Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling

Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling
Circadian Clock Genes Contribute to the Regulation of
Hair Follicle Cycling
Kevin K. Lin1,2,3, Vivek Kumar4,5,6, Mikhail Geyfman1, Darya Chudova3,7, Alexander T. Ihler7, Padhraic
Smyth3,7, Ralf Paus8,9, Joseph S. Takahashi4,5,6, Bogi Andersen1,2,3*
1 Department of Biological Chemistry, University of California Irvine, Irvine, California, United States of America, 2 Department of Medicine, University of California Irvine,
Irvine, California, United States of America, 3 Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, California, United States of America, 4 Howard
Hughes Medical Institute, Northwestern University, Evanston, Illinois, United States of America, 5 Department of Neurobiology and Physiology, Northwestern University,
Evanston, Illinois, United States of America, 6 Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America,
7 Department of Computer Science, University of California Irvine, Irvine, California, United States of America, 8 Department of Dermatology, University of Luebeck,
Luebeck, Germany, 9 School of Translational Medicine, University of Manchester, Manchester, United Kingdom
Abstract
Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence
(telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair
follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase
with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the
secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice
reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and
lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle.
Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin.
While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate
that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.
Citation: Lin KK, Kumar V, Geyfman M, Chudova D, Ihler AT, et al. (2009) Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling. PLoS
Genet 5(7): e1000573. doi:10.1371/journal.pgen.1000573
Editor: Sarah E. Millar, University of Pennsylvania School of Medicine, United States of America
Received February 26, 2009; Accepted June 23, 2009; Published July 24, 2009
Copyright: ß 2009 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants AR44882 to BA, NRSA 5 T15 LM00744 to KKL and DC, NRSA postdoctoral grant 1F32DA024556-01 to VK, NSF
grant IIS-0431085 to PS, and in part by Deutsche Forschungsgemeinschaft to RP (Pa 345/12-1). JST is an Investigator and VK was an Associate of Howard Hughes
Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bogi@uci.edu
during the telogen phase, with the caveat, however, of triggering
an injury response [3]. Although components of numerous
molecular pathways, including TGFb/BMP family members and
their antagonists, FGFs and steroid hormone receptors, have been
implicated in the control of hair follicle cycling [1,4–8], the
underlying mechanisms regulating its timing remain elusive [9].
The periodicity of the hair growth cycle is reminiscent of other
cyclic processes, such as the circadian rhythm where distinct clock
mechanisms exist. The regulation of circadian rhythm consists of
positive and negative regulatory feedback loops, having a period of
approximately 24 hours [10–12]. The positive feedback loop is
regulated by two DNA-binding basic helix-loop-helix (bHLH) PAS
domain transcription factors CLOCK and BMAL1 [13], which
form a heterodimer and transcriptionally activate genes containing
E-boxes in their regulatory regions. Among their targets are PER
(PER 1, 2 and 3) and CRY (CRY1 and 2) that form heterodimers
and repress their own transcription via direct interactions with
CLOCK/BMAL1 [14]. Additional targets include RORs (Rora,
Rorb, and Rorc) and REV-ERBs (Nr1d1 and Nr1d2), which are
members of a subfamily of orphan nuclear receptors that bind to
ROR response elements (RREs) to transcriptionally activate and
repress Bmal1, respectively [15,16]. Other CLOCK-controlled
targets, such as the transcription factors DBP and TEF, are not
Introduction
Evolutionarily conserved hair follicle cycling is thought to
provide mechanisms for controlling the length of hair in specific
body sites, and to allow the periodic shedding of fur in response to
seasonal changes in mammals [1]. The periodicity of the hair
growth cycle ranges from approximately three-weeks in synchronized hair follicles of mouse dorsal skin to several years in hair
follicles of human scalp where the follicles undergo an extended
period of hair growth [2]. In mice, hair follicle morphogenesis is
completed around postnatal day (P) 14, at which time the follicle
enters a phase called catagen. During catagen, extensive apoptosis
in the lower two-thirds of the follicle results in its dramatic
regression, leaving intact the stem cell-containing bulge region.
The hair follicle then goes through a relative quiescent phase
referred to as telogen. Following telogen, the stem cells become
activated, likely in response to inductive signals from the dermal
papilla, and the follicle enters the growth phase characterized by
active keratinocyte proliferation and differentiation known as
anagen. During the first two natural hair growth cycles in mice,
the follicles of the dorsal skin are synchronized in progressing
through the cycle, allowing the study of the mechanisms of natural
hair follicle cycling. In addition, tightly synchronized hair growth
cycle can be initiated by depilation of hair shafts (e.g., waxing)
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Clock Genes in Hair Follicle Cycling
due to initial hair follicle morphogenesis or injury response to
depilation. Given this model structure and observed data, we used
Bayesian inference techniques to infer periodic profiles and
posterior probability of periodicity for each of the probe sets
[28] (Text S1).
Applying this model to the 28,341 expressed probe sets, we
found that 8433 probe sets have posterior probability of greater
than 0.95 of being periodically expressed (Figure 1D, top panel).
Probe sets with periodic profiles over the hair growth cycle were
further narrowed to 6393 probe sets exhibiting periodic gene
expression changes that cannot be explained by changes in tissue
composition (Figure S1); we define this set of genes as hair cycleregulated genes (Figure 1B and Table S1). To assess the sensitivity
of the calculated posterior probabilities, we compiled a list of
literature-based hair cycle-dependent genes from a comprehensive
unbiased literature search of genes whose expression patterns have
been shown to be hair cycle-dependent using quantitative or semiquantitative RNA methods (Table S2). We found that over 80% of
literature-based hair cycle-dependent genes have posterior probabilities of greater than 0.95 (Figure 1D, bottom panel); for genes
that have posterior probabilities of lower than 0.95, their
expression profiles either do not visually appear to be periodic
or are detected at a low intensity level. From these findings, we
conclude that our probabilistic model is accurate and robust in
identifying periodic expression changes during hair follicle cycling.
Author Summary
The hair follicle renews itself by repeatedly cycling among
growth, regression, and rest phases. One function of hair
follicle cycling is to allow seasonal changes in hair growth.
Understanding the regulation of hair follicle cycling is also
of interest because abnormal regulation of hair cycle
control genes is responsible for several types of human
hair growth disorders and skin cancers. We report here
that Clock and Bmal1 genes, which control circadian
rhythms, are also important for the regulation of hair
follicle cycling, a biological process of much longer
duration than 24 hours. Detailed analysis of skin from
mice mutated for central clock genes indicates a significant
delay in the progression of the hair growth phase. We
show that clock genes affect the expression of key cell
cycle control genes and that keratinocytes in a critical
compartment of the hair follicles in Bmal1 mutant mice are
halted in the G1 phase of the cell cycle. These findings
provide novel insight into circadian control mechanisms in
modulating the progression of cyclic biological processes
on different time scales.
part of the central circadian mechanism, but are thought to
mediate many of its physiological effects [17,18].
In mammals, the central pacemaker that controls circadian
behavior is in the suprachiasmatic nucleus (SCN) of the
hypothalamus. It is now widely accepted that the circadian control
mechanisms are not limited to the SCN, but instead, the molecular
clock is expressed and operative in most peripheral tissues [19–21].
