The stratified epithelium of the epidermis provides the barrier
betweenthe organism and the outside world (Fuchs and Raghavan,
2002; 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
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/?-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
The chromatin remodeler Mi-2? is required for
establishment of the basal epidermis and normal
differentiation of its progeny
Mariko Kashiwagi, Bruce A. Morgan and Katia Georgopoulos*
Using conditional gene targeting in mice, we show that the chromatin remodeler Mi-2? is crucial for different aspects of skin
development. Early (E10.5) depletion of Mi-2? in the developing ventral epidermis results in the delayed reduction of its suprabasal
layers in late embryogenesis and to the ultimate depletion of its basal layer. Later (E13.5) loss of Mi-2? in the dorsal epidermis does
not interfere with suprabasal layer differentiation or maintenance of the basal layer, but induction of hair follicles is blocked. After
initiation of the follicle, some subsequent morphogenesis of the hair peg may proceed in the absence of Mi-2?, but production of
the progenitors that give rise to the inner layers of the hair follicle and hair shaft is impaired. These results suggest that the
extended self-renewal capacity of epidermal precursors arises early during embryogenesis by a process that is critically dependent
on Mi-2?. Once this process is complete, Mi-2? is apparently dispensable for the maintenance of established repopulating
epidermal stem cells and for the differentiation of their progeny into interfollicular epidermis for the remainder of gestation. Mi-2?
is however essential for the reprogramming of basal cells to the follicular and, subsequently, hair matrix fates.
KEY WORDS: Mi-2? ? (Chd4), Chromatin, Epidermis, Stem cells
Development 134, 1571-1582 (2007) doi:10.1242/dev.001750
Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard
Medical School, Charlestown, MA 02129, USA.
*Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 13 February 2007
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
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
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
Mi-2? and Mi-2? (also known as Chd3 and Chd4, respectively –
Mouse Genome Informatics) are closely related genes encoding
ATP-dependent chromatin remodelers (Seelig et al., 1996). Mi-2?
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 ofthe liverof 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. Theyreside 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.,
In the immune system, much of the Mi-2? 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-2?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-2? 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-2?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-2? 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-2? in skin development. The conditional allele of Mi-2? 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.
MATERIALS AND METHODS
The generation and characterization of the Mi-2?LoxPF/LoxPFmice have been
described previously (Williams et al., 2004). K14-Cre transgenic mice
obtained from Dr P. Chambon (Li et al., 2001) were mated to Mi-
2?LoxPF/LoxPFmice to generate mice homozygous for loss of Mi-2?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-2?+F, 5?-CTCCAAGAAGAAGACGGCA-
GATCT-3? and Mi-2 INR, 5?-GTCCTTCCAAGAAGAGCAAG-3?;
For ? F allele: Mi-2?+F and KG4R, 5?-CTTCCACAGTGACGTCCA-
For K14-Cre: K14Cre-5?-2, 5?-ACAGACATGATGAGGCGGAT-3? and
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.
For histopathology, tissue samples were frozen in OCT or fixed in 3.7%
formalin and embedded in paraffin. Sections (4 ?m) were stained with
Hematoxylin and Eosin. For immunofluorescence, TUNEL, and in situ
hybridization, tissue samples were frozen in OCT and sectioned at 6 ?m.
In situ analysis
Digoxygenin (DIG) probe synthesis was performed according to the
manufacturer’s instructions (Roche), using probes transcribed from a
plasmid containing Mi-2? cDNA (4264-5668 bp, NM145979), Mi-2?
cDNA (C-terminal 1401 bp, BC030435), ?-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 6?SSC, then
2?SSC containing 50% formamide and 10 mM EDTA, then 2?SSC, and
finally 0.2?SSC. The DIG-label was detected by anti-DIG Fab (Roche)
coupled to alkaline phosphatase using NBT/BCIP (Roche).
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-2? [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.
