Differential contributions of AF-1 and AF-2 activities to the developmental functions of RXR{alpha}

Bénédicte Mascrez, Manuel Mark, Wojciech Krezel, Valérie Dupé, Marianne LeMeur, Norbert B. Ghyselinck and Pierre Chambon*

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM-ULP-Collège de France, BP163, 67404 Illkirch Cedex, C.U. de Strasbourg, France

*Author for correspondence (e-mail: chambon{at}igbmc.u-strasbg.fr)

Accepted March 8, 2001


    SUMMARY
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have engineered a mouse mutation that specifically deletes most of the RXR{alpha} N-terminal A/B region, which includes the activation function AF-1 and several phosphorylation sites. The homozygous mutants (RXR{alpha}af1o), as well as compound mutants that further lack RXRß and RXR{gamma}, are viable and display a subset of the abnormalities previously described in RXR{alpha}-null mutants. In contrast, RXR{alpha}af1o/RAR-/-({alpha}, ß or {gamma}) compound mutants die in utero and exhibit a large array of malformations that nearly recapitulate the full spectrum of the defects that characterize the fetal vitamin A-deficiency (VAD) syndrome. Altogether, these observations indicate that the RXR{alpha} AF-1 region A/B is functionally important, although less so than the ligand-dependent activation function AF-2, for efficiently transducing the retinoid signal through RAR/RXR{alpha} heterodimers during embryonic development. Moreover, it has a unique role in retinoic acid-dependent involution of the interdigital mesenchyme. During early placentogenesis, both the AF-1 and AF-2 activities of RXR{alpha}, ß and {gamma} appear to be dispensable, suggesting that RXRs act as silent heterodimeric partners in this process. However, AF-2 of RXR{alpha}, but not AF-1, is required for differentiation of labyrinthine trophoblast cells, a late step in the formation of the placental barrier.

Key words: Nuclear receptor, Retinoic acid, Gene knockout, Transcriptional activity, Activation function, Placenta, Limb, Mouse


    INTRODUCTION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoids (vitamin A derivatives) are crucial for many aspects of vertebrate physiology and homeostasis (reviewed in Sporn et al., 1994). They also play essential roles in morphogenesis and organogenesis, as inferred from the large spectrum of developmental abnormalities displayed by vitamin A-deficient (VAD) fetuses (reviewed in Kastner et al., 1995; Dickman et al., 1997; White et al., 1998). Two families of nuclear receptors for retinoids, the RARs ({alpha}, ß and {gamma} isotypes and their isoforms; activated by all forms of retinoic acid, RA) and the RXRs ({alpha}, ß and {gamma} isotypes and their isoforms; activated by 9-cis RA only) transduce the retinoid signal. RARs and RXRs act in transfected cells in vitro as ligand-dependent transcriptional transregulators through binding as RAR/RXR heterodimers to cis-acting RA response elements present in cognate reporter genes (reviewed in Leid et al., 1992; Chambon, 1996; Mangelsdorf et al., 1995; Mangelsdorf and Evans, 1995). In addition, RXRs can act as heterodimerization partners with nuclear receptors other than RARs, such as thyroid hormone receptors, vitamin D3 receptor, peroxisome proliferator activated receptors and several orphan receptors (reviews: Mangelsdorf and Evans, 1995; Chambon, 1996; Perlmann and Evans, 1997). Importantly, RAR-specific and RXR-specific ligands synergize to activate target genes or to elicit biological responses in cell systems (Taneja et al., 1996; Taneja et al., 1997; Roy et al., 1995; Chen et al., 1996; Horn et al., 1996; Nagy et al., 1995; Vivat et al., 1997), as well as in whole embryos (Minucci et al., 1996; Minucci et al., 1997; Lu et al., 1997), indicating that both partners of the RAR/RXR heterodimer can be transcriptionally active. However, the liganded RXR is not active unless its RAR partner is itself liganded (Roy et al., 1995; Apfel et al., 1995; Chambon, 1996; Taneja et al., 1996; Taneja et al., 1997; Chen et al., 1996; Vivat et al., 1997; Botling et al., 1997).

To investigate the involvement of RARs and RXRs in the transduction of retinoid signals under physiological conditions in vivo, all RAR and RXR genes, and most of their isoforms have been knocked out in the mouse (for reviews see Kastner et al., 1995; Mark et al., 1999). Altogether, these studies have led to several important conclusions concerning the role of these receptors during morphogenesis and organogenesis: (1) RARs and RXRs mediate the developmental functions of RA; (2) functional redundancies exist among the various RARs and among the various RXRs; (3) RAR/RXR{alpha} heterodimers are most likely the main functional units that transduce the retinoid signal during development; (4) transcriptional activation of both partners in RAR/RXR{alpha} heterodimers are often required to activate target genes and to mediate under physiological conditions the retinoid effects in morphogenesis and organogenesis.

The N-terminal A/B domain of RXRs contains an autonomous ligand-independent transcriptional activation function called AF-1, whereas the C-terminal, ligand-binding domain E, contains a ligand-dependent transcriptional activation function, AF-2 (reviewed in Leid et al., 1992; Chambon, 1996; Moras and Gronemeyer., 1998). For each RXR, at least two isoforms exist, which differ in their N-terminal region (Leid et al., 1992; Fleischhauer et al., 1992; Liu and Linney, 1993; Nagata et al., 1994). As to RXR{alpha}, the major isoform RXR{alpha}1 is widely expressed in embryos and adults, whereas RXR{alpha}2 and {alpha}3 are restricted to the adult testis (Brocard et al., 1996). Furthermore, RXR{alpha} can be phosphorylated at several serine and threonine residues in its A/B domain (Adam-Stitah et al., 1999). The AF-2 activity crucially depends upon a conserved amphipathic {alpha} helix (the AF-2 AD core; Bourguet et al., 1995; Chambon, 1996; Wurtz et al., 1996; and references therein), whose deletion in the mouse has revealed its requirement for a number of RA-dependent developmental events (Mascrez et al., 1998). However, little is known about the mechanisms through which AF-1 activates transcription or about the relevance of the A/B domain in the global activity of the receptor under physiological conditions in vivo. Depending on the promoter context, the AF-1 of a given RXR modulates the AF-2 activity in cultured cells (Nagpal et al., 1992; Nagpal et al., 1993; Dowhan and Muscat, 1996; Chambon, 1996; Taneja et al., 1997). Thus, the transcriptional activity of a given RXR isoform may ultimately be determined, not only by its AF-2 activity, but also by its isoform-specific A/B domain.

