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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). |
