National Human Genome Research Institute, NIH, 49 Convent Drive, Bethesda, MD 20892, USA
* Author for correspondence (e-mail: jsegre{at}nhgri.nih.gov)
Accepted 11 March 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: KLF4, Epidermis, Permeability barrier, Perinatal mortality, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major barrier between body and environment is the exterior layer of the
epidermis, the stratum corneum, which is sloughed off and repopulated from the
inner cells. This process of differentiation is maintained throughout life as
part of epidermal regeneration and maturation
(Roop, 1995;
Steinert, 2000
).
Mammalian epidermis, a stratified epithelium, is a self-renewing tissue
composed of a population of mitotically active cells in the innermost basal
layer and their derivatives that travel upward to the skin surface in a linear
program of terminal differentiation (Fuchs
and Raghavan, 2002; Niemann
and Watt, 2002
). This process begins when basal cells
concomitantly withdraw from the cell cycle, lose adhesion to the basement
membrane and initiate differentiation. In the intermediate spinous layers, the
cells remain transcriptionally active, synthesizing and assembling a durable
cytoskeletal framework that provides mechanical strength. In the upper
granular layer, the cells flatten and the intracellular contents are degraded
(including the nuclei). Each cell leaves behind keratin macrofibrils and
lipid-containing lamellar bodies that fuse with the plasma membrane.
Subsequently, a cornified envelope (CE) is assembled directly underneath the
plasma membrane by sequential incorporation of precursor proteins. Finally, in
the outermost stratum corneum, the cells become permeable and a calcium influx
activates transglutaminase enzymes to irreversibly cross-link the CE proteins,
creating a tough, insoluble sac that surrounds the keratin fibers. The CE
serves as a scaffold for the lipids extruded from the lamellar bodies, which
in turn seal together CEs to create the barrier at the skin surface. The
structure that performs the barrier function is analogous to `bricks and
mortar' with the keratin macrofibrils and CEs forming the bricks and
extracellular lipids the mortar. Recent experimental results have also
demonstrated an essential role for tight junctions in epidermal barrier
(Furuse et al., 2002
).
Kruppel-like factor 4 (Klf4, formerly GKLF) is a zinc-finger
transcription factor expressed in the differentiated suprabasal cells of the
epidermis, crypt cells of the gastrointestinal tract and several other organs
(Garrett-Sinha et al., 1996;
Katz et al., 2002
;
Segre et al., 1999
;
Shields et al., 1996
).
Klf4 is necessary for the development of the epidermal barrier, since
mice homozygous for a null mutation in Klf4 die perinatally as a
result of water loss across the skin surface
(Segre et al., 1999
). The goal
of this study was to test if Klf4 is sufficient to accelerate
epidermal barrier acquisition with the hope that an understanding of this
pathway will lead to better methods to ameliorate this process in the
premature infant.
To test if KLF4 is sufficient to establish the epidermal barrier, we
specifically overexpressed Klf4 one stage earlier in development,
i.e. in the basal layer of the epidermis. To circumvent perinatal lethality,
we utilized the bipartite tetracycline responsive transgenic system.
Specifically, we generated `responder' lines with Klf4 transcription
directed by a minimal promoter with upstream tetracycline response elements
(TRE). These mice were crossed with a previously characterized tet-OFF
`transactivator' line expressing the tetracycline transactivating factor (tTA)
from the basal keratin 5 (K5) promoter
(Diamond et al., 2000).
Although both the K5-tTA and TRE-Klf4 lines were phenotypically
normal, crossing them together produced mice with a K5-tTA/TRE-Klf4
genotype that ectopically expressed Klf4 in the basal cells of the
epidermis earlier in development than wild-type littermates. Such mice formed
their barrier in utero one full day earlier than wild-type littermates,
providing evidence of the key role played by the transcription factor
Klf4 in the establishment of the barrier function in mice.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of TRE-Klf4 construct and mice
Klf4 cDNA (bp 273-1800 of GenBank accession no. U20344 with CDS bp
311-1762) was amplified by polymerase chain reaction (PCR) from BALB/c newborn
skin cDNA, cloned in pBS-KS+ (Promega) and sequence verified. By PCR,
BamHI restriction sites were added at both ends of the construct and
a Myc tag was added at the amino terminus. This construct was sequence
verified and subcloned into the unique BamHI site of pTRE2 vector
(Clontech).
The TRE-Klf4 construct was purified by CsCl gradient (Lofstrand).