In order to determine the importance of RXR{alpha} AF-1 domain A/B during development in vivo, we have engineered a mouse mutant line that expresses a truncated RXR{alpha} protein (RXR{alpha}{Delta}A/B). The phenotypic analysis of mice carrying this mutation indicate that RXR{alpha} AF-1 domain A/B is important for transducing RA signals in vivo. In addition, we have assessed the relative contribution of RXR{alpha} AF-1 and AF-2 activities in embryonic development and placentation.


    MATERIALS AND METHODS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Homologous recombination
The RXR{alpha} targeting vector (pB48) was designed to delete exon 2 (with the exception of its splicing acceptor site), intron 2 and 102 bp of exon 3. On the one hand, a 4.6 kb SpeI-BamHI DNA fragment (from a P19 teratocarcinoma cell genomic library; Clifford et al., 1996), containing a BglII restriction site introduced at the beginning of exon 2 through site-directed mutagenesis, was subcloned into pEMBL19+ (Dente et al., 1983), leading to plasmid pB41. On the other hand, a 3.0 kb BamHI-XbaI DNA fragment (from a 129/sv mouse genomic library; Kastner et al., 1994), containing a BglII site introduced in exon 3 by site-directed mutagenesis, was cloned into pB41, reconstituting then the 7.6 kb SpeI-XbaI region of the RXR{alpha} locus (plasmid pB42). The 4.0 kb-long DNA located between the two BglII sites of pB42 was deleted and replaced by an oligonucleotide preserving the RXR{alpha} open reading frame, and containing a NheI restriction site (plasmid pB45). The 3.6 kb SpeI-XbaI fragment of pB45 was then cloned into a pBluescript plasmid (Stratagene) containing a 5.7 kb XbaI-HindIII genomic fragment (from a 129/sv mouse genomic library), leading to pB46. Finally, a loxP site-flanked TK-NEO fusion cassette (Metzger et al., 1995) was introduced at the XbaI site located in intron 3 (leading to construct pB48).

Embryonic stem (ES) cells were transfected with NotI-linearized pB48, as previously described (Lufkin et al., 1991). Out of 87 G418-resistant clones, two targeted clones (HS23 and BL19) were obtained. Homologous recombination was confirmed by Southern blot analysis of ScaI-, NheI- and SpeI-digested genomic DNA hybridized with probes A, B and Neo (see Fig. 1 and data not shown). In order to delete the ‘floxed’ TK-NEO cassette, HS23 ES cells were transiently transfected with pSG5-Cre (Gu et al., 1993). Excision was confirmed for clone HS23.26Cre by Southern-blot analysis (Fig. 1 and data not shown). To generate the first RXR{alpha}af1o mouse line, HS23.26Cre cells were injected into C57BL/6 blastocysts. To generate a second RXR{alpha}af1o mouse line, BL19 cells were used. In this line, the selectable marker was successfully excised by crossing heterozygotes with transgenic mice expressing Cre early during embryogenesis (CMV-Cre; Dupé et al., 1997). The phenotypes illustrated here are from line HS23.26Cre. However, identical observations were made using line BL19.



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Fig. 1. Targeted deletion of the AF-1-containing A/B region of RXR{alpha}. (a) Representation of the wild-type RXR{alpha}1 isoform (WT RXR{alpha}1) and the mutant RXR{alpha} (RXR{alpha}{Delta}A/B). Exons E1 to E3 are shown. The functional domains are depicted as followed: AF-1, activation function 1; AF-2 AD, activation function 2 activating domain; DBD, DNA-binding domain (region C); LBD, ligand-binding domain (region E). The RXR{alpha}af1 mutation leads to the production of an RXR{alpha} protein truncated from amino acid 11 to 132. BglII(Bg) and NheI(N) restriction sites were introduced to allow detection of the mutant allele. E{Delta}[2-3] contains the first codon of exon 2 fused to the 3' part of exon 3. The deletion is represented by a black box. (b) The WT RXR{alpha} locus, the targeting construct and the mutated loci obtained after replacement (I), and subsequent Cre-mediated excision of the ‘floxed’ TK-NEO cassette (II). The star represents the mutation E{Delta}[2-3] described in (a). LoxP sites are represented by black arrowheads. Probes A, B and C are 0.7 kb ScaI-SpeI, 0.6 kb EcoRI-BglII and 0.3 kb BamHI-SpeI fragments, respectively. The size of the restriction fragment that allow identification of WT and targeted alleles (I) and (II) by Southern blot analysis using probes A, B, C and neo are indicated below (in kilobases). N, NheI; Bg, BglII; Sc, ScaI; S, SpeI; E, EcoRI; H, HindIII; B, BamHI; X, XbaI; Xh, XhoI. (c) Southern blotting of ES cell DNA digested with BglII or SpeI and hybridized with probe A, B and neo, as indicated. WT (+/+) and RXR{alpha}af1 mutant alleles before (+/(I)) or after (af1/+) excision of the ‘floxed’ TK-NEO cassette are indicated. (d) Detection of RXR{alpha}{Delta}A/B protein. Nuclear extracts prepared from 12.5-day-old embryos WT (+/+), af1 heterozygote (af1/+), af1 homozygote (af1o) and RXR{alpha}-null mutants (-/-) (Kastner et al., 1994) were analyzed by western blotting with the anti-RXR{alpha} monoclonal antibody 4RX3A2, directed against a C-terminal epitope.

 
Western blot analysis
Nuclear extracts were prepared from whole E12.5 embryos (Andrews and Faller, 1991). Proteins (15 µg) were separated on 10% gel by SDS-PAGE and transferred onto nitrocellulose membranes. RXR{alpha} and RXR{alpha}{Delta}A/B proteins were detected using the 4RX3A2 anti-RXR{alpha} monoclonal antibody (1/500 dilution; Rochette-Egly et al., 1994) and revealed by chemiluminescence according to the manufacturer’s instructions (Amersham).

