Unité de Biologie moléculaire du Développement, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
* Author for correspondence (e-mail: jfnicola{at}pasteur.fr)
Accepted 11 July 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Stem cell, Hair follicle, Morphogenesis, Cell lineage, Cell behavior, Clonal analysis, Mouse, Temporally induced clones, Apoptosis, Anagen, Cell competition, Developmental strategy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent advances in our understanding of HF renewal have provided insights
into key questions in morphogenesis. It has been demonstrated that some of the
major operations of development, such as cell proliferation and
differentiation, cell signaling and cell rearrangements, are involved in HF
renewal. Importantly, these operations are precisely orchestrated. It is
therefore not surprising that almost all signaling pathways involved in
embryogenesis have also been shown to be involved in HF renewal. These include
Shh (Oro and Higgins,
2003), Wnt genes (Alonso and
Fuchs, 2003
), Bmp genes
(Botchkarev, 2003
;
Kulessa et al., 2000
), Fgf
genes (Petiot et al., 2003
),
Notch family members and their ligands
(Pan et al., 2004
), together
with many transcription factors [e.g. Lef1/Tcf
(Merrill et al., 2001
),
Krox20 (Gambardella et al.,
2000
), Gata3 (Kaufman
et al., 2003
), and Msx1 and Msx2
(Ma et al., 2003
)] as well as
some belonging to the Hox complex (Hoxc12 and Hoxc13)
(Shang et al., 2002
).
The HF comprises several concentric epithelial structures. The medulla, the
cortex and the cuticle form the hair shaft, which is enveloped by two
epithelial sheaths, known as the inner root sheath (IRS) and the outer root
sheath (ORS). At the distal end of the HF, IRS cells undergo apoptosis
(Tobin et al., 2002) and
liberate the hair shaft. The matrix is a proliferative zone located at the
proximal end of the HF surrounding the dermal papilla (DP). With the exception
of the ORS, all cells are produced by the matrix. In HF renewal, morphogenesis
occurs during anagen, a period following normal or induced HF destruction.
Morphogenesis follows, resulting in the generation of a multi-tissue
structure.
Several molecules (Msx1 and Msx2, Ptc1) are expressed uniformly throughout
the matrix (Ma et al., 2003;
Oro and Higgins, 2003
),
whereas others have a restricted expression pattern (Shh, Krox20, type II
receptor of TGF ß) (Gambardella et
al., 2000
). Lef1 and P-cadherin are expressed only in the cell
layers near the DP, whereas E-cadherin is expressed in the more external
layers (Jamora et al., 2003
).
However, the expression patterns of developmental genes have not yet
identified distinct cell populations within the matrix involved in producing
the different layers. The prevalent hypothesis is that the matrix is primarily
a structure in which multipotent (Kopan et
al., 2002
) cells proliferate, perhaps owing to the control of BMP
levels (Kulessa et al., 2000
).
When cells leave this structure they acquire their final identity
(Niemann and Watt, 2002
),
according to their position (Kulessa et
al., 2000
). In accordance with this model, labeling with Ki67
(Kobielak et al., 2003
), BrdU
(Oshima et al., 2001
) and
tritiated thymidine (Epstein and Maibach,
1965
) has demonstrated that the cells of the matrix proliferate
and specific biochemical markers (Gata3, keratins) have been detected in
different layers, starting just above the bulb
(Kaufman et al., 2003
;
Kobielak et al., 2003
).
However, this view is not supported by other observations. For example, mice
expressing Noggin ectopically in the matrix have a defect specific to hair
shaft cells (Kulessa et al.,
2000
); skin reconstitution assays
(Kamimura et al., 1998
), as
well as retroviral labeling (Ghazizadeh
and Taichman, 2001
), have suggested the existence of one
progenitor cell for hair shaft lineages and one for the inner root sheath
lineages.
These discrepancies highlight the need to determine how cells are organized
in the matrix. The key questions are whether the matrix is simply a zone of
multipotent proliferating cells or of several cell populations, and whether
these populations in the matrix are spatially organized
(Langbein and Schweizer,
2005). Correct interpretation of the roles of developmental genes
and signaling molecules depends on this knowledge.