Thirty micrograms were digested to free the insert with
XhoI-SapI, purified (Qiagen) and injected into FVB/N
one-cell eggs following standard pronuclear injection
(Hogan et al., 1986). Positive
founders were identified by PCR and maintained on FVB/N background. DNA was
isolated by standard techniques. Genotyping was done by PCR on tail DNA using
the following primers: TRE-Klf4 transgene:
5'-CGC-CTGGAGACGCCATCCAC, 5'-CACCTGTGTTGCTGGCAG. Ten founders were
identified as positive by PCR and 3 lines were established. TRE-Klf4
lines were time-mated with the K5-tTA line and supplied with normal water to
obtain transactivation. The morning of vaginal plug detection was taken as
E0.5.
Immunohistochemistry
Backskin samples from double transgenic and wild-type littermates embryos
were taken at different embryonic stages laid down on a paper towel, cut in
half and either frozen on dry-ice in OCT (Tissue-Tek) or fixed overnight in 4%
PFA in PBS (later embedded in paraffin wax). Frozen sections were hybridized
using mouse monoclonal anti-Myc antibody (1:100 from Upstate Biotechnology
Inc.), and the MOM peroxidase kit or rabbit polyclonal KLF4 antibody (1:100)
and the Vectastain ABC kit with peroxidase DAB substrate (Vector
Laboratories). Paraffin sections were hybridized using rabbit polyclonal
antibodies (Covance): K1 (1:500), K14 (1:1000), loricrin (1:500) and filaggrin
(1:1000). Biotinylated secondary antibodies against rabbit (Vector
Laboratories) were used at a 1:200 dilution in combination with Vectastain ABC
kit and peroxidase DAB substrate kit (Vector Laboratories).
For the proliferation assay, pregnant females were injected intraperitoneally with 50 µg/g body weight BrdU (Sigma) and skin samples from embryos were recovered 1.5 hours after injection and processed as described above. Paraffin sections were hybridized using mouse monoclonal BrdU antibody (clone BRD.2, Neomarkers) following the manufacturer's recommendations (HCl and trypsin treatments, antibody diluted at 1:500), the MOM peroxidase and DAB substrate kit (Vector Laboratories).
RNA isolation and northern blot analyses
Sample isolation: at E10.5 and E11.5, heads of the embryos were removed and
the bodies were used for tissue sampling; at E12.5, E13.5 and E14.5, heads and
inner organs were removed prior to tissue processing; at E16.5, skin was
isolated by dissection. All samples were snap frozen in liquid nitrogen. After
genotyping, skin samples from 2-3 embryos from the same litter (same genotype)
were combined if necessary. Tissue was pulverized, homogenized in Trizol
(Invitrogen) and RNA was extracted following the manufacturer's
recommendations. Approximately 15 µg RNA was loaded in every lane and
visualized by ethidium bromide for integrity of the samples. Blots were
hybridized for 2 hours at room temperature (ExpressHyb, Clontech) with
Klf4 antisense probe (bp. 537-1471 GenBank accession no. U20344) or
with G3PDH probe as loading control. Signals were quantified using Molecular
Dynamics Phosphorimager and IQ analysis software.
Immunoblotting
Embryonic skin was isolated and snap frozen in liquid nitrogen. After
genotyping, skin samples from 2-3 embryos (same genotype) were pulverized,
placed in 250 µl boiling LDS sample buffer (without dye) and boiled for 5
minutes. Protein concentration was determined using the Lowry method (DC
protein assay, Biorad). Equivalent amounts of protein (10 µg) were
resolved using SDS-PAGE (4-12% Bis-Tris gels, Invitrogen) and electrophoresed
onto nitrocellulose (Invitrogen). Equal transfer was assayed with Ponceau S
staining (Sigma). The blots were incubated for 1 hour at room temperature with
each primary antibody: polyclonal rabbit anti-KLF4 (1:2,000), involucrin
(Covance, 1:10,000), loricrin (Covance, 1:10,000), filaggrin (Covance,
1:10,000) and mouse monoclonal anti-p84 (GeneTex, 1:10,000). This was followed
by a 1 hour incubation with HRP-conjugated secondary antibodies and detection
with ECL reagents (Amersham).
Barrier function assays
We performed dye penetration assay with X-gal at pH 4.5 for approximately 8
hours at 37°C as described previously
(Hardman et al., 1998). The
tail tips were removed for genotyping purposes. After staining, embryos were
photographed as described above.
