Vitamin A signals play critical roles during embryonic development. In particular, heart morphogenesis depends on vitamin A signals mediated by the retinoid X receptor ␣ (RXR␣), as the systemic mutation of this receptor results in thinning of the myocardium and embryonic lethality. However, the molecular and cellular mechanisms controlled by RXR␣ signaling in this process are unclear, because a myocardium-restricted RXR␣ mutation does not perturb heart morphogenesis. Here, we analyze a series of tissuerestricted mutations of the RXR␣ gene in the cardiac neural crest, endothelial, and epicardial lineages, and we show that RXR␣ signaling in the epicardium is required for proper cardiac morphogenesis. Moreover, we detect an additional phenotype of defective coronary arteriogenesis associated with RXR␣ deficiency and identify a retinoid-dependent Wnt signaling pathway that cooperates in epicardial epithelial-to-mesenchymal transformation.coronary vessels ͉ epicardium ͉ retinoids ͉ wnt ͉ FGF

We have previously identified several members of the Wnt/␤-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of ␤-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of ␤-catenin in coronary arteriogenesis, we have generated conditional ␤-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of ␤-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial ␤-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-␤-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial ␤-catenin pathway in coronary arteriogenesis. The embryonic epicardium originates from a primarily extracardiac primordium, the proepicardum, which is located at the septum transversum near the venous pole of the heart (2, 3). In mouse embryos, proepicardial cells reach the heart predominantly in the form of free-floating vesicles that traverse the pericardial cavity, adhere to the initially naked myocardial surface, and subsequently form the epicardial covering of the heart (reviewed in ref. 1). The recruitment of coronary vessel progenitor cells from the proepicardium and embryonic epicardium involves several steps of epithelial-mesenchymal transition (EMT). As a result of proepicardial EMT, the extracellular matrix of free-floating proepicardial vesicles becomes populated with mesenchymal cells (4-8). Subsequently, the primitive epicardium gives rise to the subepicardial mesenchyme, also by means of EMT.Recent data suggest that the primitive epicardium and epicardium-derived cells (EPDCs) modulate the maturation of other cardiac components, including the embryonic myocardium and the cardiac conduction system (9-14). We have previously shown that the embryonic epicardium is a key signaling tissue responsible for the transmission of the morphogenic signal derived from retinoic acid and identified several components of the Wnt/␤-catenin signaling pathway that are down-regulated upon retinoid signaling deficiency, in particular, ␤-catenin and its activator Wnt9b (15).However, it remains to be shown whether Wnt/␤-catenin signaling plays a specific role in the de...

Overexpression of adenine nucleotide translocase-1 (ANT1) is known to induce apoptosis (Bauer, M. K., Schubert, A., Rocks, O., and Grimm, S. (1999) J. Cell Biol. 147, 1493-1501), but the mechanisms involved remain unclear. In this study we show that ANT1 overexpression results in a recruitment of the IB␣-NF-B complex into mitochondria, with a coincident decrease in nuclear NF-B DNA binding activity. In this situation, NF-B transcriptionally regulated genes with antiapoptotic activity, such as Bcl-XL, MnSOD2, and c-IAP2, are down-regulated, and consequently, cells are sensitized to apoptosis. Accordingly, co-expression of p65 partially interferes with the proapoptotic effect of ANT1 overexpression. Despite the high identity of the two isoforms, overexpression of ANT2 does not exert an apoptotic effect; this lack of apoptotic activity is correlated with the absence of mitochondrial IB␣-NF-B recruitment or changes in NF-B activity. Thus, we propose that the mitochondrial recruitment of NF-B observed following ANT1 overexpression has an important role in ANT1 proapoptotic activity.Apoptosis is a form of cell death that plays a role in development, tissue homeostasis, and disease (1). The induction of apoptosis is governed by an elaborate array of checks and balances in the cell. Studies of apoptosis induction in "in vitro" systems have demonstrated that mitochondria are required for the apoptosis stimulated by a variety of different factors (2).The ANT 1 protein is localized in the inner mitochondrial membrane and exchanges cytosolic ADP for mitochondrial ATP (3). Three isoforms (ANT1, ANT2, and ANT3) with tissuespecific expression patterns have been described in humans (4). ANT interacts with several proteins of the outer mitochondrial membrane (peripheral benzodiazepine receptor, porin/VDAC, and Bax) as well as the matrix (cyclophilin D) to form the permeability transition pore (PTP) (5). The PTP appears to be an important regulator of the apoptotic process. Opening of the pore leads to a loss of mitochondrial transmembrane potential, ⌬⌿ m , which can ultimately culminate in matrix swelling and outer membrane rupture, allowing the release of apoptogenic proteins such as cytochrome c, apoptosis-inducing factor, and procaspases (6, 7). Proteins of the bcl-2 family essentially control the release of cytochrome c. Antiapoptotic members of the family (Bcl-2 and Bcl-XL) prevent cytochrome release, whereas the proapoptotic members Bax and Bak exert the opposite effect (8). Bax has been shown to interact with ANT to induce PTP opening and cytochrome c release (9). Several pharmacological compounds interfere with PTP. For instance, cyclosporin A, through its binding to cyclophilin D, prevents PTP opening, and bongkrekic acid and atractyloside are, respectively, a blocker and an inducer of apoptosis via binding of two different conformational states of ANT (10). In addition, alongside their modulation of pore formation by ANT, Bcl-2 and Bax also have been reported to influence ANT ADP/ATP antiporter activity (11). Although ...