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Differential Roles of P63 Isoforms in Epidermal Development: Selective Genetic Complementation in P63 Null Mice

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Differential Roles of P63 Isoforms in Epidermal Development: Selective Genetic Complementation in P63 Null Mice Cell Death and Differentiation (2006) 13, 1037–1047 & 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00 www.nature.com/cdd Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice E Candi1, A Rufini1, A Terrinoni1, D Dinsdale2, M Ranalli1, The skin consists of two compartments, the dermis and the A Paradisi1, V De Laurenzi2, LG Spagnoli1, MV Catani1, epidermis. The latter is a multilayered, stratified epithelium S Ramadan1, RA Knight2 and G Melino*,1,2 continuously regenerated by terminally differentiating keratino- cytes, a process called cornification1–3 (Figure 1a and b). 4 1 Biochemistry Laboratory, IDI-IRCCS, c/o University of Rome ‘Tor Vergata’, Recent evidence demonstrates a major role for p63, a 00133 Rome, Italy member of the p53 family,5–8 in this process as mutations in 2 Medical Research Council, Toxicology Unit, Leicester University, Leicester the TP63 gene cause limb and skin defects in humans,9 and LE1 9HN, UK p63À/À mice have no epidermis, no limbs and die at birth * Corresponding author: G Melino, Medical Research Council, Toxicology Unit, owing to dehydration.10,11 Hodgkin Building, Leicester University, Lancaster Road, PO Box 138, Leicester LE1 9HN, UK. Tel: þ 44 116 252 5616; Fax: þ 44 116 252 5551; The expression of p63 proteins originates from two E-mail: [email protected] promoters, giving rise to TAp63 and DNp63 isoforms. In addition, both isoforms undergo alternative splicing at the Received 03.3.06; revised 08.3.06; accepted 08.3.06; published online 07.4.06 C-terminus producing three different TAp63 and DNp63 Edited by P Vandenabeele isoforms (a, b and g). Although there is solid evidence that p63 is involved in epithelial development,10,11 for example, via regulation of PML,11–13 and in aging,13 the relative contri- Abstract bution of the different N-terminal isoforms to epidermal Epidermal development requires the transcription factor p63, formation has not been established. To address this issue, as p63À/À mice are born dead, without skin. The gene we have, therefore, generated transgenic mice expressing expresses two proteins, one with an amino-terminal transac- either TAp63a or DNp63a under the control of the keratin (K) 5 tivation domain (TAp63) and one without (DNp63), although promoter. The K5 promoter specifically drives the expression their relative contribution to epidermal development is of the gene in the basal layer, the proliferative compartment of the epithelium. TAp63a and DNp63a transgenic mice were unknown. To address this issue, we reintroduced TAp63a used to generate isoform-specific complemented mice in the and/or DNp63a under the K5 promoter into p63À/À mice by knockout background. The data presented are consistent with in vivo genetic complementation. Whereas p63À/À and a role for DNp63a in controlling the expansion of epithelial p63À/À;TA mice showed extremely rare patches of poorly cells from progenitor precursors in epidermal epithelia, to differentiated keratinocytes, p63À/À;DN mice showed sig- allow TAp63a, acting synergistically and/or subsequently to nificant epidermal basal layer formation. Double TAp63a/ DNp63a, to control epithelial differentiation. DNp63a complementation showed greater patches of differ- entiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmo- Results somes. At the molecular level, DNp63 regulated expression of Transgenic complemented mice genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. To elucidate the individual role of the TAp63a and DNp63a Conversely, TAp63 transcribed the upper layer’s genes (Ets- isoforms in the development of the epidermis, we generated transgenic mice expressing either isoform under the control of 1, K1, transglutaminases, involucrin). Therefore, the two p63 the K5 promoter and then crossed these mice into a p63À/À isoforms appear to play distinct cooperative roles in background (Figure 1c–f). Founder transgenics (four TAp63a epidermal formation. and five DNp63a) were generated by microinjecting the Cell Death and Differentiation (2006) 13, 1037–1047. purified transgene into the pronuclei of zygotes and then doi:10.1038/sj.cdd.4401926; published online 7 April 2006 implanting the zygotes into pseudo-pregnant female mice. Lines were established and maintained by backcrossing the Keywords: p63; epidermis; cornification; skin; development founders with C57/B6 mice. All founders were fertile and produced transgenic offspring at the expected Mendelian Abbreviations: TA, transactivation domain; DN, amino-terminal frequencies without any obvious abnormalities. We obtained truncated protein; H&E, haematoxylin and eosin; tg, transgenic two different mouse lines for each isoform, all of which showed mice; p63À/À;DN, mice knockout for p63, re-expressing DNp63a; similar levels of expression of TAp63a or DNp63a proteins in p63À/À;TA, mice knockout for p63, re-expressing