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Figure 1 We identified FGF target genes by microarray analysis
and subclassified them based on their sequence. (A) The data set in
the scatter plot, show the expression level of genes on a logarithmic
scale as the median value of DMSO-exposed explants on the x-axis
vs. the median value of SU5402-exposed explants on the y-axis.
(B) Out of the 43 genes including 38 down-regulated genes and
5 up-regulated genes by combinatorial microarray. (C) Out of 43
genes, 26 (60%) had unknown function or were novel and 14
genes (33%) had known functions, of which 11 were previously
identified FGF targets. Three (7%) were retrotransposon or reverse
transcriptase related genes. (D) Nine transcription factors (21%), 4
transmembrane proteins (9%), 3 cytoskeleton interaction (7%), and
3 transport-related genes (7%), and 2 growth factors (5%); however,
there were no clues to the functions of 17 genes (40%).
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Figure 2 Whole-mount in situ hybridization of several
interesting genes. Among identified genes, we picked up six
novel genes that are highly expressed around blastopore, which is
showed similar expression pattern with FGFR1 expression in
gastrula. From left to right, expression patterns of early gastrula,
late gastrula, neurula, and tailbud stage are shown. All gastrula
embryos are vegetal but late gastrula of ARL5. Late gastrula of
ARL5 is lateral view and dorsal is right. All neurula embryos are
dorsal view and anterior is left. For tailbud embryos, XL019m15
and XL027l07 are dorsal view but the others are lateral view.
Anterior is left.
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Figure 3 Semiquantitative RT-PCR confirmed FGF-dependent
regulation of the identified genes. The amplification of cDNAs
obtained from eFGF (0.01 ng), HAVø, or XFD (0.5 ng)-injected
Keller explants, and SU5402, or DMSO-exposed Keller explants
was performed. Each ratio represents the relative intensity of the
respective explants divided by the intensity of the uninjected
explants. These results are representative of three experiments.
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Figure 4 How novel genes are related to other FGF components.
(A) RT-PCR analysis of animal cap explants was performed.
cDNAs from 10 animal caps that had received injections of Xbra,
Xspry2 (1 ng) co-injected with eFGF (0.01 ng), and Xmc (1 ng)
were amplified by primers to ARL5, GPCR4, mig6, and NRH.
(B) Using a MEK inhibitor, U0126, we further examined and
confirmed that mig6 is regulated in a Ras-MAPK dependent
manner, whereas the other genes are not. RT-PCR analysis of
Keller explants was carried out in the presence of U0126 or
DMSO.
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Figure 5 Xmig6 is regulated in an eFGF, MAPK, and Xbradependent
fashion. (A) To look for the relationship among Xmig6,
eFGF (0.01 ng), and Xbra (1 ng), we performed RT-PCR analysis
MPK-1 (0.5 ng) and Xbra-EnR (Xbra repressor, 1 ng) with animal
cap assay. Animal cells that received injection were proceeded into
RT-PCR (B) In the same condition as (A), we further confirmed
that the relationship between Xmig6 and FGF-MAPK pathway.
In the presence of U0126 (50 μm) or DMSO as a control, animal
cells that received injection were proceed to RT-PCR analysis.
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Figure 6 Xmig6 depleted embryos showed muscle differentiation
defect in a Xmyf 5-dependent manner. (A) Transcription
inhibition by Xmig6 MO was confirmed by Western blotting.
Protein expression was detected by a rabbit polyclonal anti-GFP
antibody and anti-rabbit IgG-HRP antibody. (B) X-Gal-stained
embryos that received injection of Xmig6 MO (16.8 ng) or Xmig6
RNA (0.15–0.25 ng) to the one dorsal cell of 4-cell-stage were
used for WISH with a DIG-labelled Xmyf 5 anti-sense RNA
probe. Vegetal view. Xmig6 MO introduced embryos caused the
completely depleted Xmyf 5 expression, which was rescued by
mig6 RNA co-injection. (C) In eFGF induced animal caps,
Xmig6 MO (8.4–16.8 ng) specifically suppressed the expression
of Xmyf 5 and slightly suppressed XmyoD. This suppression was
rescued by Xmig6 RNA (0.2–0.25 ng). No changes were observed
in Xbra and Xnot expression. (D) In later stages, embryos that
received Xmig6 MO injection were stained by 12/101 antibody
and showed muscle differentiation defect whereas control MO
injected embryos showed well-developed somites. We counted the
embryos that proceeded 12/101 antibody staining and six of
20embryos were failed to be stained by the antibody. Xmig6MO
introduced embryos were no effect on notochord detected by
MZ15 antibody.
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Figure 7 XGPCR4 is potentially a non-canonical FGF target
gene. We investigated the role of XGPCR4 in gastrulation by
injecting its mRNA or XGPCR4 MO into the two dorsal cells
of 4-cell-stage embryos. (A) The gain-of-function effect showed
dose-dependent A-P axis truncation or spina bifida but embryos
given mRNA injection (n = 60) did not show any changes in the
differentiation of the notochord and somites. (B) The specificity
of XGPCR4 MO was confirmed by Western blotting, using
venus-tagged constructs in animal caps. (C) In XGPCR4-MO
injected embryos, neither the expression of mesodermal markers
nor the formation of the organizer was affected compared with
control MO-injected embryos. (D) Embryos given an injection of
XGPCR4 MO (16.8 ng) (n = 45) showed incomplete closure of
the dorsal blastopore and the spina bifida phenotype. However, no
changes in the formation of the notochord and somites were
observed. Using mut-GPCR4 (500 pg) construct, we partially
rescued the spina bifida phenotype caused by the GPCR4 MO
(n = 48). Mut-GPCR4 RNA itself (n = 45) exerted the same
effects on embryos as intact GPCR4 RNA.
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Table 1 The results of sequence based BLAST search and spatiotemporal expression patterns of identified genes during gastrulation stage
are described. The remarkable spatiotemporal expression patterns that we described were detected by whole-mount in situ hybridization.
No putative protein means no homologue by BLAST searches. Access our database website, XDB, for more information
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arl5c (ADP-ribosylation factor-like 5C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11, vegetal view, dorsal up.
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arl5c (ADP-ribosylation factor-like 5C) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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errfi1 (ERBB receptor feedback inhibitor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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Figure S1 The effects of SU5402 to Xenopus embryogenesis.
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Figure S2 (A) Novel genes that identified in this study showed FGF-dependent regulation. Embryos that received HAV?, or XFD (0.5 ng) injection with lineage tracer in one dorsal cell at 4-cell stage were proceed to WISH when the stage reached. (B) Novel genes are regulated by FGF-dependent manner and do not affect mesodermal genes and FGF component genes.
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Figure S3 It is shown that the amino acid sequences of human mig6, mouse mig6, and Xenopus mig6 and their phylogenic tree.
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