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Generation of the primary hair follicle pattern
Chunyan Mou*, Ben Jackson*, Pascal Schneider†, Paul A. Overbeek‡, and Denis J. Headon*§
*Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; †Department of Biochemistry, BIL Biomedical Research Center, University of Lausanne, CH-1066 Epalinges, Switzerland; and ‡Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030
Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved May 8, 2006 (received for review January 31, 2006)
Hair follicles are spaced apart from one another at regular intervals
through the skin. Although follicles are predominantly epidermal
structures, classical tissue recombination experiments indicated
that the underlying dermis defines their location during development.
Although many molecules involved in hair follicle formation
have been identified, the molecular interactions that determine the
emergent property of pattern formation have remained elusive.
We have used embryonic skin cultures to dissect signaling responses
and patterning outcomes as the skin spatially organizes
itself. We find that ectodysplasin receptor (Edar)–bone morphogenetic
protein (BMP) signaling and transcriptional interactions are
central to generation of the primary hair follicle pattern, with
restriction of responsiveness, rather than localization of an inducing
ligand, being the key driver in this process. The crux of this
patterning mechanism is rapid Edar-positive feedback in the epidermis
coupled with induction of dermal BMP4_7. The BMPs in turn
repress epidermal Edar and hence follicle fate. Edar activation also
induces connective tissue growth factor, an inhibitor of BMP
signaling, allowing BMP action only at a distance from their site of
synthesis. Consistent with this model, transgenic hyperactivation
of Edar signaling leads to widespread overproduction of hair
follicles. This Edar–BMP activation–inhibition mechanism appears
to operate alongside a labile prepattern, suggesting that Edarmediated
stabilization of _-catenin active foci is a key event in
determining definitive follicle locations.
pattern formation _ reaction–diffusion _ skin development
Periodic patterns are a recurring theme in anatomical organization.
Examples in diverse organisms include insect bristles,
mammalian hairs, the location of leaves on a plant, and the
location of stomata on those leaves. In all of these cases the
position of each element in the pattern is defined relative to
the others rather than to an absolute anatomical location. With
regular patterns found so widely in nature, a key question in
developmental biology is how an ordered array of structures can
be generated from an initially homogeneous field of cells. In
general terms, such patterns can be generated by using two
signals with different ranges of action (1, 2). These activation–
inhibition systems rely on an activator that promotes (i) its own
synthesis, (ii) assumption of a given cell fate, and (iii) synthesis
of an inhibitor of this fate. Crucial in generating a spatial pattern
is that the activator acts locally, whereas the inhibitor acts at a
distance from its site of production. These types of molecular
interactions are predicted to be capable of generating a periodic
pattern by amplifying stochastic asymmetries in initial concentrations
of activator and inhibitor (1).
Cells on the surface of mammalian embryos are competent to
become either hair follicle or surface epidermis. They coordinate
their fate choices to yield a stippled pattern of follicles,
relying on communication within the skin rather than any
external positional information. Recombination of epidermal
and dermal components of embryonic skin established that
communication between these cell layers is absolutely required
for initiation of hair and feather development and that the
dermis is responsible for inducing morphological changes in the
epidermis (3, 4).
Many molecules that play a role in hair follicle development
have been identified (5– , but the regulatory relationships
between signaling pathways involved in this process are largely
unknown. This is a particularly important problem because it is
the interactions between molecules, rather than the intrinsic
function of any individual gene product, that is responsible for
orchestrating pattern formation. One such signaling pathway,
composed of the extracellular ligand ectodysplasin (Eda), its
receptor Edar, and its cytoplasmic signaling adapter Edarassociated
death domain (Edaradd), is required for development
of a specific subset of hair follicles. Mutation of any of these three
genes, all of which are specifically expressed in the epidermis,
causes identical ectodermal dysplasia phenotypes in mouse and
human (9–12). This phenotype includes a complete absence of
primary hair follicles, which in mouse form between embryonic
day (E) 13 and E16. It appears that Edar mutant epidermis
retains its naý¨ve state until E17, when secondary follicles begin
to develop (10, 13). Secondary follicle formation has a distinct
genetic basis, with mutations in Noggin (14) or Lef1 (15) allowing
primary hair follicle initiation but blocking that of secondary
follicles.