Increasing evidence highlights the importance for the SCN in
coordinating the independent oscillation of molecular clocks in
peripheral tissues [10]. Although several studies demonstrated
circadian rhythm within human and mouse skin as well as oral
keratinocytes [22–26], a functional role for the circadian clock
genes in skin has yet to be determined. Taking a genomics
approach to study gene expression during synchronized mouse
hair follicle cycling, we discovered that the genes which control
circadian rhythm are utilized to modulate the progression of hair
follicle cycling, a biological process of much longer duration than
the diurnal period. We found a significant delay in anagen
progression in both Clock and Bmal1 mutant mice, and the
secondary hair germ cells within mutant hair follicles show
decreased levels of phosphorylated Rb and lack mitotic cells,
suggesting the circadian clock modulate anagen progression via its
effect on the cell cycle.
Distinct Functional Groups of Genes Are Activated at
Different Phases of Hair Follicle Cycling
To identify genes which are activated at specific phases of the
hair growth cycle, we performed statistical differential analysis of
hair cycle-regulated genes by comparing the expression from one
phase to the next (e.g., genes upregulated at early anagen are
significantly differentially expressed genes between telogen and
early anagen samples). We then searched for significantly enriched
Gene Ontology (GO) Biological Process categories within the
different sets of genes upregulated at specific phases of the hair
growth cycle. Among the genes upregulated at early anagen is a
significantly overrepresented number of cell cycle and DNA/RNA
metabolism genes, as well as other genes that are required during
proliferation (Figure 1E). Many of the other enriched functional
categories are also expected based on our current knowledge of
hair follicle biology, validating the value of this approach. For
example, members of the Hedgehog signaling pathway are
upregulated during early anagen (Figure S2), consistent with their
role in proliferation of keratinocytes. In contrast, TGFb/BMP
signaling pathway genes are upregulated towards the later stages of
anagen, and these genes have been reported to be involved in hair
follicle differentiation and apoptosis. In addition to identifying
several pathways known to be involved in hair follicle regulation
[1,4–7], our enrichment analysis identified functional categories
that were unexpected, potentially providing novel insights into
regulation of hair follicle cycling. For instance, telogen is often
referred to as the ‘‘quiescent phase’’ of the cycle, but we found
significantly enriched group of genes annotated with the function
of generation of precursor metabolites and energy (P = 1.97E-4) and
establishment of localization (P = 3.39E-2) that are upregulated during
telogen, suggesting that many active molecular processes are
occurring during telogen (Figure 1E).
Results
Identification of Periodically Expressed Genes during Hair
Follicle Cycling from Time-Course Expression Profiles
To investigate the molecular control of hair follicle cycling, we
profiled mRNA expression in mouse dorsal skin at multiple time
points in the synchronized second postnatal hair growth cycle and
in a depilation-induced hair growth cycle. By combining this data
with a study profiling the postnatal completion of hair follicle
morphogenesis as well as the first catagen and telogen [27], we
obtained genome-wide time-course expression profiles for three
distinct hair growth cycles (Figure 1A and 1B). To detect genes
with periodic expression changes over multiple hair growth cycles,
we constructed a probabilistic model with periodic and background components (graphical model shown in Figure 1C). The
periodic component includes a mixture model of shared periodic
expression profiles and allows deviation from perfect periodicity
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CLOCK–Regulated Genes Are Periodically Expressed in
Phase with the Hair Growth Cycle
To uncover the genome-wide landscape of transcriptional
regulation during hair follicle cycling, we performed time-course
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Figure 1. Identification of hair cycle–regulated genes using probabilistic models. (A) Representative time points selected for gene
expression profiling of the natural and depilation-induced hair growth cycles. Time points for natural cycles represent postnatal days, and time points
for depilation-induced cycle indicate the number of days following depilation. Since the three cycles have different duration, the schematic timeline is
not in actual time scale. Bottom panel consists of representative histology of dorsal skin at key phases of synchronized hair follicle cycling. (B)
Overview of data processing and statistical analyses. Note that samples for the first synchronized hair growth cycle and asynchronous time points
were profiled in an earlier study using an older generation array (Affymetrix Murine Genome U74Av2). (C) Schematic of the probabilistic model for
detection of periodic gene expression changes during hair follicle cycling. See Materials and Methods for a detailed description of the schematic.
Asynch – asynchronous cycle. (D) Histogram of the number of probe sets within each range of the indicated posterior probabilities of being
periodically expressed. Top panel is the histogram of probabilities for all present genes during the hair growth cycle. The bottom panel is the
histogram of probabilities for the literature-based hair cycle-dependent genes. (E) List of significantly enriched GO Biological Process categories
within the sets of genes specifically upregulated at the indicated phases of the hair growth cycle. Due to the redundancy of categories, not all are
listed. The number of genes upregulated at each phase of the cycle is in parentheses. Statistical enrichment of each category is shown as P-values
calculated using a modified Fisher Exact test (DAVID functional annotation analysis).
doi:10.1371/journal.pgen.1000573.g001
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described. Correspondingly, in the set of genes significantly
upregulated at telogen we also found a statistical enrichment of
the GO annotation category rhythmic processes (Figure 1E). A time
course expression profile covering hair follicle morphogenesis and
the synchronized postnatal hair growth cycles shows upregulation
of these CLOCK-regulated genes during telogen phases
(Figure 2B). In an independent Q-PCR experiment, we confirmed
up-regulation of Dbp around the first and second telogen, and
found that the expression stays elevated during early anagen
(Figure 2C). However, without a suitable DBP antibody for
western/immunohistochemistry, we were unable to determine
whether this upregulation of Dbp transcript levels is reflected in
increased protein levels. The levels of PER2 proteins in whole skin,
however, do not appear to be different between telogen and late
clustering of all hair cycle-regulated transcriptional regulators.
Remarkably, this genome-wide landscape grouped together many
of the key transcription factors previously shown to be functionally
important during hair follicle morphogenesis and/or cycling
(Figure 2A). Mice with mutations to these transcription regulators
show abnormalities in hair follicle density (Lef1, Msx1, Msx2,
Sox18, Trps1), structure (Dlx3, Foxn1, Foxq1, Hoxc13, Notch1, Ovol1,
Runx1), morphogenesis (Ctnnb1, Cutl1, Gli2, Lef1, Msx2, Smad7), and
cycling (Hr, Msx2, Stat3, Vdr).
Among the hair cycle-regulated transcriptional regulators, we
were intrigued to identify a co-expressed cluster of transcription
factors that are targets of circadian protein CLOCK, including
Dbp, Per1, Per2, Cry2, Nr1d1, Tef and Hlf (Figure 2A); a hair follicle
cycle-related expression of these genes has not been previously
Figure 2. CLOCK–regulated genes are periodically expressed during hair follicle cycling. (A) Temporal clusters (labeled 1–6 and color
coded) of hair cycle-regulated transcriptional regulators. Transcription factors that play key roles in hair follicle morphogenesis and/or cycling are
labeled with gene symbols in black. CLOCK-regulated genes are labeled in the blue box. Expression levels are from profiling data of the second hair
growth cycle and are indicated by the colorimetric ratio-scale. (B) Time-course profiles of CLOCK-controlled genes during hair follicle morphogenesis,
the first two natural and depilation-induced hair growth cycles. For each gene, the expression levels were normalized relative to the lowest expressed
time point of the second cycle; the first cycle (P1 to P23) was normalized separately because different array was used for profiling. Differences in
magnitude of change between the first and second cycles are primarily due to differential probe set efficiencies. Note the broken y-axis. E –
embryonic days; P – postnatal days; D - depilation days. (C) Q-PCR of Dbp using independent samples from the first two synchronized hair growth
cycles. Standard deviations were determined by using three replicates normalized to Gapdh and fold calculated relative to the lowest expression
sample. For (B–D), time points are mapped based on histology to the corresponding phases of the cycle: hair follicle morphogenesis (M), anagen (A),
catagen (C), and telogen (T).
