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Fig. 1. Overview of the GeneChip analysis of body skin metamorphosis.
(A) A design for a body skin microarray experiment. Premetamorphic
stage tadpoles (stage 54/55) were treated with 5 nM T3
for 7 days. Total RNA samples were extracted from body skin at the
indicated times for a GeneChip Xenopus laevis Genome array
(Affymetrix). (B) Hierarchical cluster analysis of significantly upregulated
and downregulated genes in body skin during TH-induced
metamorphosis. Green and red colors in the rows indicate that
individual genes were downregulated and upregulated compared with their
expression at 0 day, respectively. A total of 401 transcripts significantly
changed their expressions above fourfold for 7 days (161 downregulated
and 240 upregulated genes). The tree lines at the left side represent
hierarchical clustering of the identified genes. (C) Expression profiles of
larval and adult keratin clusters in body skin during TH-induced
metamorphosis. Six adult-type keratin genes and four larval-type
keratin genes were identified by the GeneChip analysis.
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Fig. 2. Validation of GeneChip-expression profiles by RT–PCR. From genes that had been identified as significantly TH-upregulated genes by GeneChip assay, we selected six genes as
those that had been already accepted as the TH-regulated genes (A) and seven genes as those that had been used as epidermal marker genes (B). The changes of mRNA expression of
these 14 genes were measured during TH-induced metamorphosis by RT–PCR. Pre-metamorphic stage tadpoles (stage 54/55) were treated with T3 for 7 days. Total RNA samples were
extracted from body skins at the indicated times. Open and closed circles represent the results of GeneChip analysis and real-time RT–PCR, respectively. rpL8 gene was used as an internal
control. Values represent the mean ± SE of three independent experiments of real-time RT–PCR test.
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Fig. 3. Expression profiles of the Gene Ontology (GO) annotated genes in body skin during TH-induced metamorphosis. Among the 401
significantly upregulated and downregulated genes, 119 genes were categorized into nine functional groups according to GO biological
process (see Table 3), and their temporal expression profiles are individually depicted; regulation of transcription (GO:6355, 31 genes),
transport (GO:6810, 23 genes), proteolysis (GO:6508, 13 genes), metabolic process (GO:8152, 12 genes), immune response (GO:6955,
12 genes), multicellular organism development (GO:7252, nine genes), cell adhesion (GO:7155, seven genes), cell cycle (GO:7049, seven
genes), and defense response (GO:6952, five genes). The change in the expression level of each gene is individually depicted by a line that
is arbitrarily colored for easy distinction. The probe annotation information was obtained from NetAffy (Affymetrix).
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Fig. 4. A schematic representation of the temporal expression
profiles of the functionally grouped genes in the body skin during
TH-induced metamorphosis. Changes of expression levels of genes
in six groups, that is, categories of transcription and proteolysis,
larval keratin, adult keratin, cell cycle, immune response, and
defense response, are depicted against time periods post T3-
treatment. The red and green bars at the top show the extent of
larval and adult phenotypes in such a manner that the color
becomes denser as the extent increases, respectively.
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Fig. 5. Promoter activity of 5′-
flanking region of xak-c gene in an
F0 transgenic frog. A brightly
EGFP-fluorescent transgenic embryo
was developed to adulthood and
observed under a fluorescent
microscope anteriorly (A), dorsally
(B), and posteriorly (C). All images
were taken from the dorsal side.
Bars, 1 mm. Skin tissues were
removed from the back and
sectioned for immunostaining with
anti-XAK-C (D) and anti-GFP
antibodies (E). The sections were
also stained with Hoechst 33342
for nuclear staining. Bars, 50 μM.
bs, basal cell; c, cornified cells;
ct, connective tissue; gl, granular
gland; gr, granular cell. Dotted lines
indicate the basement membrane.
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Fig. 6. Changes of EGFP-expression
pattern during spontaneous
metamorphosis. F1 transgenic
tadpoles were allowed to metamorphose
and were photographed at
stages 56 (A), 58 (B), 60 (C), and
63 (D) under a fluorescence (right
panels in the respective stage) and
bright-field microscope (left panels).
Strong EGFP fluorescence was
observed in the mouth, fore limb,
and hind limbs, whereas EGFP
fluorescence in the regressing tail
and reshaping face regions was
undetectable. Bars, 5 mm. FL,
forelimb; HL, hind limb; T, tail.
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Fig. 7. Expression patterns of
XLK proteins during spontaneous
metamorphosis. Tadpoles at the
indicated stages were subjected to
whole-mount immunostaining with
anti-XLK antibodies. Immunofluorescence
images (red) demonstrated
the larval skin region in tail and
head. Bar, 5 mm. FL, forelimb; HL,
hind limb; T, tail. (B) A schematic
illustration of larval to adult skin
conversion during spontaneous
metamorphosis. Red and green
colors indicate larval (XLK) and
Xenopus adult keratin (XAK-C)
skin regions, respectively. XAK-C
was specifically expressed in the
prospective adult region. In contrast,
the expression of XLK was robustly
observed in the regressing zone of
face and tail at the metamorphic
climax stage. The yellow color
represents the regions that coexpress
both XLK and adult XAK-C.
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Fig. 8. Responsiveness of xak-c
promoter to TH in vivo. F1 transgenic
tadpoles at stage 54/55 were
treated with 5 nM T3 for up to 5
days. (A) Changes of expression
of EGFP fluorescence was
undetectable at 0 day, became
detectable intensely in fore and
hind limbs at 3 days. EGFP was
uniformly expressed in body
region but not tail at 5 days. All
fluorescence images were taken
from the ventral side. The tadpole
at 5 days was photographed under
the bright field and shown at the
right. Bar, 2 mm (B) Semi-quantitative
RT–PCR analysis of expression of
endogenous xak-c gene and
exogenous EGFP gene in F1 offspring
during TH-induced metamorphosis.
F1 transgenic tadpoles were
exposed to TH for up to 3 days,
and then total RNA was sampled
from body skin at 0, 6, 12, 24, 48,
and 72 h. Both xak-c and EGFP
gene were concomitantly activated
at 48 h after T3 administration.
Ribosomal protein L8 (rpL8) was
used as an internal control.
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