Here we study the role of the Edar pathway in follicle
patterning using embryonic skin cultures. The culture system
allows experiments of short duration with a defined start point.
This feature is particularly important for studying signal responses
because it can distinguish the proximal effects of an
experimental manipulation from those that are a secondary
consequence caused by alteration of cell fates. We find that
spatial organization in the epidermis is achieved by modulation
of signal receptivity, with Edar–bone morphogenetic protein
(BMP) activation–inhibition interactions driving the patterning
process.
Results
Restriction of Eda Responsiveness Regulates Hair Follicle Density.
Although activation–inhibition systems are generally predicted
to rely on differential ligand availability, the ligand in this system,
Eda, is a poor candidate for conveying positional information.
Eda is widely expressed in the epidermis (13) and, when applied
in a diffusible form, allows pattern formation in culture (16) and
in vivo (17). Consequently, we considered dynamic Edar expression
as a means to generate a punctate cell fate pattern. Before
hair follicle initiation Edar is evenly expressed through the skin,
but as a pattern emerges it becomes up-regulated in follicle
primordia and down-regulated in surrounding cells. This dynamic
expression itself depends on Edar signal transduction
because it is not observed in Edaradd_/_ embryos (Fig. 1A).
Thus, as patterning takes place cells display one of three
expression states; those with undetectable Edar are likely excluded
from a hair follicle fate, those with high-level expression
are committed to this fate, and those with moderate expression
remain competent to assume either fate. Quantitative RT-PCR
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: BMP, bone morphogenetic protein; CTGF, connective tissue growth factor;
Eda, ectodysplasin; Edar, Eda receptor; Edaradd, Edar-associated death domain; qPCR,
quantitative RT-PCR; En, embryonic day n.
§To whom correspondence should be addressed. E-mail: denis.headon@manchester.ac.uk.
© 2006 by The National Academy of Sciences of the USA
PNAS _ June 13, 2006 _ vol. 103 _ no. 24 _ 9075–9080
DEVELOPMENTAL
BIOLOGY
(qPCR) revealed that skin of Edaradd_/_ embryos expresses the
same amount of Edar as that of their heterozygous littermates
(Fig. 1B). This result illustrates that while pattern formation
dramatically reorganizes Edar expression, it balances focal upregulation
with widespread down-regulation such that the total
level of transcript is conserved. These findings raise the possibility
that follicle patterning is controlled by restriction of
competence to respond to Eda, with nascent follicles blocking
Edar expression, and therefore follicle fate, in their surroundings.
If restriction of Edar to follicles and its down-regulation in
their surrounding cells are key events in pattern formation, then
WT skin that already contains follicle primordia should be less
competent than Eda mutant skin to produce follicles in response
to exogenous Eda. We culturedWT and Eda_/_ E14 dorsal skin
in various concentrations of recombinant Eda and assessed hair
follicle density by detecting Shh (sonic hedgehog), an early hair
follicle marker (1. WT skin contains _30 follicles per square
millimeter in the absence of exogenous Eda, rising to 50 follicles
per square millimeter at high Eda concentrations. Eda_/_ skin is
much more responsive to Eda, generating a maximum of 90
follicles per square millimeter (Fig. 1 C and D). At high
concentrations of Eda, mutant skin generated stripe-like patterns,
which were never observed in WT (Fig. 1C). Stripe
formation is predicted in activation–inhibition systems when
activator concentrations become saturating (19).
We noticed that treated mutant, but not WT, explants had
follicle primordia aligned along their edges (Fig. 1C). This
observation could be explained if cells along the margin of the
tissue have an advantage in forming a follicle by being relieved
of inhibitory factors from cells on one side. This edge effect, and
the fact that Eda_/_ skin can generate nearly twice as many
follicles as WT, argues that final follicle locations are not
predetermined in Eda mutants. These results suggest that application
of exogenous Eda in this culture system initiates pattern
formation, rather than simply revealing a preexisting, cryptic
pattern. Taken together, these results link widespread expression
of Edar with widespread competence to form a hair follicle and
indicate that existing follicles cause restriction of this developmental
potential.
