Oncogene (2009) 28, 13291338

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ORIGINAL ARTICLE

The Tcl1 oncogene denes secondary hair germ cells differentiation at

catagentelogen transition and affects stem-cell marker CD34 expression
G Ragone1, A Bresin1, F Piermarini1, C Lazzeri1, MC Picchio1, D Remotti2, S-M Kang3,
MD Cooper4, CM Croce5, MG Narducci1 and G Russo1
1
Molecular Oncology Laboratory, Istituto Dermopatico dellImmacolata, Istituto di Ricovero e Cura a Carattere Scientifico, Rome,
Italy; 2Department of Pathology, Azienda Ospedaliera San CamilloForlanini, Rome, Italy; 3Department of Microbiology and
Immunology, and Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA, USA; 4Emory Vaccine Center and
Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA and 5Human Cancer
Genetics Program, Comprehensive Cancer Center, Department of Molecular Virology, Immunology, and Medical Genetics,
College of Medicine, The Ohio State University, Columbus, OH, USA

Overexpression of the TCL1 gene family plays a role in

the onset of T-cell leukemias in mice and in humans. The
Tcl1 gene is tightly regulated during early embryogenesis
in which it participates in embryonic stem (ES)-cells
proliferation and during lymphoid differentiation. Here,
we provide evidences that Tcl1 is also important in mouse
hair follicle (HF) and skin homeostasis. We found that
Tcl1
/
adult mice exhibit hair loss, leading to alopecia
with extensive skin lesions. By analysing Tcl1 expression
in the wild-type (wt) skin through different stages of hair
differentiation, we observe high levels in the secondary
hair germ (HG) cells and hair bulges, during early anagen
and catagentelogen transition phases. The loss of Tcl1
does not result in apparent skin morphological defects
during embryonic development and at birth, but its
absence causes a reduction of proliferation in anagen
HFs. Importantly, we show the that absence of Tcl1
induces a signicant loss of the stem-cell marker CD34
(but not a6-integrin) expression in the bulge cells, which is
necessary to maintain stem-cell characteristics. Therefore,
our ndings indicate that Tcl1 gene(s) might have
important roles in hair formation, by its involvement in
cycling and self-renewal of transient amplifying (TA) and
stem-cell (SC) populations.
Oncogene (2009) 28, 13291338; doi:10.1038/onc.2008.489;
published online 26 January 2009
Keywords: Tcl1; hair follicle; bulge cells; secondary hair
germ cells; stem-cells; transient-amplifying cells

Introduction
The T-cell leukemia/lymphoma 1 (TCL1) gene has
been primarily identied for its involvement in human
Correspondence: Dr G Russo, Molecular Oncology Laboratory,
Istituto Dermopatico dellImmacolata, Istituto di Ricovero e Cura a
Carattere Scientico, Via dei Monti di Creta, 104, Rome 167, Italy.
E-mail: russo@idi.it
Received 30 July 2008; revised 25 November 2008; accepted 17
December 2008; published online 26 January 2009

T-prolymphocytic leukemia (T-PLL). Here, a reciprocal

translocation of TCL1 locus at 14q32 causes the
juxtaposition to T-cell-receptor enhancers, leading to
TCL1 ectopic overexpression (Russo et al., 1988;
Virgilio et al., 1994). Thus, TCL1 contributes to
T-PLL by increasing cell proliferation and/or cell
survival. Three transgenic mouse models carrying
TCL1 under the control of T- and B- cell promoters
develop mature T- and B-cell leukemia, thus conrming
the oncogenic role of TCL1 overexpression (Virgilio
et al., 1998; Bichi et al., 2002; Hoyer et al., 2002).
Moreover, TCL1 has been shown to be constitutively
expressed in a tightly regulated manner in lymphoid cells
of B and T origins (Narducci et al., 1997a, b; Takizawa
et al., 1998; Teitell et al., 1999; Kang et al., 2005).
Functionally, Tcl1 exerts its function by binding the
pleckstrin domain of AKT proteins, thereafter increasing AKT kinase activity and enhancing AKT nuclear
translocation in vitro (Laine et al., 2000; Pekarsky et al.,
2000; Kunstle et al., 2002) and in vivo (Zanesi et al.,
2006). In particular, the presence of Tcl1 allows
the serine-473 transphosphorylation of AKT proteins
in the oligomeric complex, thus ensuring maximal
kinase activity and facilitating an amplication loop of
the antiapoptotic, proliferative stimuli controlled by the
PI3-kinase-AKT pathway (Laine et al., 2002). More
recently, TCL1 has been shown to also bind another
type of PH domain (of the PNPase exoribonuclease)
(French et al., 2006).
Furthermore, the study of Tcl1 in murine systems
has shown that Tcl1 is expressed in the ovary, testis
and preimplantation embryos (Hallas et al., 1999;
Narducci et al., 1997a, b, 2002) and in murine embryonic stem (ES) cells (Ivanova et al., 2006). In particular,
it plays an important role in the latter as, in Tcl1
/

mice, preimplantation embryos are not able to
progress beyond the 4-cell stage, in vitro (Narducci
et al., 2002). In ES cells, Tcl1 acts in proliferation
and normal self-renewal under the direct activation
of OCT3/4, Zfx and KLF5 transcription factors
(Matoba et al., 2006; Galan-Caritad et al., 2007; Ema
et al., 2008).