Development 134 (8)
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
Skin permeability assay
Skin permeability assay was performed as described previously (Hardman
et al., 1998). 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
The number of hair follicles per unit length of epidermis was counted in
frozen and paraffin sections of Mi-2? 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-2? 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-2? mutant animals were
compared with those of 436 hair follicles from three age-matched WT
mice at P1.
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?-
?-catenin S, 5?-ACATCCTTGCTCGGGACGTT-3? and ?-catenin AS, 5?-
Mi-2? in cell fate decisions in the epidermis
Fig. 1. Expression of Mi-2? ? mRNA during epidermal development and conditional inactivation of Mi-2? ? in the skin. (A) In situ
hybridization studies reveal Mi-2? mRNA expression at E10.5, E14.5, E18.5 and P1. Mi-2? is uniformly expressed in the embryonic ectoderm
(E10.5). Mi-2? transcripts are also detected in E14.5 epidermis in the basal and stratum intermediate layers. In the differentiating hair follicle,
increased levels of Mi-2? mRNA are first detected in the placode (arrowhead) and then in the matrix (arrow). Scale bar: 50 ?m. (B) Cre-dependent
conversion of the floxed allele (LoxPF) to the mutant allele (?F) in the P1 dorsal epidermis revealed by PCR of genomic DNA (lanes 2 and 4).
(C) Wild-type (WT) and mutant littermates at P1. (D) Hematoxylin and Eosin-stained cross-sections of WT and mutant skin at P1. The dotted line
demarcates the dorsal region and the unbroken line the ventral region. The mutant skin exhibits an exacerbation of phenotypes from the dorsal to
the ventral side that includes thinning of the epidermis and reduction in hair follicles. Scale bar: 100 ?m. A further magnification of the mutant skin
is provided beneath to show the absence of the basal layer and the thinning of the suprabasal and cornified layers in the ventral region (right), and
the more normal epidermal differentiation in the dorsal region (left). (E) The presence of Mi-2? protein (green) in WT (left) and in the dorsal (middle)
and ventral (right) mutant skin was evaluated by immunofluoresence at successive stages of development. DAPI-stained nuclei are shown in red,
and the white dotted line demarcates the dermal-epidermal junction. Expression of Mi-2? protein is indicated by the presence of yellow nuclei,
depletion of Mi-2? by red nuclei. Depletion of Mi-2? protein occurs earlier (E10.5) in the ventral skin, and later (E13.5 and later) in the dorsal skin.
Scale bar: 50 ?m.
Lef1 S, 5?-CACGGACAGTGACCTAATGC-3? and Lef1 AS, 5?-
Bmp2 S, 5?-CAGGAAGCTTTGGGAAACAG-3? and Bmp2 AS, 5?-
Shh S, 5?-GACCCCTTTAGCCTACAAGC-3?
Patched S, 5?-CCTTCGCTCTGGAGCAGATT-3? and Patched AS, 5?-
Mi-2?(E11-13) S, 5?-CCTTTCCAGTTTCCGTAGCTTCAC-3? and Mi-
2?(E11-13) AS, 5?-CAGCGGAAGAATGATATGGACGAC-3?;
Mi-2? (E30-34) S, 5?-CCCGAACTGGCTGAAGTAGAGGAAAAC-3?
and Mi-2? (E30-34) AS, 5?-GGAGTTTCTCCATTCTGAAGCATCACG-
Mi-2? S, 5?-GTGTTGACCCGCATTGG-3? and Mi-2? AS, 5?-
GAPDH S, 5?-AAGGTCGGTGTGAACGGATT-3? and GAPDH AS, 5?-
and Shh AS, 5?-
Mi-2? ? expression during epidermal differentiation
To gain insight into the role of Mi-2? during epidermal
morphogenesis, we examined the pattern of Mi-2? mRNA
expression during development. At E10.5, the embryonic ectoderm
consists of a single-cell layer in which Mi-2? was uniformly and
highly expressed (Fig. 1A, E10.5). At E14.5, the ectodermal layer
had begun to differentiate into the basal and stratum intermediate
layers, which express Mi-2? (Fig. 1A, E14.5). At this stage of
development, hair placodes expressing Mi-2? were also observed.