Timing of Patterning and Morphogenetic Events. To examine the
rate of pattern formation and follicle morphogenesis we cultured
Eda_/_ skin with Eda and fixed samples at various time points.
We found an ordered pattern of Edar-expressing foci appearing
_10 h after Eda administration, after which the spots resolved
and intensified to 24 h (Fig. 1E). The first definitive morphological
indication of follicle formation, generation of a condensed
placode, did not become visible until 20 h after Eda
application (Fig. 1F). Thus, in this system a molecular prepattern
precedes the appearance of morphologically identifiable placodes
by _10 h.
Eda Is Dispensable for Pattern Formation. The transgenic line
OVE951 carries a high copy number of a yeast artificial chromosome
that includes the entire Edar locus (20) and consequently
overexpresses Edar in its endogenous pattern (10). We
quantified Edar expression in transgenic skin at E14, finding it
to be 4-fold higher than in nontransgenic skin (Fig. 2A). We
found that introducing this Edar-overexpressing locus into the
Eda_/_ line leads to rescue of primary follicle formation, as
determined by Shh expression (Fig. 2 B–D). This finding shows
that moderate Edar overexpression leads to ligand-independent
signaling and illustrates that an accurate follicle pattern can be
generated in the absence of Eda.
BMP Signaling Inhibits Edar Expression. The finding that Edar
expression is undetectable in cells close to nascent follicles,
whereas more distant cells express moderate levels of Edar,
suggests that early hair follicles produce a diffusible inhibitor of
Edar expression that restricts competence to assume this fate in
surrounding cells. Two secreted ligands have been described as
inhibitors of follicle formation: the BMPs (21, 22) and EGF (23).
Both of these molecules block Eda-mediated follicle formation
in culture (Fig. 3A), and so both are candidates for the Edarrepressing
activity. We tested these molecules for inhibition of
Edar expression in embryonic skin cultures. Importantly, the
Edar inhibitor should act before commitment to follicle fate, and
so it should repress the basal Edar expression observed before
patterning. The up-regulated Edar expression observed in follicle
primordia may be under a distinct regulatory control.
Fig. 1. Modulation of Eda responsiveness in developing skin. (A) Edar
expression in E12 WT, E14 WT, and E14 Edaradd_/_ mutant skin and semiquantitative
analysis of Edar signal intensity along the red line. (Scale bar: 100
_m.) (B) Edar expression levels in skin from Edaradd_/_ and Edaradd_/_
embryos, normalized to Keratin14. (C) Shh expression in WT and Eda_/_ skin
cultures treated with recombinant Eda protein. Eda_/_ skin displays an edge
effect, with Shh-expressing foci aligned along the margin of the explant
(Lower Center). High concentrations of Eda cause formation of stripes in
mutant skin (arrowhead). Each panel shows 1 mm2. (D) Hair follicle densities
induced by recombinant Eda in E14 WT and Eda_/_ skin. Error bars show SEM.
(E and F) Timing of patterning and morphogenetic events. (E) Edar expression
in E14 Eda_/_ skin cultures after administration of 50 ng_ml Eda. Each panel
shows 1 mm2. (F) Sectioned Eda_/_ skin after Eda administration. The first
morphological indication of hair follicle formation is condensation of cells to
form placodes (arrowheads). (Scale bar: 25 _m.)
Fig. 2. Eda is dispensable for pattern formation. (A) qPCR determination of
Edar expression in E14 Edaradd_/_ nontransgenic (NT) and Edaradd_/_
OVE951 skin. (B--D) In situ detection of Shh in WT (B), Eda_/Y (C), and Eda_/Y
(D) OVE951 E15 embryos. Expression is detected only in the vibrissae of the
mutant, but overexpression of Edar rescues the mutant phenotype, generating
an accurate follicle pattern.