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G Ragone et al

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Therefore, Tcl1 is not ubiquitously expressed, but

seems to be tightly regulated in cells that undergo
different waves of proliferative signals to complete their
differentiation programs: pro-B cells in the bone
marrow, the mantle lymphocytes of peripheral lymph
nodes in which B cells must encounter the antigen and
go through new waves of mitosis, the fertilized egg
before zygotic gene activation when the cell, still under
maternal control, must divide rapidly after having been
blocked in metaphase II for a long time and ES cells.
Since we made Tcl1
/
mice more than 5 years ago,
we have observed that the Tcl1
/
mice, other than the
lymphoid and embryonic non-lethal phenotypes (Narducci et al., 2002, Kang et al., 2005), show a skin defect
in adults with the onset of alopecia followed by skin
wounding. As the epidermis provides a model tissue to
investigate proliferation and self-renewal mechanisms,
particular attention has been focused on hair follicles
(HFs) as they go through cyclic regeneration that
comprises degenerative (catagen), resting (telogen) and
regenerative (anagen) phases (Stenn and Paus, 2001).
The bulge cells that reside in the HFs are assumed to be
the stem cells that drive the HF regeneration and
epidermal repair. Bulge cells express high levels of
CD34 (Blanpain et al., 2004; Tumbar et al., 2004), a well
known stem-cell marker of either hematopoietic or HF.
Here we provide the expression pattern of Tcl1 in the
mouse skin and an initial rst insight into the molecular
consequences of Tcl1 loss of function, in this compartment. These ndings indicate that Tcl1 has a role in the
maintenance of a normal skin homeostasis in mice,
involving both hair growth and epidermis.

Results
Tcl1
/
mice develop alopecia and skin impairment:
macroscopical and histopathological analysis
Tcl1
/
mice show no gross histological or physical
abnormality at birth, except a fertility defect and a mild
impairment of the immune system in adult life (Kang
et al., 2005; Narducci et al., 1997a, b, 2002). These mice
develop a form of mild alopecia starting from the head
and neck region (Figure 1, Supplementary Figures S1
and S2). The onset of this alopecia is variable, but we
never observe any phenotype before 2 months of age.
This phenotype result remains constant throughout the
years. Histological assessment of the affected regions
with respect to pre-alopecic condition (Figure 1b) shows
atrophy of the HFs, that is blocked at the telogen
(Figure 1c) and hypertrophy of epidermis with focal
localization (not shown). Thereafter, the alopecia
expands and the mice start to show a progressive
impairment of the skin tissue (Supplementary Figure
S2). The phenotype is rescued when decient Tcl1
/

mice are crossed to overexpressing TCL1 under keratin
14 promoter (K14TCL1; Tcl1
/
); these mice never
show alopecia and skin damages (Figure 1d).
Skin impairment shows up at around 6 months of
age, starting from regions with hair loss. Histological
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assessment at this time, shows deep-dermal inltration

by lymphocytes, plasma cells, eosinophils and mast cells,
with macrophages containing phagocytized melanotic
pigment (incontinentia pigmenti), because of continuous
damage to the basal layer of the epidermis that becomes
extensively hypertrophic. Later on, as the inltrate
intensies, ulcers and hyperplastic reparative modication of the epidermis and degeneration of subdermal fat
tissue, because of severe brosis with inltration of
inammatory cells (Figures 1ef), appear in Tcl1
/
mice
compared with normal mice. The peak of the onset of
the skin lesions is between the ages of eleven (12,58% of
Tcl1
/
) and fourteen months (62,3% of Tcl1
/
)
(n 18). Finally, 100% old Tcl1
/
mice (twenty months
old) show this phenotype.
Tcl1 is expressed in normal epidermal tissue and its
pattern is mainly restricted to early anagen and
catagentelogen transition phases of HF
These observations prompted us to investigate whether
Tcl1 is expressed in the wild-type (wt) mouse skin. We
carried out reverse transcriptasePCR analysis for
Tcl1 on wt RNA extracted from the dorsal skins of
6-weeks-old mice, 1, 3, 6 and 8 days after depilation,
corresponding to synchronised early anagen stages I, II,
III and IV of the HF development and skin differentiation (Panteleyev et al., 2000). The anagen phase involves
the complete re-growth and regeneration of the cycling
portion of HF. In all the examined samples, we found a
Tcl1-specic transcript (Figure 2a), meaning that Tcl1 is
expressed in the early anagen stages in wts.
To investigate whether the expression of Tcl1 is
cell-cycle and/or HF-phase dependent, we carried out
immunohistochemistry, immunouorescence and in situ
hybridization (with uorescent mRNA probe) on
6-weeks-old wt mice skin longitudinal sections at 1, 3,
5, 8, (anagen I to IV), 18, 21 (catagen), 22 (catagen
telogen), 23 and 24 (telogen) days after depilation. In the
epidermis, we found that Tcl1 is continuously, although
very weakly, expressed in all the examined stages
(Figure 2b, example at 8 days after depilation). In this
case, Tcl1 staining is detectable mainly by chemical, but
not by uorescent, labeling also because of autouorescence of the epidermal layers in our experimental
conditions. In the HFs, the expression is time restricted
at day 3, and from days 22 to 24 after depilation
corresponding, respectively, to early anagen (anagen II,
proliferating phase of HF) and to late catagenearly
telogen phases (the regressing-resting phases of HF)
(Muller-Rover et al., 2001). In anagen II, Tcl1 is observed
almost exclusively in the region of the outer root sheath
of the HF corresponding to the bulge (Figures 2ce), in
which HF stem-cells have been described (Blanpain et al.,
2004; Ito et al., 2004; Tumbar et al., 2004).
In catagentelogen phases, the expression pattern of
Tcl1 is more dynamic. In particular, although undetectable at day 21 (not shown), at day 22 post-depilation we
observe a strong labeling in the bulge (Figure 2f), with
also a weak expression of Tcl1 in the epithelial strand.
The epithelial strand, known to be a regressing

