TRF2 inhibition promotes anchorage-independent growth of telomerase-positive human fibroblasts.

Although telomere instability is observed in human tumors and is associated with the development of cancers in mice, it has yet to be established that it can contribute to the malignant transformation of human cells. We show here that in checkpoint-compromised telomerase-positive human fibroblasts an episode of TRF2 inhibition promotes heritable changes that increase the ability to grow in soft agar, but not tumor growth in nude mice. This transforming activity is associated to a burst of telomere instability but is independent of an altered control of telomere length. Moreover, it cannot be recapitulated by an increase in chromosome breaks induced by an exposure to gamma-radiations. Since it can be revealed in the context of telomerase-proficient human cells, telomere dysfunction might contribute to cancer progression even at late stages of the oncogenesis process, after the telomerase reactivation step.

Spatiotemporal regulation of hnRNP M and 2H9 gene expression during mouse embryonic development.

Using the HeLa cell model along with an in vitro splicing system, we have previously shown that hnRNP M and 2H9 are involved in the pre-mRNA splicing process and most interestingly also in heat shock-induced transient splicing arrest by transiently leaving the hnRNP complexes. Due to this unique regulatory function in a mechanism that turns splicing on and off, these two hnRNPs appear as important proteins for controlling gene expression. Here we investigated by in situ hybridization and immunohistochemical staining techniques the expression level of specific mRNA and protein during mouse embryonic development. HnRNP M and 2H9 are found to be expressed at all examined stages (6.5-18.5 days post-coïtum), in a differential manner, and at various levels depending on tissues, cell types and also embryonic stages; fairly high levels of both hnRNPs are always observed in the central nervous system. Furthermore, levels of colocalizing protein and transcript are not always present in the same proportion, thus suggesting a post-transcriptional regulation of hnRNP M and 2H9 gene expression. The complex spatiotemporal variations we observed might well anticipate a role for these two hnRNPs also in modulating splicing, thereby influencing gene expression and further many physiological processes.

Tissue-specific expression of retinoic acid receptor isoform transcripts in the mouse embryo.

The three murine retinoic acid receptor (RAR) genes each contain two distinct promoters which give rise to protein isoforms differing in their N-terminal regions. This study used in situ hybridization to describe the expression patterns of RARalpha1, RARalpha2, RARbeta1/3, RARbeta2/4, RARgamma1 and RARgamma2 isoform transcripts during mouse embryogenesis. RARalpha1 transcripts are widely distributed, with the exception of the central nervous system. Highest expression is found in developing muscle, pituitary gland and various epithelia. On the other hand, RARalpha2 is essentially expressed along the spinal cord up to the hindbrain 7th rhombomere and in the 4th rhombomere, pons and developing basal ganglia (corpus striatum and pallidum). RARbeta2/4 transcripts account for most of the previously described RARbeta expression features being expressed specifically, or more prominently than RARbeta1/3, in foregut endoderm and its derivatives, olfactory and periocular mesenchyme, urogenital region, proximal limb bud mesenchyme and later within interdigital regions. RARbeta1/3 is more prominently expressed in the developing heart outflow tract mesenchyme, intervertebral disks, midgut loop mesenchyme and umbilical vessel walls. RARbeta1/3 and RARbeta2/4 are coexpressed in the developing corpus striatum. They exhibit, however, distinct dorsoventral distributions along the spinal cord and caudal hindbrain. RARgamma2 is the RARgamma isoform expressed at high levels in the caudal neural groove at embryonic day 8.5. At later stages, both RARgamma isoforms are essentially coexpressed, although the progressive restriction of RARgamma1 transcripts to craniofacial or limb precartilaginous condensations appears to precede that of RARgamma2.

Identification of three major phosphorylation sites within HIV-1 capsid. Role of phosphorylation during the early steps of infection.