9076 _ Mou et al.
Because mutant skin exhibits only the widespread, moderate
expression of Edar (Fig. 1A), we used it for these experiments.
We found that the BMPs strongly repressed Edar expression,
whereas EGF did not (Fig. 3B). BMP repression of Edar is rapid
and correlates with levels of phospho-Smad1_5_8, the activated
form of intracellular transducers of BMP signals (Fig. 3C).
To determine whether endogenous BMPs influence pattern
formation we used Noggin, a specific inhibitor of BMPs 2, 4, and
7 (24). The maximum hair follicle density achieved by Eda
treatment of Eda_/_ skin was _90 per square millimeter (Fig.
1D). Cotreatment with Noggin breached this limit, allowing
formation of 140 follicles per square millimeter (Fig. 3 D and E),
without generation of stripes. This result demonstrates that
endogenous BMPs restrict Eda responsiveness during pattern
formation. Treatment with Noggin alone caused formation of
small clusters of follicles in mutant skin, indicating that relief
from BMP signaling is sufficient to allow some sporadic follicle
formation in the absence of Edar activity (Fig. 3D).
BMPs Act at a Distance from the Follicle. Because the follicle itself
produces BMPs (25) and has high Edar expression, it must
employ a mechanism to evade BMP-driven Edar downregulation.
We found that connective tissue growth factor
(CTGF), which binds to and inhibits BMPs in a manner analogous
to that of Noggin (26), is expressed in hair follicle placodes
(Fig. 4A) and is a rapidly up-regulated target of Edar signaling
(Fig. 4B). In contrast to CTGF, other inhibitors of BMP signaling
expressed in developing skin [Noggin, Smad7, and Sostdc1
(sclerostin domain-containing 1)_Ectodin_WISE (25, 27)] are
themselves transcriptional targets of BMP (Fig. 4C), likely acting
as feedback inhibitors of the signaling pathway. Consistent with
the idea that the placode is a BMP-privileged zone, the BMP
target Sostdc1 is expressed surrounding and away from, but not
within, follicle sources of BMP (Fig. 4D), and phospho-Smad1_
5_8 is detected in E15 interfollicular epidermis but is largely
absent from nascent follicles (Fig. 4E). Cotreatment of skin
cultures with Eda and BMP represses BMP induction of its target
gene Smad7, with this weakening of BMP transcriptional responses
accompanied by suppression of Smad phosphorylation
(Fig. 4F). Eda’s ability to inhibit BMP responses relies on
activation of NF-_B, because pharmacological suppression of
this transcription factor allows full BMP response in the presence
of Eda (Fig. 4 G and H). This finding is consistent with a role for
Eda target genes in BMP inhibition rather than any direct
interference between components of the Eda and BMP signal
transduction pathways. Thus, the BMPs act at a distance from
their site of synthesis, and the early follicle itself is resistant to
their action. Taken together, these experiments show that the
BMPs display the characteristics of the inhibitory arm of an
activation–inhibition loop.
Transcriptional Responses to Edar Signaling. If Edar functions in the
activation arm of this loop, then it should be able to up-regulate
its own expression, as well as that of the BMPs. To identify Edar
signaling targets we treated Eda_/_ cultures with Eda and
analyzed gene expression in isolated epidermis and dermis. We
used a high dose of Eda to achieve gross up-regulation of target
gene expression rather than the relocation of expression observed
during the physiological patterning process. Edar expres-
Fig. 3. BMPs inhibit Edar expression. (A) BMP4 and EGF inhibit follicle
formation induced in Eda_/_ skin by Eda. (B) Quantitation of Edar expression
in E13 Edaradd_/_ skin treated for 24 h with BMPs or EGF. (C) Time course
inhibition of Edar by BMP4 and corresponding phospho-Smad levels. (D) Shh
expression in Eda_/_ skin treated with Noggin only, Eda only, or Eda plus
Noggin for 24 h. (E) Hair follicle densities in Eda_/_ skin treated with Eda with
or without Noggin for 24 h. Error bars show SEM.