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anagen (Figure S3C). Thus, in murine skin, CLOCK-regulated
genes unexpectedly exhibit periodic expression in phase with the
hair growth cycle, a cyclic process with a much longer time scale
than circadian rhythms.
Activation of CLOCK–Regulated Genes in Skin Is CLOCK/
BMAL1–Dependent
To determine whether the expression of CLOCK-regulated
genes in skin is dependent on CLOCK/BMAL1 activation, we
profiled the dorsal skin of Bmal12/2 and Bmal1+/2 control
littermates at P22, when the hair follicles for both genotypes are in
the first synchronized telogen (Figure 5A). Consistent with the
mammalian circadian transcriptional circuit, functional annotation analysis of the 339 probe sets that are significantly
differentially expressed in Bmal12/2 skin identified statistical
enrichment (P = 5.7E-8) of genes annotated with the GO category
of rhythmic processes (i.e., circadian clock genes). Specifically, clock
genes with E/E9 or D-box in their regulatory regions (e.g., Nr1d1,
Nr1d2, Per1, Per2, Per3, Dbp, and Bhlhb3) are significantly
downregulated in Bmal12/2 mice (Figure 5B) [17], consistent
with attenuated transcriptional activation by the CLOCK/
BMAL1 heterodimer. The downregulation of Nr1d1 and Nr1d2
relieves the transcriptional repression of clock genes with RREs in
their regulatory regions (e.g., Clock, Bmal1, Npas2, Cry1, Nfil3, and
Rorc), resulting in significant upregulation of RRE-containing clock
genes in Bmal12/2 skin (Figure 5B) [17]. We confirmed by Q-PCR
the significant differential expression of CLOCK-regulated genes
in Bmal1 and Clock mutant skin (Figure 5C and 5D), indicating that
the activation of these genes is CLOCK/BMAL1-dependent in
skin.
A particularly intriguing finding of the microarray analysis of
Bmal12/2 skin is the significant differential expression of Rorc and
Nr1d1 (Figure 5B). Rorc, which was recently shown to be a critical
transcriptional activator of the G1 cell cycle regulator Cdkn1a (p21)
[34], is upregulated approximately 3-fold (Figure 5C). Conversely,
Nr1d1, a repressor of Cdkn1a expression [34], is downregulated
approximately 15-fold. Consistent with these changes, we found
that Cdkn1a is upregulated 2.5-fold (Figure 5C). With the exception
of Wee1, which has been shown to be a CLOCK/BMAL1 target
gene [35], other cell cycle regulators (e.g., Cdkn1b, Cdkn2b and Myc)
are not significantly differentially expressed in Bmal12/2 skin
(Figure 5C). Together, these results show that Cdkn1a, an
important inhibitor of cell cycle progression, is upregulated in
the skin of Bmal12/2 mice, suggesting the possibility that clock
genes may regulate cell proliferation within skin.
CLOCK–Regulated Genes Have Enhanced Circadian
Expression during Telogen
The expression of CLOCK-regulated genes oscillates over a
24-hour period [10–12], while we observed a hair cycleassociated oscillation over a period of several weeks (Figure 2).
Therefore, to understand the nature of the up-regulation of these
genes during telogen, we performed Q-PCR for Dbp, Nr1d1, Per2,
Bmal1, and Clock of telogen and late anagen mouse dorsal skin
over the course of 48 hours of normal light/dark cycles (Figure 3).
For Dbp, Per2, and Nr1d1, we observed circadian oscillations of
expression in both telogen and late anagen, but the amplitude of
the oscillation is significantly enhanced during telogen
(Figure 3C–3E). The peak expression of Dbp and Nr1d1 is
approximately 3-fold higher (P,0.01) in telogen than late
anagen. Similar to other peripheral tissues, such as the liver
[18], we found the peak of expression for Dbp and Per2 to be at
Zeitgeber time (ZT) 10 and 14, respectively, where ZT0 is when
light is switched on and ZT12 when light is switched off
(Figure 3C and 3D). As expected, Clock and Bmal1 have
antiphasic circadian profiles to their target genes with peak at
ZT22 (Figure 3A and 3B), suggesting that enhanced telogen
expression of CLOCK-regulated genes occurs in the context of
normal circadian expression profiles.
Prominent Expression of Clock Genes in the Secondary
Hair Germ during Telogen and Early Anagen
To identify the potential site of action for circadian mechanisms, we characterized the in situ skin expression of Bmal1 and its
target gene Dbp, a robust marker for clock output [18]. During
telogen and early anagen, the most striking site of expression for
both genes is in the secondary hair germ, an epithelial
compartment located next to the dermal papilla (Figure 4 and
Figure S3A). Interestingly, the secondary hair germ is derived
from hair follicle bulge epithelial stem cells [29] and contains
Lgr5-positive cells, a recently described multipotent stem cell
population that is actively proliferating [30]. Keratinocytes of this
compartment are the first to show robust proliferation at the onset
of anagen [3], and are the progenitor cells for the anagen hair
bulb, pigmented hair shaft, and inner root sheath [31–33]. As
expected, Dbp expression is attenuated in the secondary hair germ
of telogen hair follicles at ZT2 (Figure 4I). Bmal1 and Dbp are also
expressed in the lower regions of the late anagen and catagen hair
follicles, as well as the epidermis and dermis throughout the hair
growth cycle. Q-PCR of laser captured microdissected hair
follicles, dermis, and epidermis indicate that clock genes are
differentially expressed between ZT10 (high) and ZT18/ZT2
(low) in the dermis and epidermis during both telogen and late
anagen (Figure 4J and Figure S4), consistent with previous studies
in human skin [23,25]. Interestingly, there is more active
circadian regulation of these genes within the hair follicle proper
during telogen compared to late anagen (Figure 4J and Figure S4),
which likely contributes to the increased amplitude in expression
during telogen. In summary, these data show that Bmal1 and Dbp
are widely expressed within hair follicles and other skin
compartments throughout hair follicle cycling. However, during
the important stage of initiation of hair growth at telogen and
early anagen, the expression of these genes is prominent in the
secondary hair germ of the hair follicle.
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Delayed Anagen Progression in Bmal1 and Clock Mutant
Mice
To determine whether circadian regulators play a functional
role in the hair growth cycle, we studied the progression of
synchronized hair follicle cycling in Bmal12/2 mice [36]. While
the completion of hair follicle morphogenesis (P14) and entry into
telogen (P21) are normal, there is a clear delay at the first
synchronized anagen in Bmal12/2 mice (Figure 6A and Table S3).
