Oncogene (2002) 21, 3765 ± 3779
ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00
www.nature.com/onc
Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier
function, hair follicle development, and thymic homeostasis
Karin List1,4, Christian C Haudenschild5, Roman Szabo1, WanJun Chen2, Sharon M Wahl2,
William Swaim3, Lars H Engelholm1,4, Niels Behrendt4 and Thomas H Bugge*,1
1
Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30
Convent Drive, Bethesda, Maryland, MD 20892, USA; 2Oral Infection and Immunity Branch, National Institute of Dental and
Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda, Maryland, MD 20892, USA; 3Cellular Imaging
Core, National Institutes of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Bethesda,
Maryland, MD 20892, USA; 4Finsen Laboratory, Strandboulevarden 49, DK-2100, Copenhagen, Denmark; 5Department of
Experimental Pathology, American Red Cross, 15601 Crabbs Branch Way, Rockville, Maryland, MD 20855, USA
Matriptase/MT-SP1 is a novel tumor-associated type II
transmembrane serine protease that is highly expressed
in the epidermis, thymic stroma, and other epithelia. A
null mutation was introduced into the Matriptase/MTSP1 gene of mice to determine the role of Matriptase/
MT-SP1 in epidermal development and neoplasia.
Matriptase/MT-SP1-de®cient mice developed to term
but uniformly died within 48 h of birth. All epidermal
surfaces of newborn mice were grossly abnormal with a
dry, red, shiny, and wrinkled appearance. Matriptase/
MT-SP1-de®ciency caused striking malformations of the
stratum corneum, characterized by dysmorphic and
pleomorphic corneocytes and the absence of vesicular
bodies in transitional layer cells. This aberrant skin
development seriously compromised both inward and
outward epidermal barrier function, leading to the rapid
and fatal dehydration of Matriptase/MT-SP1-de®cient
pups. Loss of Matriptase/MT-SP1 also seriously
a€ected hair follicle development resulting in generalized
follicular hypoplasia, absence of erupted vibrissae, lack
of vibrissal hair canal formation, ingrown vibrissae, and
wholesale abortion of vibrissal follicles. Furthermore,
Matriptase/MT-SP1-de®ciency resulted in dramatically
increased thymocyte apoptosis, and depletion of thymocytes. This study demonstrates that Matriptase/MT-SP1
has pleiotropic functions in the development of the
epidermis, hair follicles, and cellular immune system.
Oncogene (2002) 21, 3765 ± 3779. DOI: 10.1038/sj/
onc/1205502
Keywords: apoptosis; cell-surface
thymus; barrier function
proteolysis;
skin;
*Correspondence: TH Bugge, Proteases and Tissue Remodeling
Unit, Oral and Pharyngeal Cancer Branch, National Institute of
Dental and Craniofacial Research, National Institutes of Health, 30
Convent Drive, Room 211, Bethesda, Maryland, MD 20892, USA;
E-mail: [email protected]
Received 25 February 2002; revised 15 March 2002; accepted 19
March 2002
Introduction
Proteolytic modi®cation of the extracellular environment is essential for all aspects of life of a multicellular
organism. Extracellular proteolysis regulates key processes such as cell growth, di€erentiation, adhesion,
migration, and programmed cell death (Werb, 1997).
Dysregulated extracellular proteolysis is also intimately
associated with the genesis and progression of a
number of chronic diseases including cancer, cardiovascular disease, and neuronal degeneration (Andreasen et al., 2000; Esler and Wolfe, 2001; Luttun et al.,
2000; Matrisian, 1999; Werb et al., 1999). Several
families of secreted or cell surface-associated proteases
have been studied extensively in the context of
physiological and pathological tissue remodeling. These
include the plasminogen activation system, the matrix
metalloproteinases, the ADAM proteases, the
ADAMTS proteases, and the cathepsins (Andreasen
et al., 1986; Blobel, 2000; Tang, 2001; Turk et al., 2000;
Vu and Werb, 2000).
The type II transmembrane serine proteases (TTSPs)
is a recently recognized and rapidly expanding family of
mammalian cell surface-associated serine proteases with
a unique modular structure (Hooper et al., 2001). The
family currently includes enterokinase, corin, human
airway trypsin-like protease, hepsin, TMPRSS2,
TMPRSS3, and Matriptase/MT-SP1 (see below).
TTSPs have also been reported in non-mammalian
vertebrates and in invertebrates (Appel et al., 1993;
Yamada et al., 2000). The common structural features
of the TTSPs include a short N-terminal cytoplasmic
tail, a signal anchor that functions as a transmembrane
domain, and a C-terminal serine protease domain. The
transmembrane and serine protease domains are
separated by a linker region consisting of a variable
series of modular protein interaction domains that
include complement factor 1R ± urchin embryonic
growth factor ± bone morphogenetic protein (CUB)
domains, low-density lipoprotein receptor (LDLR)
repeats, group a scavenger receptor domains, Frizzled
domains, and meprin-A-5 protein-mu (MAM) domains
(Hooper et al., 2001). TTSPs are likely synthesized as
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K List et al
3766
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pro-enzymes that require proteolytic cleavage for
function. Preliminary studies suggest that some TTSPs
are shed from the cell surface, either prior to activation
or, possibly, directly associated with the activation
process (Takeuchi et al., 2000). The biological functions
of most TTSPs still need to be elucidated. Enterokinase, the prototypic member of this class of proteases,
is the physiological activator of trypsinogen in the
digestive tract, and in this capacity is the initiator of a
proteolytic cascade reaction that leads to the activation
of pro-carboxypeptidase, chymotrypsinogen, and proelastase (Mann and Mann, 1994). Mutations in the
TMPRSS3 gene were recently shown to be associated
with congenital and childhood autosomal recessive
deafness (Scott et al., 2001). Corin appears to be
involved in pro-hormone processing, acting as a proatrial natriuretic peptide-converting enzyme (Yan et al.,
2000). Taken together, these ®ndings suggest that
TTSPs may have many biologically diverse functions
in development and homeostasis.
The type II transmembrane serine protease Matriptase/MT-SP1 (also known as ST14 and TAGD-15) was
®rst described as a transcript that was frequently absent
in human colon cancer (Zhang et al., 1998). Subsequently, Matriptase/MT-SP1 was independently isolated as a serine protease expressed in a prostate
carcinoma cell line, as a serine protease secreted in
human breast milk, and as a serine protease present in
ovarian cancer but not in normal ovarian tissue (Lin et
al., 1999; Takeuchi et al., 1999; Tanimoto et al., 2001).