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Figure 1 Development of skin phenotype in Tcl1
/
mice. (a) Alopecia in the skin of a 6-months old Tcl1
/
mice. (b and c)
Hematoxylin-eosin on b: normal skin region and, c: alopecia skin region, of a 3-months old Tcl1
/
mouse showing rst symptoms of
hair impairment consisting in atrophic follicles (blocked at telogen, insert c). (d) A 6-months old K14TCL1
/
; Tcl1
/
mouse normal
skin and pelage. (e, f) Giemsa at this time shows hypertrophic epidermis (arrows), hyper keratinized epidermis (open black box), with
ulcers (at the center of gure), and sub-dermal (an example in black box) and dermal inammatory inltration by lymphocytes and
plasma cells (thinner black boxes), eosinophils and mast cells (examples indicated with short arrows) together with macrophages
containing phagocytized melanotic pigment (examples indicated with arrowheads) and brotic degeneration of the sub-dermal fat
tissue (examples indicated with arrows in panel f). The later phases of the dermatitis is also characterized by hyperplastic reparative
modication of the epidermis (left side of panel f). Bars: 184 mm (92 mm in the upper c panel). All the pictures are representative
examples. Other examples of affected Tcl1
/
mice are shown in Supplementary material.

structure, contains progenitor cells called secondary hair

germ (HG) cells that are able to proliferate in response
to growth signaling. At day 23-post-depilation, the
expression becomes stronger in these cells, and is
maintained in the bulge (Figure 2g). Later on, at day
24 post-depilation, as the expression in the regressed
epithelial strand reaches its maximum, the bulgelabeling decreases (Figure 2h); a denite border is
visible between the labeled epithelial strand and the
unlabeled dermal papilla. No expression of Tcl1 is
observed in Tcl1
/
mice (not shown).
Tcl1B2, another member of the Tcl1 family is expressed
in the HF
Although Tcl1 seems to be an important molecular
marker for at least two distinct cell populations in the
HF (the bulge cells and the secondary HG-cells), the late
onset and the apparent absence of molecular and
morphological phenotypes in HF itself prompted us to

investigate the possibility that the Tcl1 function(s) could

be complemented by the expression of other members
of the Tcl1 family. Therefore, we initially carried out
reverse transcriptasePCR on wt and Tcl1
/
RNA
samples at 22 days post-depilation, using primers
specic for each member of the gene family (Tcl1, and
B1 to B5). In this way, we were able to detect the
expression of Tcl1B2 gene (Figure 2i). In particular,
when comparing Tcl1
/
with wt, we observe that
Tcl1B2 transcription is also expressed in the former.
To better understand whether Tcl1B2 is expressed in
those regions with the loss of Tcl1 mRNA, we carried
out uorescence in situ hybridization experiments using
a probe specic for Tcl1B2 both on wt and Tcl1
/
skin
samples. We found that this gene is normally expressed
in the same regions of the homolog Tcl1, both in wt
and Tcl1
/
early anagen (Figures 2l and m) and in
catagentelogen (not shown). Tcl1B2 is also present
along the entire outer root sheath up to the epidermis
(not shown).
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Figure 2 (ah) Expression of Tcl1 in wt skin during early anagen and late catagentelogen stages. (a) Reverse transcriptasePCR on
6-weeks old wt mice at 1, 3, 6 and 8 days after depilation (lanes 14; lane 5 is positive control, lane 6 negative control). (b) One example
of immunohistochemistry on wt longitudinal skin sections at day 8 after depilation, in wt (upper panel) and Tcl1
/
mice (bottom
panel). Bar: 11.5 mm. (ce) Immunouorescence on wt anagen II HF (3 days after depilation). (c) Is the total projection of a confocal
stack of images, bar: 23 mm; red is PI nuclear staining, green is Tcl1 protein mainly localized in the bulge (arrow). (d) Is a scheme
representing the HF: HS is hair shaft, Ep is epidermis, apm is arrector pili muscle, Bu is bulge (green), HG is secondary hair germ (red),
DP is dermal papilla (tallow). (e) Is a confocal single plane image, bar: 6 mm; red is propidium iodide (PI) nuclear staining. Green is
Tcl1 protein in the bulge. Upper panel is the total projection of the entire stack of images (Z plane) on the X plane, rightmost panel is
the total projection of the entire stack of images (Z plane) on the Y plane. (fh) Tcl1 expression pattern in wt late catagen, catagen
telogen transition phases HFs (22, 23, 24 days after depilation). Confocal total projection of the entire stack of images. Green is Tcl1,
red is PI nuclear staining; bars: 23 mm. Arrows indicate bulge, arrowheads the secondary hair germ cells. (im) Expression pattern of
Tcl1B2 gene in wt and Tcl1
/
mice HF at different stages of differentiation. (i) RTPCR for Tcl1B2 (up) on wt (lanes 1 and 2) and
Tcl1
/
(lane 3) RNAs from late catagen skin samples, 22 days after depilation (bottom is b-actin positive control), showing the
expression of Tcl1B2 both in Tcl1
/
and wt mice. (i and l) Confocal total projection of the entire stack of images on wt and Tcl1
/

