P1252428258UJhvH - PowerPoint PPT Presentation

Title: P1252428258UJhvH


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Temporal and Spatial Detection of Biologically
Active Retinoid X Receptor in Vivo Eric Gonzales
Peterson, Ayala Luria and J. David Furlow Section
of Neurobiology, Physiology and Behavior,
University of California, Davis

Abstract The nuclear retinoid X receptor (RXR)
functions as a ligand-activated transcription
factor. Most studies on RXR have focused on its
role as a heterodimeric partner, yet little is
known 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 RXR alpha
(RXRa) in live transgenic Xenopus laevis embryos
across a wide range of developmental stages. We
adopted a nuclear receptor Gal4 (g) fusion with
the UAS E1B minimal promoter based reporter
system for our assay. We observe strong
activation of the RXRa ligand-binding domain
(LBD) in a segment of the spinal cord just
posterior to the hindbrain. This activation is
first detected in neural stage embryos and
persists up to swimming tadpole stages, after
which activation strongly declines. Addition of
exogenous ligands, such as 9-cis retinoic acid
(9-cis RA) or all-trans retinoic acid (atRA),
expands the activation of RXRa all along the
spinal cord but not in the brain. Embryos
expressing Gal4-RXRa (gRXRa) fusion with a
deletion in the ligand-dependent activation
domain (AF2) show no reporter gene activation.
Our conclusion is that there is an endogenous
ligand for RXRa localized in the rostral region
of the developing spinal cord of Xenopus laevis
embryos.    
Fig. 7. Localized activation of gRXRa fusion
protein in the nervous system of transgenic
Xenopus laevis embryos in the presence of
exogenous ligands, 9-cis RA and atRA. Bright
field (A-D) and fluorescent images (E-H) of
transgenic embryos created with NbTg (A and E) or
NbTg RXRa (B-D and F-H) expression vectors with
the reporter gene UAS-E1B-EGFP. No neural
expression of EGFP was seen in NbTg transgenic
embryos (E) whereas EGFP was detected in the
spinal cord of embryos carrying the NbTg RXRa
transgene (F). Embryos treated with 1µM
exogenous 9-cis RA (C,G) or atRA (D,H) show EGFP
activation along the entire spinal cord but not
in the brain.  
Fig. 2. Schematic diagram of expression and
reporter constructs. A. Gal4 fusion protein
expression constructs. The DNA-binding domain of
the yeast transcriptional activator Gal4 was
fused in-frame with the ligand-binding domain of
Xenopus laevis RXR, RXRa?H12, or RARa under the
control of promoter SV40 for transfection
experiments. B. UAS reporter constructs. The
enhanced green fluorescent protein (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.  
Fig. 4. A gRXRa fusion protein regulates UAS
reporter genes in transfected XLA cells. Cells
transfected with pSG5g, pSG5gRXRa, pSG5gRXRa?H12,
or pSG5gRARa, and UAS-E1B-luciferase were treated
with vehicle alone (white bars), 100nM 9-cis RA
(black bars), 100nM all-trans RA (striped bars)
or 100nM RAR specific ligand, TTNPB (gray bars).
Molecular Mechanism of Nunclear Receptor Action
Fig. 8. Time course detection of EGFP reporter
gene in transgenic gRXRa Embryos. Pictures A-E
are bright field pictures representing stages of
development up to and including swimming tadpole
stage. Pictures F-I are under UV light showing
EGFP expression in a localized region of the
developing spinal cord. Picture J shows a
diminished level of EGFP expression by the
swimming tadpole stage.  
Comparison of Minimal Promoter Activity Between
Unregulated and Hormone Regulated Reporters
Target Cell
Protein
Co-repressors
Co-activators
Fig. 3. Minimal promoter activity was compared
using a luciferase assay, standardized by b-gal
activity. Transcription of the luciferase
reporter gene was compared using different
minimal promoters. Expression vectors pSG5gDBD
and pSG5gDBD-TRa were each introduced into XLA
cells along with UAS-luciferase reporter genes
carrying the minimal promoter SV40, TK, or E1B
separately. They were then treated with (Blue
bars) or without (Yellow bars) exogenous Thyroid
Hormone (T3). The minimal promoter that
demonstrated the least background noise in the
absence of TH was minimal promoter E1B. This
promoter was then selected to be utilized for
both transfection and transgenics.
Fig. 5. Transgenics in Xenopus laevis by
Restriction Enzyme Mediated Integration Method
(REMI). Transgenic animals carrying the plasmid
gRXRa driven by neural b tubulin (NbT) and the
reporter gene EGFP were produced using the REMI
method. Purified sperm nuclei were mixed with
the linearized plasmid and low levels of a
restriction enzyme to facilitate integration.
After incubation this mixture was injected into
Xenopus eggs. Embryos were screened for
successive cleavage after several hours and EGFP
expression was monitored under long-wavelength
ultra-violet light.
Fig. 1. Simplified model of Retinoid-X-Receptor
Action. RXRa, as a homodimer or heterodimer,
binds to its specific response elements in DNA.
In the absence of ligands, co-repressors are
bound preventing transcription. In the presence
of ligands a conformational change occurs in the
receptor complex and co-repressors are released
and co-activators become bound. As a result,
transcription of the target gene can begin.

