Lipid defect underlies selective skin barrier impairment of an epidermal-specific deletion of Gata-3.

Abstract

Skin lies at the interface between the complex physiology of the body and the external environment. This essential epidermal barrier, composed of cornified proteins encased in lipids, prevents both water loss and entry of infectious or toxic substances. We uncover that the transcription factor GATA-3 is required to establish the epidermal barrier and survive in the ex utero environment. Analysis of Gata-3 mutant transcriptional profiles at three critical developmental stages identifies a specific defect in lipid biosynthesis and a delay in differentiation. Genomic analysis identifies highly conserved GATA-3 binding sites bound in vivo by GATA-3 in the first intron of the lipid acyltransferase gene AGPAT5. Skin from both Gata-3-/- and previously characterized barrier-deficient Kruppel-like factor 4-/- newborns up-regulate antimicrobial peptides, effectors of innate immunity. Comparison of these animal models illustrates how impairment of the skin barrier by two genetically distinct mechanisms leads to innate immune responses, as observed in the common human skin disorders psoriasis and atopic dermatitis.

Full text

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T H E J O U R N A L O F C E L L B I O L O G Y
JCB: ARTICLE
The Journal of Cell Biology, Vol. 175, No. 4, November 20, 2006 661–670
http://www.jcb.org/cgi/doi/10.1083/jcb.200605057
JCB
661
Introduction
Mammalian epidermis functions as a barrier to prevent both
water loss to the terrestrial environment and entry of toxic
and pathogenic agents into the organism (Elias, 2005; Segre,
2006). Embryonic ectoderm is specifi ed to an epidermal fate at
murine embryonic day (E) 8.5, regulated by the p63 transcrip-
tion factors (Koster and Roop, 2004). At E9.5, this single layer
of basal cells express cytokeratin (K) 5 and K14 (Byrne et al.,
1994). During midembryogenesis, these basal cells continue
to divide as a single-layered epithelium to increase the sur-
face area of the developing embryo. Stratifi cation of the basal
epithelium initiates at E12.5 with asymmetric cell divisions
perpendicular to the basement membrane (Lechler and Fuchs,
2005). By E15.5, suprabasal cells initiate the differentiation
program and express K1/K10 (Byrne et al., 1994). Establish-
ment of the epidermal permeability barrier initiates at E16.5
on the dorsal surface and spreads ventrally to achieve a fully
competent barrier by E18 (Hardman et al., 1998). Barrier es-
tablishment requires cross-linking of the cells in the upper
layer, which then constrains increases to the surface area of
the embryo. Although the barrier must be acquired before the
typical end of gestation, it is not advantageous to develop a
fully competent barrier too early in development because of
the need for continued growth.
Interfollicular epidermal cells retain the ability to self-
renew under both homeostatic and injured conditions by main-
taining mitotically active cells (Blanpain et al., 2004; Morris
et al., 2004; Tumbar et al., 2004; Ito et al., 2005; Levy et al.,
2005). Terminal differentiation begins when basal cells con-
comitantly withdraw from the cell cycle and lose adhesion
to the basement membrane. In the intermediate spinous layers,
the cells assemble a durable cytoskeletal framework that pro-
vides mechanical strength to resist physical trauma. In the
upper granular layer, a cornifi ed envelope (CE) is assembled
directly underneath the plasma membrane by sequential in-
corporation of precursor proteins. Lipid-containing lamellar
bodies fuse with the plasma membrane and attach to the CE
Lipid defect underlies selective skin barrier
impairment of an epidermal-specifi c deletion
of Gata-3
Cristina de Guzman Strong,1 Philip W. Wertz,3 Chenwei Wang,1 Fan Yang,1,2 Paul S. Meltzer,1,2 Thomas Andl,4
Sarah E. Millar,4 I-Cheng Ho,5 Sung-Yun Pai,6 and Julia A. Segre1
1National Human Genome Research Institute and 2National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
3University of Iowa, Iowa City, IA 52242
4Department of Dermatology, University of Pennsylvania, Philadelphia, PA 19104
5Brigham and Women’s Hospital, Division of Rheumatology, Immunology, and Allergy and 6Dana Farber Cancer Institute and Children’s Hospital, Combined Department of
Pediatric Hematology-Oncology, Harvard Medical School, Boston, MA 02115
S
fi ed proteins encased in lipids, prevents both water loss
and entry of infectious or toxic substances. We uncover that
the transcription factor GATA-3 is required to establish the
epidermal barrier and survive in the ex utero environment.
Analysis of Gata-3 mutant transcriptional profi les at
three critical developmental stages identifi es a specifi c
defect in lipid biosynthesis and a delay in differentiation.
kin lies at the interface between the complex physi-
ology of the body and the external environment.
This essential epidermal barrier, composed of corni-
Genomic analysis identifi es highly conserved GATA-3
binding sites bound in vivo by GATA-3 in the fi rst intron of
the lipid acyltransferase gene AGPAT5. Skin from both
Gata-3−/− and previously characterized barrier-defi cient
Kruppel-like factor 4−/− newborns up-regulate anti-
microbial peptides, effectors of innate immunity. Comparison
of these animal models illustrates how impairment of the
skin barrier by two genetically distinct mechanisms leads
to innate immune responses, as observed in the common
human skin disorders psoriasis and atopic dermatitis.
Correspondence to Julia A. Segre: jsegre@nhgri.nih.gov
Abbreviations used in this paper: CE, cornifi ed envelope; ChIP, chromatin
immunoprecipitation; E, embryonic day; K, cytokeratin; Klf4, Kruppel-like factor 4;
SC, stratum corneum.