Epithin, the murine orthologue of Matriptase/MT-SP1,
was isolated from fetal thymic stromal cells (Kim et al.,
1999). Matriptase/MT-SP1 contains a transmembrane
signal anchor, separated from the chymotrypsin-like
serine protease domain by two CUB domains and four
LDLR repeats (Kim et al., 1999; Takeuchi et al., 1999;
Tanimoto et al., 2001). Matriptase/MT-SP1 has a
widespread expression in normal epithelial tissues,
including the skin, thymic stroma, gastrointestinal tract,
kidney, lung, prostate, and mammary gland (Kim et al.,
1999; Oberst et al., 2001; Takeuchi et al., 1999).
Matriptase/MT-SP1 has been proposed to play an
important role in carcinoma progression. The novel
protease is expressed in a wide variety of human tumors
of epithelial origin, is a prognostic factor in ovarian
carcinoma, and is a potential activator of key molecules
associated with tumor invasion and metastasis (see
below) (Oberst et al., 2001). Matriptase/MT-SP1 is
synthesized as a catalytically inactive single-chain
zymogen. The physiological activator of Matriptase/
MT-SP1 remains to be identi®ed, but Matriptase/MTSP1 can be activated in vitro by a factor that is present
in human serum (Benaud et al., 2001). Soluble
Matriptase/MT-SP1 is present in the conditioned
medium of cultured cells and in milk, suggesting that
proteolytic shedding of Matriptase/MT-SP1 from the
cell surface takes place. Soluble Matriptase/MT-SP1
has been found in both a free form and in association
with the Kunitz-type serine protease inhibitor, hepatocyte growth factor activator inhibitor-1 (Lin et al.,
1999). Matriptase/MT-SP1 is an ecient activator of
pro-urokinase plasminogen activator (pro-uPA), hepatocyte growth factor/scatter factor (HGF/SF), and
protease activated receptor-2 (PAR-2) in vitro, and the
protease could have pleiotropic functions in the
activation of proteolytic cascades, growth factor
activation, and activation of G-protein coupled receptors (Lee et al., 2000; Takeuchi et al., 2000).
To identify novel serine proteases involved in
epidermal remodeling, a systematic screening for serine
protease genes speci®cally upregulated in keratinocytes
during skin wound healing and squamous carcinoma
was initiated. In the study, Matriptase/MT-SP1 was
revealed to be one of several serine proteases expressed
in basal keratinocytes in the skin, and the expression of
Matriptase/MT-SP1 was strongly upregulated in migrating and proliferating keratinocytes in®ltrating the
provisional wound matrix. We also determined that
Matriptase/MT-SP1 was frequently expressed in cancer
originating from strati®ed epithelium (data not shown).
Mice de®cient in Matriptase/MT-SP1 were generated
in order to determine the function of Matriptase/MTSP1 in epidermal development and neoplasia. We
report that Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle
development, and thymocyte survival.
Results
Generation of Matriptase/MT-SP1-deficient mice
The strategy used to generate a null mutation in the
murine Matriptase/MT-SP1 gene in embryonic stem
(ES) cell is depicted in Figure 1a. No phage clones were
isolated that contained the ®rst 143 bp of the published
cDNA sequence (Kim et al., 1999). The ®rst exon
identi®ed, starting at nucleotide (nt.) 144 in the cDNA
sequence, is therefore in the following designated
`putative exon 2' and the upstream intron sequence is
designated `putative intron 1'. In the Matriptase/MTSP1 targeting vector, a 350 bp fragment containing the
3' portion of the putative intron 1 and most of the
putative exon 2 encoding the signal anchor, was
replaced by an HPRT cassette located in the opposite
transcriptional orientation relative to the transcription
of the Matriptase/MT-SP1 gene. Homologous recombination of this targeting vector into the Matriptase/MTSP1 locus removes DNA sequences encoding the Nterminal 19 of the 24 amino acids (aa) of the signal
anchor required for membrane translocation of Matriptase/MT-SP1. This deletion also introduces a frameshift mutation in the targeted Matriptase/MT-SP1 locus
through the splicing of the putative exon 1 to the
putative exon 3, leading to a truncated protein containing just the N-terminal 27 aa of Matriptase/MT-SP1.
Mice homozygous for this targeted deletion in the
Matriptase/MT-SP1 gene (Matriptase/MT-SP17/7
mice) were generated by blastocyst injection of a
targeted ES cell clone followed by interbreeding of the
o€spring of chimeric mice transmitting the targeted
allele through the germ line (Figure 1b). As would be
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K List et al
a
b
mutation, we performed RT ± PCR analysis of total
RNA from whole newborn Matriptase/MT-SP17/7 and
Matriptase/MT-SP1+/+ pups using primer pairs hybridizing to putative exons 1 and 4 (Figure 2b,c). RT ± PCR
ampli®cation with this primer pair resulted in a PCR
product of approximately 280 bp using RNA from
Matriptase/MT-SP17/7 pups, again indicative of a
putative exon 1 ± putative exon 3 splicing event. Identical
results were obtained with an independent, non-overlapping putative exon 1 ± putative exon 4 primer pair
(data not shown). The 280 bp RT ± PCR fragment
generated with RNA from Matriptase/MT-SP17/7 pups
was cloned and sequenced (Figure 2c). This sequence
analysis veri®ed the presence of a putative exon 1 ±
putative exon 3 splice event and the subsequent
introduction of a frameshift mutation in the targeted
Matriptase/MT-SP1 allele leading to a truncated protein
terminating at a TGA codon located at nt. 344 of the
Matriptase/MT-SP1 sequence (Kim et al., 1999). In
conclusion, the signal anchor is deleted from the targeted
Matriptase/MT-SP1 allele, a frameshift introduced at
position 305, and the modi®ed allele has the capacity to
express a cytoplasmic 40 aa peptide containing the Nterminal 27 of the 855 aa of the mature Matriptase/MTSP1 protein (Figure 2c). The fate of this N-terminal 27
aa cytoplasmic peptide was not studied.
3767
Postnatal lethality in Matriptase/MT-SP1-deficient mice
Figure 1 Generation of a targeted deletion in the Matriptase/
MT-SP1 gene. (a) Structure of the Matriptase/MT-SP1 targeting
vector (top), wild-type Matriptase/MT-SP1 allele (middle), and
targeted Matriptase/MT-SP1 allele (bottom). Exons are indicated
as black boxes and intron sequences as solid lines. Putative exon
2, 3 and 4 sequences, determined from the cloned Matriptase/MTSP1 gene, were identical to the published sequence of the mouse
Matriptase/MT-SP1 cDNA (Kim et al., 1999). The size of the
putative ®rst intron was not determined (denoted by / /). A PGKHPRT mini-gene cassette was introduced in the opposite
orientation to the Matriptase/MT-SP1 gene. The cassette replaced
a 350 bp fragment of the Matriptase/MT-SP1 gene containing
part of the putative intron 1 and most of the putative exon 2 that
encodes the signal anchor. The locations of the primers used for
PCR analysis of ES cell clones are indicated by arrows (see
Materials and methods). The location of the p02 primer was
outside the sequences used in the targeting vector. The probe used
for Southern blot analyses of PCR-positive ES cell clones and tail
biopsy DNA was located upstream of the Matriptase/MT-SP1
sequences used in the targeting vector (indicated by a solid line
below the targeted locus). (b) Southern blot of XhoI-digested
DNA from targeted (lane 1) and control (lane 2) ES cell clones.