early anagen HF (day 3 after depilation), respectively. Green is Tcl1B2 RNA, red is Tcl1, blue is PI nuclear staining; bars: 11.5 mm.
Tcl1B2 expression is detectable both in wt and Tcl1
/
HFs. All the pictures are representative examples.

The absence of Tcl1 affects proliferation in the HF and

differentiation in epidermis
The epidermis contains self renewing and proliferating
cells, harboring stem-cell properties in the HF and
epidermal basal layer compartments (Stenn and Paus,
2001). The former are able to regenerate a follicle at
each cycle and to re-epithelialise the epidermis during
wound repair. We then assessed if Tcl1 was involved in
proliferative/apoptotic pathways in the skin, after which
we used the depilation technique (Chase, 1954; Paus
et al., 1990, 1994a, b, 1999) on wt, Tcl1
/
and newly
generated K14TCL1; Tcl1
/
mice, as this allows the
synchronization of the HF cycle starting from the
beginning of anagen. At day 3-post-depilation we found,
in wt mice, a strong Ki67 labeling in the epithelial
strand. At this time, this is a growing structure that
contains actively proliferating secondary HG-cells, also
known as transient amplifying (TA) cells. Ki67 staining
during anagen matches exactly with the expression
pattern of Tcl1 in catagentelogen (see arrowheads in
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Figures 2h and 3a), with no proliferation taking place in

the bulge (Figure 3a). Moreover, TdT-mediated dUTPdigoxigenin DNA-nick end labeling (TUNEL) cells
are present in the proximal basal structure of the old
degenerating hair (Figure 3a). In Tcl1
/
mice, we were
not able to detect any Ki67 proliferating cells in HF,
whereas TUNEL cells are present in the old
degenerating hair as in wt (Figure 3b). As no TUNEL
cells are present in the HF, these cells do not undergo
apoptosis, although they presumably cannot start their
proliferative program, being apparently blocked at G1
phase. The proliferative pattern of the secondary HGcells seems to be completely rescued in the K14TCL1;
Tcl1
/
transgenic mice (Figure 3c). Thus, these results
indicate that Tcl1 is required (provides signal) for the
proliferation of the secondary HG-cells.
In the same experiment, the presence of the
proliferating basal layer and the degenerating
keratinocytes of the cornied layer could be assessed
(Figures 3df).

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Figure 3 (ac) Proliferative and apoptotic phenotype in wt vs Tcl1
/
and K14TCL1; Tcl1
/
phenotype in anagen II HF. (ac)
Confocal total projection on the entire stack of images on wt, Tcl1
/
and K14TCL1; Tcl1
/
anagen II HFs, respectively. Green is
TUNEL, blue is PI nuclear staining, red is Ki67; bars: 23 mm (a and b), 16,1 mm (c). (a) In wt HF high Ki67 staining is visible in the
region of secondary hair germ cells (HG, arrowhead), whereas on the other hand few cells of the old hair structure and epidermis (Ep)
are ongoing apoptosis (arrows). (b) In Tcl1
/
mice proliferation is absent in the secondary hair germ cells (HG, arrowhead), whereas
(c) proliferation pattern is restored in the secondary hair germ cells from K14TCL1; Tcl1
/
mice. Bu: bulge. Asterisks in (b) indicate
that epidermis has not been fully acquired because it stays on different focal plane. (df) Proliferative and apoptotic phenotype in wt vs
Tcl1
/
and K14TCL1; Tcl1
/
phenotype in anagen II HF epidermis. Confocal total projection on the entire stack of images on wt,
Tcl1
/
and K14TCL1; Tcl1
/
anagen II epidermis, respectively. Green is TUNEL, blue is PI nuclear staining, red is Ki67; bars
23 mm. (d) In wt HF high Ki67 staining is visible in the region of the basal cells layer whereas on the other hand cells of the outer most
layer are ongoing apoptosis. (e) In Tcl1
/
mice most of the basal proliferative keratinocytes are also positive for apoptosis (arrows)
whereas (f) proliferation and apoptotic pattern is restored in the epidermis from K14TCL1; Tcl1
/
mice. All the pictures are
representative examples.

