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The Wnt signaling pathway is conserved across species, and is essential for early development. We previously identified nucleoredoxin (NRX) as a protein that interacts with dishevelled (Dvl) in vivo to negatively regulate the Wnt/beta-catenin pathway. However, whether NRX affects another branch of the Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, remains unclear. Here we show that NRX regulates the Wnt/PCP pathway. In Xenopus laevis, over-expression or depletion of NRX by injection of NRX mRNA or antisense morpholino oligonucleotide, respectively, yields the bent-axis phenotype that is typically observed in embryos with abnormal PCP pathway activity. In co-injection experiments of Dvl and NRX mRNA, NRX suppresses the Dvl-induced bent-axis phenotype. Over-expression or depletion of NRX also suppresses the convergent extension movements that are believed to underlie normal gastrulation. We also found that NRX can inhibit Dvl-induced up-regulation of c-Jun phosphorylation. These results indicate that NRX plays crucial roles in the Wnt/PCP pathway through Dvl and regulates Xenopus gastrulation movements.
Figure 1 Bent-axis phenotype caused by NRX mRNA injection. Representative embryos with (A) no injection, (B–D) NRX mRNA
injection (100, 250 or 500 pg), and (E–G) Dvl mRNA injection (100, 250 or 500 pg). (H) The ratio of bent-axis phenotype caused by
NRX or Dvl mRNA injection. Representative embryos with “severe”, “moderate”, “weak” and “normal” phenotypes are also shown.
Figure 2 The effect of NRX mRNA co-injection with Dvl
mRNA on bent-axis phenotypes. Representative embryos with
(A) Dvl mRNA injection (100 pg) and (B) Dvl mRNA (100 pg)
and NRX mRNA (100, 500 or 1000 pg) co-injection. (C) The
ratio of bent-axis phenotype by injection of Dvl mRNA alone, or
with NRX mRNA.
Figure 3 Bent-axis phenotype caused by
NRX-MO injection. (A) Total RNAs
were collected from dissected stage 10
Xenopus embryos, and RT-PCR analyses
for NRX were carried out (B) pCS2-
NRX5′UTR-GFP (50 pg) and NRXMO
(5, 10 ng) or control MO (5, 10 ng)
were injected, and the embryos were harvested
and subjected to Western blot analyses.
(C–E) Representative embryos with
(C) no injection, (D) Idax-MO injection
(10 ng), and (E) NRX-MO injection
(10 ng). (F) The ratio of bent-axis phenotype
by Idax- or NRX-MO injection.
Figure 4 Co-injection experiments with
NRX mRNA and NRX-MO. (A–C)
Representative embryos injected with (A)
NRX mRNA alone (200 pg), (B) NRX
mRNA and NRX-MO (5 ng), (C) NRX
mRNA and Dvl mRNA (100 pg). (D) The
ratio of bent-axis phenotype by injection of
NRX mRNA alone, or with NRX-MO or
Dvl mRNA. (E–G) Embryos injected with
(E) NRX mRNA (500 pg) alone, (F) NRX
(500 pg) and β-catenin (500 pg) mRNA,
and (G) NRX (500 pg) and GSK3β
(500 pg) mRNA. (H–J) Embryos injected
with (H) Wnt11 mRNA alone (500 pg),
(I) NRX-MO alone (5 ng), (J) Wnt11
mRNA (500 pg) and NRX-MO (5 ng).
Figure 6 NRX mRNA/MO injection
inhibits animal cap elongation. Indicated
mRNAs or MOs were injected into the
animal pole of the 8 cell stage Xenopus
eggs. Animal caps were dissected, and
cultured with/without activin. Representative
images of animal caps are indicated
with elongated ratios. (B) Total
RNAs were collected from animal caps,
and RT-PCR analyses were carried out
with mesoderm markers Xbra and gsc. (C)
Representative activin-treated animal
caps from embryos injected with Xdd
mRNA and/or NRX-MO.
Figure 7 NRX suppresses c-Jun phosphorylation. NIH3T3 cells transfected with FLAG-c-Jun and indicated constructs were harvested,
and their lysates were subjected to immunoblotting (IB) with indicated antibodies. The signal intensities of IB with anti-c-Jun (phospho
Ser 63/73) were determined by densitometry and their relative values are indicated.
