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

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

View larger version (34K): [in a new window]   Fig. 1. Targeted deletion of the AF-1-containing A/B region of RXR. (a) Representation of the wild-type RXR1 isoform (WT RXR1) and the mutant RXR (RXRA/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 RXRaf1 mutation leads to the production of an RXR 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[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 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[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 RXRaf1 mutant alleles before (+/(I)) or after (af1/+) excision of the ‘floxed’ TK-NEO cassette are indicated. (d) Detection of RXRA/B protein. Nuclear extracts prepared from 12.5-day-old embryos WT (+/+), af1 heterozygote (af1/+), af1 homozygote (af1o) and RXR-null mutants (-/-) (Kastner et al., 1994) were analyzed by western blotting with the anti-RXR monoclonal antibody 4RX3A2, directed against a C-terminal epitope.

View larger version (15K): [in a new window]   Fig. 2. Weight of wild type (WT) and Xaf1o mutants at 1-2 weeks of age. To standardize between litters, the weight of each pup obtained from Xaf1/+ 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 Xaf1o and WT animals in each class is indicated on the top of the bars.

View larger version (12K): [in a new window]   Fig. 3. Weight of wild-type (WT) and Xaf1o mutant males (a) and females (b). Mean weights of offsprings (n=5 to 18) obtained from Xaf1/+ 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 Xaf1o mutants. NS, not significant.

View larger version (95K): [in a new window]   Fig. 4. Precocious differentiation of Xaf1o 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 Xaf1o 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.

View larger version (71K): [in a new window]   Fig. 5. Decreased interdigital cell death results in soft tissue syndactyly of the hindlimbs in Xaf1o/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 Xaf1o/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.

View larger version (94K): [in a new window]   Fig. 6. Comparison of ocular malformations in Xaf1o and Xaf2o 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 Xaf1o/A(ß or ) and Xaf2o/A(ß or ) mutants share the same spectrum of ocular defects, although these are systematically more severe in Xaf2o compound mutants. For example, the size of the conjunctival sac (J) and cornea (C) is only slightly reduced in Xaf1o/Aß and Xaf1o/A mutants (compare e,j,h) but markedly decreased in Xaf2o/Aß mutants (i), and absent in Xaf2o/A mutants (k). Likewise, the stroma of the iris (I), the anterior chamber (A) and the secondary vitreous body (SV) are present in Xaf1o/Aß and Xaf1o/A mutants, but not in their Xaf2o 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).

View larger version (80K): [in a new window]   Fig. 7. Comparison of E18.5 wild-type (WT), Xaf1o and Xaf2o 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 Xaf2o 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).

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