Expression and function of activin beta a during mouse cardiac cushion tissue formation

The formation of cardiac cushion tissue, which ultimately contributes to formation of the valves and septa, is dependent on the regional activation of cardiac endothelial cells to undergo an epithelial-mesenchymal transition. This endothelial transition was correlated with activin βA mRNA expression by Northern and in situ hybridization in both a temporal and spatial manner in developing mouse embryos. Activin βA was the only subunit of the inhibin family detected during the initial phase of endothelial cell transition; activin βB was detected at later stages, and inhibin α was not detectable in the heart. An in vitro assay that has been used to study mesenchymal cell formation in chick was modified for use with mammalian embryos. Conditioned media from embryonic mouse cardiocyte cultures was shown to substitute for the endogenous inductive signal in these assays. The presence of activin βA was demonstrated by Western blot analysis of the cardiocyte conditioned media (CCM). Modified antisense oligonucleotides to activin βA inhibited the endothelial-mesenchymal transition in the assay system, which was not affected by control oligonucleotides. Adapting the avian culture system for use with mice enabled the use of tissue from mice with a null allele for activin βA. CCM produced from embryos homozygous for the mutant βA allele did not contain activin βA and was used in in vitro assays. CCM lacking activin βA produced fewer mesenchymal cells from cardiac endothelial monolayers than CCM with activin βA. Localized expression of activin βA in the embryonic heart indicates a possible role in the endothelial-mesenchymal transition. Bioassays in which activin βA expression is blocked or activin βA is absent from the media indicate that activin βA promotes the formation of mesenchymal cells in the endothelial cushions, which are required for normal septation.