Histology, immunohistochemistry and skeletal analyses
Embryos and fetuses were fixed in Bouin’s fluid, embedded in paraffin, serially sectioned and stained with Groat’s Hematoxylin and Mallory’s Trichrome (Mark et al., 1993). Skeletons of E18.5 fetuses were prepared as described (Lufkin et al., 1992). Nile Blue Sulfate staining and in situ hybridization for detection of stromelysin-3 transcripts were described previously (Dupé et al., 1999).

Behavior studies
X{alpha}af1o mutant mice were in a 129/Sv/C57BL/6 (25/75%) genetic background at the time of testing. All animals were housed in cohorts of three to five mice per cage in 12 hour light/dark cycle, with freely available food and water. Behavioral testing was conducted between 14:00 and 18:00. The open field test was performed as described (Krezel et al., 1998). For the elevated plus maze, mice were placed in the central part of the maze and the percentage of time spent in the open arm, and the number of times animal stretched its head to look down were recorded for 5 minutes. To measure fine locomotor skills, the rotarod, inclined plane and cord tests were employed (Krezel et al., 1998; Wolffgramm et al., 1990; Fehlings, 1995; Perry et al., 1995). The nociception was analyzed by evaluating response to acute thermal stimuli in the tail-flick and hot-plate tests, in which the latency to remove the tail from 50°C water and the time spent on the 54° plate before licking the hindpaws were measured, respectively. Finally in taste preference, mice were presented water and 15% sucrose in different parts of the test cage. The number and the time of visits were scored for 5 minutes over 4 consecutive days, indicating preference for sucrose. For each behavioral paradigm data were analyzed by analysis of variants, ANOVA and Tukey-Kramer post hoc tests.


    RESULTS
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Targeted deletion of the RXR{alpha} AF-1 domain A/B
A targeting vector was designed to alter the RXR{alpha} locus in such a way that it encodes a mutant protein lacking amino acids 11 to 132 of RXR{alpha}1. This region contains the autonomous AF-1 activation function, several phosphorylation sites (Adam-Stitah et al., 1999), as well as the initiator codon of RXR{alpha}2 and {alpha}3 isoforms (Brocard et al., 1996). Replacement in ES cells led to a targeted allele in which most of exon 2 (with the exception of the first 3 bp), intron 2 and 102 pb of exon 3 were deleted, and in which a ‘floxed’ TK-NEO cassette was inserted into intron 3. Cre-mediated excision of the selection marker led to the mutant allele (hereafter designated as RXR{alpha}af1), containing a single intronic loxP site (see Fig. 1b,c). Chimeric males derived from two independent ES cell clones (HS23.26Cre and BL19) transmitted the mutation through their germline, and the identity of the mutation was confirmed by sequencing RT-PCR products amplified from RNA of homozygous mutants (data not shown). In nuclear extracts prepared from whole E12.5 embryos, the truncated RXR{alpha}{Delta}A/B protein was readily detected as a single species with an increased electrophoretic mobility, when compared with that of RXR{alpha} (Fig. 1d). Its overall level of expression in homozygous mutants was comparable with that of RXR{alpha} in wild type (WT) embryos (Fig. 1d, compare lanes 1 and 3). Moreover, the RXR{alpha}{Delta}A/B and RXR{alpha} proteins were present in similar amounts in heterozygous embryos (Fig. 1d, lane 2). Therefore, the present mutation does not drastically alter the steady state level of the RXR{alpha} truncated protein. Consequently, its effects are likely to reflect only the lack of RXR{alpha} A/B domain.

We describe below the effects of deleting the RXR{alpha} A/B domain in mice as well as in mutants additionally null for RXRß, RXR{gamma}, RAR{alpha}, RARß or RAR{gamma}. To simplify nomenclature, homozygote mutant mice lacking the RXR{alpha} A/B domain are designated as X{alpha}af1o, and heterozygotes as X{alpha}af1/+; RXR{alpha}, RXRß, RXR{gamma}, RAR{alpha}, RARß and RAR{gamma} homozygote null mutants are designated as X{alpha}, Xß, X{gamma}, A{alpha}, Aß and A{gamma}, respectively; for example, RXR{alpha}af1o/RAR{alpha}-/- mutants are referred to as X{alpha}af1o/A{alpha} mutants.

X{alpha}af1o mutants are growth deficient and display congenital defects
Deletion of RXR{alpha} AF-1 domain A/B does not lead to lethal developmental defects, as viable and fertile X{alpha}af1o mutants were obtained at the expected Mendelian ratio (out of 1103 littermates born from heterozygote crosses, 27% were wild type (n=299), 50% were heterozygote (n=553) and 23% were homozygote (n=251)). At embryonic day 18.5 (E18.5), the weight of X{alpha}af1o mutants (1.19 g on average; n=20) was similar to that of wild type (1.22 g on average; n=18). In contrast, between 1 and 2 weeks of birth, X{alpha}af1o mutants were on average 20% lighter than their wild-type littermates (Fig. 2). Several of the cachectic X{alpha}af1o mutants (weight ratio < 0.5) died before weaning, whereas the others lived as long as their wild-type littermates (at least 1 year), but exhibited a weight deficit of about 10% during at least 6 months after birth (Fig. 3). This growth retardation was harmonious as (1) the ratios of adult tissue weights (liver, kidney, heart, lungs, visceral fat) to the total body weight and (2) ratios of the length of skeletal elements (femur and skull) to the total body-length did not reveal significant differences between X{alpha}af1o mutants and wild-type animals. We previously showed that RXR{alpha}+/- mice are growth deficient (Kastner et al., 1994). The present data further indicate that the RXR{alpha} A/B domain is important for postnatal growth.



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Fig. 2. Weight of wild type (WT) and X{alpha}af1o mutants at 1-2 weeks of age. To standardize between litters, the weight of each pup obtained from X{alpha}af1/+ intercrosses was expressed as the ratio of its weight relative to the average weight of the WT pups from the same litter. Ratios were then grouped within classes differing by 0.1 increment (y-axis). The number of X{alpha}af1o and WT animals in each class is indicated on the top of the bars.