We previously reported the presence of two cellular serine/threonine protein kinases incorporated in human immunodeficiency virus type 1 (HIV-1) particles. One protein kinase is MAPK ERK2 (mitogen-activated protein kinase), whereas the other one, a 53-kDa protein, still needs to be identified. Furthermore, we demonstrated that the capsid protein CAp24 is phosphorylated by one of those two virion-associated protein kinases (Cartier, C., Deckert, M., Grangeasse, C., Trauger, R., Jensen, F., Bernard, A., Cozzone, A., Desgranges, C., and Boyer, V. (1997) J. Virol. 71, 4832-4837). In this study, we showed that CAp24 is not a direct substrate of MAPK ERK2. Moreover, using site-directed mutagenesis of each of the 9 serine residues of CAp24, we demonstrated the phosphorylation of 3 serine residues (Ser-109, Ser-149, and Ser-178) in the CAp24. Substitution of each serine residue did not affect viral budding, nor viral structure. By contrast, substitution of Ser-109, Ser-149, or Ser-178 affects viral infectivity by preventing the reverse transcription process to be completely achieved. Our results suggest that CAp24 serine phosphorylation is essential for viral uncoating process.

Contribution of retinoic acid receptor beta isoforms to the formation of the conotruncal septum of the embryonic heart.

To investigate the relative contribution of retinoic acid receptor (RAR)beta isoforms in conotruncal septation, RAR beta 1 and beta 3 were inactivated in the mouse. Mice lacking RAR beta 1 and beta 3 appear normal. Disruption of these isoforms in RAR alpha or RAR gamma null genetic backgrounds results in a high postpartum lethality. However, except for ocular defects found in RAR beta 1-3/RAR gamma compound mutants, the double null mutants display only abnormalities seen in single null mutants. This probably reflects a functional redundancy with other RARs, most notably with RAR beta 2 which is five- to sixfold more abundant than RAR beta 1 and beta 3 and whose domain of expression is largely overlapping. The conotruncal ridges form normally in retinoid X receptor (RXR)alpha/RAR beta compound mutants but fail to fuse, apparently as a result of excessive apoptosis of mesenchymal cells. Additionally, many cardiomyocytes in the conotruncal wall of these mutants appear necrotic. Although RAR beta 1 and beta 3 are expressed specifically in the conotruncal ridges, failure of fusion of these structures is not more frequent in RXR alpha/RAR beta 1-3 double null mutants than in RXR alpha single null mutants. Similarly, the disruption of the sole RAR beta 2 isoform in a RXR alpha null genetic background does not result in an increase of the frequency of conotruncal septum agenesis. However, this agenesis is fully penetrant in RXR alpha/RAR beta +/- mutants, which reflects distinct role of RXR alpha:RAR beta 1 (and beta 3) and RXR alpha:RAR beta 2 heterodimers in promoting the survival of conotruncal mesenchymal cells. Unexpectedly, we discovered that, in wild-type embryos, the conotruncal mesenchyme is a major site of morphogenetic cell death and that conotruncal myocytes are occasionally necrotic. Thus, excessive cell death in the conotruncus is a potential cause of ventricular septal defects in humans.

Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication.

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.

Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein.

Retinoic acid plays important roles in development, growth and differentiation by regulating the expression of target genes. A new retinoic acid-inducible gene, Stra6, has been identified in P19 embryonal carcinoma cells using a subtractive hybridization cDNA cloning technique. Stra6 codes for a very hydrophobic membrane protein of a new type, which does not display similarities with previously characterized integral membrane proteins. Stra6, which exhibits a specific pattern of expression during development and in the adult, is strongly expressed at the level of blood-organ barriers. Interestingly, in testis Sertoli cells, Stra6 has a spermatogenic cycle-dependent expression which is lost in testes of RAR alpha null mutants where Stra6 is expressed in all tubules. We suggest that the Stra6 protein may be a component of an as yet unidentified transport machinery.

The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr.

The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in human immunodeficiency virus, type 1 (HIV-1) life cycle, in particular by interrupting cells division in the G2 phase. Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the Pr55(Gag) polyprotein precursor, which includes NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding. To precisely define the Gag and Vpr sequences involved in this protein-protein interaction, NCp7, p6, and Vpr as well as a series of derived peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Binding assays were carried out by Far Western experiments and by competition studies using (52-96)Vpr immobilized onto agarose beads. The results show that interaction between NCp7 and Vpr occurs in vitro by a recognition mechanism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr. Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukemia virus but not polysine inhibits Vpr-NCp7 complexation. Interestingly enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro. In vivo mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr encapsidation. Together, these results suggest that NCp7 cooperates with p6 to induce Vpr encapsidation in HIV-1 mature particles. The NCp7-Vpr complex could also be important for interaction of Vpr with cellular proteins involved in cell division.