Isolation of cornified envelopes
Skin of E15.5 and E18.5 embryos was minced and placed in 1 ml of boiling
extraction buffer (0.1 M Tris pH 8.5, 2% SDS, 20 mM DTT, 5 mM EDTA pH 7.5) and
boiled for 5 minutes (Hohl et al.,
1991). Samples were centrifuged for 10 minutes and cornified
envelopes (CEs) were resuspended in their own volume of extraction buffer. CEs
were directly put onto a slide and photographed in phase contrast under an
Axiophot microscope (Zeiss).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of the transgenic lines K5-tTA and
TRE-Klf4
A `tet-OFF' transgenic line expressing the tetracycline transactivator tTA
from the keratin 5 promoter (K5-tTA) has been previously characterized and
shown to express tTA in the basal layer of the epidermis and in the outer root
sheath of the hair follicles of adult mice
(Diamond et al., 2000). To
characterize these mice for use in developmental studies, we crossed the
K5-tTA mice with reporter mice that express the lacZ gene downstream
from a tetracycline-regulated promoter (TRE-lacZ)
(Fig. 1A). In the absence of
doxycycline, K5-tTA/TRE-lacZ double transgenics express the
ß-galactosidase enzyme wherever the tTA molecule is expressed.
Whole-mount staining with Xgal substrate during the critical time points of
development for this study demonstrated that the K5-tTA line follows the
endogenous pattern of K5 expression faithfully. At E12.5 the
ß-galactosidase enzyme (and hence the tTA expression) is observed on the
lateral surface with distinct sites of expression in the maxillary region
(whisker pad) and a stripe on the lateral surface. At E13.5 the expression has
spread along the ventral and lateral surface with near complete expression of
the ß-galactosidase enzyme on the entire embryo surface observed by E14.5
(Fig. 1B). The developmental
pattern observed in the K5-tTA/TRE-lacZ double transgenics is
essentially identical to the endogenous K5 pattern
(Byrne et al., 1994
).
|
Analysis of Klf4 expression in bipartite transgenic mice
K5-tTA/TRE-Klf4: an allelic series of phenotypic presentation and
expression of Klf4
Of the ten TRE-Klf4 founders, we characterized three that had
single insertion sites and expressed Klf4 as double transgenics. The
K5-Klf4 DTs were an allelic series with varying phenotypic severity
(Fig. 2A). All of the
K5-Klf4 DT lines exhibited open eyes at E16.5, even though the
eyelids normally fuse at E16. Line 2 K5-Klf4 DT had subtle
abnormalities in the shape of the skull in the maxillary region. The whisker
pad was narrower and fewer whiskers were observed. Dissection of the oral
cavity revealed a cleft palate. Line 6 showed abnormalities that were more
severe in line 10. Lines 6 and 10 K5-Klf4 DTs were runted and had
taut skin, lacking the folds or wrinkles visible on normal littermates. Their
skulls are abnormally shaped with cleft palates, open mouths and almost
complete absence of whisker pads. They also had shorter protruding limbs with
normal digits. Examination of skeletal preparations stained with Alcian Blue
(to reveal cartilage) and Alizarin Red (an indicator of calcification) showed
that the DTs had almost normal sized limb bones of appropriate shape
(Fig. 2A). The failure of
complete protrusion may be due to abnormal regulation of epidermal
proliferation (see below). K5-Klf4 DT line 10 had an omphalocele
(gastrointestinal protrusion into umbilical cord).
|
To determine if these changes in Klf4 transcript level manifest as changes at the protein level, we examined the expression for the three K5-Klf4 DT lines by western blot. The endogenous KLF4 protein is barely detectable in the skin of wild-type E16.5 embryos (at 60 kDa) whereas the transgenic KLF4 protein is detected in all K5-Klf4 DT lines (Fig. 2C). Correlating with the mRNA results, expression was less in line 2 than lines 6 and 10, which were equivalent. The relative intensities of the transgenic to endogenous protein are more significant than the level of RNA. There are three possible reasons for this, (i) the KLF4 antibody has an increased affinity for the ectopic protein, (ii) this protein has increased stability or (iii) the transgenic Klf4 transcript is preferentially translated. The levels of Klf4 mRNA and protein correlate with the severity of the phenotypes observed: K5-Klf4 DT line 2 has an almost normal appearance and lower levels of transgenic Klf4 are expressed than in lines 6 and 10.