Increased levels of Mi-2? mRNA were detected in the growing tip
of the hair peg and persisted in the cells that form the matrix of the
differentiating hair follicle (Fig. 1A, E14.5, E18.5 and P1). In
contrast to the distinctive pattern of expression in the hair follicle,
Mi-2? mRNA was expressed at relatively low levels in the mature
interfollicular epidermis (Fig. 1A, E18.5 and P1).
Mi-2? ? inactivation in the epidermis causes
abnormal development of the integument
Therole of Mi-2?during development of the epidermis and hair was
evaluated using a conditional inactivation strategy. Micecontaining
an Mi-2? 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-2?remodeling activity, resulting in a mutant mRNA
that does not produce stable protein. As revealed by genomic PCR,
the floxed Mi-2?alleles (Mi-2?loxPF/loxpF) were efficiently disrupted
by K14cre in the epidermis (Fig. 1B).
Mice homozygous for the Mi-2?LoxPFallele and carrying the
K14cre transgene die within 24 hours of birth. The skin of these
mutant mice is shiny and flaky (Fig. 1C), exhibits aseverereduction
inhair follicles and shows abnormal whisker hairs (data not shown).
A curly tail is another morphological phenotype of the K14cre
deletion of Mi-2?(Fig. 1C). Histologicalanalysis of the mutant skin
demonstrated a marked difference in phenotypesbetweenthe 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-2?in the development of dorsal and ventral skin,
or by a difference in the timing of Mi-2? protein depletion.
Examination of Mi-2? protein at E10.5 revealed its dramatic
depletion in most of the ventral skin, while expression persisted in
dorsal skin (Fig. 1E). Mi-2? was still detectable at E13.5 in dorsal
epidermis (Fig. 1E). However, by E14.5, extensive depletion of Mi-
2?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-2? during the earliest stages of epidermal
development, whereas the distinct phenotypes seen in dorsal skin
occur when Mi-2? is removed after the initiation of epidermal
Effects of Mi-2? ? depletion prior to epidermal
Although Mi-2? 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-2? 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,
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-2? (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-2? 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
Development 134 (8)
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 inthe basal layer of ventral mutant skin (Fig.
3A,B). A possible effect of Mi-2? depletion on apoptosis during
epidermal differentiation was also examined. A process akin to
apoptoticcell death normallyoccurs 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-2? 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
Effects of Mi-2? ? depletion during epidermal
Mi-2? depletion occurred later in development in the dorsal
epidermis, beginning at E13.5 during the onset of epidermal
differentiation (Fig. 1E). In contrast to ventral skin, the dorsal mutant
epidermis appeared relatively normal and multilayered throughout
development, although the number and length of hair follicles was
drastically reduced (Fig. 1D). With the exception of a modest delay
in the onset of K1 expression (Fig. 4B, E13.5 and E14.5), the initial
appearance and subsequent maturation of the basal, intermediate and
Mi-2? in cell fate decisions in the epidermis
Fig. 2. Early depletion of Mi-2? ? in
the ventral epidermis results in
late depletion of the basal and
suprabasal layers. (A) Hematoxylin
and Eosin-stained sections of WT and
mutant ventral skin isolated from
E10.5-P1 stages of development. A
reduced cellularity in the ventral
epidermal layers was apparent from
E16.5 to P1. (B-D) Expression of
keratin 5 (K5) (basal epidermis),
keratin 1 (K1) (suprabasal epidermis),
and loricrin (granular epidermis) was
examined by immunofluoresence.
DAPI-stained nuclei are shown in blue.
The timing of induction and
expression of these epidermal
differentiation markers is not initially
affected. Their progressive reduction
later in development reflects a
progressive depletion of basal and
suprabasal layers. Scale bars: 50 ?m.
granular layers of the epidermis were largely indistinguishable from
WT. Even in regions where Mi-2? had been deleted throughout the
epidermis, K5, K1 and loricrin staining were similar to that in WT
skin (Fig. 4A-C).