Ontogenic expression of the Na(+)-independent organic anion transporting polypeptide (oatp) in rat liver and kidney.

BACKGROUND/AIMS: A cDNA (2.7 kb) encoding a rat liver basolateral Na(+)-independent organic anion transporter (oatp) has recently been cloned. The aim of the present study was to clarify the mechanisms of bile formation during development. METHODS: The ontogenic expression of oatp was examined by northern blot analysis and in situ hybridization in rat liver. The expression of oatp in the kidney was also studied in parallel. RESULTS: In the liver, a 2.5 kb oatp mRNA was first detected in the fetus on day 16 of gestation. The amount of this oatp mRNA remained stable during the perinatal period and increased dramatically after weaning. Other transcripts probably corresponding to oatp-related mRNAs also display a late expression pattern in the perinatal period. In contrast, Na+/taurocholate transporting polypeptide (Ntcp) mRNA was first detected on day 20 of gestation. By in situ hybridization, oatp mRNA was localized into hepatocytes and distributed without lobular heterogeneity. In the kidney, a single 2.4 kb oatp transcript was detected from birth to adult age. This transcript was exclusively distributed in the epithelial cells of the proximal tubules localized in the kidney cortex and the outer medulla. CONCLUSIONS: These results indicate that oatp undergoes a time-related expression in rat liver and kidney during development and that its gene transcription precedes Ntcp gene transcription in the liver. The delayed expression of oatp at the perinatal period may explain in part the immaturity of bile formation and the physiological neonatal cholestasis.

Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by Stra8, a novel retinoic acid-responsive gene.

The full-length cDNA corresponding to Stra8, a novel gene inducible by retinoic acid (RA) in P19 embryonal carcinoma cells, has been isolated and shown to encode a 45-kD protein. Both Stra8 mRNA and protein were induced in cells treated by all-trans and 9-cis retinoic acids. Two-dimensional gel analysis and dephosphorylation experiments revealed that the two stereoisomers of RA differentially regulate the phosphorylation status of the Stra8 protein, which was shown to exist in differently phosphorylated forms. Subcellular fractionation and immunocytochemistry studies showed that the Stra8 protein is cytoplasmic. During mouse embryogenesis, Stra8 expression was restricted to the male developing gonads, and in adult mice, the expression of Stra8 was restricted to the premeiotic germ cells. Thus, Stra8 protein may play a role in the premeiotic phase of spermatogenesis.

Retinal dysplasia and degeneration in RARbeta2/RARgamma2 compound mutant mice.

