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First published online 14 March 2007
doi: 10.1242/dev.001750
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Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
* Author for correspondence (e-mail: katia.georgopoulos{at}cbrc2.mgh.harvard.edu)
Accepted 13 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Key words: Mi-2ß (Chd4), Chromatin, Epidermis, Stem cells
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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; Watt,
1998). The cells of the basal layer of the epidermis are
comparatively undifferentiated and proliferate to generate progeny that detach
from the basement membrane and progressively differentiate as they are
displaced through the successive layers of the epidermis before being shed
from the surface. Homeostasis of the mature epidermis is thought to depend on
the cycling of stem cells in the basal layer, although it remains unclear
whether there are dedicated stem cells that self renew while giving rise to
more committed `transient amplifying' (TA) basal cells, or whether basal cells
are more generally equipotent. The differentiation of the epidermis of the
mouse begins at E8.5, as the embryonic ectoderm begins to express epithelial
markers such as the nuclear factor p63 (also known as Trp63 - Mouse Genome
Informatics) and keratins 8 and 18
(Jackson et al., 1981;
Koster et al., 2004).
Ectodermal commitment to the epidermal lineage occurs between E9.5 and E12.5,
producing a basal layer expressing keratins 5 and 14 and a second, transitory
layer known as the periderm (Byrne et al.,
2003). Further differentiation commences at E14.5, as the
intermediary layer and then the spinous and granular layers of the mature
epidermis are formed. The final stages of differentiation include the
formation of a cornified layer and the acquisition of barrier function, events
that occur just prior to birth. This progression of development in embryonic
epidermis is demarcated by the expression of structural proteins in the newly
formed layers, a pattern that is recapitulated by individual keratinocytes as
they transit from the basement membrane to the surface in mature
epidermis.
During skin development, a subset of keratinocytes is recruited to form
hair follicles. This fate decision is guided by inductive interactions with an
underlying population of mesenchymal cells, some of which eventually form the
dermal papilla (DP) (Hardy,
1992). These epithelial-mesenchymal interactions lead to the
downgrowth of a hair peg and to the formation of a hair bulb, in which
keratinocytes proliferate and differentiate into distinct concentric layers of
epithelial cells that constitute the inner root sheath and the hair shaft
(Sengel and Mauger, 1976). In
the mouse, pelage hairs consist of different types of hair follicles that are
formed in successive waves during embryogenesis
(Hardy, 1992). Primary
(tylotricht) follicles initiate development at E14.5 and are characterized by
two sebaceous glands and a large hair bulb that gives rise to a long straight
hair. Induction of secondary hair follicles that produce awls hairs begins at
E16.5. A final wave of follicle formation in late gestation and after birth
gives rise to the zigzag and auchene hairs.
Gene expression changes associated with the partially characterized genetic
hierarchy that guides follicle development serve as markers of specific steps
in follicle development (Millar,
2002). Activation of the canonical Wnt/ß-catenin pathway is
required for the initial formation of hair placodes in all three waves
(Andl et al., 2002). Another
early step in placode development is a local increase in the expression of the
ectodysplasin-A receptor (Edar), which is expressed at low levels throughout
the basal epidermis before placode formation. The local increase in Edar
expression is followed rapidly by a decrease in E-cadherin (cadherin 1)
expression and induction of both sonic hedgehog (Shh) and P-cadherin (cadherin
3) expression in the epithelial cells in contact with the forming DP
(Hardy and Vielkind, 1996;
Headon and Overbeek, 1999;
Jamora et al., 2003).
Expression of bone morphogenetic proteins 2 and 4 (Bmp2 and Bmp4) in the
mesenchyme indicates the formation of the DP, and subsequent Wnt5a expression
in the DP reflects its further maturation that is dependent on Shh expression
in the epidermal placode (Reddy et al.,
2001; Wilson et al.,
1999). Although the disruption of components of these signaling
pathways might have preferential effects on specific waves of follicle
formation, this sequence of gene expression is shared by follicles in all
three waves.
