Morpholinos for splice modificatio

Morpholinos for splice modification



An experimental system was devised to study the mechanisms by which cells become committed to the cardiac myocyte lineage during avian development. Chick tissues from outside the fate map of the heart (in the posterior primitive streak (PPS) of a Hamburger & Hamilton stage 4 embryo) were combined with potential inducing tissues from quail embryos and cultured in vitro. Species-specific RT-PCR was employed to detect the appearance of the cardiac muscle markers chick Nkx-2.5 (cNkx-2.5), cardiac troponin C and ventricular myosin heavy chain in the chick responder tissues. Using this procedure, we found that stage 4–5 anterior lateral (AL) endoderm and anterior central (AC) mesendoderm, but not AL mesoderm or posterior lateral mesendoderm, induced cells of the PPS to differentiate as cardiac myocytes. Induction of cardiogenesis was accompanied by a marked decrease in the expression of rho-globin, implying that PPS cells were being induced by anterior endoderm to become cardiac myocytes instead of blood-forming tissue. These results suggest that anterior endoderm contains signaling molecules that can induce cardiac myocyte specification of early primitive streak cells. One of the cardiac muscle markers induced by anterior endoderm, cNkx-2.5, is here described for the first time. cNkx-2.5 is a chick homeobox-containing gene that shares extensive sequence similarity with the Drosophila gene tinman, which is required for Drosophila heart formation. The mesodermal component of cNkx-2.5 expression from stage 5 onward, as determined by in situ hybridization, is strikingly in accord with the fate map of the avian heart. By the time the myocardium and endocardium form distinct layers, cNkx-2.5 is found only in the myocardium. cNkx-2.5 thus appears to be the earliest described marker of avian mesoderm fated to give rise to cardiac muscle.