Regulation of avian precardiac mesoderm development by insulin and insulin-like growth factors

Endoderm within the heart forming regions of vertebrate embryos has pronounced effects on myocardial cell development. Previous studies have suggested that these effects are mediated by soluble growth factors, in particular fibroblast growth factor 2 (FGF-2) and activin-A. Since both insulin and insulin-like growth factors (IGFs) are present in developing avian embryos at the time of heart formation, we have investigated the potential role of these molecules in promoting development of premyocardial cells in quail. Culture of precardiac mesoderm explants from stage 5 quail embryos in medium containing insulin, IGF-I, or IGF-II increased proliferation of premyocardial cells, with maximal stimulation observed at approximately 25 nM for each ligand. A direct comparison of the proliferative response of precardiac mesoderm to endoderm, fetal calf serum, insulin, IGF-I, IGF-II, activin-A, and FGF-2 showed that FGF-2 and activin-A increased proliferation of premyocardial cells approximately 2-fold, while insulin, IGF-I, and IGF-II stimulated proliferation approximately 3-fold. Insulin and IGF-I enhanced the rate of myocyte differentiation, similar to previously reported effects of endoderm. In contrast, exposure of precardiac mesoderm explants to transforming growth factor beta (TGFβ) reduced proliferation of premyocardial cells and moderated the proliferative effects of IGF-I. TGFβ did not block the differentiation of stage 5 premyocardial cells. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNAs encoding insulin, IGF-II, insulin receptor, and IGF-I receptor were present in both precardiac mesoderm and endoderm, as well as in the forming heart at stage 8. Since premyocardial cells can survive and differentiate in a defined medium lacking these factors precardiac mesoderm may produce IGF-II and insulin at levels that are sufficient to stimulate myocyte development. Taken together, these results suggest that insulin and/or IGF-II may promote cardiac development in vivo by both autocrine and paracrine mechanisms. Cardiogenesis may therefore be promoted by the combined action of several classes of growth factors.