The online version of this article contains supplemental material.

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JCB • VOLUME 175 • NUMBER 4 • 2006 662
scaffold, sealing the now enucleated cells together to create the
“bricks and mortar” barrier at the skin surface (Elias, 2005).
Recent experimental results have also demonstrated a selec-
tive role for tight junctions in establishing the epidermal barrier
(Furuse et al., 2002). This process of differentiation from a mi-
totically active basal cell to a terminally differentiated squame
is maintained throughout life as part of epidermal regeneration
and maturation.
Mouse models with targeted ablations of genes encoding
keratinocyte transcription factors have demonstrated that bar-
rier acquisition is a coordinated and regulated process (Dai and
Segre, 2004). Our earlier experiments demonstrated that the
transcription factor Kruppel-like factor 4 (Klf4) is necessary to
establish the epidermal barrier in utero (Segre et al., 1999).
To elucidate further the transcriptional networks regulating this
process, we examined the specifi c role of Gata-3, the most
highly expressed member of the GATA family of transcription
factors in interfollicular epidermis.
Results
Epidermal-specifi c deletion of Gata-3
results in perinatal lethality because
of selective barrier impairment
GATA-3 expression in interfollicular epidermis is fi rst de-
tected in the immediate suprabasal layer at E15.5 (Fig. 1 A).
At E16.5 and thereafter, GATA-3 is expressed in both basal and
immediate suprabasal layers (Fig. 1 A). Gata-3−/− embryos
die at E11 (Pandolfi et al., 1995), and pharmacologic rescue of
these embryos until E17.5 revealed a role for Gata-3 in devel-
opment of the inner root sheath of the hair follicle (Kaufman
et al., 2003). To elucidate the necessary function of GATA-3
in terminal stages of epidermal differentiation in vivo, we used
the cre–loxP system. To generate mice with an epidermal-
specifi c targeted ablation of Gata-3, homozygous fl oxed Gata-3
(Gata-3fl /fl ) mice were crossed with mice heterozygous for a dele-
tion of Gata-3 (Gata-3+/−) and expressing the Cre recombinase
under the control of the K14 promoter (K14-Cre) to generate
mice with a Gata-3fl /− K14-Cre genotype, hereafter referred to
as Gata-3 mutants (Pai et al., 2003; Andl et al., 2004). Quanti-
tative PCR of amplicons both within and outside the Gata-3
locus on mutant and control littermate epidermal genomic DNA
demonstrated that >97% of the epidermal cells had deleted
the Gata-3 locus (unpublished data). The residual amplifi ca-
tion in Gata-3 mutants may be from melanocytes or Lang-
erhans cells, resident in the epidermis. Deletion of GATA-3
mRNA and protein was also demonstrated by Northern and
immunohistochemical analysis of Gata-3 mutant newborn skin
(Fig. 1, B and C).
Gata-3 mutants, born at the expected Mendelian ratio, are
distinguishable from their littermates at birth by a lack of prom-
inent whiskers. In the perinatal period, Gata-3 mutants do not
feed and can be identifi ed by the lack of a typical “milk spot.”
Equally striking is the apparent desiccation of the Gata-3 mu-
tant skin during the perinatal period, which takes on a thin,
erythemic, wrinkled appearance (Fig. 2 A). During the fi rst 6 h
after birth, Gata-3 mutant mice lose an average of 5% of their
birth weight. By comparison, littermates who do survive >24 h
when unfed do not appear to desiccate and lose signifi cantly
less weight during a comparable period (Fig. 2 B). Specifi cally,
individual Gata-3 mutants lose weight at a rate that is >3.5- and
7.8-fold greater than the standard deviation of the control litter-
mates. Because other barrier-defi cient animal models display
a similar perinatal lethality, we tested this directly (Segre et al.,
1999; List et al., 2002).
As compared with control littermates, Gata-3 mutant new-
borns exhibit a signifi cant increase in the rate of transepidermal
water loss across their skin surface (P < 0.001; Fig. 2 C).
The increase in the rate of transepidermal water loss and
weight loss is of a similar order of magnitude (Fig. 2 B). Gata-3
mutants’ impaired skin barrier is unable to retain water in the
terrestrial ex utero environment, which results in dehydration
and, ultimately, lethality. To complement the studies that mea-
sure water loss across the skin surface, we investigated Gata-3
mutants’ competence to exclude percutaneous dye penetration.
As previously shown, dye exclusion in control littermates (visu-
alized as white areas) initiates on the dorsal surface at approxi-
mately E16.5 and spreads ventrally, resulting in complete dye
impermeability by E17.5. A transient delay of 0.5 d is observed
in both the initiation and completion of the dye exclusion of the
Gata-3 mutants (Fig. 2 D). Because the Gata-3 mutants are able
to exclude dye penetration before birth, this delay does not ex-
plain the increased rate of transepidermal water loss and sub-
sequent lethality. However, the biophysical properties of the
skin barrier that regulate the relative permeability of small mole-
cules, infectious agents, water, and gases across this surface are
still poorly understood.
Figure 1. Epidermal-specifi c deletion of Gata-3. (A) Expression of GATA-3
at E15.5 in immediate suprabasal layer and at E16.5 in both basal and
suprabasal layers. α6 marks the basement membrane of the epidermis.
(B) Deletion of Gata-3 mRNA, shown by Northern blot of skin mRNA
probed with Gata-3 and Gapdh cDNA. wt, wild type; mt, mutant. (C) Immuno-
fl uorescence of newborn epidermis, demonstrating deletion of GATA-3
in the epidermis. Nuclei were stained with DAPI. Dotted lines mark the
basement membrane. der, dermis; epi, epidermis.

