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Figure 2. In situ hybridization of mRNAs of platelet-derived growth factor A (PDGF-A) and PDGF receptor (PDGFR) - alpha on skin sections of tadpoles. Sagittal sections of the body skin were prepared from tadpoles at stage 57 (A-D), early stage 58 (E-H), late stage 58 (I-L), and stage 59 (M-P). Sections A,E,I,M; B,F,J,N; C,G,K,O; and D,H,L,P are for hematoxylin and eosin (H&E) stains, Xenopus adult keratin (XAK) -C immunostains, in situ hybridization of PDGF-A mRNAs, and in situ hybridization of PDGFR- alpha mRNAs, respectively. A,E,I,M: The epidermis at stage 57 (A) was composed of apical (ap), skein (sk), and larval basal cells (lb). XAK-C-positive preadult basal cells (pab) emerged at early stage 58 (E,F) and larval basal cells disappeared at stage 59 (M). Skein cells were well developed and an outstanding population in the epidermis at stage 59 (M). Primordial secretory glands (PSGs) were well developed at early stage 58 (E) and differentiated into secretory glands (sg) at stage 59 (M). The collagen lamella (cl) was directly attached to the basement membrane at stage 57 (A). Beneath the collagen lamella, p-ct was present where fibroblasts (f) were sparsely scattered. The s-ct began to be formed beneath PSGs at early stage 58 (E), developed at late stage 58 (I), and further developed to secretory glands (sg) at stage 59 (M). B,F,J,N: There were no immunopositive cells at stage 57 (B). XAK-C-positive preadult basal cells (pab) were observed around PSGs at early stage 58 (arrows; F), and these cells spread to other regions on the basement membrane at late stage 58 (J) and 59 (N). C,G,K,O: No cells expressed PDGF-A mRNAs at stage 57 (C). PDGF-A mRNAs were detected in basal cells (arrows) around PSGs at early stage 58 (G), and these cells spread along the basement membrane at late stage 58 (K). The basal cells (pab) ceased the expression of PDGF-A mRNAs at stage 59 (O). Some of secretory glands were positive. D,H,L,P: Fibroblasts in p-ct weakly expressed PDGFR- alpha at stage 57 (D). Fibroblasts in the newly formed s-ct at early (H) and late stage 58 (L) intensively expressed the mRNAs and continued to express them intensively until stage 59 (P). Dashed lines in photographs represent the location of the basement membrane. Scale bar = 50 mu m in H (applies to A-J), in L (applies to K,L), in P (applies to M-P).
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Fig. 1. Changes in mRNAs levels of platelet-derived growth factor A (PDGF-A) and PDGF
receptor (PDGFR) - during spontaneous and 3,3 ,5-triiodothyronine (T3) -induced metamorphosis.
Total RNA was extracted from the back skin of tadpoles, 1 g of which was used
as a template for reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified
products were electrophoresed on gels and visualized with ethidium bromide. RT-PCR was
done with 27 cycles, which produced a 510- and 444-bp product for PDGF-A and PDGFR- ,
respectively. Amplification of the rpL8 gene was performed with 28 cycles as an internal
control. A: RT-PCR for tadpoles at the stages indicated above the gel. B: RT-PCR for tadpoles
at stage 54/55 treated with T3 for the days indicated above the gel. RT-PCR was performed
two times for RNAs extracted from different samples for each determination, and similar
results were obtained reproducibly.
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Fig. 3. Culture of larval skin in the presence
of thyroid hormone (TH) or inhibitors
of platelet-derived growth factor (PDGF)
signaling. Pieces of back skin were isolated
from tadpoles at stage 54/55 (A,B)
and were cultured for 9 days in the absence
(-T3; C) or presence of ( T3; G)
100 nM 3,3 ,5-triiodothyronine (T3). A 10 M
AG1296 (K) or 100 g/ml recombinant
extracellular domain of PDGF receptor-
(rECD; O) was added for 9 days in the
absence of T3. AG1296 was dissolved in
0.1% dimethyl sulfoxide. A,C,G,K,O: Hematoxylin
and eosin (H&E) stains. B,D,H,L,P: Immunostaining
using anti-XLK antisera.
E,I,M,Q: Double immunochemical staining
using antiAK-C (shown in red) and XAK-B
(green) antisera. F,J,N,R: Skins were labeled
with 20 M bromodeoxyuridine
(BrdU) for the last 24 hr and were immunostained
by using anti-BrdU antibodies.