Commitment to the epidermal lineage and subsequent decisions between
interfollicular and follicular cell fates rely on a balance between positive
and negative events in gene expression. Sequence-specific transcription
regulators have been implicated in lineage decisions and function in part by
targeting genes whose expression supports lineage progression. p63, a member
of the p53 family of DNA-binding factors, is a key regulator of epidermal
differentiation as its ectopic expression in simple epithelia induces
expression of epidermal keratins and presumably induces the squamous cell fate
(Koster et al., 2004). The
abilities of lineage-determining DNA-binding factors to either access their
chromosomal sites and/or provide permanence to the regulation of the
associated locus is central to lineage commitment. Chromatin regulators
function in concert with lineage-specific factors to provide long-term
epigenetic regulation (Kim et al.,
1999). These include ATP-dependent remodelers, histone
deacetylases (HDACs), histone acetyltransferases (HATs) and methylases, which
are enzymes that can transiently or permanently change the accessibility of
genes to transcriptional machineries. Chromatin regulators can generate
epigenetic markings on chromatin that underlie the cell's memory and allow for
the stable propagation of lineage-specific expression profiles through
multiple divisions during development
(Georgopoulos, 2002).
Mi-2
and Mi-2ß (also known as Chd3 and Chd4, respectively -
Mouse Genome Informatics) are closely related genes encoding ATP-dependent
chromatin remodelers (Seelig et al.,
1996). Mi-2ß is expressed at significantly higher levels than
Mi-2 in developing and adult tissues and is observed in the skin,
mucosal epithelia, the thymus, the kidney, specific areas of the brain, and in
the hemopoietic foci of the liver of the mouse embryo
(Kim et al., 1999). Mi-2
proteins contain two PHD (plant homeodomain) zinc-finger domains, two chromo
domains and a SWI2/SNF2-type helicase/ATPase domain. They reside in the
nucleosome remodeling histone deacetylase (NURD) complex that includes the
histone deacetylases Hdac1 and Hdac2, two histone-binding proteins RbAp46 and
RbAp48 (also known as Rbbp7 and Rbbp4, respectively - Mouse Genome
Informatics), and the metastasis-associated proteins Mta1 and Mta2
(Hassig et al., 1998;
Xue et al., 1998;
Zhang et al., 1998). Because
of the Mi-2 association with HDAC, it was thought to be involved primarily in
establishing a repressive chromatin environment by cooperating with the HDACs
of the NURD complex. However, recent reports indicate that Mi-2 can
participate in other regulatory activities that relate to transcriptional
elongation, termination (Alen et al.,
2002; Krogan et al.,
2003), chromatid cohesion
(Hakimi et al., 2002), and
positive regulation of gene expression
(Hirose et al., 2002;
Williams et al., 2004).
In the immune system, much of the Mi-2ß is found in a stable complex
with members of the Ikaros family of lymphoid-lineage-determining factors and
HDACs in the NURD complex (Kim et al.,
1999; O'Neill et al.,
2000). However, during T-cell development, Mi-2ß also acts in
association with the E-box DNA-binding protein HEB (also known as Tcf12 -
Mouse Genome Informatics) and the HAT p300 as a positive regulator of the
Cd4 gene, a hallmark in the differentiation of the helper T-cell
lineage. Conditional inactivation of Mi-2ß during T-cell development
revealed that it is required to generate a chromatin environment at
positive-acting regulatory elements that is conducive to Cd4 expression,
thereby setting the stage for lineage-specific transcription factors to drive
this developmental decision in the T lineage. These studies also revealed a
key role for Mi-2ß in the transition from the double negative to the
double positive stage of thymocyte differentiation, and in mature T cells in
promoting antigen-mediated proliferative responses
(Williams et al., 2004).
The high levels of Mi-2ß expression in the embryonic ectoderm and its preferential expression in the hair placode and the matrix of the hair follicle (Fig. 1A) prompted us to examine the role of Mi-2ß in skin development. The conditional allele of Mi-2ß was inactivated in keratinocytes at distinct stages of epidermal development and new insights into the regulatory events that control development and homeostasis of the epidermis and its appendages were revealed.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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).
K14-Cre transgenic mice obtained from Dr P. Chambon
(Li et al., 2001) were mated
to Mi-2ßLoxPF/LoxPF mice to generate mice homozygous
for loss of Mi-2ß function in the epidermis. PCR genotyping was performed
using the genomic DNA isolated from P1 dorsal epidermis. Briefly, epidermis
was separated from dermis by treating with 0.05% collagenase overnight at
4°C. PCR primers are as follows: For WT or LoxPF allele: Mi-2ß+F, 5'-CTCCAAGAAGAAGACGGCAGATCT-3' and Mi-2 INR, 5'-GTCCTTCCAAGAAGAGCAAG-3';
For 
F allele: Mi-2ß+F and KG4R,
5'-CTTCCACAGTGACGTCCAGACGCA-3';
For K14-Cre: K14Cre-5'-2, 5'-ACAGACATGATGAGGCGGAT-3' and K14Cre3', 5'-CGACCGGTAATGCAGGCAAAT-3'.