At P24 and P28, all Bmal1+/2 mice have entered mid anagen,
while their knockout littermates are still in the first stage of anagen;
hair follicles of Bmal12/2 mice contain a thickened keratinocyte
strand between the dermal papilla and the club hair but lack the
highly proliferative matrix required for downward growth of the
hair follicle during anagen (Figure 6C). The delay in anagen
progression is confirmed by detailed quantitative hair cycle
histomorphometric analysis and downregulation of Myc, as well
as cyclins D1 and B1 (Figure 6D and 6E), which are normally
upregulated by mid-anagen. By the time Bmal12/2 mice progress
past the initial stage of anagen at P31, Bmal1+/2 mice are already
well advanced into late anagen (Figure 6A). When Bmal1+/2 mice
are in late catagen at P44, their knockout littermates are just
beginning to enter catagen; the delay in anagen progression results
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Figure 3. Enhanced circadian expression of CLOCK–regulated genes during telogen. Q-PCR of Bmal1 (A), Clock (B), Dbp (C), Per2 (D), and
Nr1d1 (E) in telogen compared to late anagen dorsal skin over the course of 48 hours (open and filled bars along the x-axis denote 12 hours light and
dark phases, respectively). Expression is normalized to Gapdh and fold calculated relative to the lowest expression time point for both telogen and
late anagen. Each error bar represents the S.E.M. for independent measurements from four mice. Asterisks denote significantly higher (P,0.01)
expression of Dbp and Nr1d1 at ZT10 in telogen.
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Figure 4. Bmal1 and its target gene Dbp are co-expressed during hair follicle cycling. In situ hybridization staining of telogen, early anagen,
late anagen, and catagen dorsal skin at ZT10 with Bmal1 (A–D) and Dbp (E–H) probes. (I) Dbp expression at ZT2 in telogen skin. Dashed lines indicate
border between epidermis and dermis. Note that the black pigment of the hair shaft in late anagen hair follicles is not hybridization signal. Bu –
bulge, CH – club hair, DP – dermal papilla, HB – hair bulb, HS – hair shaft, IRS – inner root sheath, Mx – matrix, ORS – outer root sheath, SHG –
secondary hair germ, SG – sebaceous gland. (J) Dbp expression from laser capture microdissected hair follicles, dermis, and epidermis for telogen and
late anagen dorsal skin at ZT10 and ZT18/ZT2. Standard deviations were determined by using three replicates normalized to Gapdh. Ct values
indicate detectable expression of clock genes in every sample, and fold was calculated relative to the lowest expression sample.
doi:10.1371/journal.pgen.1000573.g004
an absence of mitotic cells within the hair follicles delayed at
anagen I of P24 Bmal12/2 mice (Figure 6F); both Bmal1+/2 and
Bmal12/2 littermates have mitotic cells interspersed in the basal
layer of epidermis, indicating a hair follicle-specific effect of the
Bmal1 mutation. This is significant because hair follicles of P23
wild-type mice, which are morphologically equivalent (in anagen
I) to hair follicles of P24 Bmal12/2 mice, are molecularly distinct
in that they contain mitotic cells in the secondary hair germ
(Figure 6F, third panel), the site of high Dbp expression during
early anagen. Both highly proliferative matrix cells of P24
Bmal1+/2 hair follicles and the secondary hair germ cells of P23
wild-type hair follicles contain phosphorylated Rb (Figure 6G). In
contrast, phosphorylated Rb was not detected in the secondary
hair germ cells of P24 Bmal12/2 hair follicles of the same hair
cycle stage (Figure 6G, third panel), indicating a blockade of
secondary hair germ cells in G1. Thus, circadian clock genes may
control hair follicle cycling at least in part by regulating
proliferation in the secondary hair germ of the early anagen
follicle.
in an overall shift of the hair growth cycle, while the duration of
the entire hair growth cycle is not altered in Bmal12/2 mice. In
Clock mutant mice [37,38], we found a similar but less prominent
delay in progression into the first synchronized anagen (Figure 6B
and Table S4). We observed no obvious morphological abnormalities in the hair follicles of Bmal1 and Clock mutant mice (Figure
S5A). Hence, the delayed anagen progression in both Clock and
Bmal1 mutant mice—without abnormalities in hair follicle
morphogenesis, basic architecture, pigmentation, and hair shaft
formation—points to clock genes as important regulators of timing
in hair follicle cycling.
Circadian Clock Genes Regulate Cell Cycle Progression in
the Secondary Hair Germ of Hair Follicles
Since circadian mechanisms are implicated in cell cycle control
[34,35,39,40], and we found upregulation of Cdkn1a in Bmal12/2
mouse skin, we next examined whether Bmal12/2 hair follicles
exhibit alterations in cell cycle control. Interestingly, we observed
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Clock Genes in Hair Follicle Cycling
Figure 5. Significant differential expression of circadian transcriptional circuit and key cell cycle regulators in telogen Bmal1 and
Clock mutant dorsal skin. (A) Overview of microarray analysis of P22 Bmal12/2 and Bmal1+/2 dorsal skin. FDR – false discovery rate. (B) The
statistical differential expression of clock genes is shown with the circadian transcriptional circuit, which is represented as a matrix based on the
results of Ueda et al. [17]. In the matrix, each column represents a gene encoding a transcription factor (grouped into three classes based on their
binding sites: E-box/E9-box, D-box, and RRE), and each row represents a gene that is regulated by these transcription factors. Orange cells denote
positive regulation and blue cells denote negative regulation. (C) Q-PCR of CLOCK-regulated genes and key cell cycle regulators in P22 Bmal12/2 and
Bmal1+/2 dorsal skin. (D) Q-PCR of CLOCK-regulated genes in P23 Clock wild-type and Clock mutant dorsal skin. For (C) and (D), expression is
normalized to Gapdh and asterisks denote statistically significant (P,0.01) difference in expression between the littermates of the two genotypes.
Error bars represent the S.E.M. for independent measurements from five Bmal12/2 and four Bmal1+/2 mice, and three Clock wild-type and two Clock
mutant mice.
doi:10.1371/journal.pgen.1000573.g005
Anagen eventually proceeds in Bmal1 and Clock mutant mice,
and this could be due to the proposed modulatory role (i.e., nonobligatory) of the circadian clock in regulating the cell cycle
machinery [12,40]. A delay rather than a permanent block in
anagen progression is also found in many of the previously
described mutant mouse models of key regulators in hair follicle
cycling [41,42], which may be due to functional redundancies of
bHLH PAS transcription factors. One of these transcription
factors, NPAS2, has been shown to form heterodimers with
BMAL1 to activate circadian-controlled genes in both central and
peripheral tissues [43,44]. Intriguingly, Npas2 is regulated during
hair follicle cycling (Table S1) and is significantly upregulated in
Bmal12/2 skin (Figure 6C). A likely explanation for the less
pronounced delay in anagen progression in Clock mutant
compared to Bmal12/2 mice is the difference in the nature of
the genetic defects (reduced transcriptional activity in Clock mutant
and null-mutation in Bmal12/2 mice); similar differences were
found in previous studies showing less severe circadian phenotypes
in Clock mutant mice [12]. It is important to note that the observed
anagen progression delay in Bmal1 and Clock mutant mice is in the
context of completely normal morphological features for all
compartments of the skin, including hair follicles, sebaceous
Discussion
In summary, our findings demonstrate a novel role for circadian
clock genes in modulating the progression of the hair growth cycle,
a cyclic biological process that has a much longer duration than
the diurnal period. The fact that hair follicles of Bmal1 and Clock
mutant mice are specifically impeded in the first stage of anagen,
and that clock genes are highly expressed in the secondary hair
germ during telogen and early anagen, points to an important role
for circadian clock genes in the control of this stage of hair follicle
cycling. The delay in anagen progression is likely due to a block at
the G1 phase of the cell cycle in the secondary hair germ cells, as
these cells lack phosphorylated Rb and Cdkn1a (p21) is upregulated
in Bmal12/2 mice. Together, these results suggest that the
circadian clock modulates progression of hair follicle cycling via
its effect on the cell cycle. The recurrent cycling of hair follicles
through growth, regression, rest, and then growth again is a
classical example of a regenerating system [1]. Thus, it is
interesting that clock mechanisms in proliferation seems to become
especially important during tissue regeneration, as evident by the
impaired liver regeneration after hepatectomy in Cry-deficient
mice [35].