We investigated cellular organization in the matrix by carrying out an
extensive clonal analysis of HF morphogenesis during anagen. We developed a
novel method of clonal analysis based on the temporal induction of
ß-galactosidase labeling. This method exploits a Cre-LoxP system, in
which a Cre recombinase fused to a mutated estrogen receptor responds
specifically to hydroxytamoxifen (OHT), this fusion is under the control of a
CMV promoter in a Cre inducible mouse line CMV Cre ERT
(Feil et al., 1996). Rosa 26
reporter mice (R26R) (Soriano,
1999
) were used to detect the recombination in Cre-targeted cells
and their descendants. In CMV Cre ERTxR26R F1, the
lacZ gene is not transcribed. Hydroxytamoxifen (OHT), which binds to
the ERT, induces Cre nuclear translocation and therefore excision
of the LoxP flanked stop sequence, allowing lacZ transcription and
leading to an inheritable labeling (Fig.
1A).
Our results challenge the hypothesis that the matrix contains equivalent multipotent cells. We show that matrix cells display a highly sophisticated organization and carry out several functions controlling the shape of the HF. The inner structures are each produced by a distinct, restricted set of precursors occupying a specific position along the proximodistal axis of the matrix. These cells are capable of self-renewal and produce transient progenitors, and they therefore have attributes of stem cells. By contrast, the ORS displays a regional mode of growth, being generated by progenitors that divide locally. Based on these results, we draw a detailed map of the fate and behavior of the cells of the matrix and propose a new model of HF morphogenesis. This fate map should help us to understand the genetic basis of HF morphogenesis and the cellular operations involved. The simplicity of the HF and our precise knowledge of the genes involved in generating this structure make the HF a highly attractive model for studying the process of morphogenesis in higher vertebrates.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synchronization of HF cycles
HF cycles were synchronized in a large area on the back of the mice by
depilation with cold wax. Depilation was performed on 6- to 8-week-old mice
whose HFs were mostly in telogen. The areas that were in anagen regrew very
rapidly and were not sampled. Depilation of telogen HFs mimicks exogen and
induces synchronously the initiation of anagen in all depilated HFs
(Stenn and Paus, 2001).
Production of temporally induced ß-galactosidase+ clones
In vivo, labeling was initiated by intraperitoneal injection of OHT (67
µg/g), in CMV Cre ERt x R26R F1 mice, which gave 80-90%
unlabelled HFs. Most labeling was therefore clonal in origin. On the day of
the injection, a skin biopsy was sampled to determine the stage of the HF at
the time of induction (Muller-Rover et
al., 2001).
Treatment of biopsy samples
Pieces of skin were removed from anesthetized mice, fixed 20 minutes in 4%
PFA-PBS at 4°C then washed three times in PBS. Fat and muscles were
carefully removed with fine forceps, and biopsy samples were then incubated in
X-gal staining solution.
HF description
HFs were carefully dissected with fine forceps under a dissecting
microscope, screened and examined in toto for ß-galactosidase+
cells at 60x magnification. The low frequency of labeling in the HF
demonstrates that most labeled cells were derived from a single recombination
event. The frequency of two independent events is equal to the product of the
probability of each single event. Thus, the calculated number of observations
of two independent events is C=N*N/Nt where N is the number of
observation of events and Nt the total number of observations. For example,
the animal in which the frequency of labeling is the highest (animal 2), N=51,
Nt=225, C=11.56, the probability of more than one recombination event in the
same HF (C/Nt) is 5.1x102. Each HF was classified, and
photographed and a digital library of the clones was produced. a-EGFP-F HFs
were observed with a confocal microscope (Zeiss Axiovert 200M) and images were
obtained and processed with LSM510 software. We studied the awl and zig-zag HF
categories.
Immunostaining
Cleaved caspase 3 immunostaining was performed as described previously
(Kassar-Duchossoy et al.,
2005).