Cycling conditions for all the reactions were as follows: 35 cycles of 30 seconds at 94°C, 1 minute at 56°C and 1 minute at 72°C.
Histology
For histopathology, tissue samples were frozen in OCT or fixed in 3.7%
formalin and embedded in paraffin. Sections (4 µm) were stained with
Hematoxylin and Eosin. For immunofluorescence, TUNEL, and in situ
hybridization, tissue samples were frozen in OCT and sectioned at 6 µm.
In situ analysis
Digoxygenin (DIG) probe synthesis was performed according to the
manufacturer's instructions (Roche), using probes transcribed from a plasmid
containing Mi-2ß cDNA (4264-5668 bp, NM145979), Mi-2 cDNA
(C-terminal 1401 bp, BC030435), ß-catenin (1830-2456 bp, NM007614), Bmp2
(825-1524 bp, NM007553), Shh (120-760 bp, X76290), and Wnt5a (520-870 bp,
NM009524). Sections were fixed in 4% formaldehyde, permeabilized by proteinase
K digestion, refixed in 4% formaldehyde, acetylated in 0.1 M
triethanolamine/25% acetic anhydride, and hybridized to respective probes at
55-65°C for 16 hours. After hybridization, the sections were washed
sequentially with 6xSSC, then 2xSSC containing 50% formamide and
10 mM EDTA, then 2xSSC, and finally 0.2xSSC. The DIG-label was
detected by anti-DIG Fab (Roche) coupled to alkaline phosphatase using
NBT/BCIP (Roche).
Immunofluorescence
Sections were fixed in 4% formaldehyde and subjected to indirect
immunofluorescence. When staining with mouse mAbs, the MOM Fluorescent Kit
(Vector) was used. Primary antibodies used were: mouse monoclonal Mi-2ß
[16G4 (Kim et al., 1999)],
rabbit polyclonal K5 (BAbCo), rabbit polyclonal K1 (BAbCo), rabbit polyclonal
loricrin (BAbCo), mouse monoclonal Pcna (PC10, Santa Cruz), goat polyclonal
Edar (R&D Systems), rat monoclonal P-cadherin (PCD-1, Zymed), rat
monoclonal E-cadherin (ECCD-2, Zymed), mouse monoclonal p63 (4A4, Santa Cruz).
Fluorescence-conjugated secondary antibodies for primary antibodies developed
in rabbit, rat, or goat were obtained from Jackson ImmunoResearch
Laboratories. DAPI was used to stain nuclei.
TUNEL analysis
TUNEL was performed according to the manufacturer's protocol (Promega).
Briefly, sections were fixed in 3.7% formalin, permeabilized by proteinase K
digestion, and subjected to a TdT reaction. The TdT label was detected by
DAB.
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).
Briefly, mice were sacrificed by terminal anesthesia, incubated for 5 minutes
in methanol, rinsed in PBS, followed by incubation in 0.1% Toluidine Blue.
After extensive washing, dye penetration reveals barrier status - white skin
indicating an intact barrier, stained skin the lack of a barrier.
Quantitative histomorphometry
The number of hair follicles per unit length of epidermis was counted in
frozen and paraffin sections of Mi-2ß mutant dorsal skin (n=5)
at E18.5 and P1, and was compared with that of age-matched wild-type (WT) skin
(n=3). The percentage of hair follicles at different stages of
morphogenesis was assessed. These stages were defined on the basis of accepted
morphological criteria (Hardy,
1992). At least 151 longitudinal hair follicles in 63 microscopic
fields derived from five Mi-2ß mutant animals were compared with those of
290 hair follicles from five age-matched WT mice at E18.5. At least 412
longitudinal hair follicles in 66 microscopic fields derived from five
Mi-2ß mutant animals were compared with those of 436 hair follicles from
three age-matched WT mice at P1.