Analysis of morphology and barrier acquisition in
K5-Klf4DTs
Since Klf4 deficiency results in a failure of epidermal barrier
formation, we analyzed whether the ectopic expression of Klf4 in the
DTs conversely accelerated this process. We analyzed the state of
differentiation with a whole-mount skin permeability assay. This assay
measures the permeability of the epidermis to a dye solution which produces a
blue colored reaction in the skin. In wild-type animals, skin permeability
changes dramatically from E16 to E17 as the barrier is acquired in a dorsal to
ventral pattern (Hardman et al.,
1998).
The K5-Klf4 DTs acquire the epidermal barrier one day earlier in development than the control littermates (Fig. 3). At E15.5, the K5-Klf4 DTs have already established a 15-60% barrier, depending on the line, while the control embryos are completely blue. At E16.5, K5-Klf4 DTs are almost completely white while the control embryos are only beginning the process of barrier acquisition. By E17.5 even the control littermates have established the barrier and they appear similar to the K5-Klf4 DTs. Regressing, at E14.5 neither the controls nor K5-Klf4 DTs have initiated the process of barrier acquisition (data not shown). Although all three K5-Klf4 DT lines had accelerated barrier acquisition, the initiation and degree of coverage was more pronounced in lines 6 and 10 than in 2, correlating with the levels of Klf4 mRNA and protein expressed.
|
The K5-Klf4 DT epidermis at E15.5 is already more organized with increased stratified layers, granular cells and a precursor SC than that of the wild type. The K5-Klf4 DT epidermis at E15.5 resembles the E16.5 wild-type sample. By E16.5, the K5-Klf4 DT epidermis has already matured considerably with multiple granular layers of flattened enucleated keratohyalin containing cells, and multiple layers of SC. By E17.5, the K5-Klf4 DT and wild-type control epidermis appear identical with multiple layers of granular and SC. At the level of gross morphology, the K5-Klf4 DT epidermis appears normal, but advanced in development by at least 1 day (Fig. 4). Examination of E16.5 epidermis at the ultrastructural level gave similar results, documenting the presence of mature granular layers and SC in the K5-Klf4 DTs (data not shown).
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During normal development, barrier acquisition first appears at specific
canonical locations on the dorsal side and then spreads to the ventral surface
(Hardman et al., 1998). The
pattern of barrier acquisition in the K5-Klf4 DT line 2 recapitulates
this pattern. In these studies, the expression of Klf4 is driven from
the keratin 5 promoter, which has a pattern of expression distinct from
barrier acquisition. Specifically, K5 is expressed on the lateral and ventral
surfaces at E13.5 and over the entire embryo, except the neural tube, at E14.5
(Fig. 1A)
(Byrne et al., 1994
). Since the
pattern of barrier acquisition in the K5-Klf4 DT line 2 animals
follows the normal pattern and not that of the K5 promoter, this supports the
concept that there are fields of competence in the epidermis ready to respond
to a signal(s). K5-Klf4 DT lines 6 and 10 initiate barrier
acquisition on the dorsal surface, but have perturbed cranial patterns,
including an area on the top of the head that remains permeable to the dye
even at E17.5 when the non-transgenic mice have established full
impermeability, reflecting the developmental abnormalities associated with
lines 6 and 10. In the K5-Klf4 DT mice, the ectopic Klf4 is
detected at E10.5 although the barrier acquisition is not observed until
E15.5. We hypothesize that this intervening time is necessary to induce the
factors necessary for terminal differentiation.
The three lines of TRE-Klf4 produce an allelic series with levels of Klf4 mRNA and protein expression correlating with acceleration of barrier acquisition and associated phenotypic severity. Line 2 K5-Klf4 DTs appear phenotypically normal except for the failure of the eyelids to fuse at E16 and the cleft palate observed at E17.5. At E15.5 dye is excluded from approximately 15% of the surface area. At this same developmental time point, lines 6 and 10 K5-Klf4 DTs exclude dye from greater than 60% of the surface area and they express higher levels of Klf4 mRNA and protein. However, in addition to the failure of the eyelids to fuse, there are other more severe defects. Lines 6 and 10 K5-Klf4 DTs are runted with taut skin and craniofacial abnormalities including defects in the whisker pads, nasal pits and head shape. Line 10 K5-Klf4 DT had an omphalocele. Although the line 2 K5-Klf4 DTs appear normal at birth, except for the open eyes, there is a failure to nurse, perhaps due to the cleft palate. They appear dehydrated and die within 24 hours. Further exploration is required to determine the primary cause of death. We are currently investigating, by administering doxycycline during specific developmental windows, whether we can achieve the acceleration of barrier acquisition without the other abnormalities. In addition, we are utilizing the inducible tetracycline system to investigate the effects of ectopic Klf4 postnatally.