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GATA-3 REGULATES DEVELOPMENT OF SKIN BARRIER • DE GUZMAN STRONG ET AL. 663
To determine which of the three known components of the
barrier is disrupted in Gata-3 mutant newborns, we analyzed
tight junctions, CEs, and lipid composition. Although total lipid
content was similar, Gata-3 mutants exhibit a selective defect in
lipid synthesis. Gata-3 mutants have a decreased level of gluco-
sylceramides and its derivative ceramide EOS (Fig. 2 E).
Ceramide EOS is one of the precursors of sphingolipids, which
interact with free lipids to organize the lipid lamellar structures
in the stratum corneum (SC; Wertz and van den Bergh, 1998).
Ultrastructural analysis of Gata-3 mutant skin, preserved to
maintain lipid structures, revealed a paucity of lamellar bodies
in the SC. In addition, these lamellar bodies contain only a few
disorganized membrane leafl ets and irregular vacuoles (Fig. 2 F).
This analysis points to a specifi c defect in lipid content and
organization underlying the selective barrier impairment. In
contrast, the other two elements of the barrier appear normal.
Specifi cally, egression of a subcutaneously injected dye halted
at occludin-positive structures, indicating that the tight junc-
tions in Gata-3 mutants are fully competent (Furuse et al., 2002;
unpublished data). In addition, the CEs of the Gata-3 mutants
appear normal: mature, plump, and rigid (unpublished data).
Differentiation defects
in GATA-3–defi cient skin
To investigate the etiology of Gata-3 mutants’ barrier defect,
we examined the histology, differentiation, and proliferation
status of embryonic Gata-3 mutant skin. At E1 5.5, when GATA-3
is initially expressed, the histology of Gata-3 mutant skin
appears normal (Fig. 3 A). At E16.5, nuclei persisted in
the presumptive granular layer of Gata-3–defi cient epidermis,
consistent with a differentiation defect and delay in barrier
acquisition (Fig. 2 D and Fig. 3 A). At E17.5, granular cells of
Gata-3–defi cient epidermis were properly enucleated and differ-
entiated, again consistent with overcoming the delay in acquiring
a selective barrier (Fig. 2 D and Fig. 3 A). Gata-3 mutant new-
born epidermis appears thinner with a disorganized basal layer
(Fig. 3 A). Immunohistochemical analysis of Gata-3–defi cient
newborn epidermis demonstrated that the structural proteins
K14 (basal) and loricrin (granular) were expressed in the proper
cell layer (Fig. 3 B). Ultrastructural analysis of the Gata-3 mu-
tant newborn epidermis revealed the absence of fi laments that
connect the keratohyalin granules (Fig. 3 C), again suggesting
that the terminal differentiation program may be impaired.
Figure 2. Loss of Gata-3 in the epidermis re-
sults in perinatal lethality because of a selec-
tive barrier impairment. (A) Newborn Gata-3
mutant (mt) mice possess thin, erythemic, wrin-
kled skin. wt, wild type. (B) Weight loss as a
percentage of initial weight over time. The rate
of weight loss of mutants 1 and 2 are >3.5
and 7.8 standard deviations from the mean of
the control littermates, respectively. The asterisk
indicates the point after which mutant 1
expired. (C) Transepidermal water loss assay
measured on ventral surface of the newborn.
(D) Dye exclusion assay performed on E16.0–
E17.5 embryos. (E) Total epidermal lipid com-
ponent analysis. n = 4 mice per group. GSL,
glycosylceramides; GLA, acylglycosylceramides;
ASS, ceramide with short α-hydroxyacids
(C16) amide linked to sphingosine; ASL, ce-
ramide with long α-hydroxyacids amide linked
to sphingosine acylceramides long; NP, ce-
ramide with normal fatty acids amide linked to
phytosphingosine; NS, ceramide with normal
fatty acids amide linked to sphingosine; EOS,
ceramide with long (C30–C34) ω-hydroxyacids
amide linked to sphingosine and bearing ester-
linked linoleic acid on the ω-hydroxyl group;
FS, free sterol; CH, cholesterol. (F) Electron mi-
crographs of a region between a granulocyte
and corneocyte displaying distorted lamellar
bodies with few disorganized leafl ets (arrows)
and large, irregular vacuoles in the mutant as
compared with densely packed membrane
leafl ets (arrows) in the wild type. Bars, 0.2 μm.

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JCB • VOLUME 175 • NUMBER 4 • 2006 664
The rate of proliferation of Gata-3 mutant basal cells is similar
to controls, as measured by BrdU immunohistochemistry and
cell cycle FACS analysis (unpublished data).
To circumvent the perinatal lethality of Gata-3 mutants
and investigate the role GATA-3 plays in epidermal homeostasis,
we grafted E18.5 Gata-3 mutant and control littermate skin
onto nude mice. The gross morphology of grafted Gata-3
mutant skin confi rmed the previously reported role of Gata-3
in hair follicle specifi cation (Fig. 4; Kaufman et al., 2003).
Previous studies have established that hyperproliferation and
acanthosis (thickened epidermis) are compensatory responses to
impaired epidermal barrier (Proksch et al., 1991). Histological
analysis of the grafted Gata-3 mutant skin displayed both these
hallmark features (Fig. 4). Gata-3 mutant epidermis is ?10 cell
layers thick, with an increase in K1-positive suprabasal cells,
whereas both control grafted and hairless nude epidermis are
approximately three to four cell layers thick (Fig. 4). Although
proliferation was increased in the Gata-3 mutant epidermis, it
was restricted to the basal cells (unpublished data). These graft-
ing studies suggest that Gata-3 mutant epidermis retains an in-
herent barrier defect that extends beyond the perinatal period.
