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.
This project will generate a novel approach to discover genome-wide changes in gene expression during zebrafish development that are controlled by temporal, lineage-specific and cell-signaling response mechanisms. This project will yield mRNA translational profiles that will reveal novel pathways in heart development and cell-signaling dependent morphogenesis. Insights in these novel pathways will be useful for both basic research and in considering potential clinical therapeutics.