• Conclusion
• Our results indicate the presence of a highly
  localized RXRa ligand hot-spot in or near the
  rostral spinal cord of the developing embryo.
  This ligand is being synthesized early in neural
  stages and ceases to be made when tadpoles
  acquire the ability to swim, suggesting a
  phylogenetically conserved role for activation of
  RXRa during spinal cord neurogenesis.
• Helix 12 deletion abolishes RXRa activation in
  transfected cells and transgenic embryos.
• Activation of RXRa in the spinal cord is
  dependent on direct ligand activation of the RXRa
  LBD.
•  
•  
•  

Introduction Retinoic acid, a metabolite of
vitamin A, is a critical signaling molecule that
is essential for proper vertebrate development.
Retinoic acid deficiency or excess has been shown
to cause deformities in the developing Central
Nervous System (CNS), heart and hind limbs.
Retinoic acid and its metabolites can bind and
activate both retinoic acid receptor (RAR) and
its heterodimeric partner RXRa. Both, RAR and
RXRa have been found to be key regulatory
receptors during development. In vitro studies
show RAR can be bound and activated by atRA as
well as its isomer 9-cis RA, while RXR can be
activated by 9-cis RA only. Although the
activation pattern of RXR has been well
characterized, little is known about its specific
endogenous ligand and distribution during
development. In order to determine the
activation pattern of RXRa we have utilized a
binary nuclear receptor Gal4/UAS based reporter
assay in live Transgenic Xenopus laevis embryos.
Fig. 6. In Vivo Activation of gRXRa in Xenopus
laevis Spinal Cord. Pictures A-C are light
pictures of animals carrying NbT fused to g/EGFP,
gRXRa/EGFP and gRXRa?H12/EGFP, respectively.
Pictures D-F are the corresponding UV images
representing EGFP expression. Inserted in B and E
is a superior view of the developing spinal cord
for the NbTgRXRa transgenic animals. No reporter
gene activity was observed in animals containing
NbTg/EGFP alone, or in animals carrying
NbTgRXRa?H12.
Acknowledgements I would like to thank Dr. David
Furlow, Dr. Ayala Luria and Eric Neff for
providing me with the guidance and expertise
needed to fulfill such an involved project.
Also, I wish to thank M. Privalsky (UC Davis), S.
Frasher (Caltech) and E. Amaya (Cambridge) for
the pSG5g, UAS-E1B-EGFP and NBT GFP plasmids.
This work was funded by NIH grant RO1 DK55511 and
a UC Davis center for Environmental Health
Sciences pilot project grant. Poster printing
compliments of Hewlett Packard and the UC Davis
UC LEADS program.