Many details of cardiac chamber morphogenesis could be revealed if muscle fiber development could be visualized directly within the hearts of living vertebrate embryos. To achieve this end, we have used the active promoter of the MLC1v gene to drive expression of green fluorescent protein (GFP) in the developing tadpole heart. By using a line of Xenopus laevis frogs transgenic for the MLC1v-EGFP reporter, we have observed regionalized patterns of muscle formation within the ventricular chamber and maturation of the atrial chambers, from the onset of chamber formation through to the adult frog. In f1 generation MLC1v-EGFP animals, promoter activity is first detected within the looping heart tube and delineates the forming ventricular chamber and proximal outflow tract throughout their development. The 8-kb MLC1v promoter faithfully reproduces the embryonic expression of the endogenous MLC1v mRNA. At later larval stages, weak patches of EGFP fluorescence are found on the atrial side of the atrioventricular boundary. Subsequently, an extensive lattice of MLC1v-expressing fibers extend across the mature atrial chambers of adult frog hearts and the transgene reveals the differing arrangement of muscle fibers in chamber versus outflow myocardium. The complete activity of the promoter resides within the proximal 4.5 kb of the MLC1v DNA fragment, whereas key elements regulating chamber-specific expression are present in the proximal-most 1.5 kb. Finally, we demonstrate how cardiac and craniofacial muscle expression of the MLC1v promoter can be used to diagnose mutant phenotypes in living embryos, using the injection of RNA encoding a Tbx1-engrailed repressor-fusion protein as an example.
Copyright 2005 Wiley-Liss, Inc.