Epidermal transcriptional pathways
regulated by GATA-3 at E15.5, E16.5,
and newborn
To identify the pathways of gene expression that are affected by
the loss of GATA-3 during development, we analyzed microarray
data from Gata-3 mutant and control littermate dorsal skin isolated
from three distinct epidermal stages of development: (1) at E15.5,
Figure 3. Differentiation defects in Gata-3–defi cient epidermis. (A) Histology
of E15.5, E16.5, E17.5, and newborn (NB) dorsal skin. epi, epidermis;
der, dermis. (B) Immunohistochemical staining of newborn skin with K14
(basal) and loricrin (granular). (C) EM of the upper granular layer. Arrows
highlight the presence/absence of fi laments binding and connecting the
keratohyalin granules. wt, wild type; mt, mutant.
Figure 4. Differentiation defects and compensatory hyperproliferation in
transplanted Gata-3–defi cient skin grafts. Histological and immunohisto-
chemical staining with K14 and K1. wt, wild type; mt, mutant; H&E, hema-
toxylin and eosin.

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GATA-3 REGULATES DEVELOPMENT OF SKIN BARRIER • DE GUZMAN STRONG ET AL.665
the initial defect in barrier acquisition; (2) at E16.5, the com-
pensatory acquisition of barrier to exclude small molecules; and
(3) at newborn, the selective barrier defi ciency upon exposure to
the terrestrial environment. This tripartite experiment enabled
us to query the genes and pathways affected by GATA-3 that led
to the delay in epidermal differentiation and the persistent lipid
and barrier defect.
At all developmental stages, lipid synthesis and modifi ca-
tion was identifi ed as the most signifi cant and commonly af-
fected pathway in the Gata-3 mutants, consistent with the lipid
defect observed in these animals (Fig. 5). Down-regulated at all
epidermal developmental stages are prostaglandin-endoperoxide
synthase 1 (Ptgs1; greater than threefold), 1-acylglycerol 3
phosphate O-acyltransferase 5 (Agpat5; greater than three- to
ninefold), and sphingosine-1-phosphate phosphatase 1 (Sgpp1;
greater than two- to fi vefold). The family of Elongation of very
long fatty acids–like (Elovl) genes, Elovl1, Elovl3, Elovl4, and
Elovl6, encoding lipid biosynthetic proteins, is also down-
regulated in Gata-3 mutants.
To determine if genes in the lipid biosynthetic pathway
are direct targets of GATA-3, we used a genomic approach.
First, to identify potential cis-acting regulatory elements in the
lipid genes, we performed a multispecies alignment of the hu-
man sequences compared with mouse, rat, and dog homologues.
Between mouse and human, ?5% of the genomic sequence is
under positive selection; i.e., alignable and conserved (Waterston
et al., 2002). Only one third of these regions are predicted to
encode an exon of a gene. The other regions of alignment are
postulated to encode RNA genes or regulatory elements. An
example of the multispecies alignment of the proximal promoter
and fi rst intron of AGPAT5 with the program MultiPipMaker is
shown in Fig. 6 A (Schwartz et al., 2003). MultiPipMaker iden-
tifi es two blocks of noncoding sequence conservation: distal to
the fi rst exon and proximal to the second exon. To refi ne this
analysis, we used TRANSFAC to query whether GATA-3 bind-
ing sites were predicted within these blocks of conserved se-
quence, with a consensus binding sequence of G A T A/T A/G
(Merika and Orkin, 1993; Wingender et al., 2000). Examination
of the conservation tracks on the University California Santa
Cruz genome web browser enabled us to rapidly determine
whether these predicted GATA-3 sites are conserved between
species. Examples of two highly conserved GATA-3 sites
(GATTA and GATTG) as well as one not conserved (GATTc)
and one sequence conserved only with dog (GATTA) are given
in Fig. 6 B. Finally, to determine if GATA-3 binds in vivo to
these sites, we immunoprecipitated chromatin with a GATA-3–
specifi c antibody. Two overlapping amplicons (+0.9 and +1.0
from AGPAT5 transcription start site), which contain these
highly conserved GATA-3 binding sites, were specifi cally en-
riched 3.8- and 3.2-fold in the GATA-3 chromatin immunopre-
cipitated DNA. Sequences in the proximal promoter (−0.2) and
more distal in the AGPAT5 gene (+15.5 and +39.2) were not
enriched in the GATA-3 chromatin immunoprecipitated DNA
(Fig. 6 C). Although a similar genomic analysis of PTGS and
SGPP1 were performed, we did not identify multispecies con-
served GATA-3 binding sites, which might suggest that the cri-
teria for inclusion were very stringent. In summary, GATA-3
binds in vivo to a region in the fi rst intron of the lipid acyltrans-
ferase gene AGPAT5 that contains highly conserved GATA-3
binding sites.