Usually in the wt, (Figure 3d) it is possible to

distinguish between the proliferating keratinocytes of
the basal layer and the degenerating keratinocytes of the
cornied layer. In the Tcl1
/
mice, in the basal layer
(Figure 3e), it is possible to observe that most of the
proliferating cells are also apoptotic, indicating that
these mice have a differentiationproliferation imbalance.
Again, in the K14TCL1; Tcl1
/
transgenic mice
epidermis, the phenotype seems to be completely
rescued, although more cells of the cornied layer seems
to undergo apoptosis with respect to the wt epidermis
(Figure 3f). Thus, these results indicate that Tcl1 is
required for the correct proliferativedifferentiation
program of the keratinocytes.
The absence of Tcl1 results in loss of CD34 expression in
anagen bulge cells
So far, we have shown that Tcl1 is expressed in HF
active cell populations, and its absence affects the
proliferative rate of these cells in anagen, probably
contributing to hair loss, in Tcl1
/
mice.
The earlier observation of the presence of Tcl1 in
compartments containing stemness ability prompted us
to investigate the expression of Tcl1 in the CD34 cells

of HF. In anagen, we found that Tcl1 co-localizes with

CD34 in the bulge cells in all the observed wt HFs
(Figure 4a); the absence of Tcl1 is associated with a
markedly decreased expression of CD34 itself in the
majority (69%) of the examined HFs of Tcl1
/
mice
(Figure 4b). The expression of CD34 is at least partially
(as quantity) rescued in K14TCL1, Tcl1
/
mice HFs
(Figure 4c).
No CD34 expression could be assessed at catagen
telogen in both Tcl1
/
and Tcl1 / mice. This could be
owing undetectable expression levels of CD34 in these
stages (data not shown).
We did not nd any difference in the expression of
another marker of stemness, a6-integrin, in Tcl1
/
vs
wt animals (not shown).
These results indicate that Tcl1 may play a role in
maintaining CD34 stem-cell properties in skin HF.
Tcl1
/
mice show dramatic decrease of BrdU cells in
the secondary HG and in the bulge
To study the in vivo effects of Tcl1 depletion on the
proliferation of stem cells (SCs) in HF, we carried out
BrdU incorporation assays. It is known that the
downregulation of CD34 in bulge cells leads to the loss
of stem-cell characteristics, such as slow cycling and
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Figure 4 (ac) Absence of Tcl1 results in loss of CD34 expression in Tcl1
/
anagen II HF and is partially rescued in K14TCL1;
Tcl1
/
mice HF. Confocal total projection of the entire stack of images on wt (a), Tcl1
/
(b) and K14TCL1; Tcl1
/
(c) mice anagen
II HF. Green is Tcl1, red is CD34, blue is PI nuclear staining; asterisks in (c) indicate that Tcl1 labeling has not been carried out to
better visualize CD34 expression. Bars: 11.5 mm (a and b), 15.5 mm (c). HG: secondary hair germ; Bu: bulge. All the pictures are
representative examples. (dg) BrdU incorporation in wt and K14TCL1; Tcl1
/
vs Tcl1
/
phenotype in anagen II HF. (dg)
Confocal total projection on the entire stack of images on wt (d) and Tcl1
/
anagen II HFs (e and f), and K14TCL1; Tcl1
/
(g) mice
anagen II HF, respectively. Green is BrdU, red is PI nuclear staining; bars: 23 mm. Although in wt HF (d), high BrdU staining is visible
in the region of secondary hair germ cells (HG), and in the bulge (Bu), in Tcl1
/
HFs no (e) or very few BrdU cells (f), can be scored.
BrdU staining comes back visible in the region of secondary hair germ cells (HG), and in the bulge (Bu), in K14TCL1; Tcl1
/
HFs
(g). All the pictures are representative examples.

[3H]thymidine or BrdU label retaining (Blanpain et al.,

2004; Tumbar et al., 2004). We administered BrdU
in vivo to Tcl1
/
mice to study the cycling of both bulge
label retaining SC as well as TA cells in the anagen
secondary HG of these mice. This is important also
because it is known that at least a subset of TA cells can
dedifferentiate and repopulate the bulge SC niche during
the very rst steps of anagen (Ito et al., 2004). So far
we have described that in Tcl1
/
mice there is a loss
of proliferating Ki67 cells (without increasing of
TUNEL cells) associated to a loss of CD34 cells in
the bulge: we then tested if this is a consequence of a
functional defect of TA cells to enter the cell cycle, after
the BrdU incorporation in the HFs during the rst 3
days of anagen. We found that, whereas in the wt mice
BrdU TA cells are diffused throughout the entire
secondary HG and are well present in the bulge
(Figure 4d), in the Tcl1
/
mice BrdU TA cells and
BrdU SC in the bulge are completely lost (Figure 4e).
In a few cases, we were able to nd up to 34 BrdU
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cells in the bulge, probably because of the residual cells

from the hair plucking (Figure 4f). The number of
BrdU
HFs in Tcl1
/
is similar to those of CD34
in
the same mice described above. Conversely, BrdU
incorporation in the secondary HG-cells seems to be
completely rescued by the K14TCL1, Tcl1
/
transgenic mice (Figure 4g).
Taken together, all our observations strongly indicate
that the expression of Tcl1, in the catagentelogen
transition, contributes to the TA cell to entering the cell
cycle and, therefore, in the proliferation and repopulation of the bulge SC niche and nally, in the creation of
a new hair cycle.