Nomenclature
We define progenitor cells here as the cells for which all descendants
contribute to a single structure and have a limited potential of division. We
define permanent precursor cells as the cells that can divide during an entire
HF cycle. We define coherent growth as a mode of growth during which cells
remain close together after division. Founder cells of a structure are the
earliest cells of which all descendants participate in the structure
(Petit et al., 2005).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Restricted precursors at the origin of the different structures of the HF
In experiment 1 (Fig. 1B),
clonal labeling is induced 8 days after depilation, by injecting OHT into CMV
Cre ERT x R26R F1 mice and HFs are observed on D14. We found
that all HF structures were labeled, indicating that all structures were
accessible at D8. The most striking observation is that most clones (101 of
130) contribute to only one structure: the cortex and cuticle [referred to as
`cuticle clones' as they share common clonal origin and molecular markers such
as AE13 (Kaufman et al.,
2003)], the medulla, the IRS or the ORS
(Table 1, simple labeling). HF
growth during anagen therefore involves independent restricted precursor cells
generating the medulla, the cuticle, the IRS or the ORS. ORS clones are the
most frequent (75/100). Composite labeling was also observed (n=29,
combined+complex labeling, Table
1), frequently involving a combination of the ORS (combined
labeling, 14 out of 29 cases) and another structure. Statistical analysis
(composed probabilities) suggested that combined labeling corresponded to
independent labeling events. The composed probability is the product of the
frequency of each single event. We can approximate the frequency of labeling
of the ORS by
fO=(NO+NOIm+NOCm+NOICm+NOIC+NOImCm+NOImC)/Nt
and of the IRS by
fI=(NIm+NI+NOI+NOIm+NIC+NimCm+NimC+NcmI+
NOIC+NOImCm+NOImC)/Nt. Thus, the calculated
frequency for a double event of labeling in ORS and IRS is
fOI=fO* fI. For example, in animal
1, the calculated
fOIm=(44+3+1+2+2)/492*(10+3+2+2+2)/492=0.41% is close to
the observed fOIm (i.e. 3/492=0.61%) showing that these clones are
probably issued from two independent events of labeling.
|
|
Restricted precursor cells display self-renewal and are located in the matrix
Growth of the inner structures of the HF depends on matrix cells. If the
restricted precursors from inner structures undergo self-renewal in the matrix
and simultaneously produce differentiated cells, then the corresponding clones
must generate labeled cells in the matrix and along the proximodistal axis.
Indeed, in experiment 1, most clones restricted to the IRS
(Fig. 2A,B), the cuticle
(Fig. 2C,D) or the medulla
(Fig. 2E) contribute to the
matrix (Table 1, Im, Cm and Mm,
n=25 compared with I, C, M, n=1) in addition to generating
cells along the entire proximodistal axis of the HF
(Fig. 2). Therefore, precursor
cells in the matrix clearly undergo self-renewal and continually produce IRS,
cuticle or medulla cells from the time of clone induction (D8) until
observation (D14).
If there is a permanent pool of precursor cells in the matrix during anagen, then we would expect labeled cells to be present at all times in the D14 matrix, even after early induction. Indeed (experiment 3, Fig. 1), 98% of the clones induced at D3 that contribute to either the IRS or cuticle are also present in the matrix (n=20 out of 21).
The IRS, the cuticle and probably the medulla (see below) are therefore produced from permanent pools of restricted cells during anagen.
The permanent precursors produce transient progenitors
In experiment 1, we rarely identified clones restricted to a single inner
structure that did not also contribute to the matrix. Such clones would have
indicated the existence of transient progenitors. However, this lack of
detection may be due to the dynamics of HF growth, in which the most distal
cells continually disappear (see Tobin et
al., 2002), with only the younger ones remaining. If this is the
case, cells produced by a labeled transient progenitor, which has limited
contribution to the structure, progressively disappear at the distal part of
the HF during the time between induction and observation. Therefore, we
investigated whether transient progenitors exist by reducing the interval
between induction and observation to only three days
(Fig. 1B, experiment 2,
induction on D11, observation on D14). As in experiment 1, permanent
precursors for each structure are detected (n=16,
Table 2 and
Fig. 3G-L), but we also
detected 24 clones labeled only in the upper part of the HF
(Table 2 and
Fig. 3A-F). This labeling
pattern, found in the IRS (Fig.
3A,B), the cuticle (Fig.
3C,D) or the medulla (Fig.
3E,F), indicates that the labeled cells correspond to subclones of
clones produced by permanent precursors. The detection of these subclones
demonstrates the existence of transient progenitors for each inner structure
of the HF. For the IRS and the cuticle, most instances of labeling
(n=4/6 for the IRS and n=6/9 for the cuticle) correspond to
two cells (Fig. 3A,B),
indicating that the transient progenitor divides only once. For the medulla,
the subclones comprise more than two cells, indicating that the medulla
progenitors divide more than once before terminal differentiation.
|
|
|
Cuticle clones also have a unique, stereotyped pattern in the matrix (in experiments 1 and 2, Cm, n=10, Fig. 4E,F and Fig. 3J, Table 1) displaying a permanent precursor juxtaposed to the DP and the transient progenitor is in the second layer of matrix cells. Finally, there are medulla clones from permanent precursors that have labeled cells in the matrix close to the DP (Fig. 4G and Fig. 3L) and medulla clones from transient progenitors (Fig. 3F). Therefore, the cuticle and the medulla are clearly produced in a similar manner to the IRS.
