SUMMARYThe secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field, and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole.

Appendage regeneration in salamanders and fish occurs through formation and maintenance of a mass of progenitor tissue called the blastema. A dedicated epidermis overlays the blastema and is required for its proliferation and patterning, yet this interaction is poorly understood. Here, we identified molecularly and functionally distinct compartments within the basal epidermal layer during zebrafish fin regeneration. Proximal epidermal subtypes express the transcription factor lef1 and the blastemal mitogen shh, while distal subtypes express the Fgf target gene pea3 and wnt5b, an inhibitor of blastemal proliferation. Ectopic overexpression of wnt5b reduced shh expression, while pharmacologic introduction of a Hh pathway agonist partially rescued blastemal proliferation during wnt5b overexpression. Loss- and gain-of-function approaches indicate that Fgf signaling promotes shh expression in proximal epidermis, while Fgf/Ras signaling restricts shh expression from distal epidermis through induction of pea3 expression and maintenance of wnt5b. Thus, the fin wound epidermis spatially confines Hh signaling through the activity of Fgf and Wnt pathways, impacting blastemal proliferation during regenerative outgrowth.

SUMMARY In chick and mouse embryogenesis, a population of cells described as the secondary heart field (SHF) adds both myocardium and smooth muscle to the developing cardiac outflow tract (OFT). Following this addition, at approximately HH stage 22 in chick embryos, for example, the SHF can be identified architecturally by an overlapping seam at the arterial pole, where beating myocardium forms a junction with the smooth muscle of the arterial system. Previously, using either immunohistochemistry or nitric oxide indicators such as diaminofluorescein 2-diacetate, we have shown that a similar overlapping architecture also exists in the arterial pole of zebrafish and some shark species. However, although recent work suggests that development of the zebrafish OFT may also proceed by addition of a SHF-like population of cells, the presence of a true SHF in zebrafish and in many other developmental biological models remains an open question. We performed a comprehensive morphological study of the OFT of a wide range of vertebrates. Our data suggest that all vertebrates possess three fundamental OFT components: a proximal myocardial component, a distal smooth muscle component, and a middle component that contains overlapping myocardium and smooth muscle surrounding and supporting the outflow valves. Because the middle OFT component of avians and mammals is derived from the SHF, our observations suggest that a SHF may be an evolutionarily conserved theme in vertebrate embryogenesis.