Integration of embryonic stem cell differentiation and genomics in the identification of novel cardiac genes.

摘要

The heart is the first organ to form and function in mammalian development, the result of a complex and highly coordinated series of events. Perturbations of these processes can lead to congenital heart defects which are the most prevalent type of birth defects (∼7 in 1000 live births). In part due to the inaccessibility of the early stages of mammalian embryogenesis, the genetic programs controlling heart development are incompletely understood.;To better understand the mechanisms involved in cardiac development, we determined the transcriptional profile of mouse embryonic stem cells (mESCs) as they differentiated towards cardiac cell fates. By comparing the expression profiles of cardiac precursor cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, we have identified transcripts whose expression is enriched in cardiac populations; predicting that they will likely play a role in cardiogenesis. Of note, approximately 24% (43/176) of the cardiogenic candidate transcripts identified have previously established roles in cardiac function or development.;Evaluation of the biological relevance of a significant subset (31/133, 23%) of the remaining candidate genes by in situ hybridization at time points during development (embryonic day, E7.5, E8.5, E9.5) revealed that all are expressed in key cardiac structures during cardiogenesis. Furthermore 9/31, of which many were previously uncharacterized, are detected as early as the formation of the cardiac crescent.;One gene, Rbm24, was selected for functional evaluation in zebrafish, an excellent model for early vertebrate development. The zebrafish homolog, zgc:136803 (rbm24), displays 92.6% amino acid identity with the mouse Rbm24 protein and recapitulates the cardiac and somitic expression observed in mouse. Fish injected with translation blocking morpholinos against rbm24 displayed cardiac looping defects, reduced blood flow, and cardiac edema as well as defects in somite formation. Co-injection of the full-length rbm24 transcript with the morpholino resulted in phenotype rescue of ≥62% of injected embryos. Importantly, the human ortholog (RBM24) lies on chromosome 6p22.3, tightly linked to the segregation of congenital cardiac abnormalities (6p24.3-21.2) observed in a three-generation European family (nine affecteds; Wessels et al. 2008). Although by sequencing the RBM24 exons we have excluded coding sequence variation as the cause for this syndrome, further evaluation of non-coding, putative regulatory sequences at this locus is necessary to determine how RBM24 contributes to this phenotype.;The data presented within demonstrate the potential power of integrating genomic approaches with mESC differentiation to illuminate developmental processes, and provides a valuable resource that may be further evaluated to elucidate the genetic programs underlying cardiogenesis.