Arrows indicate the position of the wild type (6.6 kb) and
targeted 6.3 kb) alleles
predicted from the targeting strategy, Northern blot
hybridization of RNA from whole newborn Matriptase/
MT-SP17/7 pups demonstrated the presence of a single
transcript of similar or slightly increased mobility
compared to RNA from Matriptase/MT-SP1+/+ littermates. This suggests that a single putative exon 1 ±
putative exon 3 splicing event was taking place in
transcripts from the targeted Matriptase/MT-SP1 allele
(Figure 2a). To further verify the introduction of a null
Matriptase/MT-SP1 was dispensable for the development to term. Genotype analysis of newborn o€spring
of Matriptase/MT-SP1+/7 breeding pairs revealed that
the targeted Matriptase/MT-SP1 allele was present in
the expected Mendelian frequency (Figure 3a). Of 119
pups from 12 litters from Matriptase/MT-SP1+/7
parents that could be followed from the time of birth,
25 were Matriptase/MT-SP1+/+, 62 Matriptase/MTSP1+/7, and 32 Matriptase/MT-SP17/7. However, no
Matriptase/MT-SP17/7 o€spring were detected at
weaning, indicating that Matriptase/MT-SP1 is required
for postnatal survival (Figure 3b). Of 57 weaning-age
o€spring from eight litters, 41 were Matriptase/MTSP1+/7 and 16 Matriptase/MT-SP1+/+. The mortality
of newborn Matriptase/MT-SP1 pups was investigated
in detail in eight litters from Matriptase/MT-SP1+/7
parents that were monitored closely from the time of
birth. Of the 20 pups in this study cohort that died
within 48 h, 16 were Matriptase/MT-SP17/7, 4 were
Matriptase/MT-SP1+/7, and no pups were Matriptase/
MT-SP1+/+ (P50.001, Chi-square analysis, two-tailed).
No Matriptase/MT-SP17/7 pups in this cohort were
alive more than 48 h after birth. Taken together, these
data demonstrate that Matriptase/MT-SP1 is required
for survival beyond the ®rst two days after birth.
Abnormal epidermal development in Matriptase/MTSP1-deficient mice
Newborn Matriptase/MT-SP17/7 pups displayed an
abnormally dry, wrinkled, red, and shiny skin. All body
surfaces were a€ected including the trunk, limbs, tail,
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3768
Figure 2 The targeted deletion in the Matriptase/MT-SP1 gene generates a null mutation. (a) Northern blot hybridization of total
RNA from whole newborn Matriptase/MT-SP1+/+ pups (lane 1) and Matriptase/MT-SP17/7 pups (lane 2). The positions of 28S
and 18S, and 4-5S ribosomal RNAs are indicated with arrows. The blot was hybridized with a 32P-labeled EST containing the entire
murine Matriptase/MT-SP1 coding region. Comparable amounts of RNA were loaded in each lane. (b) Ethidium bromide-stained
agarose gel of DNA fragments generated by RT ± PCR of total RNA from Matriptase/MT-SP1+/+ pups (lane 1) and Matriptase/
MT-SP17/7 pups (lane 2). The RT ± PCR analysis was performed with primers complementary to sequences in the putative exon 1
(p07 (c, lower panel), nt. 59 ± 80 in the Matriptase/MT-SP1 cDNA sequence (Kim et al., 1999)) and putative exon 4 (p06 (c, lower
panel), nt. 477 ± 498). The presence of a PCR product with an approximate size of 280 bp in lane 2 demonstrates the putative exon
1 ± putative exon 3 splicing event within the targeted allele. Lane 3; 100 bp DNA ladder. (c) RT ± PCR products generated in b were
subcloned into plasmid vectors and sequenced. Upper panel, sequence of the putative exon 1 ± exon 3 border. The putative exon 2
(boxed area in bottom panel) is deleted in transcripts from the targeted Matriptase/MT-SP1 allele to generate a frameshift mutation
and translational termination at a TGA codon located at nt. 344 (shaded box)
and facial skin (Figure 4a and data not shown). This
phenotype became more pronounced within hours after
birth as a consequence of the rapid dehydration of
Matriptase/MT-SP17/7 pups (see below). The body
weight of 3 to 12 h old Matriptase/MT-SP17/7 pups was
approximately 20% lower than Matriptase/MT-SP1+/+
and Matriptase/MT-SP1+/7 (MT-SP+) littermates
(P50.0003) and the crown-rump length was 12%
shorter (P50.00006) (Figure 4b and c). In addition to
progressive dehydration beginning immediately after
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birth (see below), this di€erence in size could in part
also be attributed to selective maternal neglect of
Matriptase/MT-SP17/7 pups or to the inability of some
Matriptase/MT-SP17/7 pups to nurse properly. Thus,
milk spots in the stomachs were observed in just 6 out of
14 Matriptase/MT-SP17/7 pups (43%) versus 27 out of
29 Matriptase/MT-SP1+ pups (93%) that could be
followed closely for at least a 24 h period.
