FORMATION AND EARLY MORPHOGENESIS OF ENDOCARDIAL ENDOTHELIAL PRECURSOR CELLS AND THE ROLE OF ENDODERM

The formation of endocardial endothelium in quail embryos was investig ated using in vivo and in vitro systems. Based on the expression of an quail endothelial marker, QH-1, the initial emergence of endothelial precursor cells in the embryo occurs at stage 7(+) (two somites) in th e posterior parts of the bilateral heart forming regions. Cells that e xpressed the QH-1 antigen were mesenchymal and positioned between the mesodermal epithelium of the heart region and the endoderm. By confoca l microscopy, an asymmetrical distribution of QH-1 positive cells was observed between the two heart regions: specifically between 7(+) and 8(-), more precursor cells were seen in the right region than the left . Endothelial precursor cells did not appear outside of the heart form ing regions until stage 8(-) (three somites). Free, mesenchymal-like e ndothelial precursor cells intrinsic to the heart regions also express ed two extracellular antigens, JB3, a fibrillin-like protein, and cyto tactin, both associated with segments of the primary heart tube where endothelial cells ''re-transform'' back to a mesenchymal phenotype dur ing cardiac cushion tissue formation. Between stages 8 and 9 (four to seven somites), (1) QH-1 positive cells within the heart forming regio n established vascular-like connections with QH-1 positive cells locat ed outside of the heart region, as initially shown by Coffin and Poole (1988), (2) after fusion of the heart regions, a plexus of QH-1 posit ive cells was formed ventral to the foregut, and (3) the definitive en docardial lining of the primary heart tube formed directly from the ve ntral plexus of endothelial precursor cells. Because the QH-1 positive , endothelial precursor cells of each heart forming region were always in close association with anterior endoderm, we sought to determine i f the endoderm mediated the formation of precursor cells committed to a cardiac endothelial lineage as reflected by their expression of QH-1 , JB3 antigen, and cytotactin. To test this hypothesis, precardiac mes odermal explants were isolated from stage 5 heart forming regions prio r to their expressing of either endocardial or myocardial markers and cultured on the surface of collagen gels in the presence or absence of endoderm. In the absence of endoderm, precardiac mesoderm of each sta ge 5 explant remained epithelial, formed contractile tissue, but did n ot exhibit any QH-1 positive cells or mesenchymal cells. Conversely, w hen cocultured with endoderm or endoderm conditioned medium, in additi on to the formation of contractile tissue, the explant formed mesenchy mal cells. The latter invaded the gel lattice and, as in vivo, express ed QH-1 antigen, JB3 antigen, and cytotactin. These findings suggest t hat endoderm induces mesoderm of the heart fields to undergo an epithe lial to mesenchyme transformation that results in the segregation of m yocardial and endocardial precursor cells. (C) 1996 Academic Press, In c.