 


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Fig. 3. Weight of wild-type (WT) and X{alpha}af1o mutant males (a) and females (b). Mean weights of offsprings (n=5 to 18) obtained from X{alpha}af1/+ intercrosses are presented with s.e.m. After testing for normality and variance homogeneity, values were subjected to Student’s t tests. Asterisks indicate the significance (P<0.05) for the differences observed between WT and X{alpha}af1o mutants. NS, not significant.

 
Soft tissue syndactyly of the hindlimbs was seen unilaterally (n=33) or bilaterally (n=83) in 70% of the X{alpha}af1o adults. It affected mostly the base of the interdigital regions between digits 2-3 and 3-4 (data not shown). Hindlimb interdigital webbing was observed at a low frequency in RXR{alpha}+/- mice, but was absent in X{alpha}af2o mutants (Kastner et al., 1997a; Mascrez et al., 1998). These data indicate that RXR{alpha} AF-1 domain A/B is involved in the involution of the interdigital mesenchyme (see below).

A persistent hyperplastic primary vitreous body (PHPV) was the only ocular defect observed in X{alpha}af1o mutants (two out of 18). In contrast, eye morphogenesis is severely altered in X{alpha} and X{alpha}af2o fetuses, which display (in addition to a completely penetrant PHPV) corneal, retinal, lens and eyelid abnormalities (Kastner et al., 1994; Kastner et al., 1997a; Mascrez et al., 1998; see Table 2). X{alpha} and X{alpha}af2o fetuses also exhibit cardiac abnormalities, namely a myocardial hypoplasia and an agenesis of the conotruncal septum (Kastner et al., 1994; Kastner et al., 1997a; Sucov et al., 1994). No heart defects were detected in E18.5 X{alpha}af1o mutants analyzed by histology (n=3). However at E9.5, electron microscopic analysis of X{alpha}af1o embryos (n=5) revealed a single case of precocious differentiation of the cardiomyocytes as in X{alpha} and X{alpha}af2o mutants (Fig. 4; Kastner et al., 1994; Kastner et al., 1997b; Mascrez et al., 1998). X{alpha}af1o mutants exhibited skeletal abnormalities similar to those found in RARs and X{alpha}af2o mutants (Lohnes et al., 1993; Ghyselinck et al., 1997; Mascrez et al., 1998): (1) homeotic transformations and malformations of cervical vertebrae; (2) bilateral agenesis of the metoptic pilar (the posterior border of the optic nerve foramen); and (3) a misshapen cricoid cartilage (Table 1). Altogether, these results indicate that the RXR{alpha} AF-1 domain A/B is involved in the involution of the primary vitreous body, the differentiation of cardiomyocytes and the morphogenesis of some skeletal elements; but at first sight this domain also appears dispensable for the majority of the developmental events normally mediated by RXR{alpha}.


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Table 2.
 


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Fig. 4. Precocious differentiation of X{alpha}af1o ventricular cardiomyocytes. (a) The subepicardial myocytes of E9.5 wild-type (WT) embryos contain bundles of myofilaments, occasionally showing isolated Z lines and connected only by desmosomes. (b) In 70% of the subepicardial myocytes of this E9.5 X{alpha}af1o mutant the myofilaments are already organized into sarcomeres (S) that are connected between cells by a series of adherens junctions forming the intercalated discs (ID). D, desmosome; F, bundles of myofilaments; M, mitochondria; S, sarcomere; Z, Z line. Scale bar: 0.5 µm.

 

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Table 1.
 
Retinoid receptors regulate brain functions (Krezel et al., 1998; Chiang et al., 1998). As RXR{alpha} is almost ubiquitously expressed in the central nervous system (Krezel et al., 1999), behavioral tests were performed on X{alpha}af1o mice. Sensory faculties, motor skills, locomotion activity, stereotypic behaviours, as well as stress responses, were not affected, as X{alpha}af1o mice performed as well as wild-type littermates in all these tests (see Materials and Methods for details). Thus, no obvious non-redundant function can be readily ascribed, in the central nervous system, to the RXR{alpha} AF-1 domain A/B.

Deletion of the RXR{alpha} AF-1 domain A/B results in additional congenital defects in the absence of RXRß and RXR{gamma}
As X{alpha}af1o mutants displayed neither the prenatal lethality nor all the congenital defects previously observed in X{alpha} and X{alpha}af2o mutants, the RXR{alpha} AF-1 domain A/B might be largely dispensable during development. Alternatively, RXRß and/or RXR{gamma} may functionnally compensate for the absence of the RXR{alpha} AF-1 domain A/B. To investigate this possibility, RXR{alpha}af1o mutation was introduced into RXRß and RXRß/RXR{gamma}-null genetic backgrounds (Xß/X{gamma} mutants develop normally; Krezel et al., 1996). Steady-state levels of RXR{alpha}{Delta}A/B in X{alpha}af1o/Xß and X{alpha}af1o/Xß/X{gamma} E12.5 embryos were comparable with those of RXR{alpha} in Xß and Xß/X{gamma} embryos, respectively (data not shown).

Of the 362 mice genotyped after birth from X{alpha}af1/+/Xß+/- intercrosses, X{alpha}af1o/Xß mutants were obtained at a Mendelian ratio (23 expected, 19 obtained). However, of the 261 mice born from X{alpha}af1/+/Xß+/-/X{gamma} intercrosses, only 10 X{alpha}af1o/Xß/X{gamma} mutants were obtained (16 expected). But at E14.5, X{alpha}af1o/Xß/X{gamma} fetuses were collected at a Mendelian ratio. This indicates that the absence of all RXR AF-1 domains A/B is not lethal at a developmental stage when organogenesis is almost completed, but results in impaired viability. X{alpha}af1o/Xß and X{alpha}af1o/Xß/X{gamma} mutant females were fertile, whereas males were sterile, owing to the loss of RXRß (Kastner et al., 1996). In adults, the weight of X{alpha}af1o/Xß mutants was about 10% less than that of X{alpha}af1o mutants (data not shown).