A notable defect in the dorsal epidermis is the expression of
keratin 6 (K6). K6 is expressed in the periderm that overlies the
developing epidermis (McGowan and Coulombe, 1998). 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-2?-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-2? 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-2? had occurred demonstrated that
K6 expression was confined to cells that lacked Mi-2?(Fig. 4F). Mi-
2?-deficient cells interspersed among WT cells specifically
expressed K6, whereas cells that retained Mi-2? 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-2?.
Mosaic Mi-2? ? depletion severely affects hair
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-2?. Hair
follicle induction normally occurs during the period that Mi-2? 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
Development 134 (8)
Fig. 3. Late effects on proliferation and apoptosis
in the Mi-2? ?-depleted ventral epidermis. (A,B) The
percent of Pcna-positive cells within the basal layer (K5-
positive) of WT and mutant skin was estimated during
development. Sagittal sections were co-labeled with
antibodies against the proliferation marker Pcna and
K5. Nuclei were counterstained with DAPI (A). The data
(B) represent the mean percentage of Pcna-positive cells
within the basal layer (K5- and DAPI-positive) from five
independent animals. From E14.5 through E16.5, a
similar number of Pcna-positive basal cells were seen in
both the mutant and WT skin. However, starting at
E18.5 and through P1, a significant reduction of Pcna-
positive cells was detected in the ventral, but not the
dorsal, mutant regions. (C) Apoptotic cell nuclei (brown)
were detected by TUNEL analysis on ventral skin at P1.
In both the WT and mutant, TUNEL-positive cells were
detected in the uppermost layer of the epidermis, but
the dramatic increase in their number seen in the
mutant suggests persistence of nucleated dead cells
and a defect in terminal differentiation. Scale bars:
evaluated (Fig. 5). Such effects on hair follicle development were
even more dramatic when the analysis was confined to areas where
Mi-2? depletion was complete.
At E18.5, a later wave of hair follicle morphogenesis is
occurring and tylotrich follicles have reached stage 3-4, while awl
follicles have reached stage 1-2 in the WT (Fig. 5A). At this time,
only half the number of hair follicles seen in the WT were detected
in the mutant (Fig. 5A,B). Furthermore, hair follicles at advanced
stages of morphogenesis were greatly reduced: about 20% of hair
follicles in the WT had reached stage 3b and above, whereas only
5% were this mature in the mutant (Fig. 5C, E18.5). By P1, the
total number of follicles in the mutant was still 50% of WT, and
the distribution of developmental stages remained distinct (Fig.
5B,C, P1). In the WT, many tylotrich follicles and awl follicles had
reached stage 4-6. In addition, newly forming hair follicles at stage
1-2 were readily detected (Fig. 5A,C, P1 WT). By contrast, in the
Mi-2? in cell fate decisions in the epidermis
Fig. 4. Effects of Mi-2? ? depletion in the dorsal
epidermis. (A-D) Development of the basal and
suprabasal layers was examined by
immunofluoresence using antibodies to K5, K1,
loricrin and keratin 6 (K6). DAPI-stained nuclei are
shown in blue. Expression of K5, K1 and loricrin
was similar in WT and mutant throughout
development (A-C). The K6-positive periderm
detected at E16.5 was shed at E18.5 in both WT
and mutant (D). From E18.5 through P1, K6
induction was observed in the suprabasal layers of
the mutant but not WT skin (D). (E) Skin barrier
function was analyzed by a barrier-dependent dye
exclusion assay at E19.5. The WT and, for the
most part, the mutant dorsal epidermis prevented
dye penetration indicating intact barrier function.
By contrast, the ventral part of the mutant
epidermis was readily penetrated by the dye
indicating lack of a barrier. (F) K6 expression (red)
is confined to cells that lack Mi-2? (green). Scale
bar: 50 ?m.
mutant, follicles at stage 1 and at stages 4-6 (stage 4 and above)
were greatly reduced or even absent. The majority of the follicles
(79%) were distributed between stages 2 and 3b, whereas only
45% of the follicles were in this range in WT skin (Fig. 5A,C, P1).