In addition to the defects in lipid synthesis, Gata-3 mutants
display a developmental delay in the expression of structural
proteins (Fig. 7 A). Specifi cally at E15.5, genes encoding the
cornifi cation proteins hornerin and loricrin, as well as the differ-
entiation proteins K1 and involucrin, are down-regulated in the
Gata-3 mutants. At E16.5, expression of late CE genes (LCE 1B,
2B, 2C, 3A, and 4B) are either absent or decreased by more than
fi vefold in the Gata-3 mutants, demonstrating a continuation of
the differentiation delay. Previous work has shown that late CE
protein expression immediately precedes in utero dye imperme-
ability in a patterned fashion (Marshall et al., 2001). Therefore,
we postulate that this delay in late CE gene expression underlies
the delay in barrier acquisition visualized in Fig. 2 D. Because of
their temporal expression during development, the genes down-
regulated at E15.5 are different than E16.5, but both expression
profi les refl ect a delay in differentiation. By the newborn stage,
Gata-3 mutants express the vast majority of these genes encoding
epidermal differentiation and cornifi cation proteins at normal
levels. This transcriptional profi ling provides the molecular
underpinnings to interpret the morphological changes in the
Gata-3 mutant skin, observed during development.
The transcriptional profi le of Gata-3 mutant newborn skin
also refl ects the pathways invoked to compensate in the ex utero
Figure 5. Lipid synthesis pathway is affected in Gata-3 mutants. Heat map
representation of microarray data, which demonstrates down-regulation of
genes involved in the lipid synthesis and modifi cation in Gata-3 mutants at
E15.5, E16.5, and newborn (NB). Green indicates a more than twofold
decrease in mutants, bright green indicates a more than fi vefold decrease in
mutants, and black indicates no change between mutants and wild type.

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JCB • VOLUME 175 • NUMBER 4 • 2006 666
terrestrial environment for an intrinsic barrier defect. Classic
studies have shown that barrier defi ciency results in increased
DNA synthesis and acanthosis (Proksch et al., 1991). Gata-3
mutants express high levels of K6 in the suprabasal layers of the
interfollicular epidermis (unpublished data), consistent with many
other examples of K6/K16 in hyperproliferative conditions
(Wong and Coulombe, 2003). Repetin is a fi laggrin-like protein
that has been postulated to specifi cally aggregate K6/K16
fi laments, explaining its expression under these conditions (Pre-
sland et al., 2006). Unexpectedly, Gata-3 mutants express K13
protein in the spinous layer of the epidermis (Fig. 7 B). K13 is
normally expressed only in stratifi ed but not cornifi ed epithe-
lium, such as tongue and esophagus. K13 expression in epider-
mis has previously only been reported in papillomas at high
risk of converting to squamous cell carcinoma (Nischt et al.,
1988). The expression of K13 could suggest a role for GATA-
3 in squamous cell carcinoma progression. Alternatively, K13
expression could refl ect a similar underlying state of the skin
that is common to both barrier impairment and tumor progres-
sion, such as mounting an infl ammatory response.
The fi rst line of cutaneous defense against infection
by microorganisms is the proteinaceous/lipid skin barrier.
Augmenting this physical barrier are both the innate and adap-
tive immune systems (Zasloff, 2002; Braff et al., 2005; Lehrer,
2005). Antimicrobial peptides, effectors of innate immunity, are
expressed by keratinocytes and have distinct but overlapping
reactivity against bacteria, fungi, and enveloped viruses (Braff
et al., 2005). Antimicrobial peptides are induced to provide
a rapid defense, which is particularly important in fetal skin
Figure 6. GATA-3 binds in vivo to sites in the
fi rst intron of the lipid acetyltransferase gene
AGPAT5. (A) Comparison of human AGPAT5
sequence (−2 kb upstream of transcription
start site to second exon) with mouse, rat, and
dog Agpat5 sequences. MultiPipMaker calcu-
lates the percentage of identity using a Blastz
alignment. Percentage of identity (50–100%)
is shown on the y axis. Exons 1 and 2 are
marked above the sequence identity plot with
fi lled boxes. (B) Location of ChIP amplicons,
in proximal promoter (−0.2), in region of
sequence conservation distal to exon 1 (+0.9
and +1.0) and in region of sequence conser-
vation proximal to exon 2 (+15.5) with posi-
tions relative to AGPAT5 transcription start site.
Overlap between +0.9 and +1.0 amplicons
(Chr8:6,554,370-6,544,430) contains four
GATA-3 binding sites (boxed). (C) C hIP dem-
onstrates specifi c binding of GATA-3 in vivo
to region distal to exon 1 in the fi rst intron
of AGPAT5.
Figure 7. Delayed differentiation during embryonic development and
aberrant keratin 13 expression in Gata-3 mutant newborns. (A) Heat map
representation of microarray data, demonstrating delayed expression of
epidermal differentiation proteins at E15.5 and E16.5. Newborn (NB)
Gata-3 mutants display aberrant expression of epidermal structural
proteins. Green indicates a more than twofold decrease in mutants, bright
green indicates a more than fi vefold decrease in mutants, red indicates a
more than twofold increase in mutants, bright red indicates a more than
fi vefold increase in mutants, and black indicates no change between mu-
tants and wild type. LCE, late CE genes. (B) Immunohistochemical staining
of K13 in newborn skin. b, basal; sp, spinous; gr, granular.

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GATA-3 REGULATES DEVELOPMENT OF SKIN BARRIER • DE GUZMAN STRONG ET AL.667
before maturation of immunological memory (Marchini et al.,
2002). Epithelial defense is a signifi cantly affected pathway in
Gata-3 mutant newborns, including a strong up-regulation of
the antimicrobial peptides, secretory leukocyte proteinase in-
hibitor, adrenomedullin, S100A8, S100A9, and β-defensin 1
and 3 (Fig. 8; Braff et al., 2005; Lehrer, 2005).
