Role of the anterior endoderm in cardiac specification

The heart is the first functional organ of embryogenesis in many vertebrates, however little is known about the early specification events of cardiogenesis. Evidence in the chick and amphibian suggests a requirement for the anterior endoderm in cardiac induction to direct mesoderm toward a cardiac fate. Furthermore, the signals responsible for specification and their mode of action are unknown. Several signalling pathways, including FGF, Nodal, BMP and Wnt have been implicated. However, as these pathways have other roles in early embryogenesis a specific role in cardiac induction has been difficult to define. We have devised a model testing the cardiac-inducing activity of the anterior endoderm addressing its ability to re-specify pluripotent embryonic ectoderm upon conjugation. We have shown that the anterior endoderm is sufficient to induce robust expression of cardiac markers and formation of contractile tissue in the responder. Characterisation of the model showed the anterior endoderm produces a specific signal skeletal muscle is not induced, distinguishing it from general mesoderm induction. The cardiac-inducing capacity of the anterior endoderm was not uniform as it was restricted to the most anterior regions of the anterior endoderm, correlating with expression of Hex. The cardiac-inducing signal requires two hours of interaction with the responding tissue during gastrulation to produce an effect. Further involvement of the anterior endoderm beyond specification of cardiac precursors was not required. The model provided the basis to investigate the early signalling events of specification. Whereas BMP signalling was not necessary for cardiac induction by the endoderm, an essential requirement for FGF and Nodal pathways was shown. Timed inhibition revealed both were required during the first hour of conjugation, while sustained ERK activation was needed for at least four hours. In addition it was shown that elevated Wnt/p-catenin signalling during specification had no effect, while sustained activation antagonised cardiogenesis. Further analysis revealed Wnt/p-catenin has no direct role in specification, but suppression or low activity was required prior to the onset of cardiac differentiation. Therefore, this work established a simple and experimentally amenable assay for elucidating the mechanisms of cardiac specification