A similar bilateral and completely penetrant hindlimb interdigital webbing was observed in X{alpha}af1o/Xß and in X{alpha}af1o/Xß/X{gamma} mutants. It was more severe than in X{alpha}af1o mutants, as it often affected the full length of interdigital spaces (Fig. 5c and data not shown). E14.5 X{alpha}af1o/Xß hindlimbs showed a marked decrease in the number of dying cells within interdigital spaces, as assessed by staining with Nile Blue Sulfate (Fig. 5a). Moreover, the expression of stromelysin 3, an RA-regulated matrix metalloproteinase involved in tissue remodeling processes that accompany apoptosis in developing limbs (Lefebvre et al., 1995; Dupé et al., 1999; Ludwig et al., 2000), was markedly decreased in the interdigital spaces of E14.5 X{alpha}af1o/Xß hindlimbs (Fig. 5b). These results indicate that the soft tissue syndactyly seen in a majority of X{alpha}af1o adults, as well as in all X{alpha}af1o/Xß, X{alpha}af1o/Xß/X{gamma} adults, is caused by the persistence of the fetal interdigital mesenchyme.



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Fig. 5. Decreased interdigital cell death results in soft tissue syndactyly of the hindlimbs in X{alpha}af1o/Xß mutants. Comparison of (a) cell death assessed by Nile Blue Sulfate staining, (b) expression of stromelysin-3 assessed by in situ hybridization and (c) morphology, between E14.5 (a,b) and adult (c) wild type (WT) and X{alpha}af1o/Xß mutants. PNZ, posterior necrotic zone; I-V, digit one to five. The arrows point to the two interdigital regions, which are the most severely affected in adults.

 
All E18.5 X{alpha}af1o/Xß (n=4) and E14.5 X{alpha}af1o/Xß/X{gamma} (n=9) mutants analyzed histologically displayed a bilateral PHPV (compare Fig. 6a,b; Table 2). In addition, one X{alpha}af1o/Xß/X{gamma} mutant displayed a ventricular myocardial hypoplasia, and another one an agenesis of the conotruncal septum comparable with that of X{alpha} mutants (Table 3). These data confirm the requirement of A/B domain of RXR in the involution of the interdigital and primary vitreous body mesenchymes, and indicate that it is also involved in cardiac morphogenesis in some individuals.



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Fig. 6. Comparison of ocular malformations in X{alpha}af1o and X{alpha}af2o compound mutants. Frontal sections through the eye region of WT and mutant fetuses at E14.5 (a-d) and E18.5 (e-k). (h-k) Note that X{alpha}af1o/A(ß or {gamma}) and X{alpha}af2o/A(ß or {gamma}) mutants share the same spectrum of ocular defects, although these are systematically more severe in X{alpha}af2o compound mutants. For example, the size of the conjunctival sac (J) and cornea (C) is only slightly reduced in X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} mutants (compare e,j,h) but markedly decreased in X{alpha}af2o/Aß mutants (i), and absent in X{alpha}af2o/A{gamma} mutants (k). Likewise, the stroma of the iris (I), the anterior chamber (A) and the secondary vitreous body (SV) are present in X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} mutants, but not in their X{alpha}af2o counterparts. Note also that in some mutants at E18.5, the relative sizes of the ventral and dorsal retina is not possible to assess due the existence of retinal folds. A, anterior chamber; C, cornea; D, dorsal retina; E, eyelids; I, iris stroma; J, conjuntival sac; L, lens; M, mesenchyme replacing the eyelids and cornea; N, neural retina; R, persistant hyperplastic primary vitreous; RP, retinal pigment epithelium; SC, sclera; SV, secondary vitreous body. The large arrow points to the optic nerve exit point. The green arrowheads delimit colobomas of the optic disc. The asterisks indicate artefactual detachment of the neural retina from the retinal pigment epithelium occurring during tissue processing. Scale bar in k: 200 µm (a-d); 300 µm (e,h-k); 40 µm (f,g).

 

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Table 3.
 
The putative start codon of RXR{alpha}2 and {alpha}3, the two RXR{alpha} isoforms specifically expressed in the adult testis (Brocard et al., 1996), was removed in X{alpha}af1o mutants. Therefore, the RXR{alpha} A/B domain deletion should also result in a null mutation for RXR{alpha}2 and {alpha}3. This was confirmed by western blot analysis (data not shown). Histological analysis of the testes of X{alpha}af1o adults (n=4), which are fertile, did not reveal abnormalities, whereas X{alpha}af1o/Xß/X{gamma} adults (n=2), which are sterile because of the RXRß-null mutation, did not display any testis defect in addition to those exhibited by Xß-null mutants (Kastner et al., 1996). Thus, RXR{alpha}2 and RXR{alpha}3 isoforms are not necessary for male fertility.

In contrast to the RXR{alpha} AF-2 function, the RXR{alpha} AF-1 domain A/B is dispensable for placentation
The relative contributions of AF-1 and AF-2 activities to the functions of RXR{alpha} in placentation (Sapin et al., 1997; Wendling et al., 1999; Barak et al., 1999) were assessed by comparing wild-type, X{alpha}af1o, X{alpha}af2o, X{alpha}af1o/Xß/X{gamma} and X{alpha}af2o/Xß/X{gamma} placental morphologies. Placentas from E18.5 X{alpha}af1o (n=3) and E14.5 X{alpha}af1o/Xß/X{gamma} (n=6) mutants were macroscopically and histologically indistinguishable from wild-type placentas (data not shown). Histological defects observed at E14.5 in X{alpha}af2o (n=5) and X{alpha}af2o/Xß/X{gamma} (n=5) placentas consisted mainly in a thickening of labyrinthine trabeculae, and a lack of clear frontier between the spongiotrophoblastic and the labyrinthine zones. They were similar to those previously detected in RXR{alpha}-null placentas (Sapin et al., 1997; data not shown).

The outcome of these placental abnormalities was assessed at E18.5: X{alpha}af2o placentas were macroscopically distinguishable from wild-type and X{alpha}af2/+ placentas by their pale appearance, which reflects a deficiency in red blood cells within the labyrinthine zone (Fig. 7a,b; data not shown). Histological analysis of E18.5 X{alpha}af2o placentas (n=4) showed (1) an abnormal, wavy aspect of the interface between spongiotrophoblast and labyrinth (Fig. 7c,d); and (2) a thickening of the labyrinthine trabeculae separating fetal capillaries and maternal blood sinuses (Fig. 7e-h). Labyrinthine trabeculae represent the placental barrier across which nutrient and gas exchanges between fetal and maternal blood occur (reviewed by Cross, 2000). Limitation of the rate of fetal-maternal exchanges caused by a thickened placental barrier is probably responsible for in utero growth retardation of X{alpha}af2o mutants (Mascrez et al., 1998).