To investigate the specifi city of the transcriptional profi le
of Gata-3 mutant newborn skin, we compared these results with
a similar analysis of barrier-impaired Klf4−/− skin (Segre et al.,
1999). First, the levels of Klf4 are not altered in Gata-3 mutants
and vice versa, suggesting that these transcription factors
are not epistatic but distinct in their regulation of epidermal
differentiation (unpublished data). Second, the nature of the
barrier defi ciencies in Klf4−/− and Gata-3 mutants appears
completely distinct. Klf4−/− mutants exhibit a specifi c defect
in CE maturation, with normal synthesis but abnormal extrusion
of lipids, and a persistent dye penetration even as newborns.
Molecularly, Ptgs1 is the only “lipid synthesis pathway” gene
with decreased levels in Klf4 mutants. Both mutants do up-
regulate genes common to a hyperproliferative state, including
K6, K16, and repetin. However, the greatest similarity between
the two barrier-defi cient mutant mice is an up-regulation of the
epithelial defense genes. Klf4−/− and Gata-3 mutants show a
similar up-regulation of the innate immune effectors, secretory
leukocyte proteinase inhibitor and β-defensin 3 (Fig. 8).
However, whereas Klf4−/− mutants show a strong up-regulation
of β-defensin 6, Gata-3 mutants more strongly up-regulate ad-
renomedullin, S100A9, S100A8, and β-defensin 1. Thus, Gata-3
and Klf4 mutants, genetically distinct models of barrier impair-
ment, both activate an innate immune response, but they do so
through up-regulation of distinct antimicrobial peptides.
Discussion
These results demonstrate Gata-3’s specifi c role in epidermal
barrier acquisition. Gata-3 mutant embryos exhibit a transient
delay in differentiation, demonstrated by percutaneous dye
penetration. Similar delays in dye exclusion were observed in
mice with targeted deletions of genes encoding CE proteins,
envoplakin, and loricrin (Koch et al., 2000; Maatta et al.,
2001). However, envoplakin- and loricrin-defi cient mice sur-
vive the perinatal period, perhaps because of the compensatory
up- regulation of other structural proteins. In contrast, Gata-3
defi ciency in the epidermis results in a perinatal lethality with an
inherent barrier defect that extends postnatally, as demonstrated
by grafting experiments. Underlying Gata-3 mutant’s barrier
defect is a severe defect in lipid synthesis, in particular, ce-
ramide EOS and glucosylceramides. The electron micrographs
of Gata-3 mutant skin, postfi xed to maintain lipid structure, are
reminiscent of similar fi ndings in infants with severely affected
type 2 Gaucher disease. Mutations in β-glucocerebrosidase,
the enzyme that catalyzes the hydrolysis of glucosylceramide
to ceramide, underlie Gaucher disease (Sidransky et al., 1992).
Type 2 Gaucher disease and a mouse model, with a targeted
deletion of β-glucocerebrosidase, manifest at birth with a pri-
mary barrier defi ciency and display abnormal loosely packed
lamellar body–derived sheets in the SC (Holleran et al., 1994).
Our fi ndings suggest that GATA-3 may act to regulate this im-
portant process of lipid biosynthesis, and genes in this pathway
should be tested as a potential modifi ers to explain the wide
phenotypic variation observed among Gaucher patients (Goker-
Alpan et al., 2005). Our studies identifi ed AGPAT5, which cata-
lyzes an essential step in the synthesis of all glycerolipids, as a
direct target in vivo for GATA-3 (Lu et al., 2005). Future studies
will address the hierarchical transcriptional regulation of lipid
synthesis in the skin.
Morphological and transcriptional analyses at distinct de-
velopmental stages revealed both GATA-3’s regulation of dif-
ferentiation and lipid synthesis pathways and the compensatory
responses to impaired barrier. For example, although the new-
born barrier-defi cient Gata-3 mutant skin is hypocellular, the
grafted Gata-3 mutant skin is acanthotic or hypercellular, as
a compensatory response to the impaired barrier in the terrestrial
environment (Fig. 3 A and Fig. 4). Analysis of Gata-3 mutants
at only one developmental stage would have revealed the spe-
cifi c defect in lipid biosynthesis but would have been refractive
to elucidating the transient delay in expression of genes en-
coding differentiation and cornifi cation proteins. Transcriptional
profi ling at multiple developmental stages brings clarity to
pathways affected by and responding to Gata-3’s loss.
This process is remarkably well conserved, as GATA tran-
scription factors are also essential to specify the fate and regu-
late differentiation of epidermal cells in Caenorhabditis elegans.
Figure 8. Innate immunity is evoked in both newborn Gata-3 and Klf4−/−
mutant skin. (A) Heat map representation of microarray data, demonstrating
specifi c increased expression of genes encoding antimicrobial proteins,
effectors of innate immune, in both Gata-3 and Klf4−/− mutant newborn
skin. Black indicates no change; dark red indicates >1.3-fold up-regulated;
red indicates >1.5-fold up-regulated; and bright red indicates >2.5-fold
up-regulated in mutants. (B) Quantitative PCR confi rmation of genes en-
coding antimicrobial peptides up-regulated in Gata-3 and Klf4−/− mutant
newborn skin. β2m, β-2-microglobulin for normalization. wt, wild type.

Page 8

JCB • VOLUME 175 • NUMBER 4 • 2006 668
The cell biology of the C. elegans epidermis closely resembles
that of mammals, including intermediate fi lament networks and
cell connections through adherens and tight junctions (Hardin
and Lockwood, 2004). GATA transcription factor ELT-1 speci-
fi es epidermal cell fate (Page et al., 1997). Subsequently, ELT-5
and -6 (adjacent genes encoding GATA factors) are required
throughout development to regulate epidermal cell differentiation
(Koh and Rothman, 2001).