Discussion
The observation that Tcl1
/
mice develop a mild but
constant and unexpected phenotype in the skin,

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1335

prompted us to investigate the potential role of Tcl1 in

the epidermis and skin appendages. We found that Tcl1
is expressed in the bulge of the anagen phase and in the
secondary HG-cells in the catagentelogen phase of the
HF of wt mice, and in the Tcl1
/
mice, there is a lack
of proliferating cells following synchronization after
depilation.
The expression of Tcl1 is very high in the bulge at the
anagen stage and to a extent during the catagentelogen
transition in the secondary HG-cells. Lines of evidence
support the idea that HF-secondary germ cells have an
intrinsic, bulge-region independent, proliferative potential and are directly involved in the early events of HF
anagen induction (Cotsarelis et al., 1990; Morris and
Potten, 1999; Panteleyev et al., 2001). Furthermore, no
cell migration to the HG from the bulge region has been
reported in telogen (Morris and Potten, 1999; Ito et al.,
2004). It has also been shown that the lower portion of
the bulge collapses and undergoes gene expression
changes, giving origin to the secondary HG-cells in late
catagen (and not in telogenanagen transition), and that
these cells are able to dedifferentiate again into bulge
cells and repopulate the bulge niche at the beginning of
the anagen (Ito et al., 2004). It is interesting that the
expression pattern of Tcl1 strictly follows the events
described above (Figure 5).
In anagen, loss of Tcl1 implicates less proliferative
activity in the bulge (SC) and secondary HG-cells (TA),
as shown by BrdU incorporation and Ki67 labeling. If
the expression of Tcl1 is maintained constantly within
the same cells, as it seems to be in other tissues such as B
cells and embryo, it would implicate that proliferating
cells that derive from HG-cells, during catagentelogen

transition, are those starting the new HF cycle and

repopulating the bulge (Figure 5). As in the embryo,
where Tcl1 is necessary in the rst divisions of
proliferating cells of the preimplantation embryo, and
for self-renewal but not differentiation of their derived
ES-cells (Matoba et al., 2006; Galan-Caritad et al.,
2007) in the HF, Tcl1 is important very early during the
anagen proliferation of secondary HG-cells, as beyond
the third day after depilation the expression of Tcl1 is no
more detectable.
It is interesting to observe that in wt, CD34 and Tcl1
colocalize in the same cells (SC) and that abrogation of
Tcl1 leads to the loss of CD34, and this could be owing
to the fact that some of the SC that are repopulating the
bulge derive from TA cells as proposed (Ito et al., 2004).
it is also interesting to observe that HFs derived from
CD34
/
mice and Tcl1
/
mice are blocked at telogen
(Trempus et al., 2007).
All these ndings provide a new specic marker for
secondary HG-cell population and bulge cells, and
denitely place back in time, at late catagen and not in
telogenanagen transition, the commitment status of the
former.
We mainly studied Tcl1 phenotype in HFs of Tcl1
/

mice in those stages in which Tcl1 is expressed in wt
mice. Although no apparent major morphological
differences are visible between wt and Tcl1
/
mice
HFs, Tcl1
/
mice develop alopecia, and this alopecia is
progressive with the age and affects 100% of the Tcl1
/

mice. Moreover, in these mice, epidermis in the alopecia
area undergoes inammation, hypertrophy and nally
wounds. The observation of the rescue of the alopecia
and wounds by simply overexpressing TCL1 into

Figure 5 Schematic representation of the HF during the hair cycle. At late catagen, catagentelogen transition the bulge cells divide
and give origin to the secondary hair germ cells. At that time these cells acquire the TA potential. Once induced the anagen
(regenerative) phase by depilation, the TA cells start to actively proliferate, giving origin to a new growing structure on one side and,
in a small percentage to the repopulation of the bulge SC niche (that were destroyed by depilation) on the other side. Ep: epidermis;
HS: hair shaft; apm: arrector pili muscle; Bu: bulge; HG: secondary hair germ; DP: dermal papilla.
Oncogene