Thus, the precursors of each inner structure of the HF are all located in the cell layer juxtaposed to the DP. This layer is therefore a germinative layer, given that the precursors of the inner structures are permanent during anagen, they self-renew themselves, they produce transient progenitor exhibiting different properties, presumably by asymmetric division, they are stem of more differentiated cells and their position near the DP may correspond to a niche. Thus, they can be identified as stem cells.
Inner structures are organized into proximodistal clonal columns
Within each inner structure, clones were organized into proximodistal
columns. We did not observe cellular intercalation between adjacent columns
around the circumference. This applies to all cells of a clone, from the cell
juxtaposed to the DP in the matrix to the most distal cell, and to all
categories the IRS (Fig.
2A,B and Fig. 3G,H, Im, n=18, experiments 1 and 2,
Table 1) and cuticle
(Fig. 2C,D and
Fig. 3I,J, Cm, n=10,
experiments 1 and 2, Table 1)
and, with one exception, the medulla (Fig.
2E and Fig. 3K,L,
n=3/4).
Each column was clearly formed from several stem cells because unlabelled cells were invariably intercalated with labeled ones. Columns therefore have a polyclonal origin. We calculated the size of the polyclone at the origin of each column of the inner structures of the HF, using the medial participation of founder cells method whereby the number of founder cells corresponds to the total HF length divided by the fraction of the HF proximodistal axis to which a clone participates. The calculation indicates that IRS and cuticle columns are generated by four stem cells, and the medulla columns by about six cells (data not shown). We argued previously that the stem cell is attached to the DP, and this, together with the strict columnar organization of clones, argues that the precursors for each column are aligned along the proximodistal axis of the DP. The IRS, cuticle and medulla, are thus formed from proximodistal columns that are polyclonal in origin. This organization necessarily demands the controlled intercalation of cells produced by the transient progenitors.
|
We determined the number of cells in each sector, by measuring the percentage of the proximodistal axis on an optical section of a matrix in which the cell membranes were marked with green fluorescent protein (GFP). The IRS and cuticle sectors each contained four cells, and we estimated that the medulla sector, where the labeling was not clear enough to draw cell contour, contains five or six cells (Fig. 6).
As the cells aligned along the proximodistal axis of the DP are at the origin of a single column in each concentric layer, they should constitute the polyclone at the origin of the column. Their number must therefore be equal to the size of the polyclone. This was indeed the case for the IRS and the cuticle: the number of cells in the sectors along this dimension (four cells) was identical to the calculated size of the polyclone at the origin of the columns (four cells).
Thus, globally, the stem cells are organized in exclusive sectors around the circumference of the central DP. The cells within a given sector share the same fate. We also identified the proximal sector (0-25±5%) as a sector containing cells that do not participate in any of the inner structures of the HF.
The organization of the HF inner structures in concentric layers is prefigured in the matrix
The production of concentric layers of the HF by cells organized in sectors
along the proximodistal axis of the matrix raises the possibility that HF
organization is prefigured in the organization of the matrix. However,
projection of the organization of cells along the proximodistal axis in the
matrix to their organization in concentric layers along a perpendicular axis
would require a rotation involving a cellular strategy. We investigated
whether the spatial arrangement of the cells after the division of the
permanent precursors was part of this strategy.
In the IRS sector, the alignment of the permanent precursor and the transient progenitor form an angle of 63° with respect to the proximodistal axis of the matrix (Fig. 4I). The corresponding angle is only 20° for cells in the cuticle sector (Fig. 4I). We found that this angle depends on the sector, given that it is constant for all cells within a given sector but differs considerably between sectors (Fig. 4I; data not shown). The fixation of this angle may be part of the strategy involved in orienting cells, from their production near the DP to their final location in the upper part of the HF.
Globally this orientation is a parameter defined by the sector to which these cells belong. Differences between cells from different sectors are to be added to the specific parameters of proliferation and intercalation revealed by clonal patterns (see above). At least some of these characteristics are probably intrinsic, suggesting that the cells of the different sectors are programmed differently.