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Clock Genes in Hair Follicle Cycling
Figure 6. Bmal1 and Clock regulate anagen progression in hair follicle cycling. (A) Representative histological sections of dorsal skin from
Bmal12/2 mice and their gender-matched Bmal1+/2 littermates at the indicated postnatal age (P). (B) Representative histological sections of dorsal
skin from Clock mutant and their gender-matched wild-type (WT) littermates at the indicated postnatal age (P). For (A) and (B), time points are
mapped (indicated by dotted line) based on histology to the corresponding phases of the hair growth cycle: anagen (A), catagen (C), and telogen (T).
(C) Delayed anagen progression in Bmal12/2 mice compared to normal progression in Bmal1+/2 littermates. At postnatal day 24, hair follicles are in
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Clock Genes in Hair Follicle Cycling
anagen IIIb for the shown Bmal1+/2 dorsal skin section; matrix cells (black arrow) form the enlarged hair bulb and the dermal papilla (red arrow) is
larger than a third of bulb diameter. Note that the bulb is located in the middle of the subcutaneous adipose layer. Hair follicles are in anagen I for the
shown Bmal12/2 dorsal skin section; thickening of keratinocyte strand (blue arrow) between the dermal papilla (red arrow) and the club hair. Note
that the bulb is located in the dermis. Brackets indicate the different layers of the skin: E – epidermis, D – dermis, SC – subcutaneous adipose layer. (D)
Quantitative hair cycle histomorphometric analysis. Percentage of hair follicles at the indicated hair cycle stage is based on staging fifty unique hair
follicles for each genotype from three Bmal1+/2 and two Bmal12/2 littermates at P24. Tel – telogen. Ana – anagen (Roman numerals indicate specific
stages within anagen). (E) Q-PCR of Ccnd1, Ccnb1, and Myc in P24 Bmal12/2 and Bmal1+/2 dorsal skin. Expression is normalized to Gapdh and error
bars represent the S.E.M. for independent measurements from six Bmal1+/2 and two Bmal12/2 littermates. Asterisks denote statistically significant
(P,0.01) difference in expression between the two genotypes. Immunostaining of dorsal skin from P24 Bmal1+/2 (left panel), Bmal12/2 (central
panel) littermates using anti-phospho-histone H3 (F) and anti-phospho-Rb (Ser807/811) (G). The right panels of A and B are wild-type mice at P23
with hair follicles at equivalent stage of the hair growth cycle (anagen I) to the P24 Bmal12/2 mice. The insets are higher magnification of the lower
regions of hair follicles. Black arrowheads; cells stained positive in the epidermis. Black dashed line; border between epidermis and dermis. Red
dashed line; hair follicle bulb. Red arrowhead; cells stained positive within the hair follicle.
doi:10.1371/journal.pgen.1000573.g006
glands, epidermis, dermis, and subcutaneous adipose layer. The
young Bmal12/2 mice (3 to 4 week-old mice) examined in this
study do not exhibit the series of age-related pathologies previously
found in older Bmal12/2 mice, such as decreased hair growth after
shaving in 40-week-old mice with reduced subcutaneous adipose
tissue [45]. We found no difference in the thickness of the
subcutaneous adipose layer of Bmal12/2 and control dorsal skin at
comparable stages of hair follicle morphogenesis and cycling
(Figure S5B). Thus, the absence of morphological and systemic
abnormalities indicates a specific effect of circadian clock genes on
the timing of anagen progression.
A recent study by Tanioka et al. demonstrated that the skin has
an intrinsic oscillating circadian clock similar to other peripheral
tissues such as the liver [26]. Furthermore, their experiments with
SCN-ablated mice point to the importance of the central clock in
maintaining expression of the epidermal circadian clock [26]. This
is consistent with a number of studies demonstrating a role of the
central clock in coordinating other peripheral clocks [10].
Therefore, it is quite likely that the modulation of the hair growth
cycle by clock genes could involve both central and peripheral
mechanisms. In support of the importance of peripheral clock
mechanisms, we found prominent upregulation of clock genes
during early anagen in a specific location within the hair follicle,
the secondary hair germ. Proliferation of keratinocytes of this
compartment, which contains precursor cells to the hair follicle
and possibly stem cells [30,32], is activated at the start of anagen.
In addition, keratinocytes of the secondary hair germ lack mitotic
cells and phosphorylated Rb in Bmal12/2 hair follicles that are
halted at early anagen. Thus, the correspondence between the
temporal and spatial expression of clock genes on the one hand
and the location of the cell proliferation defect within the hair
follicle on the other hand suggests a contribution by peripheral
mechanisms. Future studies will dissect further the relative
importance of the SCN and peripheral clocks in modulation of
the hair growth cycle.
Changes in the stability of clock proteins can offset mRNA
changes, and this can potentially explain the relatively constant
PER2 protein levels between telogen and late anagen dorsal skin
(Figure S3C). Another possible explanation is that the mRNAs of
core clock genes (e.g. PER2) are fluctuating less dramatically
between the different hair cycle phases in comparison to clock
output genes (e.g. DBP, NR1D1) (Figure 3). However, without a
suitable DBP antibody for western/immunohistochemistry, we
were unable to test whether the protein level of DBP changes
similarly to the transcript during hair follicle cycling. In future
investigations, we plan to study the levels and localization of
circadian clock proteins throughout the hair growth cycle.
The initial discovery of circadian clock gene expression altering
in phase with the hair growth cycle was based on the
development of probabilistic models for detecting periodic gene
PLoS Genetics | www.plosgenetics.org
expression from time-course profiling over multiple cycles. An
important feature of our probabilistic models is the ability to take
into account of gene expression changes to due to the initial hair
follicle morphogenesis and injury response following hair
depilation. In addition, our clustering of periodically expressed
transcriptional regulators during the hair growth cycle provide a
genome-wide landscape of transcriptional regulation of hair
follicle cycling, which identifies both known and possible novel
regulators. Hence, our computational approach can be applied to
identify periodically expressed regulators for other cyclic
biological systems.