This distinct developmental stage distribution of hair follicles in
the mutant versus the WT epidermis is suggestive of discrete
defects occurring during follicle initiation and during the later
stages of follicular morphogenesis, rather than of a general delay
in appendage development.
Effects of Mi-2? ? deletion on follicular gene
The general reduction in hair follicle initiation and development was
confirmed by semi-quantitative RT-PCR. Transcripts of genes
preferentially expressed in the hair follicle epithelium – Edar, ?-
catenin, Lef1, Shh, Patched1and Bmp2– were consistently reduced
in the mutant skin (see Fig. S2A in the supplementary material).
Furthermore, no increase in the normally low levels of Mi-2?
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-2? 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-2? (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 Bmp2or Shh, confirmed the absence
of stage-0 or stage-1 follicles in the Mi-2?-depleted regions (Fig.
6A mutant, and data not shown). More mature follicles at stage 2-
3 of development were observed in regions lacking Mi-2?, but
these were assumed to be tylotricht follicles that initiated prior to
depletion of Mi-2?.
In areas of mosaic Mi-2? depletion, nascent follicular structures
expressing Edar were seen (Fig. 6B,Cb). In these incipient follicles,
most of the cells of the placode expressed Mi-2? but no nascent
follicles were forming in adjacent regions completely devoid of Mi-
2? expression. In early follicles with mosaic Mi-2? depletion, Shh
was readily detected at the growing tip of the follicular epithelium
(Fig. 6Ce), whereas in early follicles without any Mi-2?, 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-2? was completely absent (Fig. 6Db
and data not shown). This argues that although Mi-2? 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-
2?-depleted follicles, whereas normal levels of Wnt5a were
associated with Mi-2?-expressing follicles in the same animal
Taken together, these observations indicate that Mi-2?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 Mi2? can sustain some follicular
development. However, inductive signaling to and from the DP is
impaired and follicular development is ultimately arrested.
Here, we examine the role of the ATP-dependent chromatin
remodeler Mi-2? in the development of skin and its appendages.
Our experimental approach results in loss of Mi-2? in
keratinocytes of the developing integument over the period
ranging from before overt epidermal differentiation through to
late gestation. Although Mi-2? depletion occurs over a
developmental continuum, the phenotypes observed suggest that
three discrete steps in the development of the epidermis and its
appendages are critically dependent on Mi-2? activity (Fig. 7).
When Mi-2? is depleted during the early commitment of an
ectodermal progenitor to the epidermal lineage in the ventral
regions of the mouse, no immediate effect is observed. The initial
differentiation of the epidermis, several days later, occurs
Development 134 (8)
Fig. 5. Mi-2? ? depletion causes severe effects on
hair follicle morphogenesis. (A) Hematoxylin and
Eosin-stained sagittal sections of dorsal skin from WT
and mutant at E18.5 and P1. The number next to the
follicles designates their developmental stage according
to Hardy (Hardy, 1992). (B,C) The number of hair
follicles per unit length (B) and the percentage of
follicles at distinct developmental stages (C) were
evaluated from E18.5 through P1. The number of hair
follicles in the mutant was reduced by approximately
50% relative to WT from E18.5 through P1. At E18.5 in
the WT, primary follicles have developed to stage 3 or 4,
whereas secondary follicles have reached stage 1-2. In
the mutant, a reduction was detected from stage 3a
onwards. By P1 in the WT, primary, secondary and
tertiary follicles have developed to stages 5-6, 3-4, and
1-2, respectively. However, by P1 in the mutant, follicles
at stage 1 and after stage 3c were severely reduced
relative to WT.
normally. However, epidermal development is not sustained,
suggesting the capacity of the basal epidermis to self renew is
compromised. When Mi2? is deleted in the dorsal epidermis at a
later stage, after a differentiated basal epidermal cell is
established, epidermal differentiation is largely normal and is
sustained throughout embryogenesis and the perinatal period.
