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Biochem Biophys Res Commun
2004 Jan 09;3132:230-6. doi: 10.1016/j.bbrc.2003.11.113.
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Evolutionarily conserved expression pattern and trans-regulating activity of Xenopus p51/p63.
Tomimori Y
,
Katoh I
,
Kurata S
,
Okuyama T
,
Kamiyama R
,
Ikawa Y
.
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p51/p63, a member of the p53 gene family, is structurally conserved among a wide range of organisms, although the transactivator (TA) and N-terminally truncated (deltaN) isotype producing property seems to vary. Since p51/p63 is thought to play important roles in skin, limb, and craniofacial development in mammals, we examined Xenopus laevis larval and adult tissues for expression of p51/p63. Temporal analyses indicated enhanced transcription of the deltaN form of p51/p63 in premetamorphosis phase (at stage 44-48). p51/p63-positive cells in the inner layer of larval skin expanded to the suprabasal layers during the stratification. The epithelium of limb buds and the maxillofacial ectodermal tissues in tadpoles had a high level expression of p51/p63. The cloned deltaN-A/gamma type Xenopus p51/p63 exhibited a dominant-negative activity against the human TA-A/gamma isotype in a reporter assay. These results suggest that tissue-specific p51/p63-inducing mechanism and isotype-specific transcriptional regulator activities of p51/p63 are conserved between mammals and frogs.
Fig. 1. Domain structures of the p51/p63 isotypes. (A) Domain structures of the human p51/p63 isotypes, p51B/TAp63α, p51C/TAp63β, p51A/TAp63γ, ÎNp51B/ÎNp63α, ÎNp51C/ÎNp63β, and ÎNp51A/ÎNp63γ. TAD, DBD, OLID, and the COOH-terminal sequence variations (B/α, C/β, and A/γ) are shown. (B) The cloned X. laevis ÎN-A/γ type p51/p63 (bottom) and the protein structure (top) encoded by the cDNA. Nucleotide positions of the ÎN, DBD, and A/γ primers used in RT-PCR are indicated with arrows. The ÎN and A/γ probes are also aligned.
Fig. 2. Analysis of the Xenopus p51/p63 gene expression through the late phase of development and in adult tissues. (A) Temporal changes in the p51/p63 gene expression through stages 12â66. RT-PCR with the ÎN-, DBD-, and A/γ-specific primers produced 463, 185, and 292 bp products, respectively. An equal amount of total RNA obtained at each point was examined. GAPDH mRNA was analyzed as a control. A 100-bp ladder was loaded in slot M for identification of the amplified products. (B) p51/p63 expression profile in adult frog tissues.
Fig. 3. Detection of the Xenopus p51/p63 proteins by Western blotting. (A) HeLa cells transfected with human and Xenopus p51/p63 expression vectors. Cells transfected with Hu-p51A/TAp63γ (lane 1), Hu-p51B/TAp63α (lane 2), Hu-ÎNp51A/ÎNp63γ (lane 3), Hu-ÎNp51B/ÎNp63α (lane 4), and Xenopus p51/p63 (lane 6) expression vectors were analyzed. Control experiment with the vector-only plasmid is shown in lane 5. The human and Xenopus proteins were indicated by arrowheads and arrow, respectively. Apparent molecular masses are denoted in kDa in parentheses. An NHK lysate was also analyzed in lane 7. Size markers in lane M are indicated with bars. (B) Proteins in adult frog tissues. Arrow indicates ÎN-A/γ type Xenopus p51/p63 (41 kDa) found in skin, lung, gut, heart, kidney, and stomach. The 77, 70, and 52 kDa proteins found in the skin extract are indicated by arrowheads. The 70 kDa protein was also detectable in the heart.
Fig. 4. Immunohistochemical analysis of larval tissues for the p51/p63 proteins. (A) Localization of p51/p63-expressing cell in basal layer of epidermis. Xenopus, mouse, and zebrafish skin sections were stained with 4A4 and Nuclear Fast Red (Biomedia). Xenopus skin sections at indicated stages (st) and mouse sections on embryonic days (E) and that of newborn (NB) are shown. The frog sample at stage 60 displays the junction between the larval skin and the adult-type skin. Adult zebrafish epidermis with scales is shown. Bars indicate 50 μm. (B) Localization of the p51/p63 proteins in the frog tissues at stage 48. Tissues and organs are indicated. ONL, INL, IPL, and GNL in the retina indicate outer nuclear layer, inner nuclear layer, inner plexiform layer, and ganglion cell layer, respectively. The forelimb bud, gill, and velar plate sections were stained with 4A4 only.
Fig. 5. Whole mount in situ hybridization with the larvae at stage 46. Tadpoles at stage 46 were examined for expression of p51/p63 and fgf-8. Results of the hybridization with A/γ (A–F) and fgf-8 (G,H) probes with sense (A, C, E, and G) and antisense (B, D, F, and H) sequences are shown. (C,D) This show magnified views of the anterior portions of the animals in (A,B), respectively. (E,F) It show lateral views of the hindlimb buds of the samples in (A,B). Bar indicates 0.4 mm.
Xenopus skin sections at prometamorphosis NF stage 60, assayed via IHC (stained with 4A4 and Nuclear Fast Red) showing tp63 proteins localised in basal layer of epidermis in adjacent larval and adult skin areas.