RT-PCR
RNA was extracted from P1 dorsal epidermis with Trizol (Invitrogen)
according to the manufacturer's protocol. cDNA was generated using random
primers and the Superscript II Kit (Invitrogen). The cDNA was amplified by PCR
using the following conditions: 28-35 cycles of 30 seconds at 94°C, 45
seconds at 57°C and 45 seconds at 72°C. The PCR products were ligated
into the pCRII TA vector (Invitrogen) and verified by sequencing. The primers
used for PCR were:
dl (Edar) S, 5'-GTGCTGGTGGTGTCTCTGAT-3' and dl (Edar) AS, 5'-GCTGTCAGCTTCTGGAACTC-3';
ß-catenin S, 5'-ACATCCTTGCTCGGGACGTT-3' and ß-catenin AS, 5'-TACTAAGGACGATTTACAGG-3'; Lef1 S, 5'-CACGGACAGTGACCTAATGC-3' and Lef1 AS, 5'-GAAACAACCGTTTTCGGCTT-3';
Bmp2 S, 5'-CAGGAAGCTTTGGGAAACAG-3' and Bmp2 AS, 5'-CCTCCACAACCATGTCCTGA-3';
Shh S, 5'-GACCCCTTTAGCCTACAAGC-3' and Shh AS, 5'-AGAAGACCTTCTTGGCGCCT-3';
Patched S, 5'-CCTTCGCTCTGGAGCAGATT-3' and Patched AS, 5'-GGCATTCTTGACGGTACCAC-3';
Mi-2ß(E11-13) S, 5'-CCTTTCCAGTTTCCGTAGCTTCAC-3' and Mi-2ß(E11-13) AS, 5'-CAGCGGAAGAATGATATGGACGAC-3';
Mi-2ß (E30-34) S, 5'-CCCGAACTGGCTGAAGTAGAGGAAAAC-3' and Mi-2ß (E30-34) AS, 5'-GGAGTTTCTCCATTCTGAAGCATCACG-3';
Mi-2 S, 5'-GTGTTGACCCGCATTGG-3' and Mi-2 AS,
5'-TCTGTATCCAGGTTCAGG-3'; and
GAPDH S, 5'-AAGGTCGGTGTGAACGGATT-3' and GAPDH AS, 5'-TGGTGGTGCAGGATGGATTG-3'.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Mi-2ß inactivation in the epidermis causes abnormal development of the integument
The role of Mi-2ß during development of the epidermis and hair was
evaluated using a conditional inactivation strategy. Mice containing an
Mi-2ß allele with loxP sites flanking the ATPase domain were crossed to a
K14cre transgenic line that expresses cre recombinase in the basal
epidermis and the outer root sheath of the hair follicle
(Li et al., 2001). As
previously shown (Williams et al.,
2004), cre recombinase removes sequences encoding the ATPase
domain required for Mi-2ß remodeling activity, resulting in a mutant mRNA
that does not produce stable protein. As revealed by genomic PCR, the floxed
Mi-2ß alleles (Mi-2ßloxPF/loxpF) were efficiently
disrupted by K14cre in the epidermis (Fig.
1B).
Mice homozygous for the Mi-2ßLoxPF allele and carrying the K14cre transgene die within 24 hours of birth. The skin of these mutant mice is shiny and flaky (Fig. 1C), exhibits a severe reduction in hair follicles and shows abnormal whisker hairs (data not shown). A curly tail is another morphological phenotype of the K14cre deletion of Mi-2ß (Fig. 1C). Histological analysis of the mutant skin demonstrated a marked difference in phenotypes between the dorsal and ventral areas (Fig. 1D). In the dorsolateral region, a relatively normal multilayered epidermis was detected, but the number and length of hair follicles were drastically reduced. In most of the ventral region, the structure of the epidermis was greatly affected, with most layers reduced in size. Hair follicles were also absent. A stratum corneum was present in both areas (Fig. 1D). Occasional patches of multilayered skin lacking hair follicles were observed among the severely depleted ventral skin of the mutant (see Fig. S1 in the supplementary material).
The difference in phenotype between the dorsal and ventral skin could be explained either by a fundamental difference in the requirement for Mi-2ß in the development of dorsal and ventral skin, or by a difference in the timing of Mi-2ß protein depletion. Examination of Mi-2ß protein at E10.5 revealed its dramatic depletion in most of the ventral skin, while expression persisted in dorsal skin (Fig. 1E). Mi-2ß was still detectable at E13.5 in dorsal epidermis (Fig. 1E). However, by E14.5, extensive depletion of Mi-2ß protein was also observed in dorsal skin, although some areas of persistent expression were apparent at this and later stages of development (Fig. 1E and data not shown). Thus, the more severe phenotype in the ventral region of the skin is associated with depletion of Mi-2ß during the earliest stages of epidermal development, whereas the distinct phenotypes seen in dorsal skin occur when Mi-2ß is removed after the initiation of epidermal differentiation.