Dashed lines in the photographs represent
the location of the basement membrane.
Arrows with closed arrowheads in E, M, and
Q show XAK-Cositive preadult basal cells
around primordial secretory glands (PSG).
sp, spinous cells; gr, granular cells; c, cornified
cells. For other abbreviations, see
legend to Figure 2. Scale bars 50 m in B
(applies to A,B), in J (applies to C), in R
(applies to K).
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Fig. 4. Effects of 3,3 ,5-triiodothyronine (T3) on the mRNA expression of Xenopus larval
keratin (XLK), Xenopus adult keratin (XAK) -B, XAK-C, platelet-derived growth factor A
(PDGF-A), and PDGF receptor (PDGFR) - in cultures of larval skin. Larval back skins at stage
54/55 were cultured with ( T3) or without (-T3) 100 nM T3. Total RNA was extracted from them
at 0, 3, 6, and 9 days of culture and used as templates of reverse transcriptase-polymerase
chain reaction (RT-PCR) for quantifying mRNAs of XLK, XAK-B, XAK-C, PDGF-A, PDGFR- , and
rpL8 as in Figure 1. RT-PCR was performed four times for RNAs extracted from different
samples, and similar results were obtained reproducibly.
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Fig. 5. Effects of inhibitors of platelet-derived growth factor (PDGF) signaling on thyroid hormone (TH) -induced remodeling of larval skin
in vitro and in situ hybridization of PDGF receptor (PDGFR) - mRNA on the skin sections. Pieces of back skin of tadpoles at stage 54/55 were
cultured for 9 days in the presence of 100 nM 3,3 ,5-triiodothyronine ( T3). Control (A): Inhibitors were not added. AG1296 (D): 10 M
AG1296 was added through the culture. Recombinant extracellular domains of PDGFR- (rECD; G): 100 g/ml rECD was present. The
skins were subjected to hematoxylin and eosin (H&E) staining (A,D,G) or double immunohistochemical staining using antisera against
Xenopus adult keratin (XAK) -B (shown in green) and XAK-C (shown in red; B,E,H). The skin tissues in C, F, and I were exposed to
bromodeoxyuridine (BrdU) for the last 24 hr and immunostained by using anti-BrdU antibodies. Arrowheads in C point to representative
BrdU-positive cells. Arrowheads in D and G point to fibroblasts that display round and small nuclei, indicating possible cell death. Asterisks
in H and I indicate nonspecific stains. Dashed lines represent the location of the basement membrane. J: Effects of inhibitors of
PDGF/PDGFR signaling on TH-induced expression of mRNAs of XAK-B and XAK-C. Pieces of back skin of tadpoles at stage 54/55 were
cultured for up to 9 days. T3 was added to all the cultures at the concentration of 100 nM ( T3). AG1296 was dissolved in 0.1% dimethyl
sulfoxide (DMSO). CD, cultures in the presence of 0.1% DMSO; AG, in the presence of 10 M AG1296; C, control cultures for rECD
experiments; rECD, in the presence of 100 g/ml rECD. Total RNA was extracted from the cultured skin at days indicated. Reverse
transcriptase-polymerase chain reaction (RT-PCR) was carried out on 1 g of total RNA for XAK-B, XAK-C, PDGF-A, and PDGFR- , optimizing
the amplification cycles for each gene. rpL8 was used as an internal control. RT-PCR was performed three times for RNAs extracted from
independent samples and similar results were reproducibly obtained. K,L: In situ hybridization of PDGFR- mRNAs on stage 54/55 skin
cultured with 100 nM T3 for 4 (K) or 6 days (L). PDGFR- mRNAs were detected in subepidermal fibroblasts but not in epidermal cells. Dashed
lines represent the location of the basement membrane. For abbreviations, see legends to Figures 2 and 3. Scale bars 50 min I (applies
to A-I), in L (applies to K,L).
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krt5.5 (keratin 5, gene 5 ) gene expression in Xenopus laevis embryos, NF stage 54, as assayed by immunohistochemistry. Coronal section of skin, superficial up.
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krt5.5 (keratin 5, gene 5 ) gene expression in Xenopus laevis embryos, NF stage 54, as assayed by immunohistochemistry. Coronal section of skin, superficial up.
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