In conclusion, our findings support the idea that classical
circadian genes may be utilized to modulate the progression of
non-circadian cyclic processes, such as the hair growth cycle, via
cell cycle control. Since many fur-bearing mammals undergo
seasonal molting, we speculate that circadian control mechanisms
for hair follicles may have evolved to allow seasonal regulation of
hair growth. Circadian control mechanisms have previously been
suggested for seasonal breeding and reproductive cycling [46,47],
and it is likely that other cyclic biological processes on different
time scales are modulated by the circadian clock genes.
Materials and Methods
Ethics Statement
All animals were handled in strict accordance with good animal
practice as defined by the relevant national and/or local animal
welfare bodies, and all animal work was approved by the
appropriate committee.
RNA Extraction and Time-Course Microarray Experiments
For profiling of second synchronized and depilation-induced
hair growth cycle, the same upper-mid region of dorsal skin was
excised from C57BL/6 mice at representative postnatal days.
Depilation-induced hair growth cycle was performed by applying
wax/rosin mixture on the dorsal skin of seven-week old mice (all
follicles in telogen). Animals were maintained under alternating
12 hours light/dark cycles with lights on at 6AM, and dorsal skin
tissues were collected within 4-hour window (noon to 4PM).
Histological sections were used to classify each sample into specific
phases/stages of the hair growth cycle based on established
morphological guidelines [3]. Total RNA was isolated from
adjacent dorsal skin using the TRIzol method (Invitrogen) and
cleaned using RNeasy Mini Kit (Qiagen). Quality of cleaned-up
RNA was assessed using the Agilent 2100 Bioanalyzer (Agilent)
prior to hybridization to Affymetrix Mouse Genome 430 2.0
arrays as described [27]. For each time point, multiple biological
replicates were profiled, with each sample separately hybridized to
an array (a total of 69 arrays used for profiling three hair growth
cycles). The entire time-course wild-type microarray datasets are
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Clock Genes in Hair Follicle Cycling
cycle are less likely to play regulatory roles than genes that are
regulated within cells. Hence, we used a computational approach
to systematically distinguish these two types of differential
expression. To identify gene expression changes that are solely
caused by tissue composition change over the hair growth cycle,
we built a two-component Gaussian mixture model, where one
component captures the expression profiles for a set of marker
genes that are due to tissue composition change and the other
component captures the background. The profiles for the
following marker genes of different cell types were used: filaggrin
(probe set ID 1427268_at) and loricrin (1420183_at) for the
cornified cells; keratin 1 (1422481_at) and keratin 10 (1452166_at)
for the suprabasal cells; vimentin (1450641_at) and S100a4
(1424542_at) for mesenchymal cells; Mef2a (1452347_at), Mef2c
(1421027_a_at), and desmin (1426731_at) for myocytes. Their
profiles all display a characteristic U-shaped pattern during the
hair growth cycle, reflecting that these cell types make up a smaller
percentage of total skin tissue during anagen compared to telogen.
Other genes that are expressed in these cell types would display
similar profiles to these marker genes, so the mixture model was
applied to the 8433 periodically expressed probe sets. Using this
mixture model, we found that 2040 probe sets have high posterior
probabilities (.0.9) of periodic gene expression changes due to
tissue composition change over the hair growth cycle. Excluding
these probe sets results in 6393 probe sets that we define as hair
cycle-regulated genes.
deposited in the NCBI Gene Expression Omnibus (www.ncbi.nlm.
nih.gov/geo, GSE11186).
Preprocessing of Microarray Datasets
Based on Principle Component Analysis of all samples and
expression of housekeeping genes (e.g., beta-Actin, Gapdh), sample
outliers are removed from downstream analyses. To remove genes
not expressed during the hair growth cycle, we used MAS 5.0
generated present/absent calls to filter out genes that are not
expressed in the dorsal skin at any sampled time point. A twocomponent noise model (TCM) was then applied to transform the
MAS 5.0 expression data to ensure uniform replicate variance
across the range of expression intensities [27].
Probabilistic Model for Identification of Periodic
Expression Changes
To identify genes with periodic expression patterns across
multiple hair growth cycles, we construct a two-component
probabilistic model with periodic and background components.
The model includes binary indicators of periodicity that select one
of the two mixture components for each of the probe sets. We use
Bayesian inference techniques to estimate whether the observed
data for each of the probe sets is more consistent with periodic or
background expression, resulting in the final ranking of probe set
with respect to the posterior probability of periodicity (see Text
S1). Periodic component of the model is shown in Figure 1C using
the framework of directed graphical models and plate notation
[48]. Nodes within the plate (outermost rectangle labeled ‘‘n’’)
correspond to a single probe set. The plate indicates that these
nodes are repeated for each of the probe sets and they share a
dependence on a set of common periodic profiles and other
parameters W. Circular nodes are continuous variables; square
nodes are binary indicators. Large shaded rectangles visually
group variables for each of the individual cycles. For each probe
set, the hypothetical ideal cycle profile (light blue nodes) is chosen
from a mixture of possible profiles. Individual cycles (blue nodes)
follow the corresponding time points of the ideal cycle, but the
model allows for three types of systematic differences between the
cycles. First, expression measured on different generations of the
Affymetrix platform may differ by some probe set-specific additive
offset (light green node). Second, involvement in the initial hair
follicle morphogenesis for some probe sets (light orange node) may
cause altered expression during the first two time points of the first
cycle. Third, involvement of some other probe sets in injury
response to depilation (another light orange node) may cause
altered expression during the first two time points of the third
cycle. Actual replicates (red nodes) are conditionally independent
observations of the individual cycles with probe set-specific
replicate variance (green nodes). Finally, only the red nodes in
the model are observed. Parameters of the priors W are estimated
from the data using empirical Bayes framework. Bayesian
inference techniques are used to marginalize over all other latent
nodes in order to infer the posterior probability of periodic
expression for each of the probe sets, conditioned on the observed
replicate data and the prior parameters.
Statistical Differential Expression Analysis
Hair cycle-regulated expression profiles of the second synchronized cycle were used for statistical differential expression analysis
because it is not perturbed by processes such as morphogenesis
and wound healing that occur during the first and depilationinduced hair growth cycles, respectively. The time points for
second hair growth cycle are classified into different phases of the
hair growth cycle based on established morphological guidelines
[3] as follow: early anagen (P23, P25), mid anagen (P27), late
anagen (P29), early catagen (P37, P39), mid catagen (P41), telogen
(P44). One-way ANOVA was performed on the TCM-transformed values of samples from a particular phase of the cycle to
samples from the previous phase to determine upregulated genes
(P,0.01). Significant enrichment of GO Biological Process
categories within the different sets of genes upregulated at specific
phases of the cycle was determined using DAVID functional
annotation analysis [50]. Transcriptional regulators were systematically identified from the list of hair cycle-regulated genes by
searching for the annotation category ‘‘regulation of transcription’’
in the GO Biological Process annotations. Using Partek Genomics
Suite, partitioning cluster analysis with Euclidean distance was
performed to group the second hair growth cycle TCMtransformed profiles of the transcriptional regulators.