A comprehensive histological analysis was performed
on Matriptase/MT-SP17/7 pups and their associated
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K List et al
3769
Figure 3 Postnatal lethality in Matriptase/MT-SP1 de®cient embryos. Genotype analysis of o€spring from Matriptase/MT-SP1+/7
parents. (a) Distribution of genotypes in newborn pups. One hundred and nineteen o€spring from a total of 12 litters that could be
followed from the time of birth were genotyped. (b) Distribution of genotypes in weaning age o€spring. Fifty-seven o€spring from
eight litters were genotyped 21 ± 28 days after birth. **P50.001, Chi-square analysis, two-tailed
Figure 4 Skin abnormalities and growth retardation in Matriptase/MT-SP17/7 mice. (a) Representative examples of the
appearance of trunk skin from newborn Matriptase/MT-SP1+/+ pups (top) and Matriptase/MT-SP17/7 pups (bottom). Note the
abnormally red, dry, and shiny appearance of the skin from the Matriptase/MT-SP17/7 pup. Size bar, 1 mm (b) Weight and (c)
crown-rump length of Matriptase/MT-SP1+/+ (n=4), Matriptase/MT-SP1+/7 (n=13), and Matriptase/MT-SP17/7 (n=11) pups
from three newborn litters of Matriptase/MT-SP1+/7 parents. *P50.0003 and **P50.00006, Matriptase/MT-SP17/7 versus
Matriptase/MT-SP1+/7 and Matriptase/MT-SP1+/+ pups, respectively (Student's t-test, two-tailed)
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Matriptase/MT-SP1+ littermates to determine the
cause of postnatal lethality and the abnormal skin
appearance. No overt abnormalities were detected in
the lungs, urogenital tract, central nervous system,
vasculature, pituitary, pancreas, liver, adrenals, and
stomach of Matriptase/MT-SP17/7 pups. Also, no
gross developmental abnormalities were detected by Xray analysis of newborn Matriptase/MT-SP17/7 pups
(data not shown). A subset of Matriptase/MT-SP17/7
pups demonstrated a distinct subnuclear vacuolization
in the resorptive epithelium or edema of the underlying
stroma in the small intestine and colon. This
abnormality was probably secondary to starvation
and dehydration, as Matriptase/MT-SP17/7 pups with
abundant milk in the stomach presented an unremarkable resorptive epithelium (data not shown). The
epidermis, however, of Matriptase/MT-SP17/7 pups
demonstrated striking abnormalities as expected from
the macroscopic appearance of the skin (Figure 5). In
normal newborn mice, the ¯attened corneocytes of the
stratum corneum form a characteristic `basket weave'
pattern generated by a meshwork of interlocking layers
of corneocytes connected by desmosomes (Nemes and
Steinert, 1999). The intercorneocyte lacunae of the
basket weave meshwork are abundant in intercorneal
lipids that are extracted during routine tissue processing, giving rise to the appearance of empty intercorneocyte lacunae (Figure 5a). After glutaraldehyde
®xation, the intercorneal lipids are preserved and the
stratum corneum appears as the outermost highly
opaque layer of the epidermis with strati®ed layers of
¯attened corneocytes after toluidine blue staining
(Figure 5c). The basal, spinous, and granular layers
of the epidermis of Matriptase/MT-SP17/7 pups were
not conspicuously morphologically di€erent from
Matriptase/MT-SP1+ littermates when examined by
light microscopy (Figure 5b,d). However, the stratum
corneum presented striking abnormalities in all Matriptase/MT-SP17/7 pups. Very few intercorneocyte
lacunae could be observed, giving rise to a very
compact appearance of the stratum corneum after
hematoxylin and eosin (h and e) staining (compare
Figure 5a,b). At higher magni®cation, following
glutaraldehyde ®xation and toluidine blue staining of
thin sections, the corneocytes appeared clearly dysmorphic and pleomorphic, with a swollen, rather than
¯attened, appearance of individual corneocytes. Moreover, the stratum corneum of all epidermal surfaces
displayed a less organized strati®cation (compare
Figure 5c,d). Taken together, these data clearly
demonstrate an essential function of Matriptase/MTSP1 in stratum corneum development.
The stratum corneum is formed from an inner layer
of living keratinocytes that migrate upwards while
undergoing a highly complex genetic program of
terminal di€erentiation. Four distinct key events
Figure 5 Aberrant stratum corneum in Matriptase/MT-SP17/7 mice. Representative sections of newborn dorsal skin from
Matriptase/MT-SP1+/+ (a and c) and Matriptase/MT-SP17/7 (b and d) littermates. H and e staining at low magni®cation (a and b)
showing abnormal stratum corneum formation in Matriptase/MT-SP17/7 pups. e, epidermis. d, dermis. p, panniculus muscle.
Arrowheads indicate example of hair follicles. Size bar, 25 mm. Toluidine blue staining (c and d) of thin sections of the epidermis at
higher magni®cation showing dysmorphic, pleomorphic, and dysorganized corneocytes in Matriptase/MT-SP17/7 epidermis
(examples indicated with arrows). The positions of the basal (B), spinal (S), granular (G), and corneal layer (Sc) of the epidermis are
indicated. Stars, hair follicles. Size bar, 5 mm
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K List et al
characterize stratum corneum development: (1) formation of desmosomal adherence junctions between
keratinocytes, (2) intracellular accumulation of specialized keratins, (3) formation of a peripheral corni®ed
envelope, and (4) extrusion of specialized intercorneocyte lipids (Nemes and Steinert, 1999; Roop, 1995); see
Discussion for further details). Desmosomes, appearing as `spines' decorating the surface of basal and
spinous layer cells in light microscopy (Figure 5d) and
as ¯attened electron-dense disks in transmission
electron microscopy (Figure 6b), were clearly visible
in the Matriptase/MT-SP17/7 epidermis. Also, the
abnormal stratum corneum formation in Matriptase/
MT-SP17/7 pups was not directly associated with
major changes in the expression of several keratinocyte-speci®c di€erentiation markers. The onset and
level of expression of basal-layer markers (keratin 5
and 14), suprabasal layer markers (keratin 1 and 10),
and granular/corneum-layer markers (®llagrin and
loricrin) were not appreciably altered (data not
shown). Keratohyalin granules formed normally in
the granular and transitional layer of the Matriptase/
MT-SP17/7 epidermis (Figure 6b). Apoptosis was very
infrequent in the basal layer of the epidermis of both
Matriptase/MT-SP17/7 and littermate control pups,
and no signi®cant di€erence was observed in cell
proliferation as assessed by proliferating cell nuclear
antigen-staining (data not shown).
Although the corneocytes of the Matriptase/MTSP17/7 epidermis were strikingly dysmorphic when
visualized by light microscopy, the corni®ed envelope
itself presented no obvious morphological changes
when examined by transmission electron microscopy
(Figure 6). However, striking di€erences in transitional
cell vesicular body content were immediately evident.
In control mice, numerous intracytoplasmic vesicular
bodies were visible in Matriptase/MT-SP1+ transitional cells. These vesicular bodies, which gives rise to
the majority of the intercorneocyte lipids of the normal
epidermis, were located just below the plasma
membrane and also accumulated in the extracellular
space between transitional layer cells and corneocytes
(Figure 6a). In Matriptase/MT-SP17/7 pups, these
vesicular structures were entirely absent in transitional
cells of the epidermis and in the extracellular space
(Figure 6b). The absence of vesicular bodies was not
limited to the epidermis, but was also observed in
transitional cells within the hair follicles of Matriptase/
MT-SP17/7 mice, suggesting a generalized defect in
transitional cell function (data not shown).
3771
Loss of epidermal barrier function in Matriptase/
MT-SP1-deficient skin causes neonatal death
The epidermal water barrier that prevents catastrophic
¯uid loss from the surface of terrestrial vertebrates
Figure 6 Transmission electron microscopy of the transitional cell-®rst corneocyte interface of Matriptase/MT-SP1+/+ (a) and
Matriptase/MT-SP17/7 (b) pups. Transitional cells with keratohyalin bodies (k) are present in the bottom of the panels followed
upwards by layers of corneocytes (stars). Examples of desmosomes are indicated with arrows. Vesicular bodies (some examples
indicated by arrowheads in (a)) are highly abundant at the cytoplasmic face of transitional cells and in the extracellular space
between the transitional cell and ®rst layer of corneocytes. The Matriptase/MT-SP17/7 epidermis lacks vesicular bodies. Size bar,
0.5 mm
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3772
resides in the stratum corneum. Barrier function is
conferred by a combination of the corni®ed envelope
and intercorneocyte lipids (Nemes and Steinert, 1999).