However, the conversion of a basal epidermal cell to a progenitor
of the pilosebaceous unit does not occur. Finally, when follicular
anlagen are established in the presence of Mi-2?, they can
continue to develop in its absence. However, depletion of Mi-2?
after follicular specification results in arrest of follicular
development at stage 3, a period when follicular progenitors are
transformed to progenitors of the hair shaft and inner root sheath
in the forming hair bulb.
Mi-2? in cell fate decisions in the epidermis
Fig. 6. Effects of Mi-2? ? depletion on the signaling network that controls hair follicle morphogenesis. (A-D) Immunofluorescence of E18.5
dorsal skin labeled with antibodies against Edar or Edar and E-cadherin (A), or against Mi-2? and Edar (B,C), or against Mi-2? and E-cadherin (D).
(C,D) In situ hybridization of E18.5 dorsal skin with probes against Shh and Wnt5a. The depletion of Mi-2? in follicles was confirmed by
immunofluoresence staining of adjacent serial sections using antibodies to Mi-2? and E-cadherin. DAPI-stained nuclei are blue. (A,B) In the WT, a
local increase in Edar expression was detected among basal epithelial cells (asterisks) that give rise to the hair placode, as well as within the hair
follicle (arrowhead). Edar upregulation was followed by a decrease in E-cadherin expression (A, WT). In the mutant, no Edar upregulation or E-
cadherin downregulation was seen in areas of the mutant skin in which Mi-2? was absent (A, mutant). By contrast, in areas with mosaic Mi-2?
depletion, follicular structures expressing Edar were detected in the Mi-2? mosaic area in the mutant (arrowhead in B mutant, Cb). Shh expression
was seen at the tip of stage-2 and stage-3a follicles in the WT (Cc and Da). In the mutant, Shh transcript was seen in stage-2 follicles with mosaic
Mi-2? depletion (Cd), but was significantly reduced in the Mi-2?-null counterparts (Ce). By contrast, Shh was seen in Mi-2?-null stage-3a follicles
(Db). Expression of Wnt5a was observed in the dermal condensate of stage-3a follicles in the WT and in Mi-2?-positive stage-3a follicles in the
mutant (Dc and De). Wnt5a was, however, significantly reduced in the Mi-2?-null counterparts (Dd).
The most severe phenotype associated with early deletion of Mi-
2? is observed in ventral epidermis, whereas the phenotypes
associated with later deletion of Mi-2? predominate in dorsal
epidermis. It is formally possible that the difference in phenotype
reflects a fundamental difference in the physiology of the
keratinocytes of the dorsal and ventral compartments, rather than the
timing of Mi-2? deletion. However, the timing of deletion in the
ventral region is also somewhat variable. Patches of epidermis
exhibiting the intermediate phenotype of relatively normal epidermis
but lacking hair follicles, are also observed on the ventrum of some
embryos. We interpret these to be regions where Mi-2? is deleted
later in the ventral region, and conclude that although the different
phenotypes observed on the dorsum and ventrum may be influenced
in part by differences in these keratinocyte populations, the timing
of deletion appears to play a more central role in the phenotype
observed. However, the timing of the deletion in the patches that give
rise to the ‘dorsal’ phenotype in ventral epidermis has not been
directly assessed, and a more significant role for differences in the
physiology of dorsal and ventral keratinocytes in the observed
differences in phenotype remains a possibility.
Distinct transitions in the differentiation of a hair
follicle are dependent on Mi-2? ?
Of the three transitions in keratinocyte behavior that reveal a crucial
requirement for Mi-2?, only the formation of the epidermal placode
of the hair follicle represents an empirically defined transition in
developmental fate. The follicular epithelium is a lineage
compartment that segregates from the surrounding epidermis at the
time of epidermal placode formation (Levy et al., 2005). In the
dorsal epidermis, Mi-2?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-2?-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-2? 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-2? is required in the keratinocytes that
must respond to these inductive signals. Although Mi-2?-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,
Although Mi-2? 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 ?-cateninare
all found in cells lacking Mi-2?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-2?. Although the levels of Shh expression are apparently
decreased in less mature follicles lacking Mi-2?, 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-2?in the DP of Mi-2?-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
Development 134 (8)
Fig. 7. The role of Mi-2? ? in skin
development: a model for the
development of the skin and its
appendages and the role of Mi-2? ?
in this process. Successive stages in
the development of wild-type skin are
depicted on the upper time line. We
propose that ectodermal cells (yellow)
are first committed to an epidermal
TA cell (blue) that can make epidermis
but has limited proliferative potential
and developmental plasticity. This cell
type is then converted to an
epidermal stem cell (green) with
extensive proliferative capacity and
plasticity to adopt alternative fates.