Effects of Mi-2ß depletion prior to epidermal differentiation
Although Mi-2ß depletion in the ventral epidermis occurs at E10.5,
ventral skin from embryos at E14.5 was histologically normal
(Fig. 2A, mutant E14.5). The
effects of Mi-2ß depletion were not detected until E16.5, when expansion
and maturation of the stratified layers became apparent
(Fig. 2A, mutant E16.5). At
this and subsequent stages, the epidermis was markedly reduced in thickness
and no appendages were detected (Fig.
2A, mutant E16.5-P1). A thin, cornified layer could be discerned
in ventral skin even in the most severely affected areas
(Fig. 1D;
Fig. 2A, mutant P1).
Keratin 5 (K5), keratin 1 (K1) and loricrin serve as markers of the basal, suprabasal and granular layers, respectively, in mature skin and are also indicative of the formation of these layers during embryonic development. K5 was expressed in a wild-type (WT) pattern in the innermost layer of the mutant epidermis up to E14.5-16.5 (Fig. 2B). However, after E16.5, a progressive reduction in the K5-expressing layer was seen. Strikingly, by P1, some areas of the skin failed to express any K5 (Fig. 2B, mutant P1-2). K1 expression in the suprabasal layer of WT skin was readily detected by E13.5 (Fig. 2C, WT). In the mutant skin, induction of K1 expression was delayed to E14.5 (Fig. 2C, mutant). A progressive reduction in the K1-expressing layer was seen from E16.5 to P1, with some areas completely lacking the suprabasal layer (Fig. 2C, mutant P1-2). Finally, loricrin, which demarcates the granular layer, is normally detected by E14.5 and its induction was not influenced by the absence of Mi-2ß (Fig. 2D, mutant). Nonetheless, by P1, the loricrin-expressing layer was greatly reduced in the mutant skin with very little staining detected (Fig. 2D, mutant).
Thus, Mi-2ß depletion at an early stage of development, prior to or during ectodermal commitment to the epidermal lineage, severely affects epidermal differentiation. The initially normal induction and expression of squamous-cell-layer markers indicates that there is no major defect in the differentiation of the cell types of the epidermis. However, the progressive depletion of the lower layers of the skin during development suggests either a defect in the ability of epidermal stem cells to renew themselves or to continue to generate their more differentiated progeny. Defects in proliferation, survival or differentiation of the cells of the basal layer of the epidermis could give rise to this general thinning of the epidermis and depletion of the lower layers. However, at both E14.5 and E16.5, a similar number of Pcna-positive cells were detected in the basal layer of the ventral mutant as compared with WT skin (Fig. 3A,B), indicating that there is no initial defect in the proliferation of the basal cells. By contrast, from E18.5 through P1, a significant reduction of Pcna-positive cells was seen in the basal layer of ventral mutant skin (Fig. 3A,B). A possible effect of Mi-2ß depletion on apoptosis during epidermal differentiation was also examined. A process akin to apoptotic cell death normally occurs during terminal differentiation in the uppermost layer of the WT skin, but little or no apoptosis is normally observed in the basal layer (Fig. 3C, WT ventral). In the mutant skin, the persistence of nuclei in the uppermost layers of the skin accounts for the increased number of TUNEL-positive cells observed (Fig. 3C, mutant ventral). However, no increase in apoptosis was observed in the basal or immediate suprabasal layers of many of those areas where the depletion of Mi-2ß lead to a dramatic thinning of the epidermis. Thus, the abnormal differentiation of the skin can be attributed in part to a defect in renewal of the basal epidermis, rather than to the death of this cell population.
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).
After the cornified layer has fully developed around E17.5, the periderm
sloughs off and K6 expression is confined to the hair follicle. In both the WT
and Mi-2ß-depleted skin, a K6-expressing periderm was detected at E16.5
and was shed by E18.5 (Fig. 4D,
E16.5 and E18.5). In sharp contrast to the situation in the WT, cells in the
suprabasal layers that lacked Mi-2ß expressed K6 starting at E18.5
(Fig. 4D, mutant). A barrier
defect could explain the K6 induction in the suprabasal layers
(Paladini et al., 1996).