Due to the conspicuous stratum corneum defects, the
functional integrity of the epidermis of Matriptase/MTSP17/7 pups was investigated using a standard dye
permeability assay (Koch et al., 2000). The ability to
exclude histological dyes from penetrating through the
epidermis requires an intact epidermal barrier and dye
penetration is indicative of abrogated barrier function.
Newborn litters from Matriptase/MT-SP1+/7 intercrosses were stained by submersion in toluidine blue.
As expected, Matriptase/MT-SP1+ pups showed minimal epidermal dye penetration, showing that the
neonatal epidermis is highly impermeable and functional (Figure 7a). In contrast, Matriptase/MT-SP17/7
littermates showed extensive dye penetration across the
entire epidermal surface demonstrating a generalized
loss of `outwards in' epidermal barrier function. To
ascertain whether the abnormal epidermal maturation
in Matriptase/MT-SP17/7 pups also impaired `inwards
out' epidermal barrier function, the rate of ¯uid loss
through evaporation was determined in Matriptase/
MT-SP17/7 and Matriptase/MT-SP1+ pups. Newborn
litters of Matriptase/MT-SP1+/7 parents were separated from their mothers to prevent ¯uid intake, and
the loss of ¯uids at 378C was recorded over a ®ve-hour
period in individual pups by monitoring the loss of
body weight. The pups were genotyped at the
termination of the experiment (Figure 7b). Overall,
the loss of ¯uids in Matriptase/MT-SP1+ animals was
minimal, with an average reduction in body weight of
0.39+0.05% per hour. Remarkably, the ¯uid loss in
Matriptase/MT-SP17/7 pups was accelerated by more
than ®vefold, demonstrating a dramatic loss of
`inwards out' epithelial barrier function (2.09+0.55%
per hour). The diminished barrier function of Matriptase/MT-SP17/7 skin was not a consequence of a
general developmental retardation or reduced body
weight, as Matriptase/MT-SP1+ pups of similar or
even smaller size than their Matriptase/MT-SP1de®cient littermates in all cases demonstrated minimal
¯uid loss (data not shown). In conclusion, Matriptase/
MT-SP1 is required for the development of epidermal
barrier function. Loss of Matriptase/MT-SP1 leads to
uncontrolled ¯uid loss and rapid postnatal lethality.
Hair follicle hypoplasia and dysgenesis in Matriptase/
MT-SP1-deficient mice
Vibrissal hairs, which are normally fully developed at
birth (Kaufmann and Bart, 1999), were absent in all
Matriptase/MT-SP17/7 pups, immediately revealing a
role of Matriptase/MT-SP1 in hair follicle development. Examination of the facial area of Matriptase/
MT-SP17/7 pups by scanning electron microscopy
showed a general absence of both erupted vibrissal hair
shafts and vibrissal hair canals (Figure 8a,b). Interestingly, the snouts of Matriptase/MT-SP17/7 pups
instead displayed a large number of ellipsoid epidermal
Figure 7 Disruption of epidermal barrier function in Matriptase/MT-SP17/7 mice. (a) Loss of `outwards in' barrier function.
Newborn Matriptase/MT-SP1+ (top) and Matriptase/MT-SP17/7 (bottom) pups were submerged in toluidine blue, brie¯y
destained with PBS and photographed. Representative sections of trunk skin are presented. Matriptase/MT-SP17/7, but not
Matriptase/MT-SP1+, pups display extensive dye penetration of all epidermal surfaces. Size bar, 1 mm (b) Loss of `inward out'
barrier function. Newborn pups from a litter of Matriptase/MT-SP1+/7 parents were separated from their mother to prevent ¯uid
intake, and the rate of epithelial ¯uid loss was estimated by measuring the reduction of body weight as a function of time. The data
are expressed as the average body weight as a percent of initial body weight in Matriptase/MT-SP1+ (squares; n=3) and
Matriptase/MT-SP17/7 (diamonds; n=3) pups. Error bars indicate standard deviations. One Matriptase/MT-SP17/7 pup urinated
after 210 min and was excluded from further measurements. *P50.028 to 0.022. **P50.014 to 0.005 (Student's t-test, two-tailed)
Oncogene
Type II transmembrane serine protease in development
K List et al
protrusions that were uniformly absent in Matriptase/
MT-SP1+ littermate controls (Figure 8c,d). Although
erupted vibrissae were not present, a histological
analysis revealed that vibrissal follicles were nevertheless abundant in Matriptase/MT-SP17/7 pups
(Figure 8h). The vibrissal follicles were distinctly
hypomorphic (compare Figure 8e,f, and Figure 8g,h),
but did contain vascular sinuses, inner and outer root
sheaths, follicular papillae, hair bulbs, and hair shafts
(Figure 8f,h). In accordance with surface scanning
data, no vibrissal hair canals were detected in a
comprehensive analysis of serial frontal sections of
the facial area of Matriptase/MT-SP17/7 pups and the
hair shaft of all isolated follicles was uniformly curved
and ingrown (Figure 8f,h). Likely as a directly
consequence of this, the nasal area displayed an
unusually high number of aborted vibrissal follicle
remnants presenting as large follicles in various stages
of degeneration embedded in reactive hyperplastic
epidermis. The speci®c requirement of Matriptase/
MT-SP1 for the formation of pelage hair could not
be determined directly, as all Matriptase/MT-SP17/7
pups died before the eruption of body hairs in the
littermates. However, a comprehensive histological
analysis of skin demonstrated that pelage hair follicles
of Matriptase/MT-SP17/7 pups displayed marked
hypoplasia when compared to hair follicles of
Matriptase/MT-SP1+ littermates (Figure 9). Speci®cally, Matriptase/MT-SP17/7 skin contained fewer
pelage hair follicles that generally penetrated less
deeply into the underlying dermis and overlying
epidermis, and were more poorly di€erentiated (Figure
9a,b). Taken together, the data show that Matriptase/
MT-SP1-de®ciency is associated with follicular hypoplasia and dysgenesis.