Quantitative Real-Time PCR
For cDNA synthesis of RNA (1 mg as input) extracted from
dorsal skin, High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems) was performed as described [27]. For cDNA
synthesis of RNA isolated from LCM samples, Sensiscript RT Kit
(Qiagen) was performed following manufacture’s protocol. The
following TaqMan Gene Expression Assays (Applied Biosystems)
were used. For circadian-regulated genes: Bmal1 (Arntl,
Mm00500226_m1),
Clock
(Mm00455941_m1),
Cry2
(Mm00546062_m1),
Dbp
(Mm01194021_m1),
Nr1d1
(Mm00520708_m1), Per2 (Mm01285621_m1), and Rorc
(Mm01261019_g1). For cell cycle-related genes: Ccnd1
(Mm00432359_m1),
Ccnb1
(Mm00838401_m1),
Cdkn1a
Determination of Gene Expression Changes Due to
Tissue Composition Changes
Previous studies have shown that the thickness of different layers
of skin (epidermis, dermis, subcutaneous adipose tissue, and hair
follicle depth) is significantly altered during the hair growth cycle
[3,49]. Genes whose expression in skin changes simply due to
differences in the tissue composition of skin during the hair growth
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Clock Genes in Hair Follicle Cycling
CAATGTCCTGGAAGG R-GCGATGACCCTCTTATCCTG.
Dbp forward (F-) and reverse (R-) primers: F-CCCACAGTTGCAAAGAGACA and R-ATATGTCAGTCACCCGCACA. The
resulting PCR products were cloned into pSTP19 vector (Roche
Applied Science) for generation of digoxigenin-labeled (Roche
Applied Science) antisense and sense probes using SP6 and T7
RNA polymerases, respectively. The probe generation, hybridization, washing and signal detection (using NBT/BCIP alkaline
phosphatase substrate solution) procedures were performed as
described [51].
(Mm00432448_m1),
Cdkn1b
(Mm00438167_g1),
Cdkn2b
(Mm00483241_m1), Myc (Mm00487803_m1), and Wee1
(Mm00494175_m1). Three measurement replicates was performed to determine the expression level (critical threshold value)
per sample, and the expression for each sample is normalized to
the endogenous control gene, Gapdh (Mm99999915_g1).
Western Blotting
Whole cell lysates from telogen (P20) and late anagen (P30)
mouse dorsal skin were collected at ZT16. The following primary
antibodies were used for overnight incubation: rabbit anti-mouse
PER2 (Affinity Bioreagents, 1:500 dilution) and rabbit anti-mouse
GAPDH (Ambion, 1:1000 dilution). Secondary antibody used is
peroxidase-conjugated affinity pure goat anti-rabbit polyclonal
antibody (Jackson ImmunoResearch, 1:10000 dilution).
Clock and Bmal1 Mutant Mice
Total of 80 Clock mice and 93 Bmal1 mice (both in C57BL/6J
genetic background [36–38]) were genotyped and grouped by
gender. Each mouse is classified into specific phases/stages of the
hair growth cycle based on the majority of hair follicles using
established morphological guidelines [3].
Circadian Experiments
Two groups of C57BL/6 mice (104 mice total) were used for
circadian experiments: P30 (late anagen; anagen IV to VI) and
P46 (telogen). These two time points were selected because the
follicles stay in late anagen and telogen phases for several
consecutive days (verified with histological sections). Prior and
during the experiments, the mice were carefully housed under
alternating 12 hours light/dark cycles. For each group, four mice
were sacrificed every four hours over the course of 48 hours, and
total RNA was extracted from the same upper-mid region of
dorsal skin as well as the liver for control. Similar circadian Dbp
expression levels were found for liver taken from P30 and P46
mice.
Quantitative Hair Cycle Histomorphometric Analysis
Analysis was performed as previously described with minor
modifications [52].
Expression Profiling of Dorsal Skin from Bmal12/2 Mice
Histological sections were used to verify that the hair follicles are
in the first synchronized telogen based on established morphological guidelines [3]. RNA extraction from dorsal skin, clean-up, and
array hybridization were the same as that described for the timecourse microarrays above. We profiled three Bmal12/2 and three
Bmal1+/2 littermates, with each sample separately hybridized to an
array (Affymetrix Mouse Gene 1.0 ST arrays). Statistical analyses
were performed using Cyber-T program [53]. The cutoff for
significant differential expression is set at P-value of 0.001, which
corresponds to a false discovery rate of within 7% based on the
calculated posterior probability of differential expression. The
wild-type and Bmal1 mutant microarray datasets are deposited in
the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/
geo, GSE14006).
Laser Capture Microdissection
The excised dorsal skin was immediately embedded in O.C.T.
Compound (Sakura Finetek USA, Torrance, CA), frozen in dry
ice and stored at 280uC. Serial cryostat sections (8 mm thickness)
were cut and mounted on autoclaved polytarthalene (PET) foil
stretched on a metal frame (Leica). Tissue was fixed in cold
acetone for 2 min and stained using Arcturus HistoGene LCM
Frozen Section Staining Kit (Arcturus, Mountain View, CA)
according to manufacturer’s protocol. PET foil metal frames were
mounted on a Leica AS LMD system (Leica) with the section
facing downwards. Laser and microscope settings were as follow:
1506 objective, aperture 6, intensity 46. The pulsed UV laser
beam was carefully directed along the borders of each of the
structures: whole hair follicles, epidermis, and dermis. Crosscontamination is very minimal in LCM samples as verified by QPCR of the following specific markers: loricrin (epidermis), vimentin
(high in dermis, low in hair follicle), and keratin 14 (epidermis and
hair follicle). The collection tube cap was filled with a guanidine
isothiocyanate (GITC)-containing buffer (Buffer RLT, RNeasy
Mini Kit, Qiagen, Hilden, Germany) for cell lysis and preservation
of RNA integrity. Tissue collection was verified by inspecting the
tube cap. Post collection microcentrifuge tubes were immediately
vortexed for 1 min. Total RNA was isolated using the RNeasy
Micro Kit (Qiagen) according to the manufacturer’s recommendations, including the DNA digestion step, with an elution volume
of 14 ml and the addition of poly-A carrier RNA to the lysate.
Shown are the results from one of three independent LCM
experiments that yielded similar quantitative measurements.
Immunohistochemistry
Dorsal skin was fixed in 10% formalin, paraffin-embedded and
sectioned at 6-mm. Antigen-retrieval was performed by heating
slides in 0.01 M citrate buffer (pH6) for 20 min using an autoclave
oven. After quenching endogenous peroxidase activity with 3%
hydrogen peroxide for 5 min, sections were permeabilized using
0.2% Triton-X for 5 min. We then applied DakoCytomation
Protein Block Serum-Free (Dako) to the sections for 30 min. Next,
the slides are incubated overnight at 4uC with the following
primary antibodies: phospho-histone H3 (Upstate, 1:1000),
phospho-Rb Ser807/811 (Cell Signaling, 1:100), Keratin 5
(Covance, 1:1000), AE13 and AE15 (kindly provided by Dr. T.
T. Sun, 1:100). We applied 1:500 biotinylated IgG secondary
antibodies (Vector) for 1 hr, and then incubated in Vectastain elite
ABC Reagent (Vector) for 30 min. The slides were stained using
the DakoCytomation Liquid DAB+ Substrate Chromogen System
(Dako), and counterstained using 1:5 diluted hematoxylin followed
by bluing reagent.