In vitro, biochemical studies in CHO cells have suggested that KLF4 binds
to and activates its own promoter (Mahatan
et al., 1999). Since KLF4 has both activation and repression
domains, it may function differently in various cell types
(Yet et al., 1998
). The
decreased expression of the endogenous Klf4 transcript observed in
the epidermis of the K5-Klf4 DTs counters the hypothesis that KLF4
activates its own promoter, but rather suggests that in vivo KLF4 represses
its own transcription.
Various studies have implicated Klf4 in various growth-related and
proliferation pathways of the intestine
(Chen et al., 2001;
Dang et al., 2000
;
Dang et al., 2001
). In vivo,
expression of Klf4 is restricted to the differentiated cells in the
epidermis, thymic epithelium and intestine
(Garrett-Sinha et al., 1996
;
Panigada et al., 1999
;
Shields et al., 1996
).
Analysis of the Klf4-/- mice does not show a change in
proliferation status in the epidermis or intestine
(Katz et al., 2002
;
Segre et al., 1999
). At E16.5
and E17.5, K5-Klf4 DTs exhibit approximately a 40% decrease in
proliferation. As the rates of proliferation from E16.5 to E17.5 do not appear
to change substantially in either the transgenics or wild type, this appears
to be specific for the Klf4 expression. The observed change in
Klf4 expression in cancer cell lines and intestinal and colonic
adenomas in mice and humans is consistent with Klf4 regulating the
balance between proliferation and differentiation. Analysis of Klf6
in primary human prostate tumors identified loss of heterozygosity at the
locus and mutations in the coding sequence, suggesting that Klf6 is a
tumor suppressor gene in this tissue
(Narla et al., 2001
).
Prenatal maternal injections of pharmacological levels of glucocorticoids
are also known to accelerate barrier acquisition
(Horbar et al., 2002;
Outcomes, 1995). This regime accelerates intestinal, lung and epidermal
maturation prior to an anticipated premature delivery and has been shown to
reduce morbidity. Physicians do not treat a premature infant with
corticosteroids owing to the deleterious side effects, including arrest of
lung branching morphogenesis and immune suppression. Developing an
understanding of the mechanisms by which glucocorticoids accelerate barrier
function development should lead to more specific compounds to achieve this
end. With the ectopic expression of Klf4 we observe patches of
hypoplastic skin or underdeveloped dysplastic hair follicles. These defects
may be a direct result of the expression of KLF4 or a result of the
accelerated differentiation and concomitant decreased proliferation. Mice with
ectopic expression of the glucocorticoid receptor from the K5 promoter
demonstrate a range of developmental defects ranging from epidermal hypoplasia
with underdeveloped dysplastic hair follicles to skin lesions in the cranial
and umbilical regions (Perez et al.,
2001
). The milder phenotype of the K5-Klf4 DTs suggests
that it may be beneficial to enhance the levels of Klf4 to achieve
normal epidermal differentiation in premature infants. However, the highest
expression of KLF4 in the K5-Klf4 DT line 10 with the defects in the
cranial regions and the omphalocele are highly reminiscent of the defects seen
in the K5-glucocorticoid receptor transgenic mice.
Since the Klf4-/- mutants and the K5-Klf4 DTs
have complementary phenotypes, the overlap of genes misexpressed in
Klf4-/- mutant and K5-Klf4 DTs is a powerful way
to identify genes that may be direct targets of KLF4 in regulating the
establishment of epidermal barrier. We examined the mRNA expression of the
epidermal genes previously published as misregulated in newborn
Klf4-/- mutants, Sprr2a, repetin (Rptn)
and Planh2/Serpinb2 at E16.5 in Klf4-/-,
K5-Klf4 DT and their respective controls
(Segre et al., 1999).
Rptn mRNA is not expressed at detectable levels at E16.5 in any of
the four samples. Levels of Sprr2a and Planh2 mRNA show no
significant changes at E16.5 between the Klf4-/-,
K5-Klf4 DT and their respective controls. However, other targets have
been identified on microarrays and subtractive hybridization that are
misregulated in opposite directions between the Klf4-/-
mutants and the K5-Klf4 DTs (data not shown). In the future, this
strategy should prove fruitful to identify and examine primary targets of KLF4
in establishing the epidermal barrier.