The eye is the organ whose development is the most frequently altered in response to maternal vitamin A deficiency [VAD; Warkany, J. and Schraffenberger, S. (1946). Archs Ophthalmol. 35, 150-169]. With the exception of prenatal retinal dysplasia, all the ocular abnormalities of the fetal VAD syndrome are recapitulated in mouse mutants lacking either RARalpha and RARbeta2, RARalpha and RARgamma, RARgamma and RARbeta2, or RXRalpha [Lohnes, D., Mark, M., Mendelsohn, C., Dolle, P., Dierich, A., Gorry, P., Gansmuller, A. and Chambon, P. (1994) Development 120, 2723-2748; Mendelsohn, C., Lohnes, D., Décimo, D., Lufkin, T., LeMeur, M., Chambon, P. and Mark, M. (1994) Development 120, 2749-2771; Kastner, P., Grondona, J. Mark, M., Gansmuller, A., LeMeur, M., Décimo, D., Vonesch, J.L., Dollé, P. and Chambon, P. (1994) Cell 78, 987-1003], thus demonstrating that retinoic acid (RA) is the active vitamin A metabolite during prenatal eye morphogenesis. Whether retinoids are also involved in postnatal eye development could not be investigated, as VAD newborns are not viable and the above RAR double null mutants and RXRalpha null mutants died in utero or at birth. We report here the generation of viable RARbeta2/RARgamma2 double null mutant mice, which exhibit several eye defects. The neural retina of newborn RARbeta2gamma2 mutants is thinner than normal due to a reduced rate of cell proliferation, and from day 4 shows multiple foci of disorganization of its layers. These RARbeta2gamma2 mutants represent the first genetically characterized model of retinal dysplasia and their phenotype demonstrates that RARs, and therefore RA, are required for retinal histogenesis. The RARbeta2gamma2 retinal pigment epithelium (RPE) cells display histological and/or ultrastructural alterations and/or fail to express cellular retinol binding protein I (CRBPI). Taken altogether, the early onset of the RPE histological defects and their striking colocalisation with areas of the neural retina displaying a faulty laminar organization, a reduced neuroblastic proliferation, and a lack of photoreceptor differentiation and/or increased apoptosis, make the RPE a likely target tissue of the RARbeta2gamma2 double null mutation. A degeneration of the adult neural retina, which may similarly be secondary to a defective RPE, is also observed in these mutants, thus demonstrating an essential role of RA in the survival of retinal cells. Moreover, all RARbeta2gamma2 mutants display defects in structures derived from the periocular mesenchyme including local agenesis of the choroid and of the sclera, small eyelids, and a persistence of the primary mesenchymal vitreous body. A majority of the RARbeta2 single null mutants also exhibit this latter defect, thus demonstrating that the RARbeta2 isoform plays a unique role in the formation of the definitive vitreous body.

Immunohistochemical analysis of retinoic acid receptor-alpha in human breast tumors: retinoic acid receptor-alpha expression correlates with proliferative activity.

Retinoids are known to prevent mammary carcinogenesis in rodents and inhibit the growth of human breast cancer cells in vitro. Previously we demonstrated that retinoid inhibition of proliferation of human breast cancer cell lines is largely mediated by retinoic acid receptor (RAR)-alpha. In this study we describe for the first time the histological distribution of RAR-alpha in 33 breast lesion specimens as determined by immunostaining with RAR-alpha antibody. Nuclear staining was observed in tumor tissue and normal portions of the breast samples. Connective tissue exhibited relative uniform staining, whereas a wide range of RAR-alpha expression was found in the epithelial tumor cells. RAR-alpha protein was expressed at significantly higher levels in tumors with greater proliferative activity as determined by immunostaining with Ki-67 antibody. This suggests that RAR-alpha expression may be altered with tumor progression. Although a positive correlation between RAR-alpha mRNA levels and estrogen receptor status of breast tumors has previously been documented, we did not find such a relationship at the protein level. As RAR-alpha plays a major role in retinoid-mediated growth inhibition of human breast cancer cell in vitro, our findings suggest that patients with highly proliferating tumors could be responsive to retinoid independently of their responsiveness to (anti)-estrogens.

Abnormal spermatogenesis in RXR beta mutant mice.

We have generated mouse lines in which the RXR beta gene was disrupted by homologous recombination. Approximately 50% of the RXR beta homozygous mutants died before or at birth, but those that survived appeared normal except that the males were sterile, owing to oligo-astheno-teratozoospermia. Failure of spermatid release occurred within the germinal epithelium, and the epididymis contained very few spermatozoa that, in addition, exhibited abnormal acrosomes and tails. There was a progressive accumulation of lipids within the mutant Sertoli cells, which were histochemically characterized as unsaturated triglycerides. In old mutant males, progressive degeneration of the germinal epithelium occurred, ending with the formation of acellular lipid-filled tubules. The selective expression of RXR beta in Sertoli cells, together with the timing of appearance of the histological abnormalities, suggests that the primary defect resulting from the mutation resides in these cells.

AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis.

