Retroviral analysis of cardiac morphogenesis: discontinuous formation of coronary vessels.

Abstract

Cellular progenitors of the coronary vasculature are believed to enter the chicken heart during epicardlal morphogenesis between stages 17 and 27 (days 3-5) of egg incubation. To trace cells which give rise to the coronary arteries in vivo, we applied retroviral cell tagging procedures and analyzed clonal populations of vascular smooth muscle, endothelium, and connective tissue in the hearts of post-hatch chickens. Our data provide direct proof that (t) vascular smooth muscle progenitors begin to enter the heart at stage 17, substantially after the heart begins propulsive contractions; (ii) cardiac myocytes, vascular smooth muscle, perivascular fibroblasts, and coronary endothelial cells all derive from independent precursors when these cells migrate into the heart; (ON) endothelial cells of the coronary vessels have a different clonal origin than endothelial cells of the endocardium; (iv) coronary arteries form by the coalescence of discontinuous colonies (i.e., in situ vasculogenesis), each derived from a founder cell tagged at the time of retroviral injection (stages 17-18); and (v) coronary arteries contain discrete segments composed of "polyclones." These studies indicate the feasibility of gene targeting to coronary progenitors through the use of recombinant retroviruses.The embryonic chicken heart begins rhythmic contractions at Hamburger-Hamilton stage 10 ("'28 hr of incubation) (1, 2), but for the first 6 days of chicken embryogenesis the myocardial wall is avascular and nourished by diffusion through the endocardium (3)(4)(5). Such diffusion is facilitated by extensive trabecular channels which markedly increase endocardial surface area (6). Overt coronary vasculogenesis begins on day 6 of incubation (stage 29), first as venous sinusoids in communication with the trabecular channels, and secondarily as coronary arterial vessels which anastomose with the sinusoids (7,8). A closed coronary vasculature is completed after day 14 ofembryogenesis (3-5). Up to stage 15, the heart is composed of only two cell types: myocytes and endothelial cells; neither connective tissue, coronary vessels, neural elements, nor the conduction system is evident histologically (9, 10). Progenitor cells of the coronary vessels along with connective-tissue precursors are believed to enter the heart as the epicardial mantle envelopes the myocardium at stages 17-27 (10-12). Except for one study using chicken-quail chimeras to trace endothelial cells in the developing avian vasculature (13), no lineage analyses of coronary progenitors have been published. In this report we present an application of retroviral cell tagging (14,15) procedures for tracking smooth muscle, endothelial, and connective-tissue progenitors during coronary morphogenesis. We demonstrate the independence offibroblast, smooth muscle, and endothelial lineages and prove that coronary vessels form by in situ vasculogenesis rather than by angiogenic outgrowth from the root of the aorta. 106 active virions per ml, were concentrated by ultracentrifugation (...

The epicardium and epicardially derived cells (EPDCs) as cardiac stem cells

“…The differentiation of proepicardial/epicardial cells into CoSMCs was first reported in a series of studies based on a retroviral in vivo labeling (Mikawa and Fischman, 1992;Mikawa and Gourdie, 1996). Subsequently, a number of studies using quail-to-chick proepicardial transplantations confirmed and expanded these observations (Fig.…”

“…In a series of elegant retroviral labeling studies, Mikawa and colleagues (Mikawa and Fischman, 1992;Mikawa and Gourdie, 1996) clearly showed that the coronary system does not derive from the myocardium or from the endocardium, but that instead the proepicardium was the source of coronary endothelium. The retroviral studies did not, however, provide any data on the specific origin of the coronary progenitors found within the proepicardium.…”

Evidence for an extracellular matrix bridge guiding proepicardial cell migration to the myocardium of chick embryos

“…Morphologic and experimental studies point to a transitory structure, the proepicardium (PE), a grape-like cluster of mesothelial cells derived from the pericardial serosa that extends to and migrates over the myocardial surface during the looping, tubular stage of heart development (Manasek, 1969;Ho and Shimada, 1978;Viragh and Challice, 1981;Komiyama et al, 1987;Kuhn and Liebherr, 1988;Fransen and Lemanski, 1990;Manner, 1992;Mikawa and Fischman, 1992;Viragh et al, 1993;Munoz-Chapuli et al, 1994;Mikawa and Gourdie, 1996).…”

“…Common to all vertebrate species, once the epicardial sheet has formed, some its cells undergo an epithelial-mesenchymal transformation that gives rise to connective tissue cells of the subepicardium and myocardium, and the angiogenic precursors of the coronary vessels (Mikawa and Fischman, 1992;Dettman et al, 1998;Vrancken Peeters et al, 1999).…”

Multiple growth factors regulate coronary embryonic vasculogenesis

“…The first step in establishing a coronary vasculature involves the migration of angioblasts from the subepicardium into the myocardium, their differentiation into endothelial cells, and their assembly to form vascular tubes (Mikawa and Fischman, 1992;Poelmann et al, 1993;Rongish et al, 1994;Viragh et al, 1993). This process is termed vasculogenesis and also consists of fusion of the vascular tubes (a process reviewed by Drake and Little, 1999).…”