Targeted ablations of epidermal proteins can perturb early events in
epidermal differentiation, resulting in a subsequent defect in terminal
differentiation. There are at least three essential components of epidermal
permeability barrier: proteins cross-linked to form the cornified envelope,
extruded lipids, and tight junctions. Ablations in the transglutaminase 1
enzyme, matriptase, and claudin 1 loci have all produced mice with red, shiny,
wrinkled skin and subsequent perinatal lethality due to water loss
(Furuse et al., 2002;
List et al., 2002
;
Matsuki et al., 1998
). The
Klf4-/- mice exhibited similar epidermal permeability
defects but the skin had an outwardly normal appearance. Although there are
several genetic ways to perturb the barrier, the K5-Klf4 DT mouse is
the first to accelerate the acquisition of the barrier. As such, these mice
have tremendous potential as animal models to understand how to accelerate
this process for a premature infant ex utero.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Byrne, C., Tainsky, M. and Fuchs, E. (1994).
Programming gene expression in developing epidermis.
Development 120,2369
-2383.
Cartlidge, P. (2000). The epidermal barrier. Semin. Neonatol. 5,273 -280.[CrossRef][Medline]
Chen, X., Johns, D. C., Geiman, D. E., Marban, E., Dang, D. T.,
Hamlin, G., Sun, R. and Yang, V. W. (2001).
Kruppel-like factor 4 (gut-enriched Kruppel-like factor) inhibits cell
proliferation by blocking G1/S progression of the cell cycle. J.
Biol. Chem. 276,30423
-30428.
Dang, D. T., Bachman, K. E., Mahatan, C. S., Dang, L. H., Giardiello, F. M. and Yang, V. W. (2000). Decreased expression of the gut-enriched Kruppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients. FEBS Lett. 476,203 -207.[CrossRef][Medline]
Dang, D. T., Mahatan, C. S., Dang, L. H., Agboola, I. A. and Yang, V. W. (2001). Expression of the gut-enriched Kruppel-like factor (Kruppel-like factor 4) gene in the human colon cancer cell line RKO is dependent on CDX2. Oncogene 20,4884 -4890.[CrossRef][Medline]
Diamond, I., Owolabi, T., Marco, M., Lam, C. and Glick, A.
(2000). Conditional gene expression in the epidermis of
transgenic mice using the tetracycline-regulated transactivators tTA and rTA
linked to the keratin 5 promoter. J. Invest. Dermatol.
115,788
-794.
Fuchs, E. and Raghavan, S. (2002). Getting under the skin of epidermal morphogenesis. Nat. Rev. Genet. 3,199 -209.[CrossRef][Medline]
Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A.,
Sugitani, Y., Noda, T., Kubo, A. and Tsukita, S.
(2002). Claudin-based tight junctions are crucial for the
mammalian epidermal barrier: a lesson from claudin-1-deficient mice.
J. Cell Biol. 156,1099
-1111.
Garrett-Sinha, L. A., Eberspaecher, H., Seldin, M. F. and de
Crombrugghe, B. (1996). A gene for a novel zinc-finger
protein expressed in differentiated epithelial cells and transiently in
certain mesenchymal cells. J. Biol. Chem.
271,31384
-31390.
Hardman, M. J., Sisi, P., Banbury, D. N. and Byrne, C.
(1998). Patterned acquisition of skin barrier function during
development. Development
125,1541
-1552.
Hoath, S. B. and Narendran, V. (2000). Adhesives and emollients in the preterm infant. Semin. Neonatol. 5,289 -296.[CrossRef][Medline]
Hogan, B. L., Constantini, F. and Lacy, E. (1986). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Hohl, D., Mehrel, T., Lichti, U., Turner, M. L., Roop, D. R. and
Steinert, P. M. (1991). Characterization of human
loricrin. Structure and function of a new class of epidermal cell envelope
proteins. J. Biol. Chem.
266,6626
-6636.
Horbar, J. D., Badger, G. J., Carpenter, J. H., Fanaroff, A. A.,
Kilpatrick, S., LaCorte, M., Phibbs, R. and Soll, R. F.
(2002). Trends in mortality and morbidity for very low birth
weight infants, 1991-1999. Pediatrics
110,143
-151.