However, the barrier was largely intact on the dorsal side
(Fig. 4E). Analysis of regions
where mosaic depletion of Mi-2ß had occurred demonstrated that K6
expression was confined to cells that lacked Mi-2ß
(Fig. 4F). Mi-2ß-deficient
cells interspersed among WT cells specifically expressed K6, whereas cells
that retained Mi-2ß in largely deleted areas lacked K6 despite its
expression in immediately adjacent cells. Furthermore, aberrant K6 expression
was confined to the suprabasal layers. Although the induction of K6 might
depend on a signal induced in response to a subtle defect in epidermal
structure or barrier function (Fig.
4E), this signal is only adequate to induce K6 in suprabasal cells
that lack Mi-2ß.
Mosaic Mi-2ß depletion severely affects hair follicle morphogenesis
Although differentiation of the dorsal interfollicular epidermis was for
the most part intact in the mutants, the development of hair follicles was
severely compromised in the absence of Mi-2ß. Hair follicle induction
normally occurs during the period that Mi-2ß is being deleted in a mosaic
fashion in the dorsal epidermis. Despite the variable nature of this deletion
pattern, dramatic differences in hair follicle development were evident when
whole skin was evaluated (Fig.
5). Such effects on hair follicle development were even more
dramatic when the analysis was confined to areas where Mi-2ß depletion
was complete.
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expression was detected in
the mutant skin by either RT-PCR or in situ hybridization (see Fig. S2 in the
supplementary material). Augmented expression of this closely related gene
does not ameliorate the consequences of deleting Mi-2ß in embryonic
skin. Examination of marker gene expression on tissue sections revealed more dramatic defects in follicle formation. At E18.5 and P1, Edar was detected at low levels in the basal layer prior to placode formation and expressed more highly in the developing hair placode. This higher level of expression persisted in cells at the leading edge of the hair peg as it invaded the dermis, whereas cells in the rest of the peg exhibited a lower level similar to that in the interfollicular epidermis (Fig. 6A,B, WT). At E18.5, many stage-0 and stage-1 follicles exhibiting bright Edar expression were observed in WT skin (Fig. 6A,B). By contrast, although the levels of Edar in the basal epidermis were similar to those in the WT, no patterned expression of Edar indicative of the initiation of hair placodes was seen in epidermal regions lacking Mi-2ß (Fig. 6A, mutant). Analysis of other early molecular markers of follicle induction, including the downregulation of E-cadherin, induction of P-cadherin, or expression of Bmp2 or Shh, confirmed the absence of stage-0 or stage-1 follicles in the Mi-2ß-depleted regions (Fig. 6A mutant, and data not shown). More mature follicles at stage 2-3 of development were observed in regions lacking Mi-2ß, but these were assumed to be tylotricht follicles that initiated prior to depletion of Mi-2ß.
In areas of mosaic Mi-2ß depletion, nascent follicular structures expressing Edar were seen (Fig. 6B,Cb). In these incipient follicles, most of the cells of the placode expressed Mi-2ß but no nascent follicles were forming in adjacent regions completely devoid of Mi-2ß expression. In early follicles with mosaic Mi-2ß depletion, Shh was readily detected at the growing tip of the follicular epithelium (Fig. 6Ce), whereas in early follicles without any Mi-2ß, Shh was greatly reduced (Fig. 6Cd). By contrast, in more mature follicles (stage 2/3 or 3a), expression of Shh (and Edar) was observed, albeit at lower levels, even when Mi-2ß was completely absent (Fig. 6Db and data not shown). This argues that although Mi-2ß activity is required for the induction of genes involved in follicular morphogenesis, it is not required for maintenance of their patterned expression at later stages of development.
The growth of a follicle is dependent on the continued inductive interactions between DP and the follicular epithelium. Wnt5a was examined as a marker of the DP that is dependent on expression and signaling of Shh in the follicular epithelium. Significantly, a marked reduction in Wnt5a expression was seen in the DP of Mi-2ß-depleted follicles, whereas normal levels of Wnt5a were associated with Mi-2ß-expressing follicles in the same animal (Fig. 6Dd,De).
