoints in development. To accomplish this we have built a two-component system in which Biotin Ligase Recognition Peptide (BLRP) epitope is attached to accessible regions of two independent ribosomal proteins (Rpls), Rpl18a and Rpl23 and the second component drives expression of BirA in a lineage or cell-signaling responsive manner. BirA is an enzyme that covalently adds biotin to the BLRP-tagged construct. Using stable transgenic zebrafish lines, the goal is to drive BLRP-Rpl ubiquitously, and to drive BirA in a cell-lineage restricted manner with tissue-specific transcriptional promoter elements (for example, specifically in cardiac cells, or temporally regulated by a heat shock promoter, or in response to specific cell signals). Thus, only defined cell lineages will have both the BRLP-tagged ribosomes and the BirA enzyme will have ribosomes that are tagged with biotin. Immunopurification (IP) of these biotin-labeled ribosomes will allow us to purify actively translated mRNAs. As proof of principle, we tested the hypotheses that we can use BirA to biotinylate specific Rpl's in fish and that we can use a heart specific (cmlc2) promoter-driven BirA expression to label and IP polysomes from the heart lineage. Our first proposed aim is to investigate the cardiac-specific expression profiles of IP'ed polysomes using RNA-Seq at critical early developmental timepoints. Further, it is known that members of the TGF¿ superfamily, such as Bone Morphogenic Proteins (BMPs), control distinct cellular processes such as cell growth and differentiation to regulate development. Therefore, our second aim is to capture the gene expression profile of cells that are actively signaling via the BMP pathway, and perform comparative analysis versus other cell-signaling pathways. This project will generate a novel approach to discover gene expression profiles and translational control in the whole heart and specific heart lineages. The training environment is superb, with expertise in cardiovascular research and bioinformatics and genome-wide analyses. The research aims will yield translational profiles that will identify candidate genes and novel pathways in heart development and cell-signaling dependent morphogenesis. These results will have significant impact in basic research, provide tools for the zebrafish and cardiovascular research communities, and suggest new pathways for potential targets of clinical therapeutics.