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Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands. The aim of this study is to visualize the distribution of activated RXRalpha in live transgenic Xenopus laevis embryos across a wide range of developmental stages. We adopted a nuclear receptor-Gal4 fusion/upstream activation sequence-based reporter system for our assay. Strong activation of the RXRalpha ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain. This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines. Addition of exogenous ligands, such as 9-cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4-RXR activation into the brain and olfactory epithelia. Treatment with the RAR-specific ligand 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4-RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions. Embryos expressing a Gal4-RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation. The results shown in this paper reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence of RXR ligand "hot-spots" in this region.
Fig. 1. Schematic diagram of expression and reporter constructs. (A) Gal4 fusion protein expression constructs. The Gal4 DNA-binding domain (amino acids 1–147) was fused in-frame with the ligand-binding domain of X. laevis RXRα (gRXR, amino acids 229–488), RXRα δH12 (gRXRδH12, amino acids 229–469), or RARα (gRAR, amino acids 157–458) under the control of a ubiquitously expressed SV40 early promoter (pSG5) for transfection experiments or the X. laevis neural-β tubulin (NβT) promoter for transgenic experiments. (B) UAS reporter constructs. The EGFP reporter construct UAS-E1b-EGFP contains 14 Gal4 upstream activation sequences (UAS, black boxes) and a minimal promoter (E1b) driving the expression of EGFP. UAS-E1b-Luc is identical to UAS-E1b-EGFP except luciferase is the reporter gene.
Fig. 2. gRXR fusion proteins regulate UAS reporter genes in transfected XLA cells. Relative luciferase activity was used to quantitate the transcriptional responsiveness of the gRXR or gRAR fusion proteins in response to added ligands. (A) Cells transfected with pSG5gRXR and treated with increasing concentrations of 9-cis RA (squares) or atRA (circles). (B) Cells transfected with pSG5g, pSG5gRXR, pSG5gRAR, or pSG5gRXRδH12 with UAS-E1b-Luc and treated with vehicle alone (DMSO, white bars), 100 nM 9-cis RA (black bars), or RAR selective agonist TTNPB (striped bars) as indicated. (C) Cells transfected with PSG5g or PSG5gRXR and UAS-E1b-Luc and treated with vehicle alone (DMSO), 9-cis RA (100 nM), LG100268 (1, 10, and 100 nM LG268, as indicated), or 9-cis RA with increasing concentrations of LG100208 (0.1, 1, and 10 μM LG208, as indicated). LG268 increased luciferase activity, whereas LG 208 inhibits 9-cis RA effect only at 10 μM. Error bars represent mean ± SEM of triplicates from a representative experiment.
Fig. 3. A gRXR fusion protein expressed in the nervous system activates GFP expression specifically in the rostral spinal cord. Transgenic X. laevis tadpoles were created with UAS-E1b-EGFP along with one of the following expression vectors: NβTg (A and D;g/EGFP), NβTgRXR (B and E; gRXR/EGFP), or NβT gRXRδH12 (C and F; gRXRδH12). Bright field images are shown in A–C; fluorescent images are shown in D–F. Endogenous activation of gRXR was observed only in embryos carrying gRXR/EGFP transgene, and not in g/EGFP alone or mutated gRXRδH12/EGFP. (G and H) Overlaid picture of light and fluorescent images. Cross sections of midbrain (G) and rostral spinal cord (H) of gRXR/EGFP tadpole stained with an anti-EGFP antibody. (I and J) Dorsal view of light (I) and fluorescence (J) images. (Bar, 100 μm.) This pattern was observed in all positive embryos, with few intensity changes along the spinal cord.
Fig. 4. Developmental activation of the gRXR fusion protein in the X. laevis spinal cord. EGFP expression is specifically induced in the rostral spinal cord of NβTgRXR/UAS-E1b-EGFP transgenic embryos beginning at stage 24 (A and F), and continuing through stages 26 (B and G), 35 (C and H), and 42 (D and I, overlaid image), but disappears by stage 46 (E and J). Bright field images are shown in A–E; fluorescent images are shown in F–J. Arrows indicate location of fluorescence induced above background autofluorescence, including both spinal cord and eye (in I).
Fig. 5. Exogenous ligand treatment expands GFP expression in gRXR fusion protein expressing embryos. Bright field (AâD and I) and fluorescent images (EâH and J) of transgenic embryos created with NβTgRXR (AâH) or NβTgRAR (I and J) expression vectors and the UAS-E1b-EGFP reporter gene. No expression of reporter gene was observed in untreated swimming tadpoles (A and E). Reporter gene expression was seen in gRXR tadpoles that were treated with exogenous 1 μM 9-cis RA (B and F), atRA (C and G), or LG100268 (D and H). TTNPB induced activation only in gRAR transgene embryos (I and J). Embryos were monitored daily, and activation was observed after 3 days of treatment.
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