Taken together, these observations indicate that Mi-2ß is required for the initial patterning of the expression of signaling molecules involved in follicular morphogenesis. Once committed to a follicular fate, epidermal cells lacking Mi2ß can sustain some follicular development. However, inductive signaling to and from the DP is impaired and follicular development is ultimately arrested.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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). In the
dorsal epidermis, Mi-2ß is progressively depleted from E14 through birth,
when hair follicles normally form in successive waves. The general reduction
in hair follicle number, and the more specific perturbation of gene expression
and structure within the follicles that do form, must be interpreted in the
context of this mosaic and progressive depletion. Newly initiating placodes
were not observed in the Mi-2ß-depleted regions at the early and later
stages of embryogenesis examined in this study. The absence of locally
increased Edar expression, of decreased expression of E-cadherin, or of
induction of Shh expression in these regions, demonstrates that placode
induction is blocked at the very first steps of this process. The fact that
isolated groups of cells expressing Mi-2ß within a mosaically deleted
epithelium can form epidermal placodes demonstrates that the development of
inductive signals in the dermis is not impaired, and that Mi-2ß is
required in the keratinocytes that must respond to these inductive signals.
Although Mi-2ß-deficient basal cells cannot initiate follicle formation,
they can nonetheless sustain the development and differentiation of the
epidermis. Thus, the apparent defect in the basal mutant epidermis is in the
plasticity of these cells to assume the pilosebaceous (follicular) fate
(Fig. 7, phenotype 2). Although Mi-2ß is required for the activation of a battery of markers in the context of follicle initiation, it does not seem to be directly required for the maintenance of gene expression patterns once they have been established. Edar, Shh, Bmp2 and ß-catenin are all found in cells lacking Mi-2ß in follicles that presumably formed and activated gene expression in its presence. In a similar fashion, the suppression of E-cadherin expression is stable in the absence of Mi-2ß. Although the levels of Shh expression are apparently decreased in less mature follicles lacking Mi-2ß, this is likely to be an indirect effect of a failure in inductive signaling. A more consistent and presumably previous decline in Wnt5a expression despite normal levels of Mi-2ß in the DP of Mi-2ß-depleted follicles suggests that inductive signaling to the papilla is compromised in the mutant follicles. This might in turn reduce the levels of expression of genes in the follicular epithelium that are dependent on inductive signaling from the DP. The principal exception to the observed maintenance of gene expression in the absence of Mi-2ß is the behavior of P-cadherin, which appears to decline rapidly in Mi-2ß-depleted cells (data not shown).
During follicle neogenesis, the follicular bulge stem cells arise from
within the follicular epithelium (Levy et
al., 2005), but the timing of this event remains unknown. In a
similar fashion, the segregation of transient matrix stem cells from the cells
that will constitute the permanent portions of the follicular epithelium
during this first hair cycle, is ill defined. It is assumed to occur during
stage 3, when differentiated cell types begin to appear within the follicular
epithelium (Hardy, 1992).
During this stage, the hair matrix is generated and the follicle begins the
transition to an organized structure of concentrically arranged,
differentiated cell types. It is thus noteworthy that a preponderance of
Mi-2ß-depleted follicles is arrested in mid-stage 3. As observed in the
initial formation of the epidermal placode, Mi-2ß appears to be
preferentially required during the specification of a progenitor population
with a characteristic developmental potential, rather than for the expansion
of cell populations with common developmental potential. Finally, follicles
lacking Mi-2ß at later stages of development are observed. These
comparatively rare follicles are likely to represent those that completed the
establishment of the hair matrix stem cells before Mi-2ß was
depleted.
Determination of the self-renewal capacity of epidermal precursors by Mi-2ß
Skin development begins from a single layer of embryonic ectoderm that
gives rise to a self-renewing epidermis and its appendages. Mi-2ß is
highly expressed in the E10.5 ectoderm, when it begins to commit to an
epidermal/appendage lineage. This suggests that the capacity of Mi-2ß to
modify chromatin might be actively required to reprogram the cell fate of
these early progenitors. Nonetheless, depletion of Mi-2ß in the E10.5
ectoderm does not interfere with the initial differentiation of the epidermis
that begins a few days later. The differentiation of the successive layers of
the epidermis occurs on schedule. Instead, Mi-2ß depletion at this early
stage in skin development appears to alter the properties of the emerging
epidermal precursors allowing them an apparently reduced capacity for
self-renewal and maintenance of the differentiated cell types of the
epidermis. The progressive depletion of squamous layers observed in the
ventral part of the skin, where Mi-2ß is depleted early, is consistent
with a defect in the ability of a basal epidermal precursor to regenerate
itself (Fig. 7, phenotype 1).
