INTRODUCTION Atrial and ventricular muscle cell lineages display distinct electrophysiological (Maltsev et al., 1994; Davies et al., 1996; Chuck et al., 1997; Thomas et al., 1997), biochemical (Zeller et al., 1987; Seidman et al., 1988; Argentin et al., 1994) and contractile properties (Parker-Thorneburg et al., 1992; Gulick et al., 1997), which are largely dependent on the expression of distinct subsets of chamber-specific genes during the course of cardiogenesis (Lyons, 1994; Fishman and Chien, 1997; Fishman and Olsen, 1997). Relatively little is known regarding the positional and molecular cues that regulate this panel of proteins. Although the initial diversification of atrial and ventricular lineages occurs shortly after gastrulation, a large number of chamber-specific genes acquire a regionally restricted pattern of expression relatively late during cardiac development. In certain cases, these changes are observed post-septation or in the neonatal period (Edmondson et al., 1994; Lyons, 1994; Fishman and Chien, 1997; Fishman and Olsen, 1997). Thus, while the initial commitment to atrial and ventricular lineages occurs relatively early during vertebrate cardiogenesis, a series of maturational steps within the ventricular muscle lineage must occur for appropriate chamber development. Subsequent steps, such as the expansion of the compact layer of the ventricular chamber, formation of the spongy myocardium, initiation of trabeculation and cues for the appropriate onset of conotruncal and endocardial cushion formation, are critical in the maintenance of normal cardiac morphogenesis. Mutations in specific signaling pathways that impair these various steps of ventricular chamber morphogenesis can result in embryonic death. Recent studies using gene-targeted approaches of specific retinoid receptors in mice have now provided direct evidence that retinoids are required for normal cardiogenesis (Kastner et al., 1994; Sucov et al., 1994, 1996). Retinoid X receptor alpha (RXRα29 gene- deficient mice display a persistent atrial-like phenotype that is characterized by a lack of expansion of the compact zone, abnormal ventricular trabeculation and persistent expression of an atrial marker myosin light chain 2 (MLC-2a), which is normally downregulated during the course of murine 4427 Development 125, 4427-4438 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 DEV4881 Vertebrate cardiogenesis is a complex process involving multiple, distinct tissue types which interact to form a four- chambered heart. Molecules have been identified whose expression patterns co-segregate with the maturation of the atrial and ventricular muscle cell lineages. It is not currently known what role intrinsic events versus external influences play in cardiac chamber morphogenesis. We developed novel, fluorescent-based, myocardial, cellular transplantation systems in order to study these questions in murine embryos and report the irreversible nature of chamber specification with respect to the downregulation of atrial myosin light chain 2 (MLC-2a) and alpha myosin heavy chain (α-MHC). Grafting ventricular cells into the atrial chamber does not result in upregulation of MLC-2a expression in ventricular cells. Additionally, wild-type ventricular muscle cells grafted into the wild-type background appropriately downregulate MLC-2a and α-MHC. Finally, grafting of RXRα gene- deficient ventricular muscle cells into the ventricular chambers of wild-type embryos does not rescue the persistent expression of MLC-2a, providing further evidence that ventricular chamber maturation is an early event. These studies provide a new approach for the mechanistic dissection of critical signaling events during cardiac chamber growth, maturation and morphogenesis in the mouse, and should find utility with other approaches of cellular transplantation in murine embryos. These experiments document the irreversible nature of the downregulation of atrial markers after the onset of cardiogenesis during ventricular chamber morphogenesis and temporally define the response of cardiac muscle cells to signals regulating chamber specification. Key words: Ventricular chamber, Myosin light chain, Cardiac morphogenesis, RXRα, Heart, Mouse SUMMARY Downregulation of atrial markers during cardiac chamber morphogenesis is irreversible in murine embryos Peter J. Gruber*, Steven W. Kubalak ‡ and Kenneth R. Chien § Department of Medicine, Center for Molecular Genetics, and the American Heart Association-Bugher Foundation Center for Molecular Biology, University of California, San Diego, La Jolla, California 92093-0613, USA * Present address: Johns Hopkins Hospital, 844 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA ‡ Present address: Medical University of South Carolina, Department of Cell Biology and Anatomy, 171 Ashley Avenue, Charleston, SC 29425, USA § Author for correspondence (e-mail: kchien@ucsd.edu; pgruber@welchlink.welch.jhu.edu) Accepted 20 August; published on WWW 20 October 1998