Expression of exogenous protein and analysis of morphogenesis in the developing chicken heart using an adenoviral vector

Abstract

Recombinant retroviral vectors have been shown to be useful tools for marking cells so as to follow their fates during development. The aim of this study was to determine the utility and advantages of an adenoviral vector as a tool to study the heart as it develops from a simple tube into a complex four-chambered organ. Replication-defective adenovirus (10⁷ pfu) expressing β-galactosidase (β-gal) under the control of the RSV-LTR was applied to the external surface of embryonic stage 13–21 chick hearts in ovo. Embryos were incubated for up to an additional 96 h. Hearts were harvested at 12–24 h intervals and (1) whole-mount-stained for β-gal and sectioned, (2) examined by electron microscopy and (3) homogenized and β-gal activity measured with a luminescent assay. β-gal expression peaked at 48 h, when a significant percentage of the myocytes in the atrial and ventricular walls expressed the protein, and it comprised 0.5% of total heart protein. Significant levels were still expressed at 96 h. When applied to early-stage (13-6) embryos, expression occurred predominantly in cardiomyocytes. β-gal marking of cells enabled us to identify the following morphogenic patterns: (1) cells of the conus region compact into the bulbis cordis; (2) by applying the virus at later stages, e.g. 21–22, it was evident that the epicardium invests the heart, after stage 17, in a dorsal to ventral and caudal to rostral direction; (3) at lower titers (10⁵ pfu), the virus serves as a clonal marker through several cell divisions, with an estimated cell doubling time of 24 h. Application of an adenoviral vector to early-stage embryonic chick heart results in substantial expression of exogenous protein in a significant percentage of cardiomyocytes without grossly affecting heart development. Adenoviral vectors are useful for following the fate of cells as the heart develops from a simple tube into a complex four-chambered organ and hold promise for enabling the expression of exogenous proteins which might alter cell behavior.