Primary culture of avian embryonic heart forming region cells to study the regulation of vertebrate early heart morphogenesis by vitamin A

Background: Important knowledge about the role of vitamin A in vertebrate heart development has been obtained using the vitamin A-deficient avian in ovo model which enables the in vivo examination of very early stages of vertebrate heart morphogenesis. These studies have revealed the critical role of the vitamin A-active form, retinoic acid (RA) in the regulation of several developmental genes, including the important growth regulatory factor, transforming growth factor-beta2 (TGF beta 2), involved in early events of heart morphogenesis. However, this in ovo model is not readily available for elucidating details of molecular mechanisms determining RA activity, thus limiting further examination of RA-regulated early heart morphogenesis. In order to obtain insights into RA-regulated gene expression during these early events, a reliable in vitro model is needed. Here we describe a cell culture that closely reproduces the in ovo observed regulatory effects of RA on TGF beta 2 and on several developmental genes linked to TGF beta signaling during heart morphogenesis. Results: We have developed an avian heart forming region (HFR) cell based in vitro model that displays the characteristics associated with vertebrate early heart morphogenesis, i.e. the expression of Nkx2.5 and GATA4, the cardiogenesis genes, of vascular endothelial growth factor (VEGF-A), the vasculogenesis gene and of fibronectin (FN1), an essential component in building the heart, and the expression of the multifunctional genes TGF beta 2 and neogenin (NEO). Importantly, we established that the HFR cell culture is a valid model to study RA-regulated molecular events during heart morphogenesis and that the expression of TGF beta 2 as well as the expression of several TGF beta 2-linked developmental genes is regulated by RA. Conclusions: Our findings reported here offer a biologically relevant experimental in vitro system for the elucidation of RA-regulated expression of TGF beta 2 and other genes involved in vertebrate early cardiovascular morphogenesis.