The mode of production of ORS cells
The ORS, like other HF structures, grows from restricted precursors during
anagen (Table 1). However, ORS
clones differ radically from inner structure clones in other properties. The
labeling induced on D8 and observed on D14 is surprisingly heterogeneous in
distribution (Fig. 5A-D). The
clonal complexity index [the number of clones that participate to an
elementary region (Petit et al.,
2005)] (Fig. 5N)
shows a maximum of 13, which represents only 22% of the clones. Therefore, the
analysis of clonal complexity index failed to identify a family of clones
systematically contributing to one region of the HF. These data suggest that
ORS progenitors are dispersed throughout the structure and that there is no
pool of permanent stem cells at the origin of the ORS. They suggest instead a
mode of coherent growth, consistent with the observed systematic arrangement
of the labeled cells in coherent clusters
(Fig. 5A-D).
Clone size (cell number) and clonal extension were extremely disparate (Fig. 5O,P). This indicates that the cells labeled on D8 do not have an equivalent outcome. These differences may be due to differences in generation time, division potential or apoptosis. If only differences in generation time and/or division potential were involved, then the number of ORS clones would remain constant during anagen. We tested this hypothesis (Fig. 1B, experiment 3). Remarkably, 70% of the labeling observed on D9 had disappeared by D14 (Fig. 5Q). This decrease in labeling indicates that a large fraction of ORS cells undergo apoptosis, leading to the eventual disappearance of many clones. To test this point directly, we have performed an immunostaining against cleaved caspase 3 on cryostat sections of HF in anagen. We have detected apoptotic cells in the ORS (Fig. 5E-M). We investigated whether this apoptosis specifically affected certain regions of the ORS by analyzing the distribution of clones as a function of their size (Fig. 5O). Regardless of cell number (1 to 121) the clones were evenly distributed in the HF (Fig. 5N) and we therefore found no evidence for preferential regional apoptosis of ORS cells.
|
The ORS therefore differs from the IRS, cuticle and medulla as it follows a regional, coherent mode of growth from progenitors of polyclonal origin and the proliferation of these progenitors is limited by apoptosis. Apoptosis affects all progenitors, whatever their position in the ORS, but the clones are affected to different extents. Overall the ORS remains somewhat static with a balance between apoptosis and mitosis.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altogether, this map of the fate and behavior explains the complex morphology of the HF in terms of a few operations controlled and executed in the matrix: (1) an absence of circumferential intercalation within and above the matrix, resulting in the organization of the HF into proximodistal columns; (2) an intercalation in the matrix of the cells produced by the transient progenitors that accounts for the polyclonal origin of these columns; (3) an arrangement of cells after stem cell division that relates to the rotation that converts the proximodistal axis in the matrix to the radial axis in the HF; and (4) an absence of radial intercalation in the matrix that maintains the organization in concentric layers. As a consequence of these cell operations, the proximodistal sectors in the germinative layer prefigure the radial order of the inner concentric layers of the HF.
This fate map bridges the gap in our knowledge regarding the way in which multipotent epithelial stem cells in the bulge renew the multi-tissue HF. The remaining missing step is how the multipotent epithelial stem cells diversify when the matrix reforms. This step can be analyzed further by using the method of clonal analysis presented in this study.
|
The HF has a linear growth (Fig. 1B) that fits with the stem cell mode of growth of the IRS, cuticle and medulla that progress in concert during the entire duration of anagen. However, this linear growth does not fit with a simple proliferative mode of growth of the ORS. Therefore the non-regional apoptosis detected in the ORS probably adapts the growth of the outer layer to the linear growth of the inner layers of the HF by modulating a proliferative mode of growth that gives an exponential increase in cell number. This coordination is essential for balanced HF morphogenesis.
The organizations in the matrix
Radial organization of cell behaviors
Our results confirm the view that the matrix is the only proliferative
region of the inner structures of the HF. However, they show that the matrix
is not composed of equivalent cells, a finding that complements the results of
Kopan and collaborators (Kopan et al.,
2002). We also show that each HF inner structure is produced by
two categories of precursors: permanent precursors with attributes of stem
cells [self-renewal, asymmetric division and their location in the germinative
layer, whose proximity with the DP provides a specific micro-environment that
may constitute a niche (Fuchs et al.,
2004
)] and transient progenitors that divide symmetrically and
produce postmitotic cells (Fig.
