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The tumor-suppressor protein p53 belongs to a small gene family that includes p63 and p73. While p53 and p73 regulate cell cycle progression and apoptosis, the major role of p63 appears to be in promoting ectodermal proliferation and differentiation. In this report we describe the cloning of a Xenopus orthologue of mammalian p63 that is extraordinarily conserved in sequence. The major sites of expression of Xenopus p63 mRNA are the epidermis and some neural crest and crest derivatives such as the branchial arches and tailfin. Expression is also observed in the neural plate and in the stomodeal-hypophyseal anlage. Antibodies against p63 detect a nuclear protein that is distributed in a manner similar to that of Xp63 mRNA. Both mRNA and protein are conspicuously absent from regions of the epidermal sensorial layer that are induced to form a number of (but not all) ectodermal placodes and Xp63 protein levels are particularly dynamic in the epidermis of the eye as the lens forms.
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11287210
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Fig. 2. Expression of Xp63 mRNA and protein. Panels within the box labeled ‘mRNA’ illustrate examples of wholemount in situ hybridization using a digoxygenin labeled Xp63 anti-sense RNA probe. Panels within the box labeled ‘protein’ illustrate antibody 4A4 (anti-p63) immunoreactivity in wholemounts. (A) Dorsal view of mid-neurulaXp63 RNA distribution. (B) Anterior view of mid-neurulaXp63 RNA distribution. (C) Dorsal view of mid-neurulaXp63 protein distribution. (D) Mid-neurulaXp63 protein. (E). Stage 20/21 dorsal view of Xp63 RNA. (F) Stage 20/21 anterior view of Xp63 RNA. (G) Stage 19/20 dorsal view of Xp63 protein. (H)Stage 19/20 anterio-dorsal view of Xp63 protein. (I) Stage 24 dorsal view of Xp63 RNA. (J) Stage 24, anterior view of Xp63 RNA. (K) Stage 24 dorsal view of Xp63 protein. (L) Anterior view of stage 24 Xp63 protein expression. (M) Lateral view of stage 33 Xp63 RNA distribution. (N) Stage 31 tail, lateral view of Xp63 RNA is indicated by blue stain. (O) Stage 31 lateral view of headXp63 protein expression. (P) Anterior view of stage 31 head, Xp63 protein pattern. (Q) Lateral view of stage 30 tail, Xp63 protein expression pattern. Black arrow, epidermal staining; tailed black arrow, medial neuroblasts; red arrow, Xp63 negative region in brachial ectoderm. Blue arrow, stomodeal-hypophyseal anlage; red tailed arrow, cranial neural crest; black arrow with circular tail; branchial arches; black arrow with double headtailfin; yellow arrow, olfactory placode; blue tailed arrow, cement gland. In panels A,C,E, G, I, K, M, N, O and Q anterior is to the left. In panels B, D, F, H, J, L and P dorsal is up.
Fig. 3. Localization of p63 protein within the epidermis. Transverse optical sections through ventralepidermis. 4A4 binding was detected using a Cy5 labeled secondary antibody, and the sample counterstained with propidium iodide. (A) Fluoresence in the rhodamine channel. Bright nuclear labeling (e.g. white arrow) and weaker autofluorescence are detected. (B) Fluorescence in the Cy5 channel. The flattened nuclei of the sensorial ectoderm are labeled (e.g. yellow arrow). (C) Merging of the two channels shows nuclei labeled with propidium alone in green and p63 positive nuclei in yellow. Yolk autofluorescence appears olive-brown in color.
Fig. 4. Downregulation of p63 protein in epidermis of the lens placode. 4A4 immunohistochemistry was used to detect Xp63 in eyeepidermis. Forming eyes are circled to mark their boundaries. Note the few Xp63 negative cells above the lens in the stage 35–36 embryo (white arrow).
Tp63 Antibody (Tp63 Ab1) detected in the flattened nuclei of the basal layer of the ventralepidermis. 4a4 primary antibody detected with a Cy5 coupled secondary. faint autofluorescence from yolk platelets is also visible.
tp63 (tumor protein p63) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17/18, dorsal view, anteriorleft.
tp63 (tumor protein p63) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17/18, anterior view, dorsal up.