This effect is unlikely to be due to a defect in the general ability of basal
keratinocytes to enter the cell cycle as normal numbers of proliferating cells
are seen in the basal layer early in development. Reduced proliferation is
only observed after depletion of the basal, suprabasal and granular layers has
begun. Similarly, increased cell death does not account for depletion of the
basal cells. Later deletion of Mi-2ß (after E13.5) does not interfere
with the development and maintenance of a multilayered epidermis. Once
established, basal cells subsequently deleted for Mi-2ß can sustain
epidermal differentiation and expansion throughout fetal development to the
postnatal stage.
In the parlance of stem cells and TA cells, these studies suggest that as ectodermal progenitors acquire an epidermal/appendage progenitor fate, they require Mi-2ß to achieve the extended proliferative and self-renewal capacities of an epidermal stem cell. In its absence, they acquire the more restricted proliferative capacity and generative potential of a transit-amplifying cell (Fig. 7, phenotype 1).
Epigenetic regulation in the development of epidermal lineages
The specific blocks at follicular lineage specification, and the subsequent
transition of a follicular progenitor to a matrix stem cell, suggest a crucial
role for Mi-2ß and its associates in restructuring a chromatin
environment permissive for the gene expression changes required in the
specified path of differentiation. The brahma (Brm) and brahma-related Brg1
(also known as Smarca4 in mouse - Mouse Genome Informatics)
ATP-dependent nucleosome remodelers act in the context of the SWI2/SNF2
complex and have also been deleted in developing epidermis
(Indra et al., 2005). Ablation
of Brg1, or of both Brg1 and Brm, causes
progressively more severe defects in the later terminal differentiation of the
stratum corneum and in its barrier function. However, defects in earlier
stages of epidermal differentiation or follicular development were not
observed. Thus, the requirement for Mi-2ß and possibly the NURD complex
to mediate chromatin remodeling during early differentiation of the integument
is distinct from the functions of the SWI/SNF complex.
Perhaps less clear is how Mi-2ß activity might be required to instil
the self-renewal capacity that is lost upon early deletion during epidermal
development. The phenotype of early depletion of Mi-2ß is, in some
respects, similar to disruption of p63 activity
(Mills et al., 1999;
Yang et al., 1999). Distinct
isoforms of p63 are thought to be required for the commitment to formation of
stratified epidermis and subsequent maintenance of the proliferative potential
of basal keratinocytes (Koster et al.,
2004; Koster et al.,
2005; Koster and Roop,
2004; McKeon,
2004; Suh et al.,
2006). No gross deregulation of p63 expression is observed in
Mi-2ß-depleted skin (data not shown). However, genome-wide analysis of
p63 binding has suggested that chromatin states might regulate p63 access to
cognate sites, and it is possible that Mi-2ß activity is permissive for
p63 function (Yang et al.,
2006). Whether mediated by p63 or other factors, a unifying
hypothesis that provides a common mechanistic explanation for the effects of
Mi-2ß depletion on the plasticity and self-renewal capacity of
keratinocytes, is that extended self-renewal capacity is actively conferred on
a progenitor with more limited proliferative capacity. In this model, the
self-renewal defects observed in the basal epidermis in ventral skin could be
ascribed to a lack of plasticity in the epidermal progenitor, precluding the
imposition of this aspect of epidermal stem cell character that normally
occurs between E10 and E14 (Fig.
7, phenotype 1).
Defects in terminal differentiation
Although the most dramatic effects of Mi-2ß depletion are observed at
critical transitions in cell fate and potential, more modest defects in the
execution of terminal differentiation programs are also detected. Depletion of
Mi-2ß in epidermal precursors does not interfere with their ability to
give rise to a multilayered epidermis, as exemplified by the normal expression
of basal and suprabasal layer markers such as K5, K1 and loricrin in the
dorsal side of the mutant skin. However, abnormal expression of K6 was
detected in the suprabasal layers of the mutant animals, but only in cells
that lack Mi-2ß. K6 induction occurs in response to defects in terminal
differentiation and/or barrier function of the epidermis. Barrier function
studies indicated a severe defect in the ventral side, but not in the dorsal
side, of the skin where K6 induction is also observed. Although signals
associated with a modestly affected barrier not revealed by the permeability
assay might be responsible for K6 induction, they are only sufficient to
activate K6 in Mi-2ß-depleted cells. Whether this reflects a direct
influence of Mi-2ß on the keratin gene cluster, or an indirect
consequence of its effects on the physiology of the cell, remains to be
determined.
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Conclusion |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/8/1571/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionREFERENCES |
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