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,
A Paradisi1, V De Laurenzi2, LG Spagnoli1, MV Catani1,
S Ramadan1, RA Knight2and G Melino*,1,2
1Biochemistry Laboratory, IDI-IRCCS, c/o University of Rome ‘Tor Vergata’,
00133 Rome, Italy
2Medical Research Council, Toxicology Unit, Leicester University, Leicester
LE1 9HN, UK
* Corresponding author: G Melino, Medical Research Council, Toxicology Unit,
LE1 9HN, UK. Tel: þ44 116 252 5616; Fax: þ44 116 252 5551;
Received 03.3.06; revised 08.3.06; accepted 08.3.06; published online 07.4.06
Edited by P Vandenabeele
Epidermal development requires the transcription factor p63,
as p63?/? mice are born dead, without skin. The gene
expresses two proteins, one with an amino-terminal transac-
tivation domain (TAp63) and one without (DNp63), although
their relative contribution to epidermal development is
unknown. To address this issue, we reintroduced TAp63a
and/or DNp63a under the K5 promoter into p63?/? mice by
in vivo genetic complementation. Whereas p63?/? and
p63?/?;TA mice showed extremely rare patches of poorly
differentiated keratinocytes, p63?/?;DN mice showed sig-
nificant epidermal basal layer formation. Double TAp63a/
DNp63a complementation showed greater patches of differ-
entiated skin; at the ultrastructural level, there was clear
reformation of a distinct basal membrane and hemidesmo-
somes. At the molecularlevel, DNp63regulated expression of
genes characteristic of the basal layer (K14), interacting (by
Chip, luc assay) with the third p53 consensus site.
Conversely, TAp63 transcribed the upper layer’s genes (Ets-
1, K1, transglutaminases, involucrin). Therefore, the two p63
isoforms appear to play distinct cooperative roles in
Cell Death and Differentiation (2006) 13, 1037–1047.
doi:10.1038/sj.cdd.4401926; published online 7 April 2006
Keywords: p63; epidermis; cornification; skin; development
Abbreviations: TA, transactivation domain; DN, amino-terminal
truncated protein; H&E, haematoxylin and eosin; tg, transgenic
p63?/?;TA, mice knockout for p63, re-expressing TAp63a;
p63?/?;DN;TA, mice knockout for p63, re-expressing both
DNp63a and TAp73a
The skin consists of two compartments, the dermis and the
epidermis. The latter is a multilayered, stratified epithelium
continuously regenerated by terminally differentiating keratino-
cytes, a process called cornification1–3(Figure 1a and b).
Recent evidence demonstrates a major role for p63,4a
member of the p53 family,5–8in this process as mutations in
the TP63 gene cause limb and skin defects in humans,9and
p63?/? mice have no epidermis, no limbs and die at birth
owing to dehydration.10,11
The expression of p63 proteins originates from two
promoters, giving rise to TAp63 and DNp63 isoforms. In
addition, both isoforms undergo alternative splicing at the
C-terminus producing three different TAp63 and DNp63
isoforms (a, b and g). Although there is solid evidence that
p63 isinvolved in epithelial development,10,11for example, via
regulation of PML,11–13and in aging,13the relative contri-
bution of the different N-terminal isoforms to epidermal
formation has not been established. To address this issue,
we have, therefore, generated transgenic mice expressing
either TAp63a or DNp63a under the control of the keratin (K) 5
promoter. The K5 promoter specifically drives the expression
of the gene in the basal layer, the proliferative compartment of
the epithelium. TAp63a and DNp63a transgenic mice were
used to generate isoform-specific complemented mice in the
knockout background. The data presented are consistent with
a role for DNp63a in controlling the expansion of epithelial
cells from progenitor precursors in epidermal epithelia, to
allow TAp63a, acting synergistically and/or subsequently to
DNp63a, to control epithelial differentiation.
Transgenic complemented mice
To elucidate the individual role of the TAp63a and DNp63a
isoforms in the development of the epidermis, we generated
transgenic mice expressing either isoform under the control of
the K5 promoter and then crossed these mice into a p63?/?
background (Figure 1c–f). Founder transgenics (four TAp63a
and five DNp63a) were generated by microinjecting the
purified transgene into the pronuclei of zygotes and then
implanting the zygotes into pseudo-pregnant female mice.