Using a differential subtractive hybridization cloning procedure we have recently identified the AP-2.2 gene as a novel early retinoic acid-induced gene in murine P19 embryonal carcinoma cells. We have also shown that the AP-2.2 protein, which is highly related to the AP-2 transcription factor, can activate transcription when bound to an AP-2 consensus binding site [Oulad-Abdelghani et al. (1995) Mol. Cell. Biol., submitted]. We report here the in situ hybridization pattern of expression of AP-2.2 transcripts during mouse embryogenesis. At 7.5 days post-coitum, AP-2.2 transcripts were detected in the boundary region between neural plate and surface ectoderm, as well as in extra-embryonic tissues. By 8.0-8.5 gestational days, AP-2.2 transcripts appeared to be expressed in premigratory and migrating neural crest cells. Over the following days, the AP-2.2 gene displayed region-restricted expression in the facial mesenchyme, especially around the embryonic mouth cavity and the nasal cavities, as well as in the surface ectoderm, nasal and oral epithelia. AP-2.2 RNA was also specifically expressed in the presumptive cortical region of the forebrain vesicles. AP-2.2 transcripts were restricted to the distal mitotic area (the 'progress zone') of the limb buds and of the genital bud. AP-2.2 expression also appeared to be specific for primordial germ cells in the genital ridges. Thus, the AP-2.2 gene is expressed in several embryonic areas whose development can be affected by retinoids, such as the forebrain, face and limb buds.

Developmental roles of the retinoic acid receptors.

Retinoic acid, one of the principle active metabolites of vitamin A (retinol), is believed to be essential for numerous developmental and physiological processes. Vitamin A deprivation (VAD) during development leads to numerous congenital defects. Previous studies of retinoic acid receptor (RAR) deficient mice failed to reveal any of these VAD-induced defects. This finding suggested that either the RARs are functionally redundant or that they are not critically required during development. In order to address these possibilities, we derived a number of RAR compound mutants. Unlike RAR single mutants, these compound null mutants died either in utero or shortly following birth. Histological analysis revealed essentially all of the defects characteristic of fetal VAD. A number of additional malformations, not described in previous VAD studies, were also observed. These included defects of the ocular and salivary glands and their ducts, the skeletal elements of the fore- and hindlimbs, and the cervical region of the axial skeleton. In addition, with the exception of derivatives forming within the first pharyngeal arch, most of the elements derived from mesectoderm emanating from cranial and hindbrain levels were affected. A number of these mutants also exhibited supernumerary cranial skeletal elements characteristics of the reptilian skull. A summary of the defects found in these RAR double mutants is presented.

Retinoic acid regulates the development of oligodendrocyte precursor cells in vitro.

Cultures of oligodendrocyte precursor cells can be grown from brain hemispheres of newborn rats. These cells, also called O-2A progenitor cells, can differentiate in vitro into oligodendrocytes or type 2 astrocytes. Basic FGF and PDGF are known to stimulate their proliferation and delay their differentiation. Lack or excess of retinoic acid (RA) has been known for a long time to alter brain development suggesting that this compound is involved in normal brain development. Here we report that RA partially inhibits both the proliferation and the differentiation of oligodendrocyte precursor cells. It also down-regulates the mitogenic effect of bFGF on these cells while keeping them in an immature stage. RA is more effective than bFGF in inhibiting myelin basic protein mRNA expression in these cells, and like bFGF, it preserves their bipotential character. RA nuclear receptors RAR-alpha and their transcripts are expressed in oligodendrocyte precursor cells as seen by Western blot, Northern blot and in situ hybridization. The expression of RAR-alpha transcripts is stimulated transiently by RA alone or associated to bFGF. The expression of RAR-beta transcripts is not constitutive and is induced by RA alone or associated to bFGF and to a lesser extent by bFGF alone. These results suggest that retinoids participate in the control of the development of glial cells of the oligodendrocyte lineage.

Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities at various stages of organogenesis in RAR double mutants.

