Serial analysis of gene expression (SAGE) yields a quantitative, representative and comprehensive differential gene expression profile. Using several time points in the precise developmental profile of embryonic stem and embryonic carcinoma cell differentiation to cardiomyocytes, SAGE analysis has been used to generate a quantitative transcript assessment that has proven to be much more rapid and economical than other techniques. We used an RT-PCR based technique to determine the time points where a number of mesodermal and cardiac-restricted gene products are expressed in in vitro differentiated EC derived cardiomyocytes. Results show that the earliest contractions occur on Day 5.5 of differentiation (3+2.5 or 4+1.5). Some contractile protein genes (beta MHC) are expressed before the third day of differentiation, but we show that a clear induction of expression of alpha MHC and MLC2V starts relatively later (5+0.5 and 3+1 for alpha MHC and MLC2V respectively). GATA-4, a transcription factor involved in the regulation of cardiac contractile protein gene expression is highly present in the earliest stages of differentiation, and form 3+1.5 days of differentiation, its expression is reduced to the level of adult heart tissue. This indicates that this time period corresponds to an early stage of cardiac differentiation. We have now used the protocols of SAGE to analyze expressed sequence profiles during these time periods. A total of 150,354 tags have been sequenced from three P19 EC cell-based libraries (undifferentiated P19 cells, differentiation Days 3+0.5 and 3+3.0). Ten percent of the 43,432 unique tags matched sequences in the NR GenBank database, while more than 30% of the unique tags did not match any known mouse sequence. Temporally-restricted induction or repression of transcripts at each differentiation stage examined was a common feature of the developmental profiles, suggesting that differentiation-specific processes are necessary for pre- and early cardiac development. Analysis of the normalized tag frequency with differentiation revealed significant changes (p<0.01) in expression of 357 gene products, including the known cardiac differentiation factor Thing1 (HAND1). We have utilized quantitative PCR and in situ hybridizations to analyze the temporal and spatial distribution of a number of the most differentially regulated transcripts. Several of the products have shown a cardiac predominance in either fetal or adult heart. These genes are now being evaluated for any functional significance in the induction or differentiation of pluripotent cells to cardiac myocytes, through exploitation of a variety of model systems. The novel gene products identified in this study provide a framework for the analysis of pre- and early cardiac developmental processes in human and mouse.
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