UC San Diego

The mature heart is comprised of multiple types of specialized cardiomyocytes, each with distinct functional attributes. However, the mechanisms that specify discrete populations of cardiac progenitors are not well understood. For example, it is clear that cardiac pacemaking activity is confined to a specialized population of cells in the cardiac venous pole, but the signals that create the appropriate number of pacemaker cardiomyocytes remain unknown. We have therefore sought to understand how pacemaker cells develop in the zebrafish embryo. First, we have investigated pacemaker cells in the inflow tract (IFT) of wild-type zebrafish embryos. We have observed that IFT cardiomyocytes express a suite of molecular markers that are reminiscent of mammalian pacemaker cells and that confer attributes specific to this population. Furthermore, we have determined that IFT progenitors are localized to discrete areas at the edges of the heart fields, prior to their differentiation. Next, we have shown that the specification of this IFT progenitor population is influenced by opposing inputs from two signaling pathways: Hedgehog (Hh) signaling and Bmp signaling. Given our prior finding that Hh signaling promotes cardiomyocyte production, we were surprised to discover that Hh signaling also acts to delimit the number of IFT cardiomyocytes. Using both genetic and pharmacological manipulations of the Hh pathway, we have shown that loss of Hh signaling results in dramatically expanded expression of IFT markers. Conversely, Bmp signaling drives IFT formation, as embryos with reduced Bmp signaling have a diminished IFT. Timed manipulations of Hh and Bmp activity have demonstrated that both signals act during early steps of cardiac patterning to define IFT size. Intriguingly, reducing both Hh and Bmp signaling restores a nearly normal number of IFT cells. We therefore propose a model in which IFT specification relies on both limited amounts of Hh signaling and robust levels of Bmp signaling, which together set appropriate boundaries for the IFT progenitor population. Our findings reveal novel mechanisms of cardiac patterning; in the long term, these studies could contribute to our understanding of congenital heart disease and improve efforts to generate pacemaker cells in vitro for use in regenerative medicine.