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Fig. 7. Comparison of E18.5 wild-type (WT), X{alpha}af1o and X{alpha}af2o placentas. (a,b) Halved placentas from the same litter fixed in glutaraldehyde. (c-d) Histological sections from paraffin-embedded placentas. (e-h) Semithin sections: labyrinthine trabeculae consist of three layers of trophoblast cells (designated I, II and III counting from the maternal towards the fetal blood spaces) and of the endothelium lining fetal capillaries; the trabecular thickening in X{alpha}af2o placenta affects mainly layers I and II. E, nuclei of endothelial cells; F, fetal (allantoic) capillary; L, labyrinthine zone; M, maternal blood sinuses; S, spongiotrophoblast; T, nuclei of trophoblast cells; TI, nuclei of trophoblast cells from layer one; I, II and III, first, second and third layer of labyrinthine trabeculae. The green brackets delimit the placental barrier. Modified Mallory’s trichrome (c,d), Toludine Blue (e,f) and periodic acid-schiff (g,h). (a) and (b) are displayed at the same magnification. Scale bar: 500 µm (c,d); 25 µm (e,f); 12 µm (g,h).

 
In absence of a RAR partner, the RXR{alpha} AF-1 domain A/B becomes essential for embryonic development
The RXR{alpha}af1o mutation was introduced into RAR({alpha}, ß or {gamma})-null genetic backgrounds to assess the functional contribution of the RXR{alpha} AF-1 domain A/B in RAR/RXR heterodimers. Steady-state levels of RXR{alpha}{Delta}A/B in E12.5 X{alpha}af1o/A({alpha}, ß or {gamma}) mutant embryos were comparable with those of RXR{alpha} in A{alpha}-, Aß- and A{gamma}-null embryos, respectively (data not shown). Therefore, the abnormal phenotypes seen in X{alpha}af1o/A({alpha}, ß or {gamma}) mutants most probably reflect the lack of the RXR{alpha} A/B domain.

A{alpha}, Aß and A{gamma} mutants are viable (Kastner et al., 1995; Ghyselinck et al., 1997). In contrast, no living X{alpha}af1o/A{alpha} mutants were recovered from X{alpha}af1/+/A{alpha}+/- intercrosses, even though they could be collected at Mendelian ratio at E18.5. Similarly, only few X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} adults were viable (two and one obtained versus ten and seven expected, respectively). The lethality of X{alpha}af1o/A({alpha}, ß or {gamma}) mutants clearly show that the RXR{alpha} AF-1 domain A/B is essential, at least in some RAR genetic settings.

X{alpha}af1o/A({alpha}, ß or {gamma}) compound mutants were analyzed by histology at E14.5 and E18.5. X{alpha}af1o/A{alpha} mutants displayed, with a low penetrance, the majority of the cardiovascular, respiratory and urogenital defects previously observed in X{alpha}/A{alpha} mutants (Tables 3, 4; Kastner et al., 1994; Kastner et al., 1997a). Similarly, X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} mutants reproduced a milder form of the ocular defects observed in X{alpha}/Aß and X{alpha}/A{gamma} fetuses (Table 2; Kastner et al., 1994; Kastner et al., 1997a), including closer eyelid folds (E in Fig. 6a,c,d), thickening of the ventral portion of the cornea (C), shortening of the ventral retina (V), ventral rotation of the lens (L), coloboma of the optic disc (green arrowheads in Fig. 6d) and absence of the sclera (SC, compare Fig. 6f with 6g). Thus, in a genetic background null for either RAR{alpha}, RARß or RAR{gamma}, the RXR{alpha} A/B domain becomes indispensable for a large subset of RA-dependent functions involved in morphogenesis.

It is interesting to note that the defects observed in X{alpha}af1o/A({alpha}, ß or {gamma}) mutants were less penetrant or less severe than those observed in X{alpha}af2o/A({alpha}, ß or {gamma}) mutants (Mascrez et al., 1998). For example, lung hypoplasia was present in about two thirds of X{alpha}af1o/A{alpha} mutants, whereas it is completely penetrant in X{alpha}af2o/A{alpha} mutants. One third of X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} mutants displayed a coloboma of the optic disc, an abnormality observed in all X{alpha}af2o/Aß and X{alpha}af2o/A{gamma} mutants. Ocular abnormalities found with the same penetrance in X{alpha}af1o/RAR-null and in X{alpha}af2o/RAR-null mutants, were less severe in X{alpha}af1o/RAR-null mutants. For example, shortening of the ventral retina, ventral rotation of the lens, thickening of the corneal stroma and closer eyelid folds, are less severe in X{alpha}af1o/Aß and X{alpha}af1o/A{gamma} mutants than in X{alpha}af2o/Aß and X{alpha}af2o/A{gamma} mutants (Table 2; Fig. 6c,d; for additional examples, compare Fig. 6h and 6j with 6i and 6k). Altogether, these observations indicate that, for a large fraction of the RAR-dependent events, the functions of the RXR{alpha} AF-1 domain A/B are less crucial than those of the AF-2 activity.

RARß2 promoter activity in X{alpha}af1o mutant mice
We also investigated the possible involvement of the RXR{alpha} AF-1 domain A/B in the regulation of a transgene, whose expression is under the control of the RARß2 promoter that contains a RA-reponse element (Mendelsohn et al., 1991). To this end, the RARß2 promoter-lacZ reporter transgene was introduced into the RXR{alpha}af1o genetic background. At E13.5, lacZ expression was similar in X{alpha}af1o (n=9) and wild-type (n=13) fetuses (data not shown). In order to eliminate a functional compensation by RXRß (Mascrez et al., 1998; Wendling et al., 1999), lacZ expression was also studied in X{alpha}af1o/Xß mutants. At E13.5, expression of lacZ was similar in wild-type (n=13) and X{alpha}af1o/Xß (n=9) fetuses, including the interdigital regions (data not shown). Altogether, these data indicate that the RXR{alpha} AF-1 domain A/B, in contrast to RXR{alpha} AF-2 (Mascrez et al., 1998), is dispensable for in vivo transactivation by RXR{alpha}, at least in the context of the RARß2 promoter.


