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In vertebrates, nodal-related genes are crucial for specifying mesendodermal cell fates. Six nodal-related genes have been identified in Xenopus, but only one, nodal, has been identified in the mouse. The Xenopus nodal-related gene 3 (Xnr3), however, lacks the mesoderm-inducing activity of the other five nodal-related genes in Xenopus, and can directly induce neural tissue in animal caps by antagonizing BMP signals. In this study, we isolated three clones of the Xenopus (Silurana) tropicalis nodal-related gene 3 (Xtnr3) and analyzed their function. The Xtnr3 genes show high homology to Xnr3 and have the same activity. Southern blot and genomic PCR analyses indicate that the X. tropicalis genome has duplications in the Xtnr3 gene sequences and our three clones represent separate gene loci. We also found a partial clone of Xtnr3 that coded for the N-terminal part of its pro-region. Surprisingly, this sequence also induced neural tissue by antagonizing BMP signals, and its coded protein physically associated with BMP4 mature protein. Furthermore, we showed that the pro-region of Xnr5 has the same activity. Together, these findings indicate that the pro-region of nodal-related genes acts antagonistically towards BMP signals, which identifies a novel mechanism for the inhibition of BMP signaling.
Fig. 2. Temporal and spatial expression patterns of Xtnr3s during X. tropicalis development. (A) Temporal expression was analyzed by RT-PCR. Lane numbers
indicate stages according to Nieuwkoop and Faber (1956). The Xtnr3 transcripts were first detected at the blastula stage (stage 8), and peaked through the midblastula
(stage 8.5) to early gastrula stage (stage 10). ODC RT+ was used as an internal control. ODC RT indicates no genomic DNA contamination. RT
indicates that reverse transcriptase was omitted from the RT reaction. (B–E) Whole-mount in situ hybridization shows the spatial expression patterns of the
Xtnr3s. (B) Vegetal view of stage 9 embryo; transcripts were detected around the dorsal region. (C) Vegetal and (E) dorsal views of stage 10 embryo; transcripts
were restricted to the Spemann organizer region. (D) Vegetal view of stage 11 embryo; transcripts were decreased.
Fig. 3. Genomic Southern blot and genomic PCR analyses for Xtnr3. (A)
The copy number of Xtnr3 was examined by Southern blot analysis.
Genomic DNA from the liver of an adult male X. tropicalis was digested
with EcoRI, HincII, or PstI. PCR fragments derived from the presumptive
exon-1 region of Xtnr3 were used as probes. The positions of the DNA size
markers are indicated in kilobases. (B) Genomic PCR with gene-specific
primers from 3VUTR reveal all three forms, Xtnr3-A–C, present in multiple
X. tropicalis individuals that were not siblings. Genomic DNAs were
extracted from the liver of six adult X. tropicalis (five females and one
male).
Fig. 4. Effects of injecting Xtnr3 mRNAs into X. tropicalis and X. laevis embryos. X. tropicalis embryos were injected into the animal pole of one blastomere at
the two-cell stage with 150 pg of synthetic mRNA and cultured until stage 35. (A) Control injection with h-galactosidase (b-gal) mRNA, (B) Xnr3 mRNA, (C)
Xtnr3-A mRNA, and (D) pXtnr3DC mRNA. X. laevis embryos were injected with 1 ng of synthetic mRNA in the same manner as X. tropicalis embryos and
cultured until stage 35. (E) b-gal mRNA, (F) Xnr3 mRNA, (G) Xtnr3-A mRNA, and (H) pXtnr3DC mRNA. Xtnr3-A and Xnr3 induced the same phenotype
with anterior defects and tail-like protrusions in X. tropicalis and X. laevis embryos, but pXtnr3DC, lacking the mature region and the C-terminal part of the
pro-region, did not. X. laevis embryos injected with pXtnr3DC (short form) developed a bump on the head. The blue and red arrowheads indicate the protrusion
and the bump on the head, respectively. The black arrows indicate anterior defects. (I, J) Section of X. laevis embryo injected with Xnr3 mRNA into the animal
pole. The protrusion contains mesenchymal tissue (arrow). The boxed area in I corresponds to J.
Fig. 5. Secondary axis formation by Xtnr3s and Xnr3. X. laevis embryos were injected into the marginal zone of both ventral-vegetal blastomeres at the eightcell
stage with 500 pg of synthetic mRNA and cultured until stage 35. (A) Control injection with b-gal mRNA, (B) Xnr3 mRNA, (C) Xtnr3-A mRNA, and (D)
pXtnr3DC mRNA. Not only Xtnr3-A and Xnr3, but also pXtnr3DC, lacking the mature region and the C-terminal part of the pro-region, induced secondary
axis. X. laevis embryos were injected (E) into the animal pole of one blastomere at the two-cell stage with 1 ng of pXtnr3DC mRNA and 150 pg of b-gal
mRNA or (F) into the marginal zone of both ventral-vegetal blastomeres at the eight-cell stage with 500 pg of pXtnr3DC mRNA and 150 pg of b-gal mRNA.