The Tcl1 gene and hair follicle cycling in mice

G Ragone et al

1336

keratinocytes in K14TCL1; Tcl1
/
mice indicates that

dermal immune inltrate is the consequence, more than
the cause, of the skin impairment. Whether it is owing to
problems of the innate immune response (leaded by skin
integrity), a higher susceptibility to external agents or
simply to intrinsic cellular defects of keratinocytes is the
goal of further investigations, even though the rescue of
proliferation, CD34 expression and BrdU incorporation
in transgenic mice supports the last hypothesis. Here we
show that CD34 cells are diminished in Tcl1
/
mice.
We do not know if and how Tcl1 regulates CD34
expression; however, it has already been reported that
Tcl1 and CD34 are co-expressed in the lymphoid
compartment in the very early stage of B-cell differentiation (pro-B cells) (Narducci et al., 2000), and that
this compartment of pro-B cells is also less numerous in
the Tcl1
/
mice (Kang et al., 2005).
Together with the inammation and hypertrophy, the
CD34 bulge cell pool derived from the secondary
HG-cells dedifferentiation could easily nish and
explain the onset and progression of alopecia with age
in these mice. It is known that from the bulge arise,
among others, those cells involved in epidermal wound
repair (Ito et al., 2005; Trempus et al., 2007). In the
absence of secondary HG-cells with proliferative status,
bulge cells are progressively lost after some cycles, and
this ultimately increases the CD34 depletion pool.
Tcl1 function is exerted in HF stem and proliferating
cells, as its absence affects CD34 expression. The
mechanism(s) through which this takes place will be
the subject of further investigations. Consequently in
Tcl1
/
mice, we observe a loss of bulge cells and a
decreased, if not absent, proliferative rate in early
anagen, when a new structure needs to be formed after
the wounding stimulus of depilation.
The variability of the skin phenotype could be owing
different overlapping or redundancy. Given the high
similarity with Tcl1, these genes could act as functional
homologs in the HF. In fact, we observed transcriptional levels of Tcl1b2 but not of Tcl1b1, 3, 4 and 5,
which were not detectable neither by reverse transcriptasePCR nor by uorescence in situ hybridization, both
on wt and Tcl1
/
mice HFs.
We were unable, on these mutant mice, to show by
immunohistochemistry any direct effect on AKT phosphorylation variation (not shown), although this should
not be excluded by observing the role that PI3/Akt
signaling exerts in postnatal epidermal development and
SC-self renewal, and production of committed cells in
hematopoiesis. Work here might be complicated by
redundant functions of the family members of Akt1/2/3
and Tcl1 gene family. However, it is interesting to
observe that in the epidermis, deletion of Akt1 causes
thinner epidermal layers and retarded-HF development,
(Di-Po et al., 2005) whereas double mutants (Akt1
/

Akt2
/
and Akt1
/
Akt3
/
) (Peng et al., 2003; Yang
et al., 2005) show more severe defects, and transgenic
mice expressing AktMer fusion proteins induces
epidermal hyperplasia and proliferation of epidermal
progenitors leading to epidermal and follicular hyperplasia (Murayama et al., 2007). As already pointed out
Oncogene

before, Tcl1 is involved in the normal-self renewal of ES

cells (Glover et al., 2006; Matoba et al., 2006; GalanCaritad et al., 2007) and upstream genes such as
transcription factor Oct4, which directly activates Tcl1
in ES cells and is expressed in human HFs (Yu et al.,
2006), should also be taken into account.
In conclusion, we show that Tcl1 has a novel,
important role in skin homeostasis. This adds to many
other hematopoietic factors that are expressed in the
HFs: CD34, but also Tcf-3/Lef-1, NFATC1, S100A4,
LTBP1, b1-integrin and so on. (Tiede et al., 2007). We
believe that this study may contribute to a better
understanding of the cellular mechanisms underlying
the physiology of a population of adult stem cells
and to the opening of a novel eld of study for TCL1
function.

Materials and methods

Animals
Wild type, Tcl1
/
and K14TCL1; Tcl1
/
mice background
is C57BL/6NCrlBR (C57BL/6) (Narducci et al., 2002). K14
hTCL1 transgenic mice was obtained by cloning the human
TCL1 cDNA (Bichi et al., 2002) into a pG3Z vector containing a
K14 cassette (pG3ZK14, gift from E Fuchs), with BamHI as
insertion site. The plasmid was microinjected into C57BL/
6NCrlBR (C57BL/6) ES cells at the Thomas Jefferson University
Animal Facility at Philadelphia (PA, USA). Founders and pups
were genotyped by PCR using the primers: b-globin 1: 50 -CTG
CTA ACC ATG TTC ATG CC-30 and 210 R: 50 -GAT CGG
TAT CGT CCA TCA GG-30 . K14hTCL1 genotypes into the
Tcl1
/
background also were determined by using the primers:
G418for: 50 -ATT GTC TTC CCA ATC CTC CC-30 ; rev:
50 -CGA CTG TGC CTT CTA GTT GC-30 and INTR-2for:
50 -GGG TGA TCT TGA GTG ATC CTG-30 ; rev: 50 -GTG
GCT AGC CCT AAC TG-30 . Amplication was carried out
using Taq DNA polymerase (Promega Co., Madison, WI, USA)
and an automated thermal cycler (PTC-200; MJ Research Inc.,
Waltham, MA, USA). Each cycle consisted of denaturation at
94 1C (45 s), annealing at 60 1C (1 min) and extension at 72 1C
(1 min). Synchronized HF cycling on wt, K14TCL1; Tcl1
/
and
Tcl1
/
mice was induced as already described (Chase, 1954; Paus
et al., 1990, 1994a, b, 1999). Depilated skin was harvested as
described (Paus et al., 1999).
RTPCR
Total RNA was isolated from back skin samples using TRIREAGENT protocol (Sigma-Aldrich, St Louis, MO, USA).
3 mg total RNA was used to produce cDNA. Tcl1 was
amplied for 40 cycles followed by 40 cycles of nested PCR
amplication. Primers used to amplify cDNAs are listed in
Supplementary materials.
Immunofluorescence
Sections were xed in 4% paraformaldehyde and permeabilized with phosphate buffered saline/0.1%TritonX-100. Incubations for 2 h at room temperature with primary antibodies
were followed by 1 h incubations with the correspondening
secondary antibodies. Blocking was 10% normal goat serum
or 3% bovine serum albumin. Nuclei counterstain: 2 mg/ml of
propidium iodide (PI) dye. Antibodies used: monoclonal rat
anti-Tcl1 (5A4) 1:50 and polyclonal rabbit anti-CD34 1:100
(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