    DISCUSSION
 TOP
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The RXR{alpha} deletion created in the present study encompasses most of the N-terminal A/B domain. This region contains the autonomous ligand-independent transcriptional activation function, AF-1, which exhibits some specificity in transfected cells in vitro, depending upon the cell-type and promoter context (Nagpal et al., 1992; Nagpal et al., 1993). Little is known about the molecular mechanisms through which RXR{alpha} AF-1 domain A/B exerts its transcriptional activity. For other members of the nuclear receptor superfamily, e.g. PPAR{gamma}, the A/B domain has been shown to interact directly with co-activators, e.g. PGC-2 (Castillo et al., 1999). Along the same lines, phosphorylation of the A/B domain may modulate either the interaction of receptors with co-activators or their affinity for ligands. For example, phosphorylation of ERß A/B domain promotes recruitment of the SRC-1 co-activator (Tremblay et al., 1999), whereas phosphorylation of the A/B domain reduces the ligand-binding affinity of PPAR{gamma}, thus negatively regulating the transcriptional activity of PPAR{gamma} (Shao et al., 1998). In F9 cells, phosphorylation of the RAR{gamma} A/B domain is required not only for induction of several RA-target genes (Taneja et al., 1997; Rochette-Egly et al., 1997; Bastien et al., 2000), but also for receptor degradation by the ubiquitin-proteasome pathway (Kopf et al., 2000). At least some of these features could probably be extended to RXR{alpha}, which is phosphorylated at several serine and threonine residues in the A/B domain (Adam-Stitah et al., 1999). The present investigation was designed to establish the developmental role of the RXR{alpha} A/B region, as a prerequisite to further genetic and molecular studies aimed at elucidating the molecular mechanisms that underlie physiological events in vivo.

The AF-1 domain A/B of RXR{alpha} is required for the transduction of retinoic acid signals during development
Involution of the primary vitreous body and of the hindlimb interdigital mesenchyme, and cardiomyocyte differentiation and morphogenesis of some cranial skeletal elements and vertebrae are impaired in X{alpha}af1o mutants (this study), in RAR({alpha}, ß and {gamma}) mutants (Lohnes et al., 1993; Ghyselinck et al., 1997; Kastner et al., 1997b) and in animals with vitamin A and RA deficiency (Wilson et al., 1953; Lussier et al., 1993). It appears therefore that in RXR{alpha}/RAR heterodimers, the AF-1 domain A/B of RXR{alpha} participates in the transduction of retinoid signals normally required for the completion of these developmental events. The observation that the developmental defects exhibited by the X{alpha}af1o/Xß/X{gamma} mutants can all be attributed to abnormalities in the RAR/RXR signaling pathway suggest that RXR partners other than RAR do not exert any developmental function that require the RXR{alpha} A/B region.

During development, RXR{alpha} is the most important RXR, as X{alpha} fetuses die in utero and display severe cardiac and ocular defects, whereas Xß/X{gamma} mutants are viable and do not exhibit any congenital defects (Kastner et al., 1994; Krezel et al., 1996). However, a lack of RXR{alpha} or its AF-2 activity can be functionally compensated by RXRß and RXR{gamma} in mouse embryos, as the spectrum of defects observed in X{alpha}/Xß and X{alpha}af2o/Xß/X{gamma} mutants is much broader than that of X{alpha} mutants (Mascrez et al., 1998; Wendling et al., 1999). RXRß and RXR{gamma} also compensate for the deletion of RXR{alpha} A/B domain, as the PHPV and the hindlimb interdigital webbing become completely penetrant and eventually more severe in X{alpha}af1o/Xß/X{gamma} mutants; however, no additional abnormalities are generated in these latter mutants, except for single cases of myocardial hypoplasia and conotruncal septum agenesis. Thus, the functional compensation by RXRß or RXR{gamma} cannot account for the ‘weak’ phenotype of the X{alpha}af1o mutants.

Two scenarios could account for the paucity of the developmental defects in X{alpha}af1o and X{alpha}af1o/Xß/X{gamma} mutants. First, the A/B domain might in fact be dispensable for the majority of the developmental events that require RXR{alpha}. Given the phenotypes of the various X{alpha}af1o/RAR({alpha}, ß or {gamma}) mutants (see below and Tables 2-4), this possibilty is unlikely. Alternatively, the RXR{alpha} A/B domain might be necessary for optimal transactivation of several developmental target genes, through RXR{alpha}/RAR heterodimers. According to this second scenario, the contribution of the RXR{alpha} A/B domain to the transactivation potential of the heterodimers might not be detectable in the protected environment of the animal facility, unless the phenotyping techniques are sophisticated enough, or the examined sample size becomes very large (Brookfield, 1992; Thomas, 1993). Our findings that one out of nine X{alpha}af1o/Xß/X{gamma} mutants displays an hypoplasia of the ventricular myocardium and that another one of these nine has an agenesis of the conotruncal septum, defects that are characteristic of the RXR{alpha}-null phenotype but have never been observed in the several dozens of wild-type embryos that we have analyzed over the past 10 years, support the second scenario (Kastner et al., 1994; Sucov et al., 1994). The requirement of RXR{alpha} A/B domain for myocardial growth or fusion of conotruncal ridges in some fetuses might then reflect stochastic inter-individual variations in the expression of other synergistic factors involved in RA-dependent cardiac morphogenesis. This hypothesis predicts that more VAD-related developmental abnormalities might be detected if a larger sample of X{alpha}af1o/Xß/X{gamma} fetuses were examined.


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Table 4.
 