Lines were established and maintained by backcrossing the
founders with C57/B6 mice. All founders were fertile and
produced transgenic offspring at the expected Mendelian
frequencies without any obvious abnormalities. We obtained
similar levels of expression of TAp63a or DNp63a proteins in
the basal layer. The density of the hair follicles, and outer
appearance of the skin and fur were normal. Efficient
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
Cell Death and Differentiation (2006) 13, 1037–1047
& 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00
epidermal basal layer and in tissues where K5 is normally
expressed (thymus, eye, lung), but not in other tissues
including bone, muscle, liver (not shown). Figure 1d shows
the immunoblot staining with antibodies specific for p63 (left
panel) and for the haemagglutin-antigen (HA) tag (right
panel). The transgene was overexpressed as a nuclear
protein, as shown in Figure 1e, in the basal layer of the
epidermis, Figure 1f, consistent with the known specificity of
the K5 promoter. To obtain double mutant mice, the
transgenic mice overexpressing TAp63a/DNp63a were back-
crossed with p63þ/? mice.11
The p63?/?;TA (mice knockout for p63, re-expressing
TAp63a) and p63?/?;DN (mice knockout for p63, re-expres-
sing DNp63a) transgenic mice died at birth, like the p63?/?
mice (Figure 2). TAp63a complemented mice, like p63?/?
animals, had only very limited areas of epithelialisation with
abnormal visibility of the vasculature through the skin
(Figure 2d) due to the absence of the epidermis. In contrast,
limited, macroscopical formation of the epidermis (Figure 2c).
In agreement with this observation, the DNp63a complemen-
ted animals expressed greater amounts of the characteristic
basal layer proteins, K5 and K14, than either the p63?/? or
the p63?/?;TA mice (Figures 3a, b and 4a). No significant
was detected in p63?/?;TA and p63?/?;DN transgenic mice
(Figure 3c, d and 4b, c).
At the ultrastructural level, unlike wild-type (wt) mice
(Figure 5a), these three groups (p63?/?, p63?/?;TA,
p63?/?;DN) were all devoid of fully keratinised squamous
corneocytes, intercellular lipid or corneodesmosomes, and
there were no recognisable filaggrin granules or keratin fibrils
(Figure 5b–d). No traces of hemidesmosomes/basement
membrane were found in any of these animals. The surface
layers of KO mice were dominated by fibroblast-like cells
interspersed by irregular cell profiles that contained a few
randomly arranged keratin filaments. These cells were not
restricted to the outer surface but showed localised cornifica-
tion of their cell envelopes and many contained some
enlarged, osmiophilic granules (Figure 5b). The contents of
these granules were extracted, from resin sections, and were
identified as loricrin granules (not shown). TAp63a comple-
mented mice had only very limited areas of epithelialisation
and the resulting cell profiles usually contained enlarged
loricrin granules together with accumulations of a moderately
electron-dense material, presumably filaggrin (Figure 5c). In
contrast, the epidermis of the p63?/?;DN mice contained
and signs of nuclear disintegration or cornification of the cell
envelope, but these cells were often overlaid by non-cornified
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
beingpredominant. (b)Therole ofthe p63proteinisstillcontroversial,23,24regulatingeitherthe stemcells/transientamplifying (TA)cells11(1) ortheir 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 5.2kb 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
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. Bar¼15mm. (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.
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
p63 in epithelial development
E Candi et al
Cell Death and Differentiation
Assay System (Promega, Madison, WI, USA); light emission was
measured over 10s using an OPTOCOMP I luminometer. Efficiency of
transfection was normalised using Renilla luciferase activity.
We thank Dr. F McKeon for providing us with the p63?/? mice, Dr. C
Tiveron for crucial help and advice in the generation of the transgenic
mice, and Mr. A Colangeli and J McWilliam for ultramicrotomy. This work
Telethon, FIRB, MIUR, MinSan, TelethonGGP04110 and MRC to GM.
Note added in proof
After this paper was accepted, Laurikkala et al. (Laurikkala J, Mikkola ML,
James M, Tummers M, Mills AA and Thesleff I (2006) P63 regulates
multiple signaling pathways required for ectodermal organogenesis and
differentiation. Development 133: in press. Published online on March 8,
2006. doi: 10.1242/dev.0235) demonstrated that DNp63 is the main
development, accounting for 100%of allp63 expressed up toE9 and 99%
at E13. TAp63 expression starts at E13, and only accounts for 1% of total
is predominant over that of TAp63, at a time before any epidermal
stratification occurs. These results obtained by Laurikkala et al. are fully in
agreement with our studies, using a completely different experimental
approach, and confirm that the DNp63 isoform is crucial for the formation
of the epidermis, whereas TAp63 contributes to the control of epithelial
differentiation by acting synergistically and/or subsequently to DNp63.
Moreover, Laurikkala etal. also demonstrated that Bmp7, Fgfr2b,Jag1
and Notch1 transcripts are coexpressed with DNp63 and are absent in
p63?/?mice. Additional support for this complex pathway of regulations
also comes from a further independent paper in press (Nguyen BC, Lefort
Wang J, Tommasi di Vignano A, Kitajewski J, Chiorino G, Roop DR,
in keratinocyte commitment to differentiation. Genes Dev. in press.
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of epidermal homeostasis.
p63 in epithelial development
E Candi et al
Cell Death and Differentiation