Fig. 4. BMPs act at a distance from the nascent follicle. (A) CTGF is expressed
in hair follicle primordia in E15 WT, but not Eda_/_, embryos. Expression is also
seen in the eyelid. (Scale bar: 1 mm.) (B) Quantitation of CTGF mRNA in
separated epidermis and dermis of mutant skin treated with 1,000 ng_ml Eda
for 4 h. Edar stimulation induces CTGF expression in the epidermis, with little
dermal expression observed. (C) Quantitation of gene expression in epidermis
5 h after addition of BMP4 to whole skin. Noggin, Smad7, and Sostdc1 are induced by BMP treatment, whereas CTGF is not. (D) Sostdc1 is expressed
surrounding and away from follicle primordia 24 h after administration of
Eda. (Scale bar: 100 _m.) (E) Immunodetection of phospho-Smad1_5_8 in the
epidermis of E15 WT skin, with low levels in follicle placodes (arrowhead).
(Scale bar: 100 _m.) (F) Smad7 expression in epidermis 5 h after BMP4 with or
without Eda application and corresponding epidermal Smad1_5_8 phosphorylation
levels. Cotreatment with Eda suppresses BMP-induced Smad7 activation
and Smad1_5_8 phosphorylation. (G) Smad7 expression in isolated epidermis
5 h after BMP4 with or without Eda application in the presence of the
NF-_B inhibitor BAY 11-7082. (H) Bay 11-7082 blocks Eda-mediated I_B_
phosphorylation in the epidermis. Error bars show SEM.
Mou et al. PNAS _ June 13, 2006 _ vol. 103 _ no. 24 _ 9077
DEVELOPMENTAL
BIOLOGY
sion was modestly activated by Eda within 4 h, with suppression
of BMP signaling by cotreatment with Noggin enhancing this
autoregulation (Fig. 5A). Analysis of BMP expression at 4 and
10 h found no significant changes in BMP2 levels (data not
shown), whereas BMP4 was strongly activated in the dermis by
10 h (Fig. 5B). BMP7 displayed the most rapid response to Eda,
with initially low dermal levels up-regulated within 4 h (Fig. 5A)
and strongly up-regulated at 10 h (Fig. 5B). The Eda-induced
BMPs were focally expressed (Fig. 5B). Because Edar expression
is restricted to the epidermis (Fig. 5A), dermal up-regulation of
BMP4 and BMP7 must be an indirect effect. Interestingly, in
addition to increasing overall BMP levels, up-regulation of
BMP7 in the dermal compartment would be predicted to enable
formation of BMP4_7 heterodimers, which have been shown to
be much more potent signals than BMP homodimers (2.
Based on these findings we propose a model for primary hair
follicle patterning (Fig. 5C) in which the naý¨ve embryonic
epidermis evenly expresses molecules that activate (Eda and
Edar) and inhibit (BMP) hair follicle identity. Edar undergoes
local autoregulation and signal amplification, induces CTGF,
and indirectly up-regulates BMP expression in the dermis. Local
inhibition of BMP signaling forces their action at a distance to
repress epidermal Edar and hence follicle fate. These interactions
serve to amplify deviations in the initial conditions to
generate a spatially organized follicle array.
We sought to incorporate _-catenin into this model because
its activation is essential for follicle patterning and morphogenesis
(29). We analyzed expression of Axin2, a direct _-catenin
target gene (30), and found an ordered array of Axin2-positive
foci in WT E15 epidermis. Eda_/_ skin had occasional clusters
of these Axin2 foci, suggesting that some punctate _-catenin
activity is present in the absence of Edar signaling. In culture
these foci were suppressed by BMP and enhanced or stabilized
by Eda. Thus, some prepatterned _-catenin activity appears
independent of Edar function. This observation apparently
contradicts our model in which Edar function regulates patterning
decisions. One possibility is that these Axin2 foci are proposed
follicle locations that have the potential to be stabilized by
Edar. To determine when a final follicle pattern becomes fixed
we cut cultured skin at different times after Eda application and
looked for alignment of follicles along the newly generated edge.