Compound null mutations of retinoic acid receptor (RAR) genes lead to lethality in utero or shortly after birth and to numerous developmental abnormalities. In the accompanying paper (Lohnes, D., Mark., M., Mendelsohn, C., Dollé, P., Dierich, A., Gorry, Ph., Gansmuller, A. and Chambon, P. (1994). Development 120, 2723-2748), we describe malformations of the head, vertebrae and limbs which, with the notable exception of the eye defects, were not observed in the offspring of vitamin A-deficient (VAD) dams. We report here abnormalities in the neck, trunk and abdominal regions of RAR double mutant mice, which include: (i) the entire respiratory tract, (ii) the heart, its outlow tract and the great vessels located near the heart, (iii) the thymus, thyroid and parathyroid glands, (iv) the diaphragm, (v) the genito-urinary system, and (vi) the lower digestive tract. A majority of these abnormalities recapitulate those observed in the fetal VAD syndrome described by Joseph Warkany's group more than fourty years ago [Wilson, J. G., Roth, C. B. and Warkany, J. (1953) Am. J. Anat., 92, 189-217; and refs therein]. Our results clearly demonstrate that RARs are essential for vertebrate ontogenesis and therefore that retinoic acid is the active retinoid, which is required at several stages of the development of numerous tissues and organs. We discuss several possibilities that may account for the apparent functional redundancy observed amongst retinoic acid receptors during embryogenesis.

Expression of nuclear retinoic acid receptors during mouse odontogenesis.

The developmental expression of retinoic acid (RA) nuclear receptors RAR(alpha, beta, gamma) and RXR(alpha, beta, gamma) was analysed during mouse odontogenesis by in situ hybridization on frozen sections and compared with the expression patterns of the cellular retinoic acid binding proteins CRABPI and II. The transcripts distribution of each RAR and RXR was basically similar in developing molars and incisors. RAR alpha and RXR alpha were preferentially expressed in dental epithelia, whereas RAR gamma and RXR gamma were transcribed in the dental mesenchyme. RAR beta, RAR gamma and RXR beta displayed both epithelial and mesenchymal expression. RAR beta expression was initiated during bell stage. RXR gamma transcripts were observed only at day 19.5 post coitum in the mitogenic mesenchyme facing the epithelial loops. Odontoblasts expressed RAR beta and RAR gamma, RXR alpha and RXR beta. Preameloblasts expressed RXR alpha and RXR beta and ameloblasts RXR gamma, RXR alpha and RXR beta. RAR alpha transcription in the incisor preameloblasts and ameloblasts was not observed in the first molar. The coexpression between RARs and RXRs might be important to form RAR/RXR heterodimers which are necessary to activate the transcriptions of target genes. CRABPI and CRABPII demonstrated graded variation of expression during odontogenesis in the mesenchyme and in the inner dental epithelium respectively. The pattern of CRABPI transcripts overlapped at least partially with expressions of all the studied nuclear receptors whereas CRABPII epithelial expression was superimposed with the transcription of RAR alpha, RXR alpha and RXR beta. These cytoplasmic proteins might participate in the storage and/or metabolism of RA and then distribute RA to colocalized nuclear receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The cellular retinoic acid binding protein I is dispensable.

The cellular retinoic acid binding proteins I and II (CRABPI and CRABPII) bind retinoic acid with high affinity, exhibit distinct patterns of expression during embryonic development, and are thought to play important roles in the RA signaling pathway. We have generated a targeted mutation of the CRABPI gene using the "hit-and-run" strategy and shown that it prevents the production of a functional CRABPI protein. Homozygous mutant mice were normal, indicating that CRABPI does not play a crucial role in the RA signaling pathway.

Genetic analysis of RXR alpha developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis.

A null mutation was generated in the mouse RXR alpha gene by targeted disruption. Growth deficiency occurred in heterozygote mice. Null mutants died in utero and displayed myocardial and ocular malformations. These malformations belong to the fetal vitamin A deficiency syndrome, supporting the idea that RXR alpha is involved in retinoid signaling in vivo. A phenotypic synergy was observed when the RXR alpha mutation was introduced into RAR alpha or RAR gamma mutant backgrounds: RXR alpha null mutants and RXR alpha +/-/RAR gamma-/- double mutants displayed similar ocular defects, which became more severe in RXR alpha-/-/RAR gamma+/- and RXR alpha-/-/RAR gamma-/- mutants. Furthermore, RXR alpha/RAR double mutants exhibited several malformations not seen in single mutants. This functional convergence strongly suggests that RXR alpha/RAR heterodimers mediate retinoid signaling in vivo.