TAp63a; the basal layer. The density of the hair follicles, and outer p63À/À;DN;TA, mice knockout for p63, re-expressing both appearance of the skin and fur were normal. Efficient DNp63a and TAp73a expression of the transgenes was evident both by immuno- staining on skin biopsies and by Western analysis on cultured primary keratinocytes. Expression was restricted to the p63 in epithelial development E Candi et al 1038 Figure 1 Expression of TAp63a or DNp63a under the control of the K5 promoter in transgenic mice. We obtained two different mouse lines for each isoform, all of which showed similar levels of expression of TAp63a or DNp63a proteins in the basal layer. (a) DNp63 and TAp63 are both expressed in the basal layer, with DNp63 being predominant. (b) The role of the p63 protein is still controversial,23,24 regulating either the stem cells/transient amplifying (TA) cells11 (1) or their differentiation10 (2) or cell death28 (3). The data herein reported are compatible with the first hypothesis, with distinct roles for DNp63 and TAp63. (c) The 5.2 kb K5 constructs used to express TAp63a or DNp63a in basal keratinocytes. Mouse cDNAs are fused in-frame at the N-terminal end with an HA epitope. The distances in kb are indicated in the figure. (d) Expression of the transgene in cultured primary keratinocytes. Western blots for p63 (left, showing endogenous and transgenes) or HA tag (right, showing only transgenes). TA and DN indicate the protein expression in representative transgenic mice (DNp63a or TAp63a). The two lanes on the right show a marker control for both TAp63a and DNp63a proteins. (e) Immunofluorescence for the transgene (stained using an antibody against the HA tag) shows a nuclear localisation for both the TAp63a and DNp63a proteins in primary keratinocytes cultured from the transgenic mice. Bar ¼ 15 mm. (f) Immunohistochemistry of p63 in epidermis, using anti-HA antibody for transgenic mice and anti-p63 (Ab4 clone) for wt mice, showing overexpression of the transgene (brown colour) in the basal layer of the epidermis only. Bar ¼ 120 mm epidermal basal layer and in tissues where K5 is normally was detected in p63À/À;TA and p63À/À;DN transgenic mice expressed (thymus, eye, lung), but not in other tissues (Figure 3c, d and 4b, c). including bone, muscle, liver (not shown). Figure 1d shows At the ultrastructural level, unlike wild-type (wt) mice the immunoblot staining with antibodies specific for p63 (left (Figure 5a), these three groups (p63À/À, p63À/À;TA, panel) and for the haemagglutin-antigen (HA) tag (right p63À/À;DN) were all devoid of fully keratinised squamous panel). The transgene was overexpressed as a nuclear corneocytes, intercellular lipid or corneodesmosomes, and protein, as shown in Figure 1e, in the basal layer of the there were no recognisable filaggrin granules or keratin fibrils epidermis, Figure 1f, consistent with the known specificity of (Figure 5b–d). No traces of hemidesmosomes/basement the K5 promoter. To obtain double mutant mice, the membrane were found in any of these animals. The surface transgenic mice overexpressing TAp63a/DNp63a were back- layers of KO mice were dominated by fibroblast-like cells crossed with p63 þ /À mice.11 interspersed by irregular cell profiles that contained a few The p63À/À;TA (mice knockout for p63, re-expressing randomly arranged keratin filaments. These cells were not TAp63a) and p63À/À;DN (mice knockout for p63, re-expres- restricted to the outer surface but showed localised cornifica- sing DNp63a) transgenic mice died at birth, like the p63À/À tion of their cell envelopes and many contained some mice (Figure 2). TAp63a complemented mice, like p63À/À enlarged, osmiophilic granules (Figure 5b). The contents of animals, had only very limited areas of epithelialisation with these granules were extracted, from resin sections, and were abnormal visibility of the vasculature through the skin identified as loricrin granules (not shown). TAp63a comple- (Figure 2d) due to the absence of the epidermis. In contrast, mented mice had only very limited areas of epithelialisation the p63À/À;DN transgenic mice showed greater, although still and the resulting cell profiles usually contained enlarged limited, macroscopical formation of the epidermis (Figure 2c). loricrin granules together with accumulations of a moderately In agreement with this observation, the DNp63a complemen- electron-dense material, presumably filaggrin (Figure 5c). In ted animals expressed greater amounts of the characteristic contrast, the epidermis of the p63À/À;DN mice contained basal layer proteins, K5 and K14, than either the p63À/À or many cells with traces of randomly dispersed keratin filaments the p63À/À;TA mice (Figures 3a, b and 4a). No significant and signs of nuclear disintegration or cornification of the cell increase in the expression of upper layer markers (K1, loricrin) envelope, but these cells were often overlaid by non-cornified Cell Death and Differentiation p63 in epithelial development ECandiet al 1039 Figure 2 Reintroduction of TAp63a or DNp63a in p63À/À mice by genetic complementation.
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