The RXRalpha gene functions in a non-cell-autonomous manner during mouse cardiac morphogenesis

Chanh M. Tran Henry M. Sucov ) cardiac morphogenesis SUMMARY Germline mutation in mice of the retinoic acid receptor gene RXRa results in a proliferative failure of cardiomyocytes, which leads to an underdeveloped ventricular chamber and midgestation lethality. Mutation of the cell cycle regulator N-myc gene also leads to an apparently identical phenotype. In this study, we demonstrate by chimera analysis that the cardiomyocyte phenotype in RXRa - /- embryos is a non-cell-autonomous phenotype. In chimeric embryos made with embryonic stem cells lacking RXRa , cardiomyocytes deficient in RXRa develop normally and contribute to the ventricular chamber wall in a normal manner. Because the ventricular Most aspects of vertebrate organogenesis begin with the assembly of cells of different lineage into a tissue primordium. Thereafter, a progressive series of inductive interactions between these lineages results in morphogenesis and maturation into a differentiated and functional organ. In the developing heart, cells of the cardiomyocyte and endothelial (endocardial) lineages are segregated within the cardiac crescent of anterior lateral mesoderm prior to the assembly of a beating heart. Cells which form the epicardium (the outer layer of the heart) and tissue of the aorta and pulmonary outflow vessels then migrate to the heart from the sinus venosus and neural crest, respectively. These lineages are assembled in the mouse heart by approximately embryonic day 10.5 (E10.5). Thereafter, each domain of the developing heart undergoes further maturation. Morphogenesis of the ventricular chamber involves proliferation and accumulation of cardiomyocytes in the ventricular chamber wall, resulting in a thickened muscular compact zone, which is essential for pumping blood through the rapidly growing embryo. The vitamin A derivative retinoic acid (RA) is a critical signaling molecule in numerous developmental and physiological processes, including several aspects of cardiovascular morphogenesis. The biological effects of RA are mediated by members of the nuclear receptor family of ligand-dependent transcription factors (Evans, 1988). Germline mutation (Sucov et al., 1994; Kastner et al., 1994) in hypoplastic phenotype reemerges in highly chimeric embryos, we conclude that RXRa functions in a nonmyocyte lineage of the heart to induce cardiomyocyte proliferation and accumulation, in a manner that is quantitatively sensitive. We further show that RXRa is not epistatic to N-myc, and that RXRa and N-myc regulate convergent obligate pathways of cardiomyocyte maturation. mice of the retinoic acid receptor gene RXRa results in a prominent and completely penetrant ventricular chamber phenotype. In RXRa - /- embryos, all lineages of the developing heart are present and normally organized up to E11.5. However, further proliferation of the myocardium in the compact zone of the ventricular chamber wall fails to occur, so that by E14.5 a persistently thin-walled and hypoplastic ventricle remains. Some 50 years ago, nutritional studies established that expansion of the compact zone requires vitamin A (Wilson and Warkany, 1949), which is now recognized as a requirement for provision of RA as a signaling molecule and which is received at least in part by RXRa . The RXRa - /- phenotype is embryonic lethal around E15.5 because of insufficient cardiac performance (Dyson et al., 1995). Mutation of several other genes, including the cell-cycle regulator N-myc (Moens et al., 1993), results in a similar, if not identical, hypoplastic ventricular chamber phenotype. Although this phenotype is ultimately a proliferative deficiency of cardiomyocytes, it cannot be assumed that the function of any of the genes that affect this process is required in cardiomyocytes. RXRa is ubiquitously expressed prior to E15.5 (Mangelsdorf et al., 1992; Dolle et al., 1994) and, while fetal (unpublished observations) and neonatal (Zhou et al., 1995) cardiomyocytes are able to respond to RA signaling (as evidenced by transcriptional activation of reporter genes), almost all cell types have this same capacity (Sucov et al., 1990). Another lineage of the heart could in principle respond to RA signaling through RXRa and in a secondary process then inductively direct cardiomyocyte proliferation and accumulation. RXRa , N-myc and other genes might function within or external to the ventricular cardiomyocyte lineage and possibly regulate pathways of cardiac development that are epistatic or convergent. In this study, we have undertaken a direct investigation of the site of RXRa action. We show in chimeric embryos, made by introduction of RXRa - /- embryonic stem (ES) cells into wild-type recipient blastocysts, that cardiomyocytes that lack RXRa proliferate normally in the compact zone of the ventricular chamber wall. Thus, RXRa functions in a lineage external to the cardiomyocyte population and the RXRa - /cardiomyocyte phenotype is cell non-autonomous. This same conclusion has been reached by a completely different methodology, as described in the accompanying paper (Chen et al., 1998). We furthermore demonstrate that RXRa and Nmyc regulate convergent obligate pathways of cardiomyocyte maturation. MATERIALS AND METHODS Derivation of embryonic stem cell lines Mice bearing the ROSA-26 transgene (Friedrich and Soriano, 1991) were obtained from Jackson Laboratories at the 6th generation of breeding into the C57Bl/6 background. These were crossed to RXRa - /+ partners that had been inbred on the C57Bl/6 background at least seven generations, and transgenic male heterozygotes were used in matings with nontransgenic heterozygous females from the same strain background. Females were superovulated prior to mating and morula isolated from the oviduct-uterine junction at E2.5. Following overnight culture, blastocysts were plated on a layer of mitotically inactive primary embryonic fibroblast feeder cells in conventional ES cell media supplemented with 1000 units/ml LIF. Blastocyst outgrowths were picked and expanded, and then genotyped and characterized for the presence of the ROSA-26 transgene. Production and analysis of chimeric embryos Morula were isolated from matings of wild-type CD-1 (ICR) mice at E2.5 and cocultured with ES cells essentially as described (Wood et al., 1993). For low percentage chimeras, ES cells were plated at a concentration of 1.5 105 cells/ml; for highly chimeric embryos, the ES cell concentration was raised to 5 105 cells/ml. Coculture was for 3.5 hours, after which embryos were removed and allowed to incubate overnight and viable blastocysts were implanted into pseudopregnant ICR females the following day. Embryos were isolated at midgestation, with chimeric embryos identified initially on the basis of eye pigmentation chimerism. Generally, hearts were isolated, fixed and stained in X-gal by standard procedures, then paraffin embedded, sectioned at 3 m m thickness and counterstained with nuclear fast red. For deter (...truncated)