Other evidence supporting the conclusion that the RXR{alpha} AF-1 domain A/B could be necessary for transactivation by the various RXR{alpha}/RAR heterodimers is suggested by the observation that the corresponding X{alpha}af1o/RAR({alpha}, ß or {gamma}) mutants display most of the VAD-related defects observed in the cognate X{alpha}/RAR({alpha}, ß or {gamma}) double mutants (Kastner et al., 1994; Kastner et al., 1997a). In fact, in the absence of a given RAR, the RXR{alpha} A/B domain becomes essential for enabling the remaining RARs to functionally replace the missing one (discussed by Kastner et al., 1997a; Chiba et al., 1997a; Chiba et al., 1997b; Mascrez et al., 1998; see Tables 2- 4). Altogether, these data favor the involvement of RXR{alpha} AF-1 domain A/B in most of the developmental events that are mediated through the various RAR/RXR{alpha} heterodimers, under conditions where either RAR and/or retinoid levels become limited.

The RXR{alpha} AF-1, but not the AF-2 activity appears dispensable for placentation
Thickening of the labyrinthine trabeculae and lack of definite frontier between the labyrinth and spongiotrophoblast are hallmark features of RXR{alpha}-/- placentas, and probably reflect a failure of a late step of trophoblast cells differentiation (Sapin et al., 1997). Our data show that the RXR A/B domain appears largely unnecessary for the differentiation of trophoblast cells. In contrast, the AF-2 of RXR{alpha} is clearly essential for this differentiation process, as (1) X{alpha}af2o placentas are histologically indistinguishable from placentas that lack RXR{alpha}; and (2) defects of X{alpha}af2o/Xß/X{gamma} placentas are not more severe than in X{alpha}af2o. Furthermore, as X{alpha}af2o/RAR({alpha}, ß or {gamma}) mutants are obtained at the expected Mendelian ratio at E18.5, and are not more growth deficient than X{alpha}af2o mutants (B. M., M. M., N. B. G. and P. C., unpublished), it is likely that terminal differentiation of labyrinthine trophoblast cells involves heterodimers in which RXR{alpha} is transcriptionally active, but distinct from RXR{alpha}/RAR heterodimers. On the other hand, the formation of the labyrinthine zone of the placenta is totally inhibited in X{alpha}/Xß mutants, and severely impaired in PPAR{gamma} mutants, leading to embryonic death before E10.5 (Wendling et al., 1999; Barak et al., 1999). Thus, RXR{alpha} and RXRß, probably in the form of PPAR{gamma}/RXR heterodimers, have an early role in placentogenesis. We have shown that X{alpha}af1o/Xß/X{gamma} and X{alpha}af2o/Xß/X{gamma} mutants do not display these early defects. Thus, neither the RXR{alpha} AF-1 domain A/B nor the AF-2 activity is required on their own in early placentogenesis, suggesting that RXRs could act in this process as silent partners with PPAR{gamma}. Studies with RXR{alpha} mutants that lack both AF-1 and AF-2 are in progress to investigate this possibility further.

The RXR{alpha} AF-1 domain A/B is specifically required for involution of the interdigital mesenchyme
Among the various RA-dependent morphogenetic events involving the RXR{alpha} AF-1 domain A/B, separation of the digits is the only one that specifically requires this region, as X{alpha}af1o and X{alpha}af1o/Xß/X{gamma} mutants display soft tissue syndactyly of the hindlimbs, whereas X{alpha}af2o and X{alpha}af2o/Xß/X{gamma} mutants never display this defect (Mascrez et al., 1998). Phosphorylation of RXR{alpha} at a specific serine residue located in the A/B domain is necessary for the antiproliferative response of F9 teratocarcinoma cells to RA (Rochette-Egly and Chambon, 2001). It is noteworthy that RA-dependent involution of the interdigital mesenchyme results from arrest of cell proliferation, as well as from apoptosis (Dupé et al., 1999). Therefore, the possibility exists that phosphorylation of the RXR{alpha} A/B domain might have important functions in the cascade of molecular events that, in vivo, leads to the normal disappearance of the interdigital mesenchyme.

The AF-1 and AF-2 activities of RXR{alpha} are differentially required for transducing retinoic acid signals during development
AF-1 and AF-2 activities do not have the same importance in the transcriptional activity of RXR{alpha} during embryonic development: (1) X{alpha}af2o/Xß/X{gamma} fetuses display a large array of congenital defects not found in X{alpha}af1o/Xß/X{gamma} fetuses; and (2) X{alpha}af2o/RAR({alpha}, ß or {gamma}) compound fetuses are more severely affected than X{alpha}af1o/RAR({alpha}, ß or {gamma}) fetuses (Mascrez et al., 1998; see Tables 2-4). Moreover, the AF-2, but not the AF-1 of RXR{alpha} is crucial for the transcription of the RARß2-lacZ transgene (Mascrez et al., 1998; this study). At the molecular level, little is known about the mechanism through which AF-1 activates transcription. In the case of ER{alpha}, AF-1 and AF-2 appear to interact simultaneously in vitro with distinct interfaces of the TIF2 co-activator, leading to synergistic activation of transcription (Benecke et al., 2000). Our present data showing that similar abnormalities are exhibited by X{alpha}af1o/RAR({alpha}, ß or {gamma}) and X{alpha}af2o/RAR({alpha}, ß or {gamma}) compound mutants, although more severe in the latter case, not only suggest that RXR{alpha} AF-1 and AF-2 may synergize in vivo as they do in vitro (Nagpal et al., 1993), but also that AF-2 is more important than AF-1 for most of the developmental events that require RXR{alpha}/RAR heterodimers.


    ACKNOWLEDGMENTS
 
We thank C. Birling-Ziegler, I. Michel, B. Weber, C. Dennenfeld and B. Féret; the staff of IGBMC common services, ES cell culture and animal facility for their excellent technical assistance; P. Kastner for the gift of plasmids and helpful discussions; J. Brocard for CMV-Cre mice; C. Rochette-Egly for gift of the antibodies; M. C. Rio for stromelysine-3 probe; N. Messaddeq for transmission miscroscopy; C. Werlé and the secretarial staff for their help in the preparation of this manuscript. This work was supported by funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Hôpital Universitaire de Strabourg, the Collège de France, the Institut Universitaire de France, the Association pour la Recherche sur le Cancer (ARC) and Bristol-Myers Squibb. B. M. was the recipient of fellowships from the French Ministry of Research, the Ligue Nationale contre le Cancer, the Association pour la Recherche contre le Cancer and the Université Louis Pasteur.


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