Accelerated thymocyte apoptosis in Matriptase/MT-SP1deficient mice
Matriptase/MT-SP1 is abundant in the developing
thymic epithelium (Kim et al., 1999). Moreover,
mutations a€ecting the epidermis often also a€ect
thymic function due to the close ontogenic relationship
between the two organs and the expression of a
number of epidermal structural genes in the thymic
epithelium (Davisson et al., 1994; Flanagan, 1966;
Manley, 2000; Nakagawa et al., 1998; Reske-Kunz et
al., 1979; Shultz, 1988). Indeed, the thymus is rich in
keratinized epithelium in the form of terminally
di€erentiated swirls of corni®ed epithelial cells, termed
Hassall's bodies (Manley, 2000). Interestingly, the
thymus of all examined Matriptase/MT-SP17/7 pups
was grossly abnormal as compared to Matriptase/MTSP1+ littermates (Figure 10). Thymocyte apoptosis was
widespread throughout both the cortex and the
medulla of the thymus of Matriptase/MT-SP17/7 pups
when analysed histologically by in situ labeling of
fragmented DNA by Tunel staining (Figure 10b). In
contrast, the thymus of Matriptase/MT-SP1+ pups
demonstrated few apoptotic cells (Figure 10a). Quantitative ¯ow cytometry analysis of immature
CD4+CD8+ double positive thymocytes, which
represent about 90% of T-lymphocytes in the thymus,
revealed a ®vefold increase in the apoptotic index
(P50.016), and a corresponding almost 50% reduction
in the total number of double positive cells residing in
the thymus (P50.003) (Figure 10c,d). The accelerated
thymocyte apoptosis in Matriptase/MT-SP17/7 pups
appeared to be intrinsic to the thymic environment and
not a primary defect in lymphocytes. Thus, other
lymphoid tissues examined in Matriptase/MT-SP17/7
pups, including the spleen and gut-associated lymphoid
tissue, were similar to Matriptase/MT-SP1+ pups
presenting no appreciative lymphocyte apoptosis. In
conclusion, Matriptase/MT-SP1 is critical for the
survival of developing lymphocytes within the thymic
microenvironment.
3773
Discussion
This paper shows that the type II transmembrane
serine protease Matriptase/MT-SP1 is required for
postnatal survival and has an essential function in
epidermal, hair follicle and thymic development. The
epidermis is a self-renewing, strati®ed epithelium that
serves as a ®rst line of defense against the external
environment by providing a protective barrier against
mechanical, chemical, and biological insults. The
epidermis also provides a water-impermeable barrier
that prevents excessive loss of body ¯uids, a function
that is critical for the survival of all terrestrial
vertebrates immediately after birth. The epidermal
barrier function resides in the stratum corneum, which
chie¯y consists of an interlocking meshwork of
¯attened corneocytes connected by desmosomes and
intercorneocyte lipids. Water impermeability is conferred to the stratum corneum by an insoluble corni®ed
envelope located just beneath the plasma membrane of
the corneocytes, and by the surrounding intercorneocyte lipids (Nemes and Steinert, 1999; Roop, 1995).
The stratum corneum is maintained by the constant
reproduction of the inner layer of living keratinocytes
that migrate upwards while undergoing a complex
genetic program of terminal di€erentiation. This
program includes the formation of desmosomal
adherence junctions, the accumulation of specialized
keratins that provide mechanical strength to the skin,
the accumulation of a complex array of structural
proteins, lipids, and enzymes that constitute the
corni®ed envelope, and the synthesis, extrusion, and
processing of intercorneocyte lipids (Nemes and
Steinert, 1999; Roop, 1995). The data presented here
show that Matriptase/MT-SP1 is required for the
formation of a functional stratum corneum and the
establishment of epidermal barrier function. This
de®ciency does not appear to be the consequence of
a general developmental delay, as other organ systems
such as the musculoskeletal system, the central nervous
system, the gastrointestinal tract, the respiratory tract,
and the urogenital tract did not demonstrate any signs
of immaturity in newborn Matriptase/MT-SP17/7
Oncogene
Type II transmembrane serine protease in development
K List et al
3774
Figure 8 Vibrissal follicular hypoplasia and dysgenesis in Matriptase/MT-SP17/7 mice. (a ± d; scanning electron microscopy
images of the facial area of Matriptase/MT-SP1+/+ (a and c) and Matriptase/MT-SP17/7 (b and d) pups. a and b; lateral view at
low magni®cation of parts of the upper (U) and lower (L) lip showing the absence of erupted vibrissae and hair canals in
Matriptase/MT-SP17/7 pups. Size bar, 200 mm. (c) high magni®cation of vibrissal hair shaft and hair canal in a Matriptase/MTSP1+/+ pup and (d) corresponding epidermal protrusion in a Matriptase/MT-SP17/7 pup. Size bar, 20 mm. (e and f) representative
examples of isolated vibrissal follicles from newborn Matriptase/MT-SP1+/+ (e) and Matriptase/MT-SP17/7 (f) pups. Matriptase/
MT-SP17/7 hair shafts are hypomorphic and ingrown. Size bar, 100 mm. (g ± i) Representative longitudinal sections of vibrissal
follicles (stars) from newborn Matriptase/MT-SP1+/+ (g) and Matriptase/MT-SP17/7 (h and i) pups after h and e staining. (h)
Follicular hypoplasia and hair canal agenesis. (i) Aborted vibrissal follicle remnant (star) with reactive epidermal hyperplasia
(arrows). Size bar, 50 mm
pups. Pathological abnormalities in the stratum
corneum, with the subsequent breakdown of epidermal
Oncogene
barrier function, is seen in an extraordinary variety of
skin conditions of diverse etiology that are collectively
Type II transmembrane serine protease in development
K List et al
Figure 9 Pelage hair follicle hypoplasia in Matriptase/MT-SP17/7
mice. Parasagittal sections of newborn mid-dorsal skin from
Matriptase/MT-SP1+/+ (a) and Matriptase/MT-SP17/7 (b) littermates after toluidine blue staining. Pelage hair follicles (arrowheads) are hypoplastic in Matriptase/MT-SP17/7 skin, penetrating
less deeply into the dermis and epidermis. e, epidermis. d, dermis.
Size bar, 50 mm
referred to as ichthyosis. At the genetic level, the loss
of barrier function can be caused by an array of defects
in the complex machinery required for the proper
assembly of keratin ®laments in the corneocyte, the
assembly of corni®ed envelope components, the
formation of corneocyte adherens junctions, or the
assembly of intercorneocyte lipids, as observed in
numerous inherited skin diseases or in transgenic mice
with dominant or recessive mutations in epidermal
components (Nemes and Steinert, 1999; Presland and
Dale, 2000; Roop, 1995). Null, frameshift, or point
mutations in genes encoding structural proteins of the
corneocyte (e.g., keratins, loricrin), in components of
the enzymatic machinery that process and cross-link
these structural proteins (e.g., transglutaminase 1), in
desmosomal cadherins linking corneocytes together
(e.g., desmoglein), or in the enzymes that manufacture
and process corni®ed envelope and intercorneocyte
lipids (e.g., fatty aldehyde dehydrogenase, arylsulfatase
C/cholesterol sulfatase) have all been reported to
disrupt epidermal barrier function (De Laurenzi et
al., 1996; Elias et al., 2001; Huber et al., 1995; Koch et
al., 2000; Kubilus et al., 1979; Matsuki et al., 1998;
Russell et al., 1995; Shapiro et al., 1978). The data
presented in this paper strongly suggest that the
abrogation of barrier function is related to abnormal
extrusion of vesicular bodies in the transitional layer of
the cornifying epidermis, which likely a€ects the
formation of the specialized set of crosslinked intercorneocyte lipids that confer epidermal barrier function
in conjunction with the corni®ed envelope.