The Tcl1 gene and hair follicle cycling in mice

G Ragone et al

1337
TdT-mediated dUTP-digoxigenin DNA-nick end labeling
was carried out using the in situ cell death detection, POD
(Roche Corp., Indianapolis, IN, USA), according to the
manufacturers protocol, followed by Ki67 immunodetection
using a rabbit antiserum 1:1500 (Novocastra Labs Ltd.,
Newcastle upon Tyne, UK). Fluorescein isothiocyanate
(FITC)- or CY5-conjugated anti-rat, anti-rabbit or anti-goat
IgG was obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA). The specimens were
observed by laser scanning confocal microscopy using a Zeiss
LSM 510 Meta microscope. The experiments were carried out
twice on different mice (males and females).
Immunohistochemistry
Incubation for 2 h at room temperature with rat anti-Tcl1
primary antibody 5A4 (Narducci et al., 2002) 1:50 was carried
out after xation in 4% paraformaldehyde and permeabilization with phosphate buffered saline/0.1%TritonX-100. Endogenous peroxidase was blocked with 0.3% hydrogen
peroxide in methanol followed by 1 h incubation with
biotinylated rabbit anti-rat IgG 1:200 and 150 incubation with
horseradish peroxidesstreptavidin (LSAB system, DAKO
Corp., Glostrup, DK). Staining was carried out with 3-30
diamino benzidine reaction and counterstaining with hematoxylin. Giemsa and hematoxylin-eosin experiments were
carried out following standard procedures. The experiments
were carried out twice on different mice (males and females).
In situ hybridization
The 249 bp mouse Tcl1B2 cDNA obtained by PCR was
subcloned into pCRII. FITC labeled RNA probes were
synthesized with SP6 and T7 RNA polymerases, according
to the manufacturers instructions (Roche).
Hybridizations were carried out at 56 1C O/N after 4%
paraformaldehyde xation and 0.1 M triethanolammine/0.25%
acetic anhydride post-xation. Then O/N incubation at 4 1C with
a rabbit anti-FITC 1:100 (Zymed Inc., San Francisco, CA, USA)
was carried out followed by a 4 h incubation at room temperature
with a FITC-conjugated goat anti rabbit IgG. Nuclei were
counterstained with PI dye. Sense strands were used as negative

controls. Acquisitions and number of experiments are those as

indicated above.
BrdU labeling and detection
8-weeks-old C57BL/6NCrlBR wt, Tcl1
/
and K14TCL1;
Tcl1
/
mice were plucked as mentioned. At 24 h later, the mice
were subcutaneously injected with BrdU (50 mg/g body weight)
twice daily for 4 days. Three days after plucking the dorsal
skins were excised, embedded in OCT and frozen.
Sections were xed with 30 ml of 50 mM glycine added to
70 ml of absolute ethanol, pH 2.0 for 20 min at
20 1C, washed
with phosphate buffered saline and incubated with an antiBrdU working solution (Roche) containing nucleases. Animal
research kits were used (DAKO Corp., Glostrup, DK). The
sections were incubated with 1:50 diluted FITC-conjugated
streptavidin (Vector Labs, Burlingame, CA, USA) for 30 min
at room temperature; nuclei were counterstained using PI dye.
Acquisitions and number of experiments were those as
indicated above.

Abbreviations
HF, hair follicle; ORS, outer root sheath; SC, stem cells
TA, transient amplifying; TCL1, T-cell leukemia/lymphoma 1.
Acknowledgements
We thank M Helmer Citterich and Paolo Fadda for technical
assistance, sequencing and microarrays services and Dr G
Zambruno for helpful comments during the preparation of the
paper. This work was supported by grants from the
Associazione Italiana Ricerca sul Cancro (AIRC) and from
the Ministero della Sanita`.

Conict of interest
The authors declare no conict of interest

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Oncogene