Mammalian lung and skin are both epithelia at the inter-
face between the body and the environment that form protein-
aceous lipid barriers. Although lung is a branched simple
epithelium and the composition of the barriers is distinct, there
are remarkable similarities between the systems. At the tran-
scriptional level, corticosteroids and thyroid hormone accelerate
barrier maturation in utero of both epidermis and alveoli
(Aszterbaum et al., 1993). Just as Klf4 is necessary for the ter-
minal stages of epidermal development, Lklf (Klf2) plays an im-
portant role in the terminal stages of lung development (Wani
et al., 1999). GATA-6 is the only known GATA factor expressed
in the distal epithelium of the developing lung. Expression of a
dominant-negative form of GATA-6 in these alveolar cells re-
sulted in a defect in terminal differentiation and proximal air-
way development. These GATA-6 transgenic mice die perinatally
with defects in lipid (surfactant protein) synthesis and decreased
expression of Aquaporin 5, a gene encoding a water channel.
Similar to GATA-3’s role in epidermal barrier, GATA-6 is nec-
essary for maturation of the proteinaceous lipid barrier that
regulates alveoli gas exchange (Yang et al., 2002).
Extending the well-established paradigms from hemato-
poietic cells, it is intriguing to speculate whether GATA-3 will
have similar interactions with family members of other tran-
scription factors in the skin. GATA-1 acts upstream of EKLF
(KLF1) during erythroid development, and GATA-3 acts up-
stream of LKLF (KLF2) during lymphocte development (Kuo
and Leiden, 1999; Anderson et al., 2000). The expression of
GATA-3 and KLF4 in basal and suprabasal cells, respectively,
is consistent with GATA-3 acting upstream of KLF4. Klf4 levels
are unchanged in Gata-3 mutants, which could refl ect compen-
satory autoregulation or parallel pathways.
Both Klf4 and Gata-3 mutants exhibit an epidermal bar-
rier defi ciency, but each activates distinct antimicrobial pep-
tides, effectors of innate immunity. Innate immunity is important
before the adaptive immune system mounts a response and
particularly during the fi rst year of human life, as the adaptive
immune system is maturing. These fi ndings demonstrate that new-
born skin can mount a robust activation of an innate immunity.
Moreover, an analysis of Gata-3 and Klf4 mutant newborns
demonstrates that genetically distinct barrier impairments acti-
vate overlapping but distinct innate immune responses. The
comparison of Klf4- and Gata-3–defi cient newborn epidermis
will be very informative to unravel the complex immune
response to barrier impairment.
Barrier disruption is a hallmark characteristic of common
infl ammatory skin disorders, such as atopic dermatitis (more
commonly known as eczema) and psoriasis (Segre, 2006). Recent
work has examined the distinct innate immune responses of
psoriasis and atopic dermatitis (de Jongh et al., 2005). In particular,
patients with atopic dermatitis have an increased tendency to
develop both disseminated viral skin infection after smallpox
vaccine inoculation and recurrent Staphylococcus aureus
infections because of inadequate innate immune response
(Howell et al., 2006). In contrast, barrier-impaired keratitis-
ichthyosis-deafness patients develop recurring Candida albicans
yeast infections. Mutations in the epidermal cornifi cation protein
fi laggrin were recently reported to underlie atopic dermatitis,
focusing attention on the role that barrier impairment plays in
this disorder (Palmer et al., 2006). Because of the naive state of
T and B cells in newborn mice, a full investigation into this
complex innate/adaptive immune response requires adult
epidermal-specifi c targeting of Gata-3 and Klf4.
Materials and methods
Generation of Gata-3fl /fl K14-Cre mice and skin grafts
Mice carrying the Gata-3fl allele (Pai et al., 2003) were crossed with mice
expressing germline Cre recombinase (Scheel et al., 2003) to generate a
Gata-3− allele. These mice were then crossed onto mice transgenic for
human K14-driven Cre recombinase (Andl et al., 2004) to generate
Gata-3+/− K14-Cre. Gata-3−/fl K14-Cre mice were generated by crossing
Gata-3fl /fl mice with Gata-3+/− K14-Cre mice. Genotyping was done as
previously described (Pai et al., 2003; Andl et al., 2004). The morning of
the plug was 0.5 d after coitum. E18.5 dorsal skin was grafted onto nude
mice in an area that the mice could not scratch, and these mice were indi-
vidually housed. All animal studies were approved by the National Human
Genome Research Institute animal care and use committee, and all mice
were housed in our Association for Assessment of Laboratory Animal
Care–accredited facility.
Barrier function assays
Dye penetration assays were performed with X-gal at pH 4.5 for 4 h at
37°C as previously described (Hardman et al., 1998). After staining, em-
bryos were photographed under a dissecting scope (MZFLIII; Leica) using
a digital camera (AxioCam; Carl Zeiss MicroImaging, Inc.), and images
were acquired with OpenLab software (Improvision). Transepiderrmal
water loss was measured using a Tewameter (Courage + Khazaka).
Histology and immunohistochemistry
Routine histology and paraffi n staining were performed as described previ-
ously (Jaubert et al., 2003). For immunofl uorescence, frozen sections were
fi xed in 10% formalin/PBS and stained with primary antibodies: rabbit
polyclonal antibodies against GATA-3 (Segre 379-2b; 1:100), K14
(1:1,000; Covance,), K1 (1:1,000; Covance), Loricrin (1:500; Covance),
K13 (1:500; a gift from S. Yuspa, National Cancer Institute, Bethesda, MD),
and α6 integrin rat polyclonal antibodies (MAB1982; 1:100; Chemicon).