Although the short lifespan of Matriptase/MT-SP17/7
mice did not permit a comprehensive initial description
of the role of this protease in hair biology of adult
mice, it is clear from the data presented in this paper
that Matriptase/MT-SP1 plays a critical role in hair
follicle development. Matriptase/MT-SP17/7 mice
presented with marked, generalized follicular hypoplasia and agenesis of the vibrissal hair canal. Despite
their hypoplastic appearance, many follicular structures
could, nevertheless, be identi®ed in the vibrissal
follicles of Matriptase/MT-SP17/7 mice, including the
inner and outer root sheaths, follicular papillae, hair
bulb, and hair shaft. An exception was the hair canal,
which was generally absent. As a consequence of this,
the growing vibrissal hair shaft was unable to erupt,
instead growing backwards into the vibrissal follicle,
likely causing wholesale follicle abortion. Hair canal
formation is a poorly understood process, but is
believed to occur prior to the emerging hair shaft
pushing through the follicle and may include apoptosis
of follicular epithelial cells (Davisson and Hardy, 1952;
Magerl et al., 2001). Although a speculative assertion,
it is tempting to propose that a common molecular
defect underlies the epidermal and hair follicle
abnormalities in Matriptase/MT-SP17/7 mice.
The thymus ful®lls a critical function in Tlymphocyte development through the selection of
mature T-lymphocytes from a repertoire of immature
T-cell precursors. Only a small proportion of T-cell
precursors survives thymic selection and is exported to
secondary lymphoid tissues (Sebzda et al., 1999). The
thymic microenvironment provides negative selection
against thymocytes that express a T-cell receptor that
binds with high anity to self-peptide-MHC complexes (negative thymocyte selection) or are incapable
of interacting with self-peptide-MHC complexes (thymocyte death by neglect). Both groups of thymocytes
are eliminated through apoptosis, but the exact nature
of the stimuli that trigger apoptosis in the thymocytes
is not fully understood (Williams and Brady, 2001).
The data presented in this paper show that Matriptase/MT-SP1 has a pivotal role in thymocyte survival
by suppressing apoptosis. The thymus of Matriptase/
MT-SP17/7 pups was profoundly depleted of immature CD4+CD8+ double positive thymocytes.
This ®nding opens several questions to be addressed
in future studies as to the function of Matriptase/MTSP1 in T-cell immunity. Does Matriptase/MT-SP1
promote thymocyte survival by activating or liberating
cytokines that favor thymocyte survival, or by
3775
Oncogene
Type II transmembrane serine protease in development
K List et al
3776
Figure 10 Thymocyte depletion in Matriptase/MT-SP17/7 mice. Tunel-stained sections of the thymus of newborn Matriptase/MTSP1+/+ (a) and Matriptase/MT-SP17/7 (b) mice. Multiple apoptotic cells are seen in the Matriptase/MT-SP17/7 thymus (examples
indicated by arrowheads in b). Size bar, 50 mm. (c) Percent apoptotic CD4+CD8+ double positive lymphocytes in the thymus of
Matriptase/MT-SP17/7 pups (white bar, n=5) and Matriptase/MT-SP1+ littermates (black bar, n=4) determined by ¯ow
cytometry of total thymocyte populations triple-stained with CD4 antibodies, CD8 antibodies, and 7-amino-actinomycin D as a
marker for apoptosis. *P50.016, Wilcoxon rank sum test, two-tailed. (d) Total numbers of CD4+CD8+ double positive
lymphocytes in Matriptase/MT-SP17/7 pups (white bar, n=5) and Matriptase/MT-SP1+ littermates (black bar, n=5) enumerated
by ¯ow cytometry. *P50.0079, Wilcoxon rank sum test, two-tailed
eliminating pro-apoptotic signals within the thymic
microenvironment? Does Matriptase/MT-SP1 suppress
apoptosis during negative thymocyte selection or
suppress the process of thymocyte death by neglect?
Most importantly, what is the overall contribution of
Matriptase/MT-SP1 to the establishment of a functional cellular immune system?
The future identi®cation of the speci®c molecular
defects underlying the pleiotropic e€ects of loss of
Matriptase/MT-SP1 may represent a challenging
undertaking. These defects could include the lack of
appropriate growth factor processing, growth factor
inactivation, shedding of growth factor receptors, or
the direct modi®cation of the extracellular matrix to
create the appropriate extracellular environment for
keratinocyte di€erentiation and thymocyte survival.
The essential activities of Matriptase/MT-SP1 are likely
to take place directly on the surface of keratinocytes
and thymic epithelium due to the high levels of
expression of the protease on these cells in particular,
and the strict expression of Matriptase/MT-SP1 on
epithelial, and not mesenchymal cells, in general.
Several possible substrates for Matriptase/MT-SP1
have been proposed from biochemical studies, including the cleavage and activation of pro-uPA, HGF/SF,
and PAR-2 (Lee et al., 2000; Takeuchi et al., 2000).
The speci®c relationship between the lack of activation
of these factors by Matriptase/MT-SP1 and the e€ects
Oncogene
of Matriptase/MT-SP1 de®ciency described here is not
obvious. uPA-de®cient mice have been reported to
display reduced epidermal proliferation in the neonatal
period (Jensen and Lavker, 1999). However, uPAde®cient mice display no obvious histological abnormalities in the skin and have normal epidermal function
in the absence of other challenging factors (Carmeliet
et al., 1994). Complete de®ciency in PAR-2 has also
not been reported to be associated with skin
abnormalities (Damiano et al., 1999; Lindner et al.,
2000b). Thus, inappropriate processing of pro-uPA and
PAR-2 can essentially be ruled out as a contributing
factor to the epidermal and thymic phenotype of
Matriptase/MT-SP17/7 mice described here. HGF/SF
is a pleiotropic regulator of cell growth and di€erentiation with possible functions in keratinocyte biology.