Starting at E14.5, some of these cells
are induced to become follicular
progenitor cells (pink), and sometime
thereafter other epidermal stem cells
give rise to TA cells of the epidermis
with more restricted proliferative and
developmental potential. Follicular
progenitors proliferate to make the
hair peg, while epidermal stem and
TA cells generate the stratified
epidermis. Finally, a subset of follicular progenitors at the base of the follicle are specified as the matrix stem cells (orange) that give rise to the hair
shaft and inner root sheath over the anagen phase of the hair cycle (right). These matrix stem cells are distinct from the follicular bulge stem cells
(purple) that regenerate the lower follicle in the adult. The three phenotypes resulting from deletion of Mi-2? at different stages of development
are shown (1-3, indicated by a red cross) and interpretations of these phenotypes in the context of the model are shown beneath.
Deletion in Ectodermal
Progenitor or TA
of TA to Epidermal
Deletion in Epidermal
Stem Cell Prevents
Deletion in Follicular
Cell Prevents Conversion
to Matrix Stem Cell
in the Basal Layer
leads to Depletion
of Basal and
Follicles Arrest Prior to
Matrix Stem Cell Differentiation
maintenance of gene expression in the absence of Mi-2? is the
behavior of P-cadherin, which appears to decline rapidly in Mi-2?-
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-2?-depleted follicles is
arrested in mid-stage 3. As observed in the initial formation of the
epidermal placode, Mi-2? 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-2?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-
2? was depleted.
Determination of the self-renewal capacity of
epidermal precursors by Mi-2? ?
Skin development begins from a single layer of embryonic
ectoderm that gives rise to a self-renewing epidermis and its
appendages. Mi-2? 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-2? to modify chromatin might be
actively required to reprogram the cell fate of these early
progenitors. Nonetheless, depletion of Mi-2? 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-2? 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-2? 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-2? (after E13.5) does not interfere with
the development and maintenance of a multilayered epidermis.
Once established, basal cells subsequently deleted for Mi-2? 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-2? 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,
Epigenetic regulation in the development of
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-2? 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 Smarca4in 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 Brg1and 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-2? 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-2?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-2?
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-2?-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-2? 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-2? 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-2?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-2? 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-2?. 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-2?-depleted
cells. Whether this reflects a direct influence of Mi-2?on the keratin
gene cluster, or an indirect consequence of its effects on the
physiology of the cell, remains to be determined.
Mi-2? in cell fate decisions in the epidermis
DEVELOPMENT Download full-text
In summary, the progressive depletion of Mi-2? during the
development of the epidermis in this experimental model has
revealed critical transition points at which this chromatin remodeler
is required for the normal development of the skin and its
appendages. Significant changes in the developmental potential and
regenerative capacity of the progenitor cells that give rise to the
epidermis and its appendages depend on the activity of Mi-2?. Once
these changes have been imposed, more modest deficits in the
execution of developmental programs are observed in the absence
of Mi-2?. Further investigation of the mechanisms by which Mi-2?
exerts these effects in this tractable system will provide additional
insight into the role that chromatin remodelers play in the
specification of stem cell identity and potential.
This work was supported by a CBRC Director’s Fund and by NIH R01 AI380342
to K.G. We thank Pierre Chambon for providing the K14-Cre mice. We also
thank Bob Czyzewski for mouse husbandry, Janice Brissette and the K.G.
laboratory for discussions and critical comments on the manuscript.
Supplementary material for this article is available at
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