6B). Their lineage is highly stereotyped. In this study, we were
able to locate the three types of cells in the matrix, including the permanent
precursor cells that are juxtaposed to the DP (the germinative layer), the
transient progenitors that are in the next radial layer and the postmitotic
cells, which exist in the layers. Clonal analysis also revealed intercalation
of the postmitotic cells, at the level of the more external layers. This
intercalation applies to the IRS, the cuticle and the medulla. It will be
interesting to determine which type of intercalation is involved. Radial and
mediolateral intercalation types are used several times during vertebrate
development, for example for gastrulation movements, and they rely on
different basic mechanisms (Keller,
2002
). Whatever the case may be, cell behavior (symmetric or
asymmetric division, intercalation, and probably adhesion and cell polarity)
are organized radially in the HF matrix.
Interestingly, several molecular markers and signaling patterns have been
shown to vary across the radial plane. Lef1 and Tcf3, two transcription
factors activated by the canonical Wnt pathway, are expressed close to the DP
(probably in the first two layers) (Jamora
et al., 2003) and in the outer cell layer
(DasGupta and Fuchs, 1999
),
respectively. The adhesion molecule P-cadherin is found in the cells closest
to the DP (layers 1 and 2) and E-cadherin is found in the more external
layers. The genetic circuitry controlling this differential expression of
cadherins has been identified: Noggin secreted by the DP inhibits BMP,
inducing the production of Lef1, which represses E-cadherin in the layers near
the DP (Jamora et al., 2003
).
The shift from one type of cadherin to another may be involved in the control
of cell intercalation, as maybe the non-canonical Wnt pathway (reviewed by
Myers et al., 2002
).
Thus, both cell behaviors and the genetic markers probably involved in their control are organized radially. The DP may be responsible for this organization. For example, a radial gradient of BMPs may be established as a result of the homogeneous expression of Noggin in the DP. The relationship between cell behavior, signaling pathways in the matrix and the DP and other molecular aspects remains to be elucidated. However, the radial dimension seems to correspond to the control of many cellular behaviors, regardless of the cell fate.
Proximodistal organization of cell fates
Several analyses have suggested the existence of different precursors in
the matrix (Ghazizadeh and Taichman,
2001; Kamimura et al.,
1998
; Kopan et al.,
2002
; Kulessa et al.,
2000
). Our analysis demonstrates the existence of three distinct
sectors in the matrix corresponding to IRS precursors, precursors for the
cuticle and the cortex and precursors for the medulla. These sectors are
organized along the proximodistal axis in the germinative layer. Wnt activity,
revealed by a Wnt reporter gene (TOPGAL), located hair shaft precursors in the
distal part of the matrix (DasGupta and
Fuchs, 1999
). Our clonal analysis is consistent with these
findings. We demonstrated that the structure to which a precursor contributes
depends on its proximodistal position. In the fate map, we suggest that the
hair shaft (medulla, cortex and cuticle) is produced by cells more distal than
those that produce the IRS. These sectors probably remain static after their
establishment at the beginning of anagen because over the longest observable
periods, a labeled stem cell produces only one type of differentiated cell.
The strict proximodistal organization of the germinative layers into sectors
raises the possibility that the cells of these sectors are different. These
sectors cannot be differentiated with known biochemical markers, but do differ
in the cell arrangement after the permanent precursor division.
Thus, there are at least three different populations of stem cells in the matrix, in terms of properties, location and, most importantly, fate. We suggest that the proximodistal axis of the matrix corresponds to the organization of stem cell fate. Cell fates and cell behaviors therefore seem to be uncoupled and are probably organized by means of two orthogonal systems.
Conclusion: the uncoupling of developmental operations
In summary, the matrix seems to be organized by two systems working in
orthogonal dimensions and controlling two key operations of HF morphogenesis,
notably cell diversification and cell behavior. This combination of two
systems, exploiting the three dimensions of the matrix, might have greatly
simplified evolution of the morphogenetic strategy used to create HFs and to
diversify them (Wu et al.,
2004) in mammals. The basis of this simplification is the
uncoupling of cell diversification and morphogenesis suggested by the fact
that the same cell behavior (for instance a stem cell mode of division) is
shared by cells with different fate (IRS, cuticle and medulla precursors). The
HF matrix thus provides a paradigm for elucidating more complex embryonic
structures in which the existence of three dimensions may have made it
possible to uncouple various developmental operations, thereby considerably
simplifying their evolution.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alonso, L. and Fuchs, E. (2003). Stem cells in
the skin: waste not, Wnt not. Genes Dev.