Fluorescent secondary antibodies were Alexa 488 goat anti–rabbit
(1:400) and Alexa 594 goat anti–rat (1:200). Slides were mounted with
DAPI glycerol media, containing SlowFade Gold antifade, to counterstain
nuclei (Invitrogen). Fluorescent staining was imaged with a microscope
(Axioplot; Carl Zeiss MicroImaging, Inc.) and photographed with a camera
(CoolSNAP; Photometrix).
RNA isolation, Northern blot analysis, and microarray
RNA was isolated from the dorsal skin of newborns and embryos, incubated
in RNALater (Ambion), snap frozen, homogenized in TRIzol (Invitrogen)
using tissue lyser (QIAGEN), and processed according to the manufacturer’s
instructions. Northern blot was hybridized with probes for Gata-3 and
Gapdh. Microarrays were done on independent samples for newborns
(n = 4) and E15.5 and E16.5 embryos (n = 3). Control littermates are
Gata-3fl /+ or Gata-3fl /fl , and mutant mice are Gata-3fl /− K14-Cre. Compli-
mentary RNA was labeled according to the manufacturer’s recommenda-
tions and hybridized onto Affymetrix 430 2.0 A+B mouse arrays. These
arrays contain 45,000 probe sets, representing 34,000 well-substantiated
mouse genes. We identifi ed ?20,000 probes as present in mouse skin dur-
ing the developmental windows analyzed in these experiments. Microarray
results were analyzed by Genesifter using a t test (P < 0.05) and Benjamini
and Hochberg correction (VizX Labs). Confi rmation of fold changes was
made with quantitative PCR on cDNA from Gata-3 and Klf4 mutants on a

Page 9

GATA-3 REGULATES DEVELOPMENT OF SKIN BARRIER • DE GUZMAN STRONG ET AL. 669
TaqMan light cycler (Applied Biosystems) with SYBR Green mix (Invitrogen),
and primers spanning exon boundaries are listed in Table S1 (available at
http://www.jcb.org/cgi/content/full/jcb.200605057/DC1).
Genomic analysis
Pipmaker and MultiPipmaker were performed with repeat masked
sequences (http://pipmaker.bx.psu.edu/pipmaker/)
(Chr8:18,841,481-18,861,523), human (Chr8:6,548,286-6,569,868),
rat (Chr16:75,769,607-75,791,434), and dog (Chr16:61,666,596-
61,692,310) sequences (Schwartz et al., 2003). Coordinates for blocks 1
and 2 are human (Chr8:6,553,816-6,554,997 and Chr8:6,568,473-
6,569,799, respectively). The overlap of amplicon +0.9 and +1.0 in
which the GATA-3 conserved sequences are identifi ed is Chr8:6,554,370-
6,544,430. Mouse and human sequence coordinates are relative to
February 2006 and March 2006 releases, respectively. TRANSFAC was
accessed through a National Human Genome Research Institute site license
(Wingender et al., 2000).
with mouse
Chromatin immunoprecipitation (ChIP) studies
ChIP was performed on human MCF-7 cells, an epithelial cell line that
expresses GATA-3 and the lipid biosynthetic genes, including AGPAT5.
Chromatin was immunoprecipitated with a GATA-3 antibody (SC-9009;
Santa Cruz Biotechnology, Inc.) binding to the endogenous protein. Other
reagents were provided in the ChIP-IT kit (Active Motif), and we followed
the manufacturer’s instructions. DNA/GATA-3 antibody complexes were
immunoprecipitated with protein G and A beads. DNA was quantifi ed
with QuantiTect SYBR Green PCR kit (QIAGEN). Primers are listed in Table S2
(available at http://www.jcb.org/cgi/content/full/jcb.200605057/DC1),
and amplification was quantified on a TaqMan light cycler (Applied
Biosystems). Binding of GATA-3 to chromatin immunoprecipitated DNA
was measured as the change in the number of cycles required to cross a
threshold, normalized to sonicated, reverse-cross-linked input DNA.
Ultrastructural and lipid analysis
Whole backskin was removed, placed on a paper towel, and fi xed in
modifi ed Karnovsky’s fi xative (2% paraformaldehyde, 2% glutaraldehyde,
0.1 M cacodylate buffer, pH 7.3, and 0.06% CaCl2) overnight at 4°C.
Samples were washed twice in 0.1 M cacodylate buffer after fi xation be-
fore embedding. Lipids were extracted into chloroform: methanol mixtures
and analyzed by thin-layer chromatography as previously described (Law
et al., 1995). Lipid masses were used to calculate weight percentages.
Ruthenium tetroxide transmission EM was performed as previously described
(List et al., 2003).
Online supplemental material
Tables S1 and S2 provide the sequences of the primers used for quantita-
tive RT-PCR and ChIP, respectively. Online supplemental material is avail-
able at http://www.jcb.org/cgi/content/full/jcb.200605057/DC1.
We thank Cherry Yang for genotyping; Abdel Elkahloun for microarray analysis;
Travis Moreland for building the database; Qian-Chun Yu and Neelima
Shah for EM expertise; Arturo Incao for performing the graft surgeries; Julia
Fekecs and Darryl Leja for assistance with preparing the fi gures; Stuart Yuspa
for insightful discussions; and David Bodine, Tiffany Scharschmidt, and Paul Liu
for critical evaluation of the manuscript. We also thank members of the labora-
tory, in particular, Satyakam Patel, Jennifer Yang, and Christina Feng for their
underlying contributions.
This work was supported by the National Human Genome Research
Institute intramural program.
Submitted: 9 May 2006
Accepted: 13 October 2006
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