Edge effects were observed when skin had been exposed to Eda
for _10 h, whereas after this time the ability of the pattern to be
reconfigured in response to perturbation of the field is lost (these
data are in Fig. 6, which is published as supporting information
on the PNAS web site). These findings indicate that a labile
prepattern exists in the absence of Edar signaling but that it takes
_10 h of molecular negotiation in the presence of Eda to fix a
definitive pattern.
The proposed model considers restriction of Edar activity as
a pivotal event in patterning. A prediction that it makes,
therefore, is that widespread Edar activation should lead to
widespread assumption of hair follicle fate. We generated transgenic
mice expressing a cDNA composed of the intracellular
domain of Edar fused to the transmembrane domains of LMP1,
a viral protein that confers ligand-independent signaling when
fused to cell-surface receptors (31). This cDNA was expressed in
the basal layer of the epidermis by using the Keratin14 promoter.
Three independent founder mice displayed thickened, scaly skin
and because of ill health had to be killed within 20 days of birth.
Sectioning revealed that the skin of these animals was consumed
with hair follicle down-growths, the follicles packed against one
another with essentially no intervening spaces (Fig. 5D). Quantitation
of follicles in transgenic dorsal skin showed that it has a
density _40% greater than that of nontransgenic littermates
(Fig. 5E). This generation of supernumerary follicles confirms
the importance of restricting Edar signaling in generation of an
appropriately patterned hair follicle array.
Discussion
We propose a receptivity-driven model for hair follicle formation
in which regulation of Edar expression is pivotal. Other
activation–inhibition systems that have been studied at a molecular
level, such as determination of the branched feather structure
(32) or vertebrate left–right asymmetry (33), rely on differential
diffusion properties of two secreted ligands. In contrast,
modulation of a receiving cell’s responsiveness to a widely
available signal is central to our model.
Fig. 5. Timing of transcriptional events, patterning model, and Edar hyperactivation. (A) Eda_/_ mutant explants were cultured with or without Eda with or
without Noggin for 4 h, and expression of Edar, BMP4, and BMP7 was determined in separated epidermis and dermis. (B) BMP expression levels and location
in mutant skin cultured with Eda for 10 h. The induced expression of BMP4 and BMP7 is punctate. (C) Proposed molecular interactions that generate the primary
hair follicle pattern. Solid lines indicate cell-autonomous local interactions, and dotted lines indicate action at a distance. (D) Keratin14 immunostained
longitudinal sections, and hematoxylin and eosin stained cross-sectioned dorsal skin of 10-day-old K14::LMP1-Edar transgenic and nontransgenic littermates. (E)
Quantitation of hair follicle density in transgenic and nontransgenic mouse dorsal skin. Error bars show SEM.
9078 _ Mou et al.
Such patterning mechanisms rely on a differential range of
activating and inhibitory molecules. Edar and _-catenin are
restricted to the cell in which they are produced, whereas CTGF
and the BMPs are secreted molecules. This property suggests
that CTGF action must be spatially restricted. Restriction could
be achieved by CTGF immobilization on extracellular matrix
components or by diffusion of CTGF–BMP complexes with
subsequent release of active BMPs. The observation that mutant
andWT embryonic skin have the same levels of Edar expression
(Fig. 1B) is best explained by the fact that, although Eda
up-regulates Edar expression, it also induces BMPs, which feed
back to inhibit Edar.
The culture method we used allows synchronization of follicle
formation and makes the skin accessible to experimental manipulations.
However, it is important that observations from
such ex vivo systems are correlated with the findings made in
intact animals. In particular, application of recombinant proteins
mimics transgenic gain of function approaches, whereas loss-offunction
experiments are essential to understanding endogenous
functions. In whole animals ablation of Edar signaling specifically
blocks primary hair follicle formation (10), whereas suppression
of _-catenin activation prevents formation of all follicle
types (29). Consistent with an inhibitory role for BMPs in the
patterning process, deletion of BMP receptor genes in embryonic
epidermis causes an increase in follicle density by the end
of the primary wave of follicle formation at E16 (34). In addition,
deletion of Noggin, presumably leading to enhanced BMP signaling,
reduces hair follicle numbers. However, Noggin mutation
specifically ablates secondary follicles while allowing primary
follicles to form (14). This mutant phenotype may indicate that
Noggin is the chief BMP inhibitor used by secondary follicles to
avoid BMP autostimulation, while primary hair follicles instead
employ CTGF. CTGF-null mice display skeletal abnormalities
and die at birth (35), but their skin phenotype has not been
described.
