Epicardial retinoid X receptor ␣ is required for myocardial growth and coronary artery formation Esther Merki*†,Mo´ nica Zamora*†‡, Angel Raya§¶, Yasuhiko Kawakami§, Jianming Wang‡, Xiaoxue Zhang*, John Burchʈ, Steven W. Kubalak**, Perla Kaliman††, Juan Carlos Izpisua Belmonte§, Kenneth R. Chien*‡‡§§, and Pilar Ruiz-Lozano*‡§§ *Institute of Molecular Medicine, University of California, San Diego, CA 92093; §Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92186; ʈFox Chase Cancer Center, Philadelphia, PA 19111; **Cardiovascular Developmental Biology Center, Medical University of South Carolina, Charleston, SC 29425; ††Department de Bioquimica i Biologia Molecular, Universitat de Barcelona, 08028 Barcelona, Spain; ‡The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037; and ‡‡Massachusetts General Hospital, MGH Cardiovascular Research Center CPZN 3208, 185 Cambridge Street, Boston, MA 02114-2790 Edited by Pierre Chambon, Institut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Illkirch-Cedex, France, and approved November 3, 2005 (received for review May 26, 2005) Vitamin A signals play critical roles during embryonic development. phogenesis. This is an important consideration, because muta- In particular, heart morphogenesis depends on vitamin A signals tion of other nuclear receptors expressed in the placenta have mediated by the retinoid X receptor ␣ (RXR␣), as the systemic severe implications in cardiac development (16). mutation of this receptor results in thinning of the myocardium Here, we analyze a series of tissue-restricted mutations of the and embryonic lethality. However, the molecular and cellular RXR␣ gene in the cardiac neural crest, endothelial, and epicar- mechanisms controlled by RXR␣ signaling in this process are dial lineages, for which we have generated transgenic mice unclear, because a myocardium-restricted RXR␣ mutation does not driving Cre expression selectively into each of these derivatives. perturb heart morphogenesis. Here, we analyze a series of tissue- We show that RXR␣ signaling in the epicardium is critical for restricted mutations of the RXR␣ gene in the cardiac neural crest, proper morphogenesis of the heart. Moreover, we detect an endothelial, and epicardial lineages, and we show that RXR␣ additional phenotype of defective coronary arteriogenesis asso- signaling in the epicardium is required for proper cardiac morpho- ciated with RXR␣ deficiency in the epicardium, and identify a genesis. Moreover, we detect an additional phenotype of defective retinoid-dependent Wnt signaling pathway that cooperates in coronary arteriogenesis associated with RXR␣ deficiency and iden- epicardial epithelial-to-mesenchymal transformation (EMT). tify a retinoid-dependent Wnt signaling pathway that cooperates in epicardial epithelial-to-mesenchymal transformation. Methods Generation of Epicardial-Restricted RXR␣ Mutants. Transgenic mice coronary vessels ͉ epicardium ͉ retinoids ͉ wnt ͉ FGF expressing Cre-recombinase in an epicardial-restricted manner (G5 line) were generated by the use of a construct in which a etinoids exert their functions by binding to the nuclear 4.8-kb NotI͞KpnI fragment of the GATA-5 promoter (17) was Rreceptors, retinoid A receptor and retinoid X receptor ligated upstream of the cDNA encoding phage P1 Cre- (RXR), both of which are members of the superfamily of recombinase. ROSA26-indicator mice were obtained from The ligand-inducible transcriptional regulators (1, 2). The ligand- Jackson Laboratory. activated receptors differentially regulate gene expression Epicardial-restricted RXR␣ mutant mice were generated through specific hormone responsive DNA elements, where the upon crossing G5 with a previously described mouse line that receptors can bind as heterodimers or homodimers (3). Knock- carries a RXR␣ floxed allele (RXR␣fl/fl) (9). out mice carrying single or combined mutations of retinoid receptors (4) demonstrated that the only single mutation in its Generation of Neural Crest-Restricted and Endothelial-Restricted homozygous state that confers a cardiac phenotype and embry- RXR␣ Mutants. Neural crest-restricted RXR␣ mutant mice were onic lethality is that of RXR␣. Midgestation lethality in the RXR␣ generated by cross breeding RXR␣fl/fl with Wnt-1-Cre trans- mutant is due to heart failure (5) that results, in part, from genic mice (18). Double transgenic mice were characterized by thinning of the myocardial compact zone (6–8). The specific PCR using the following primers: Wnt1P1, 5Ј-GAG CAC CCT partner for RXR␣ responsible for the cardiac defect is currently GGA TGT GAA GT-3Ј and Wnt1P2, 5Ј-ATT CTC CCA CCG unknown. Despite the major cardiac muscle defect of the TCA GTA CG-3Ј; RXR␣fl: RXRp1, 5Ј-ACC AAG CAC ATC systemic RXR␣ mutants, ventricular-specific RXR␣ mutant mice TGT GCT ATC T-3Ј and RXRp3, 5Ј-ATG AAA CTG CAA are viable and indistinguishable from their wild-type littermates GTG GCC TTG A-3Ј. (9), and in chimeric embryos made with embryonic stem cells Endothelial-restricted RXR␣ mutants were generated by lacking RXR␣, cardiomyocytes deficient in RXR␣ developed crossing the RXR␣fl/fl line with a tie2-cre transgenic line (19). normally and contributed to the ventricular chamber wall in a For breeding, RXR␣fl/fl females were crossed with males het- normal manner (10). Together, these data demonstrated that erozygous for the floxed RXR␣ allele (RXR␣fl/ϩ) and heterozy- thinning of the myocardium is secondary to arrest of RXR␣ gous for the Cre transgene. Primers for genotyping were: Tie2 signaling in neighboring tissues. cre: Tie2creP1, 5Ј-CCC TGT GCT CAG ACA GAA ATG Recent reports point to a putative role of the epicardium in the transduction of retinoid signaling. First, studies using retinoic BIOLOGY acid response elements in transgenic mice indicated that epi- Conflict of interest statement: No conflicts declared. DEVELOPMENTAL cardial cells have the potential to respond to retinoids (11). This paper was submitted directly (Track II) to the PNAS office. Second, some mutations that affect the epicardium (12–14) Abbreviations: RXR, retinoid X receptor; EMT, epithelial-to-mesenchymal transformation; result in a thin myocardial wall, and finally, combined epicardial͞ En, embryonic day n. placental expression of a dominant-negative retinoid A receptor †E.M. and M.Z. contributed equally to this work. in transgenic mice induces prominent cardiac defects (15). ¶Present address: Institucio´Catalana de Recerca i Estudis Avanc¸ats i Centre de Medicina However, the absence of epicardial-restricted promoters to Regenerativa de Barcelona, Dr. Aiguader 80, 08003 Barcelona, Spain. target Cre expression made it impossible to discriminate the role §§To whom correspondence may be addressed. E-mail: [email protected] or of the epicardium from the function of placental tissues in [email protected]. transducing retinoid signaling and direct proper cardiac mor- © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504343102 PNAS ͉ December 20, 2005 ͉ vol. 102 ͉ no. 51 ͉ 18455–18460 Downloaded by guest on October 1, 2021 Table 1. Survival of mice after cell lineage-specific gene targeting of the RXR␣ floxed allele Genotype Survival, no. Age, observed͞no. Strain n nCreϩ days Breeding cre RXR␣ expected tie2-cre 56 39 P20 Tie2cre͞ϩRXR␣f͞ϩ ϫ RXR␣f͞f tie2 RXR␣ f͞ϩ 22͞14 tie2 RXR␣ f͞f17͞14 wnt1-cre 97 66 P20 W1cre͞ϩRXR␣f͞ϩ ϫ W1cre͞ϩRXR␣f͞ϩ wnt1 RXR␣ ϩ͞ϩ 20͞18 wnt1 RXR␣ f͞ϩ 27͞36 wnt1 RXR␣ f͞ϩ 19͞18 tie2-cre mlc2V-cre 57 46 P20 Tie2cre͞ϩRXR␣f͞f ϫ M2vcre͞ϩRXR␣f͞f tie2 RXR␣ f͞f13͞14 tie2 mlc2v RXR␣ f͞f10͞14 mlc2v RXR␣ f͞f23͞14 tie2-cre wnt1-cre 21 15 P20 Tie2cre͞ϩRXR␣f͞f ϫ W1cre͞ϩRXR␣f͞f tie2 RXR␣ f͞f3͞5 tie2 wnt1 RXR␣ f͞f8͞5 wnt1 RXR␣ f͞f4͞5 g5-cre 100 51 E13 G5cre͞ϩRXR␣f͞ϩ ϫ RXR␣f͞f g5 RXR␣ f͞ϩ 26͞25 g5 RXR␣ f͞f25͞25 g5-cre 64 31 E16 g5 RXR␣ f͞ϩ 20͞16 g5 RXR␣ f͞f11͞16 g5-cre 43 18 P20 g5 RXR␣ f͞ϩ 14͞11 g5 RXR␣ f͞f4͞11 Animals hemizygous for either tie2-cre (endothelial-specific), wnt-1-Cre (neural crest-specific), a combination of tie2-cre and mlc2v-cre (ventricular myocyte- specific), a combination of tie2-cre and wnt-1-cre, or the epicardial-specific G5-cre were crossed with the RXR␣ floxed allele mouse to obtain each of the above cre animals that were also hemizygous for RXR␣-flox allele. These animals were then bred to the homozygous RXR␣-flox to obtain offspring with lineage-specific removal of RXR␣. Survival rates and respective genotypes were scored. Genotypes corresponding to CreϪ animals are not indicated. n, the total number of animals resulting from each breeding scheme; nCreϩ, the number of Cre positive animals, Pn, postnatal day n. AGA-3Ј; Tie2creP2, 5Ј-CGC ATA ACC AGT GAA ACA GCA staged according to Hamburger and Hamilton (23). Recombi- TTG C-3Ј. nant adenoviruses encoding full-length chicken Wnt9b (Ad- Wnt9b), a constitutively active form of Xenopus ␤-catenin (24) Histology, Immunohistochemistry, and ␤-Galactosidase Staining. His- containing the internal Armadillo repeats (Ad-act␤cat) and egfp tological analysis was performed according to standard proto- (Ad-egfp) were produced and purified as previously described cols for paraffin embedding. For immunohistochemistry, em- (25). Recombinant FGF2 (R & D Systems) was loaded into bryos were cryopreserved in 30% sucrose and embedded in heparin acrylic beads (Sigma) for 1 h. Concentrated adenovi- OCT. Cryosections were cut at a thickness of 15 ␮m, unless ruses or FGF2 beads were microinjected or grafted, respectively, described otherwise. Antibodies used were as follows: 1:100 into