An in vitro assay has been developed to investigate tissue interactions regulating myocardial cell specification in birds. Explants from the posterior region of stage XI-XIV blastulas were found to form heart muscle at high frequency with a timing that corresponded to onset of cardiac myocyte differentiation in vivo. Isolation and recombination experiments demonstrated that a signal from the hypoblast was required to induce cardiac myogenesis in the epiblast, and regional differences in epiblast responsiveness and hypoblast inductiveness restrict appearance of cardiac myocytes to the posterior region. Explantation studies provided evidence that myocardial cell specification is underway by stage 3, indicating that the hypoblast-derived signal occurs shortly before specification is detected. Recombinations were also performed to compare cardiac-inducing capacities of pregastrula hypoblast and stage 5 anterior lateral endoderm. The hypoblast possessed broad capacity to induce heart muscle cells in pregastrula and mid-gastrula epiblast, and modest ability to induce cardiac myogenesis in stage 4 posterior primitive streak. Stage 5 anterior lateral endoderm, in contrast, showed no ability to induce heart development in epiblast cells but was a potent inducer of cardiac myogenesis in cells from stage 4 posterior primitive streak. These findings suggest that the hypoblast-derived signal likely acts upstream of proposed heart-inducing signals provided by anterior lateral endoderm. Experiments were also performed to investigate whether activin, or an activin-like molecule, is involved in regulating cardiac myogenesis. Follistatin blocked cardiac myogenesis in stage XI-XIV posterior region explants and activin induced cardiac myogenesis in a dose-dependent fashion in posterior epiblast. These findings indicate that activin, or an activin-like molecule, is required for and is sufficient to stimulate cardiac myogenesis in posterior region pregastrula epiblast. Three models are presented to explain these results.

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