Red-gal stain showed that the injected location affected the resultant phenotype. The red and black arrowheads in A through F indicate the secondary axis and
the bump on the head, respectively. (G, H) Section of X. laevis embryo injected with Xtnr3 into the VMZ. The secondary axis possessed a neural tube (black
arrow) and an otic vesicle (black arrowhead). The boxed area in G corresponds to H.
Fig. 6. pXnr3 (pro-region of Xnr3) mRNA induces neural but not mesodermal markers in animal caps, by blocking BMP signaling. (A) Control injection with
b-gal mRNA, and (B) secondary axis induced by pXnr3 mRNA. X. laevis embryos were microinjected into the marginal zone of both ventral-vegetal
blastomeres at the eight-cell stage with 500 pg of mRNA and cultured until stage 35. The red arrowhead indicates the secondary axis. (C) Four-cell-stage
embryos were injected with 1 ng of mRNA into the animal pole of each blastomere. At the late blastula stage, injected and uninjected animal caps were
dissected and cultured until sibling embryos reached stage 30. RT-PCR analysis shows that all Xtnr3s and the pro-region of Xnr3 (pXnr3) induce the general
neural marker NCAM and the cement gland marker XAG-1. The lack of ms-actin showed that neural induction occurred without a mesodermal intermediary. (D)
Four-cell-stage embryos were injected with mRNA encoding BMP4 (1 ng) and BMP4 (1 ng) mixed with Xnr3, pXnr3, Xtnr3-A, Xtnr3-B, Xtnr3-C, and
pXtnr3DC (1 ng of each) into the animal pole of each blastomere. Animal cap explants were dissected at stage 9 and harvested at stage 10.5 for RT-PCR
analysis. In each assay, expression of Xvent1 and Xmsx1, which are induced by BMP signaling, was blocked, suggesting that Xnr3, pXnr3, Xtnr3s, and
pXtnr3DC induce neural tissue by antagonizing BMP signaling.
Fig. 7. The N-terminal part of the pro-region of Xtnr3 interacts with BMP
protein. Interaction between the pro-region of Xtnr3 and BMP4 was
determined by coimmunoprecipitation and Western blot analysis. RNAs
encoding pXtnr3DC/myc (1 ng) and BMP4/HA (1 ng) were injected into X.
laevis embryos. The lysates from stage 10 embryos were immunoprecipitated
for either pXtnr3DC/myc (A) or BMP4/HA (B) with anti-Myc or anti-
HA antibodies, respectively.
Fig. 8. The pro-region of Xnr5 inhibits BMP signaling. (A) Schematic drawing of two constructs: the pro-region of Xnr5 (pXnr5) and the pro-region of a
cleavage mutant of Xnr5 (pcmXnr5). pXnr5 encodes only the pro-region of Xnr5, which has a putative cleavage site, ‘‘RRHRR’’. pcmXnr5 also encodes only
the pro-region of Xnr5, but its cleavage site was changed from ‘‘RRHRR’’ to ‘‘GVDGG,’’ corresponding to cmXnr5. (B) Two-cell stage embryos were injected
into both blastomeres with 2 ng RNA of pXnr5 (p5) or pcmXnr5 (pc5). Animal cap explants were dissected at stage 9 and harvested at stage 30 for RT-PCR
analysis. Injection of pXnr5 or pcmXnr5 RNA induced the expression of the general neural marker NCAM without that of dorsal mesodermal marker ms-actin.
(C) Two-cell stage embryos were injected into both blastomeres with 1 ng of BMP4 RNA plus 1 ng of pXnr5 (p5) or pcmXnr5 (pc5) RNA. Animal cap explants
were dissected at stage 9 and harvested at stage 10.5 for RT-PCR analysis. Both pXnr5 and pcmXnr5 suppressed the expression of Xvent1 and Xmsx1, which are
induced by BMP4. Injection of neither 1 ng of pXnr5 nor pcmXnr5 RNA induced the expression of these marker genes. un, uninjected explants control; WE,
whole embryo positive control; ( ), RT negative control. (D) Interaction between pXnr5 and BMP4 was determined by coimmunoprecipitation and Western
blot analysis. Synthetic mRNAs encoding pXnr5/myc (1 ng) and BMP4/HA (1 ng) were injected into X. laevis embryos. Lysates from stage 10 embryos were
immunoprecipitated for pXnr5/myc with anti-myc antibody.