17,1189
-1200.
Botchkarev, V. A. (2003). Bone morphogenetic proteins and their antagonists in skin and hair follicle biology. J. Invest. Dermatol. 120, 36-47.[CrossRef][Medline]
Cotsarelis, G., Sun, T. T. and Lavker, R. M. (1990). Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61,1329 -1337.[CrossRef][Medline]
DasGupta, R. and Fuchs, E. (1999). Multiple
roles for activated LEF/TCF transcription complexes during hair follicle
Development and differentiation. Development
126,4557
-4568.
Epstein, W. L. and Maibach, H. I. (1965). Cell renewal in human epidermis. Arch. Dermatol. 92,462 -468.[CrossRef][Medline]
Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D. and
Chambon, P. (1996). Ligand-activated site-specific
recombination in mice. Proc. Natl. Acad. Sci. USA
93,10887
-10890.
Fuchs, E., Merrill, B. J., Jamora, C. and DasGupta, R. (2001). At the roots of a never-ending cycle. Dev. Cell 1,13 -25.[CrossRef][Medline]
Fuchs, E., Tumbar, T. and Guasch, G. (2004). Socializing with the neighbors: stem cells and their niche. Cell 116,769 -778.[CrossRef][Medline]
Gambardella, L., Schneider-Maunoury, S., Voiculescu, O., Charnay, P. and Barrandon, Y. (2000). Pattern of expression of the transcription factor Krox-20 in mouse hair follicle. Mech Dev. 96,215 -218.[CrossRef][Medline]
Ghazizadeh, S. and Taichman, L. B. (2001).
Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of
adult mouse skin. EMBO J.
20,1215
-1222.
Hardy, M. H. (1992). The secret life of the hair follicle. Trends Genet. 8, 55-61.[Medline]
Hendriks, R. W., Nawijn, M. C., Engel, J. D., van Doorninck, H., Grosveld, F. and Karis, A. (1999). Expression of the transcription factor GATA-3 is required for the Development of the earliest T cell progenitors and correlates with stages of cellular proliferation in the thymus. Eur. J. Immunol. 29,1912 -1918.[CrossRef][Medline]
Jamora, C., DasGupta, R., Kocieniewski, P. and Fuchs, E. (2003). Links between signal transduction, transcription and adhesion in epithelial bud development. Nature 422,317 -322.[CrossRef][Medline]
Kamimura, J., Lee, D., Baden, H., Brissette, J. and Dotto, G. (1998). Primary mouse keratinocyte cultures contain hair follicle progenitor cells differentiation potential. J. Invest. Dermatol. 109,534 -540.
Kassar-Duchossoy, L., Giacone, E., Gayraud-Morel, B., Jory, A.,
Gomes, D. and Tajbakhsh, S. (2005). Pax3/Pax7 mark a novel
population of primitive myogenic cells during development. Genes
Dev. 19,1426
-1431.
Kaufman, C. K., Zhou, P., Pasolli, H. A., Rendl, M., Bolotin,
D., Lim, K. C., Dai, X., Alegre, M. L. and Fuchs, E. (2003).
GATA-3: an unexpected regulator of cell lineage determination in skin.
Genes Dev. 17,2108
-2122.
Keller, R. (2002). Shaping the vertebrate body
plan by polarized embryonic cell movements. Science
298,1950
-1954.
Kobielak, K., Pasolli, H. A., Alonso, L., Polak, L. and Fuchs,
E. (2003). Defining BMP functions in the hair follicle by
conditional ablation of BMP receptor IA. J. Cell Biol.
163,609
-623.
Kopan, R., Lee, J., Lin, M. H., Syder, A. J., Kesterson, J., Crutchfield, N., Li, C. R., Wu, W., Books, J. and Gordon, J. I. (2002). Genetic mosaic analysis indicates that the bulb region of coat hair follicles contains a resident population of several active multipotent epithelial lineage progenitors. Dev. Biol. 242, 44-57.[CrossRef][Medline]
Kulessa, H., Turk, G. and Hogan, B. L. (2000).
Inhibition of Bmp signaling affects growth and differentiation in the anagen
hair follicle. EMBO J.
19,6664
-6674.
