Investigation of zebrafish homologues to specifically upregulated TUFs in cardiomyocytes derived from murine ES cells

Heart failure due to loss of functional cardiomyocytes is one of the most frequent cardiovascular diseases. Understanding the genetic network that leads to functional cardiomyocytes is the first step to develop future therapies.A global transcriptome analysis yielded up-regulated genes in cardiomyocytes, which were derived from murine embryonic stem (ES) cells (Doss et al., 2007). In the present study we were on the one hand interested in a fast screen for the functional role of transcripts with unknown function (TUFs) for an intact activity of the heart. Therefore we searched for homologues in the zebrafish genome and performed a morpholino-based knockdown approach. We tested several TUFs of the zebrafish and found most of them to be expressed in the cardiovascular system. Morpholino-oligonucleotide injections caused highly specific cardiovascular defects in the majority of them such as altering of heart morphology, vascular defects or accumulation of blood cells to a different extent and penetrance. This pilot approach thus shows the potential of the zebrafish to identify TUFs in the cardiovascular system.Based on that, on the other hand we want to investigate promising candidate genes from the zebrafish approach in ES cells. ES cells are pluripotent cells which were isolated from early mouse embryos and because of their pluripotency able to differentiate into derivatives of the three primitive layers: ectoderm, endoderm and mesoderm. Using ES cells which were differentiated into three-dimensional structures, so-called embryoid bodies (EBs), we want to study early developmental stages. For this we have generated stable knockdown clones of mouse ES cells expressing a shRNA construct against the desired genes. These clones were also labeled with a green fluorescent protein (GFP) reporter to track the silencing effect. Clones, expressing GFP, were initiated to generate EBs and will be reviewed via PCR, western blotting and beating activity.