Kalia, Y. N., Nonato, L. B., Lund, C. H. and Guy, R. H. (1998). Development of skin barrier function in premature infants. J. Invest. Dermatol. 111,320 -326.[Abstract]
Katz, J. P., Perreault, N., Goldstein, B. G., Lee, C. S.,
Labosky, P. A., Yang, V. W. and Kaestner, K. H.
(2002). The zinc-finger transcription factor Klf4 is
required for terminal differentiation of goblet cells in the colon.
Development 129,2619
-2628.
Lewandoski, M. (2001). Conditional control of gene expression in the mouse. Nat. Rev. Genet. 2, 743-755.[CrossRef][Medline]
List, K., Haudenschild, C. C., Szabo, R., Chen, W., Wahl, S. M., Swaim, W., Engelholm, L. H., Behrendt, N. and Bugge, T. H. (2002). Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis. Oncogene 21,3765 -3779.[CrossRef][Medline]
Mahatan, C. S., Kaestner, K. H., Geiman, D. E. and Yang, V.
W. (1999). Characterization of the structure and regulation
of the murine gene encoding gut-enriched Kruppel-like factor (Kruppel-like
factor 4). Nucleic Acids Res.
27,4562
-4569.
Matsuki, M., Yamashita, F., Ishida-Yamamoto, A., Yamada, K.,
Kinoshita, C., Fushiki, S., Ueda, E., Morishima, Y., Tabata, K.,
Yasuno, H. et al. (1998). Defective stratum corneum and early
neonatal death in mice lacking the gene for transglutaminase 1 (keratinocyte
transglutaminase). Proc. Natl. Acad. Sci. USA
95,1044
-1049.
Narla, G., Heath, K. E., Reeves, H. L., Li, D., Giono, L. E.,
Kimmelman, A. C., Glucksman, M. J., Narla, J., Eng, F. J., Chan, A. M.
et al. (2001). KLF6, a candidate tumor suppressor gene
mutated in prostate cancer. Science
294,2563
-2566.
Niemann, C. and Watt, F. M. (2002). Designer skin: lineage commitment in postnatal epidermis. Trends Cell Biol. 12,185 -192.[CrossRef][Medline]
NIH Consensus Developmental Panel. (1995). Effect of corticosteroids for fetal maturation on perinatal outcomes. J. Am. Med. Assoc. 273,413 -418.[Abstract]
Panigada, M., Porcellini, S., Sutti, F., Doneda, L., Pozzoli, O., Consalez, G. G., Guttinger, M. and Grassi, F. (1999). GKLF in thymus epithelium as a developmentally regulated element of thymocyte-stroma cross-talk. Mech. Dev. 81,103 -113.[CrossRef][Medline]
Perez, P., Page, A., Bravo, A., Del Rio, M., Gimenez-Conti, I.,
Budunova, I., Slaga, T. J. and Jorcano, J. L. (2001).
Altered skin development and impaired proliferative and inflammatory responses
in transgenic mice overexpressing the glucocorticoid receptor.
FASEB J. 15,2030
-2032.
Roop, D. (1995). Defects in the barrier. Science 267,474 -475.[Medline]
Rutter, N. (2000). Clinical consequences of an immature barrier. Semin. Neonatol. 5, 281-287.[CrossRef][Medline]
Segre, J. A., Bauer, C. and Fuchs, E. (1999). Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat. Genet. 22,356 -360.[CrossRef][Medline]
Shields, J. M., Christy, R. J. and Yang, V. W.
(1996). Identification and characterization of a gene encoding a
gut-enriched Kruppel-like factor expressed during growth arrest. J.
Biol. Chem. 271,20009
-20017.
Steinert, P. M. (2000). The complexity and redundancy of epithelial barrier function. J. Cell Biol. 151,F5 -8.[Medline]
Wilson, D. R. and Maibach, H. I. (1980). Transepidermal water loss in vivo. Premature and term infants. Biol. Neonate 37,180 -185.[Medline]
Yet, S. F., McA'Nulty, M. M., Folta, S. C., Yen, H. W.,
Yoshizumi, M., Hsieh, C. M., Layne, M. D., Chin, M. T., Wang, H., Perrella, M.
A. et al. (1998). Human EZF, a Kruppel-like zinc finger
protein, is expressed in vascular endothelial cells and contains
transcriptional activation and repression domains. J. Biol.
Chem. 273,1026
-1031.