Supporting Information
Figure S1 Exclusion of gene expression changes that correspond
In Situ Hybridization
to tissue composition changes. (A) Expression profiles of marker
genes that are altered as the tissue composition changes during the
hair growth cycle. The marker genes for the different cell types are
as follows: filaggrin and loricrin for the cornified cells, keratin 1
Probes specific for Bmal1 and Dbp was generated by PCR using
total cDNA derived from C57BL/6 mouse dorsal skin. Bmal1
forward (F-) and reverse (R-) primers: F-TTAGCPLoS Genetics | www.plosgenetics.org
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Clock Genes in Hair Follicle Cycling
and 10 for the suprabasal cells, vimentin and S100a4 for
mesenchymal cells, and Mef2a, 2c, and desmin for myocytes. (B)
Mixture model identified genes that can be explained by tissue
composition changes over the hair growth cycle. The x-axis is the
posterior probability of gene expression changes due to tissue
composition changes, and the y-axis is the number of probe sets
within each range of posterior probabilities.
Found at: doi:10.1371/journal.pgen.1000573.s001 (0.07 MB PDF)
ed hair shaft reveals expected AE15 staining in the medulla. (B) No
difference in the thickness of the subcutaneous adipose layer of
Bmal12/2 and control dorsal skin. Note we measured thickness for
comparable stages of hair follicle cycling; Bmal12/2 mice reaches
late anagen at P34-P35, and Bmal1+/+ and Bmal1+/2 mice reaches
late anagen at P30-P31.
Found at: doi:10.1371/journal.pgen.1000573.s005 (0.80 MB PDF)
Table S1 Time-course profile clustering of hair-cycle regulated
genes. Probe set ID corresponds to the array used for the second
and depilation-induced hair growth cycles (Mouse Genome 430
2.0). Old probe set ID corresponds to the array used for the first
hair growth cycle (Murine Genome U74Av2).Columns labeled 1–
9 correspond to log-transformed, zero-mean gene expression
profiles for the ‘‘ideal hair cycle.’’
Found at: doi:10.1371/journal.pgen.1000573.s006 (2.04 MB
XLS)
Figure S2 Time-course profiles of genes belonging to represen-
tative GO Biological Process categories found to be significantly
enriched. The heat map was generated using profiling data from
the second synchronized hair growth cycle. Expression levels are
indicated by colorimetric ratio-scale. Time points are mapped
based on histology to the corresponding phases of the hair growth
cycle: anagen (A), catagen (C), and telogen (T).
Found at: doi:10.1371/journal.pgen.1000573.s002 (0.23 MB PDF)
Figure S3 Expression of circadian clock genes and proteins in
mouse dorsal skin at different phases of the hair growth cycle. (A)
In situ hybridization staining of telogen, early anagen, late anagen,
and catagen dorsal skin at ZT10 with Bmal1 (left column) and Dbp
(right column) anti-sense probes. Note the black pigment of the
hair shaft in late anagen hair follicles is not hybridization signal. (B)
As negative control, in situ hybridization staining of telogen, early
anagen, and late anagen dorsal skin at ZT10 with Dbp sense
probes. Dashed lines indicate border between epidermis and
dermis. Brackets indicate the different layers of the skin: E epidermis, D - dermis, SC - subcutaneous adipose layer. Bu bulge, CH - club hair, DP - dermal papilla, HB - hair bulb, HS hair shaft, IRS - inner root sheath, Mx - matrix, ORS - outer root
sheath, SHG - secondary hair germ, SG - sebaceous gland. (C)
Levels of PER2 proteins are not significantly different between
telogen (P20) and late anagen (P30). Shown are two independent
whole cell lysates from mouse dorsal skin collected at ZT16.
Found at: doi:10.1371/journal.pgen.1000573.s003 (0.75 MB PDF)
Table S2 List of genes previously reported to have hair cycledependent gene expression changes. Probe set ID corresponds to
the array used for the second and depilation-induced hair growth
cycles (Mouse Genome 430 2.0). Old probe set ID corresponds to
the array used for the first hair growth cycle (Murine Genome
U74Av2). pHC is the posterior probability of being periodically
expressed during the hair growth cycle. Note some genes have
multiple probe sets, which have different hybridization signals and
thus result in differences in pHC values.
Found at: doi:10.1371/journal.pgen.1000573.s007 (0.08 MB PDF)
Table S3 Hair cycle staging of Bmal1 knockout mice (2/2) and
their control littermates (+/+ and +/2). For each postnatal day
(P), mice are grouped by genotype and each mouse is classified into
specific phases/stages of the hair growth cycle based on the
majority of hair follicles using established morphological guidelines. In general, we noted a slightly more advanced hair cycle
progression in male mice; the table includes both genders which
accounts for most of the variation within each genotype, but does
not explain the observed differences between genotypes. Hence,
for hair cycling progression comparison across genotypes, we
matched littermates by gender.
Found at: doi:10.1371/journal.pgen.1000573.s008 (0.06 MB PDF)
Figure S4 Expression of circadian clock genes from laser capture
microdissected skin compartments. Q-PCR of Bmal1 (A), Per2 (B)
and Cry2 (C) from LCM-hair follicles, dermis, and epidermis for
telogen and late anagen dorsal skin at ZT10 and ZT18/ZT2. (D)
Q-PCR of Dbp from laser capture microdissected hair follicles at
telogen, early anagen (anagen I–II), mid anagen (anagen III), late
anagen (anagen IV–VI). For all panels, standard deviations were
determined by using three replicates normalized to Gapdh. Ct
values indicate detectable expression of clock genes in every
sample, and fold was calculated relative to the lowest expression
sample.
Found at: doi:10.1371/journal.pgen.1000573.s004 (0.06 MB PDF)
Table S4 Hair cycle staging of Clock mutant mice (Cl/Cl) and
their control littermates (+/+ and Cl+). Methodology same as
Table S3.
Found at: doi:10.1371/journal.pgen.1000573.s009 (0.06 MB PDF)
Text S1 Probabilistic model for detection of periodic profiles.
Found at: doi:10.1371/journal.pgen.1000573.s010 (4.01 MB PDF)
Acknowledgments
Figure S5 No morphological abnormalities in skin and hair
follicle in Clock and Bmal1 mutant mice. (A) Hair follicle structures
are normal in Clock and Bmal1 mutant mice. The top row show the
H&E sections of hair follicles in dorsal skin of Clock and Bmal1
mutant mice and their control littermates at late anagen. The
bottom three rows are the corresponding immunostainings of the
following specific hair differentiation markers: AE13 (cortex and
cuticle of the hair shaft), AE15 (inner root sheath and medulla of
the hair shaft), K5 (outer root sheath). Note that this particular
Bmal12/2 mouse has white fur coat and therefore the unpigment-
We thank Xing Dai, Peter Kaiser, Ambica Bhandari, and Amelia Soto for
advice and review of the manuscript. We acknowledge UCI DNA
Microarray Facility for performing microarray hybridizations.
Author Contributions
Conceived and designed the experiments: KKL MG RP JST BA.
Performed the experiments: KKL VK MG. Analyzed the data: KKL
MG DC ATI. Contributed reagents/materials/analysis tools: VK DC ATI
PS RP JST. Wrote the paper: KKL DC BA.
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