CLONAL ANALYSIS OF CARDIAC MORPHOGENESIS IN THE CHICKEN-EMBRYO USING A REPLICATION-DEFECTIVE RETROVIRUS .1. FORMATION OF THE VENTRICULAR MYOCARDIUM

Cells of the precardiac mesoderm (stages 4-6) and dividing myocytes of early hearts (stages 10-15) were tagged with a replication-incompetent retrovirus (CXL) (Mikawa et al., 1991b) encoding bacterial beta-galactosidase (beta-gal). Two protocols were used to infect the cardiogenic cells. (1) Small blocks (approximately 50-mu-m2) of anterolateral mesoderm were dissected from gastrula-stage embryos (stages 4-6) and incubated in liquid medium containing the retrovirus. After removal of CXL, the tissues were dispersed into single-cell suspensions and pressure injected into the precardiac areas of recipient embryos (stages 4-6). Such embryos were then incubated in vitro at 37-degrees-C for 2 days (New, 1968), and those embryos with beating hearts were fixed for X-gal histochemistry and paraffin serial sectioning. (2) CXL was pressure injected in ovo (embryonic stages 4-15) into cardiogenic tissues and the eggs subsequently returned to an incubator. At selected stages of development embryos or whole hearts were fixed, stained with X-gal, and serially sectioned after paraffin embedding. The first method showed that (1) cells of the precardiac mesoderm could be infected with the retrovirus, (2) the transplanted cells would differentiate into beating myocytes, and (3) beta-gal expression was sufficiently high to be detected histochemically. With the second procedure we could show that (1) beta-gal-tagged cells formed colonies in the myocardium, (2) the labeled cells were exclusively myocytes, (3) the number of cells per colony increased with increasing age of embryonic development, (4) the size of colonies was larger in the left than the right ventricle, (5) many of the colonies were transmural, i.e., they extended from epicardial to endocardial layers of the myocardium and generally exhibited a cone or funnel-shape with the base of the cone nearest the epicardium, (6) the orientation of myocytes within each colony changed at different layers of the myocardium, and (7) the cones contained both beta-gal+ and beta-gal- myocytes. DNA labeling studies with [H-3]thymidine indicated that cardiogenic cells divided every 16-18 hr during the first week of development and that the CXL-labeled cells divided indistinguishably from unlabeled myocytes. Based on these observations a model for the growth of the myocardium is presented.