In our experiments, manipulation of signaling activities enabled
modulation of placode densities over a wide range, from 30 per
square millimeter to 140 per square millimeter. This plasticity, as
well as the alignment of follicles along the boundary of dissected
skin explants, indicates that their ultimate locations have not been
defined in Eda mutant skin. However, we did find sporadic Axin2
expression, indicative of patterned_-catenin activity, in the absence
of Eda. These foci may be the same cells that were recently
identified as initiating, but failing to maintain, very early follicle
morphogenesis in Edar mutant skin (36). The malleability of follicle
position in response to experimental perturbation suggests that this
prepattern does not necessarily represent the final hair follicle array.
Axin2-expressing foci might be ‘‘proposed’’ follicle locations that
become fixed or otherwise as Edar function is restricted in the skin.
Although the relationship between _-catenin and Edar remains to
be fully elucidated, one link between these signaling modules is the
finding that BMP inhibits formation of Axin2 foci, whereas Edar
suppresses BMP responses. Thus, Edar could enhance _-catenin
function indirectly by shielding it from BMP action. The placodes
induced by Noggin in Eda mutant skin (Fig. 3D) may represent such
a stabilization of Axin2 foci.
The up-regulation of Edar observed in early placodes is likely
to influence its signaling properties, as illustrated by its overexpression
under control of native regulatory elements in the
OVE951 line. The ability of moderately elevated receptor levels
to compensate for Eda deficiency suggests that receptor upregulation
confers ligand-independent signaling. Thus, the autoactivation
of Edar expression that normally occurs in early
placodes may be sufficient to allow Eda-independent signal
transduction, helping establish commitment to a follicle fate.
Our model predicts that ectopic follicles are not produced in this
case, despite autonomous Edar signaling, because its expression
remains susceptible to BMP inhibition. The K14:LMP1-Edar
line was engineered to have ligand-independent signaling but
produced a much more dramatic phenotype of ectopic follicle
formation. This observation is in accordance with our model
because the promoter driving expression is not susceptible to
down-regulation by BMPs. The skin of this transgenic line is
essentially unpatterned in the sense that follicles simply pack all
available space rather than spatially regulating their locations.
The Edar-induced phenotype contrasts with the effects of widespread
transgenic activation of _-catenin, which causes growth of
new follicles only in adult mouse skin, but does not lead to
formation of ectopic follicles during the embryonic period (37).
This finding suggests that restriction of _-catenin activation is
not limiting in defining hair placode locations, although its
activity is clearly necessary for follicle formation.
In this work, we provide a framework onto which to build other
factors involved in hair follicle development as their relationships
to the Edar and BMP pathways are uncovered. More
broadly, it is likely that variations on this molecular network
underlie pattern formation in scale and feather development and
generation of tooth morphology. In addition, our findings of
epidermal–dermal communication from the earliest stages of
patterning indicate a decisive role for both tissues and contradict
the simple view that positional information is first generated in
the dermis and then conveyed to a passive epidermis.
Methods
Animals.WT, EdaTa/Ta, and Edaraddcr/cr lines were on the FVB_N
background. OVE951 transgenic animals were used to detect
Edar by in situ hybridization. Eda is on the X chromosome; for
brevity Eda_/_ is used in the text to refer to female Eda_/_ and
male Eda_/Y animals. For timed matings the day on which a
vaginal plug was detected was counted as day 0. K14:LMP1-Edar
transgenic mice were generated as described (3.