Langbein, L. and Schweizer, J. (2005). Keratins of the human hair follicle. Int. Rev. Cytol. 243, 1-78.[Medline]
Ma, L., Liu, J., Wu, T., Plikus, M., Jiang, T. X., Bi, Q., Liu,
Y. H., Muller-Rover, S., Peters, H., Sundberg, J. P. et al.
(2003). `Cyclic alopecia' in Msx2 mutants: defects in hair
cycling and hair shaft differentiation. Development
130,379
-389.
Merrill, B. J., Gat, U., DasGupta, R. and Fuchs, E.
(2001). Tcf3 and Lef1 regulate lineage differentiation of
multipotent stem cells in skin. Genes Dev.
15,1688
-1705.
Moreno, E., Basler, K. and Morata, G. (2002). Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416,755 -759.[CrossRef][Medline]
Morris, R. J., Liu, Y., Marles, L., Yang, Z., Trempus, C., Li, S., Lin, J. S., Sawicki, J. A. and Cotsarelis, G. (2004). Capturing and profiling adult hair follicle stem cells. Nat. Biotechnol. 14,14 .
Muller-Rover, S., Handjiski, B., van der Veen, C., Eichmuller, S., Foitzik, K., McKay, I. A., Stenn, K. S. and Paus, R. (2001). A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Invest. Dermatol. 117,3 -15.[CrossRef][Medline]
Myers, D. C., Sepich, D. S. and Solnica-Krezel, L. (2002). Convergence and extension in vertebrate gastrulae: cell movements according to or in search of identity? Trends Genet. 18,447 -455.[CrossRef][Medline]
Niemann, C. and Watt, F. M. (2002). Designer skin: lineage commitment in postnatal epidermis. Trends Cell. Biol. 12,185 -192.[CrossRef][Medline]
Oliver, R. F. (1966). Whisker growth after removal of the dermal papilla and lengths of follicle in the hooded rat. J. Embryol. Exp. Morphol. 15,331 -347.[Medline]
Oro, A. E. and Higgins, K. (2003). Hair cycle regulation of Hedgehog signal reception. Dev. Biol. 255,238 -248.[CrossRef][Medline]
Oshima, H., Rochat, A., Kedzia, C., Kobayashi, K. and Barrandon, Y. (2001). Morphogenesis and renewal of hair follicles from adult multipotent stem cells. Cell 104,233 -245.[CrossRef][Medline]
Pan, Y., Lin, M. H., Tian, X., Cheng, H. T., Gridley, T., Shen, J. and Kopan, R. (2004). gamma-secretase functions through Notch signaling to maintain skin appendages but is not required for their patterning or initial morphogenesis. Dev. Cell 7, 731-743.[CrossRef][Medline]
Petiot, A., Conti, F. J., Grose, R., Revest, J. M.,
Hodivala-Dilke, K. M. and Dickson, C. (2003). A crucial role
for Fgfr2-IIIb signalling in epidermal development and hair follicle
patterning. Development
130,5493
-5801.
Petit, A. C., Legué, E. and Nicolas, J. F. (2005). Methods in clonal analysis and applications. Reprod. Nutr. Dev. 45,321 -339.[CrossRef][Medline]
Shang, L., Pruett, N. D. and Awgulewitsch, A. (2002). Hoxc12 expression pattern in developing and cycling murine hair follicles. Mech. Dev. 113,207 -210.[CrossRef][Medline]
Simpson, P. (1979). Parameters of cell competition in the compartments of the wing disc of Drosophila. Dev. Biol. 69,182 -193.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stenn, K. S. and Paus, R. (2001). Controls of
hair follicle cycling. Physiol. Rev.
81,449
-494.
Tobin, D. J., Foitzik, K., Reinheckel, T., Mecklenburg, L.,
Botchkarev, V. A., Peters, C. and Paus, R. (2002). The
lysosomal protease cathepsin L is an important regulator of keratinocyte and
melanocyte differentiation during hair follicle morphogenesis and cycling.
Am. J. Pathol. 160,1807
-1821.
Tumbar, T., Guasch, G., Greco, V., Blanpain, C., Lowry, W. E.,
Rendl, M. and Fuchs, E. (2004). Defining the epithelial stem
cell niche in skin. Science
303,359
-363.
Wu, P., Hou, L., Plikus, M., Hughes, M., Scehnet, J., Suksaweang, S., Widelitz, R., Jiang, T. X. and Chuong, C. M. (2004). Evo-Devo of amniote integuments and appendages. Int. J. Dev. Biol. 48,249 -270.[CrossRef][Medline]