In Situ Hybridization. Samples were fixed in 4% paraformaldehyde
in PBS overnight at 4°C. Hybridization was performed as
described (39). In situ hybridizations were photographed, and
follicle density was determined by counting the number of
Shh-expressing foci in a square of side 1 mm. Data from at least
three independent skin explant cultures were used for each
follicle density determination. Skin edges were not included in
the analysis. Stripes were included in counts as a single follicle.
qPCR. RNA was reverse-transcribed by using random primers
and AMV reverse transcriptase (Roche) in a 20-_l reaction.
Reactions were diluted 10-fold, and 5 _l was used as template
for each qPCR. TaqMan probes were supplied by Applied
Biosystems. The probes used were as follows: _Actin
(4352341E), BMP2 (Mm01962382_s1), BMP4 (Mm00432087_m1),
BMP7 (Mm00432102_m1), CTGF (Mm00515790_g1), Edar
(Mm00839685_m1), Keratin14 (Mm00516876_m1), Noggin
(Mm00476456_s1), Smad7 (Mm00484741_m1), and Sostdc1
(Mm00840254_m1). Twenty-microliter reactions were performed
in triplicate by using an OpticonII thermocycler, with
at least three biological replicates used to determine each data
point. We did not observe changes in the total amount of
_Actin expression across the different experimental treatments.
For each experiment control and treated samples came
from the same litter. Relative or absolute amounts of normalizer
and test transcripts were calculated from a standard curve.
Skin Organ Culture and Treatments. Dorsal skin was dissected,
placed onto an MF-Millipore filter on a metal grid, and submerged
in DMEM plus 5% FBS in a center-well dish (Falcon)
at 37°C and 5% CO2. Epidermal–dermal separations were
performed by incubating skin samples at 37°C for 10 min with 2
mg_ml dispase (GIBCO). Tissues were homogenized in TRI
reagent (Sigma) to isolate total RNA and proteins. Recombinant
Mou et al. PNAS _ June 13, 2006 _ vol. 103 _ no. 24 _ 9079
DEVELOPMENTAL
BIOLOGY
EdaA1 (17) was used at 50 ng_ml for in situ hybridizations and
histology and at 1,000 ng_ml for analysis of transcriptional
targets by qPCR. Recombinant BMPs and EGF were used at 500
ng_ml, and Noggin was used at 1,000 ng_ml. Human BMP2,
human BMP4, human BMP7, mouse EGF, and mouse Noggin
proteins were from R & D Systems. For experiments involving
cotreatment with Noggin the cultures were pretreated with
Noggin for 2 h before the addition of Eda. BAY 11-7082
(Calbiochem) was used at 20 _M.
Histology and Immunohistochemistry. Samples were fixed in 4%
paraformaldehyde in PBS at 4°C overnight and then dehydrated
and embedded in paraffin wax. Six-micrometer sections
were stained with hematoxylin and eosin, 1_1,000 FITCconjugated
anti-Keratin14 (Covance), or 1_100 rabbit antiphospho-
Smad1_5_8 (Cell Signaling Technology). Rabbit
primary antibody was detected by using 1_200 biotinylated
goat anti-rabbit (Upstate Biotechnology) and ABC peroxidase
(Vector Laboratories).
Western Blotting. Protein samples were run on a 12% SDS_
polyacrylamide gel and transferred to a nitrocellulose membrane.
Blots were blocked in 5% skimmed milk in TBS_0.1%
Tween 20 for 1 h and then probed with primary antibody
[1_25,000 mouse monoclonal anti-_Actin-horseradish peroxidase
AC-15 (Sigma), 1_1,000 anti-phospho-Smad1_5_8, and
1_2,000 mouse anti-phospho-I_B_ 5A5 (Cell Signaling Technology)]
in TBS_0.1% Tween 20 overnight. Signal was detected
by using horseradish peroxidase-conjugated secondary antibodies
and chemiluminescent substrate.
We thank C. M. Chuong, M. Dixon, D. Garrod, E. Harris, A. Hurlstone,
H. Meinhardt, C. Thompson, and R. Widelitz. This work was supported
by Wellcome Trust Grant 075220_Z.
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