Keratinocytes express c-met, the receptor for HGF/SF,
and display increased mobility in response to HGF/SF
in culture (McCawley et al., 1998). Transgenic overexpression of HGF/SF, intradermal injection of HGF/
SF, or administration of HGF/SF to organ cultures all
stimulate hair follicle formation, accelerate hair follicle
di€erentiation, promote hair growth, and prolong the
anagen phase of the hair cycle (Jindo et al., 1998;
Lindner et al., 2000a). HGF/SF7/7 mice die early in
embryonic development due to inappropriate epithelial-mesenchymal interactions (Stella and Comoglio,
1999) and the function of HGF/SF in epidermal
Type II transmembrane serine protease in development
K List et al
development cannot be determined directly. HGF/SF is
secreted by follicular papilla cells and acts as a
paracrine factor on neighboring follicular epithelial
cells to promote follicle growth. However, both
follicular papilla cells and outer root sheath cells
express HGF activator, a highly ecient activator of
single chain HGF/SF (Lee et al., 2001; Yamazaki et
al., 1999). Moreover, no studies have indicated an
e€ect of HGF/SF on epidermal di€erentiation and
stratum corneum formation. It is, thus, altogether
conceivable that Matriptase/MT-SP1 functions in
epidermal development by a novel mechanism that is
independent of uPA, PAR-2, or HGF/SF activation.
In conclusion, this study demonstrates that Matriptase/MT-SP1 is required for postnatal survival and has
pleiotropic functions in epidermal di€erentiation, hair
follicle development, and thymic homeostasis.
Materials and methods
Construction of targeting vector and generation of transgenic
mice
A mouse genomic DNA fragment was isolated from a 129/
SvJ bacteriophage library using a 169 bp 32P-labelled
synthetic oligonucleotide probe corresponding to nt. 83 to
252 of the published Matriptase/MT-SP1 cDNA sequence
(Kim et al., 1999). The targeting vector was generated from a
7.5 kb EcoRI-NotI fragment containing the putative intron 1
through the putative exon 4, that was subcloned and
characterized extensively by restriction mapping, Southern
blotting, and sequencing of the intron/exon boundaries. The
targeting vector was constructed by placing a PGK-HPRT
cassette into the AscI site of the pKO ScramblerV924 gene
targeting vector (Stratagene, La Jolla, CA, USA). A 4.5 kb
BamHI-SmaI fragment located in intron 1 was thereafter
inserted between the BglII and XhoI sites after inserting a
SalI linker into the SmaI site. A PCR-generated 1 kb BamHIHindIII fragment that includes the 3' part of the putative
exon 2 through the putative exon 4 was next inserted between
the BamHI and HindIII sites of the vector. The Matriptase/
MT-SP1 targeting vector was further furnished with a
¯anking Herpes simplex virus-thymidine kinase expression
cassette inserted into the RsrII site to provide a means of
selection against random insertion of the targeting vector.
The targeting vector was introduced into HM-1 ES cells
(Magin et al., 1992) by electroporation and ES cell clones
were selected in HAT medium (Gibco ± BRL, Gaithersburg,
MD) and 2 mM ganciclovir (Syntex, Clarecastle, Ireland).
Resistant ES cell clones were isolated, expanded, and
screened for homologous recombination of the targeting
vector into the Matriptase/MT-SP1 locus by PCR using a
primer complementary to the promoter region of the PGKHPRT minigene (p01, 5'-GTGCGAGGCCAGAGGCCACTTGTGTAGCG-3') and a primer complementary to a
sequence of the Matriptase/MT-SP1 gene located downstream of the short arm of the targeting vector (p02, 5'CTCAGTGCCACAGAAAATAAATGA-3').
Matriptase/
MT-SP1 gene targeting was veri®ed by Southern blot
hybridization of XhoI-digested genomic DNA using a 32Plabeled 700 bp XhoI-BamHI probe that was external to the
targeting vector sequences. Matriptase/MT-SP1-targeted ES
cells were injected into the blastocoel cavity of C57B1/6Jderived blastocysts and implanted into pseudopregnant
females. Chimeric male o€spring was bred to NIH Black
Swiss females (Taconic Farms, Germantown, NY, USA) to
generate heterozygous o€spring. These mice were subsequently interbred to generate homozygous Matriptase/MTSP17/7 progeny. Genotyping of mice was performed by
PCR ampli®cation of DNA from ear or tail biopsies with the
primers p03 (5'-GCATGCTCCAGACTGCCTTG-3') and
p04 (5'-GTGGAGGTGGAGTTCTCATACG-3') to detect
the presence of the targeted Matriptase/MT-SP1 allele, and
p05 (5'-CAGTGCTGTTCAGCTTCCTCTT-3') and p04 (5'GTGGAGGTGGAGTTCTCATACG-3') to detect the wild
type Matriptase/MT-SP1 allele).
3777
RNA preparation and Northern blot analysis
Whole newborn pups were euthanized, snap-frozen in liquid
nitrogen, and ground to a ®ne powder with a mortar and
pestle. Total RNA was prepared by extraction in Trizol
reagent (Gibco ± BRL), as recommended by the manufacturers. Samples (1 mg) were fractionated electrophoretically
on formaldehyde agarose gels, blotted onto NytranSuper
Charge nylon membranes (Schleicher and Schuell, Keene,
NH, USA), hybridized to a 32P-labeled 3.5 kb Matriptase/
MT-SP1 expressed sequence tag (EST) probe (I.M.A.G.E. ID
2609399) that contains the complete full-length murine
Matriptase/MT-SP1 cDNA, and subjected to PhosphorImage
analysis using ImageQuant software from Molecular Dynamics (Molecular Dynamics, Sunnyvale, CA, USA).
RT ± PCR
Total RNA from newborn pups was ampli®ed by reverse
transcription followed by PCR ampli®cation using Ready-togoTM RT ± PCR beads (Amersham Pharmacia Biotech Inc,
Piscataway, NJ, USA), as recommended by the manufacturers. First strand cDNA synthesis was performed using a
Matriptase/MT-SP1-speci®c primer (p06, 5'-AGGCAGTTACAGCCGACTTCTT-3') complementary to the putative exon
4 sequences (nt. 477 ± 498 of the murine Matriptase/MT-SP1
cDNA sequence, (Kim et al., 1999). The subsequent PCR
ampli®cation (annealing temperature 558C, denaturation
temperature 928C, extension temperature 728C, 40 cycles)
was performed with the ®rst strand primer in combination
with a putative exon 1-speci®c primer (p07, 5'-AACCATGGGTAGCAATCGGGGC-3') corresponding to nt.
59 ± 80 of Matriptase/MT-SP1 (Kim et al., 1999). The RT ±
PCR products were excised from agarose gels following
electrophoresis, puri®ed, and cloned using the TopoTA
cloning vector system (Invitrogen, Carlsbad, CA, USA) and
subsequently analysed by DNA sequencing.
Histological analysis
Newborn to 48 h old pups were ®xed for 24 h in 4%
paraformaldehyde (PFA) in PBS, processed into paran,
sectioned into parallel sagittal section, and stained with h and
e, or periodic acid-Schi€. For toluidine blue staining, pups
were ®xed by intracardial perfusion with 1% glutaraldehyde,
®xed for further 24 h in the same ®xative, and embedded in
epon. One mm sections were stained with hot toluidine blue
and examined by light microscopy. For transmission electron
microscopy, the tissues were post®xed in 1% aqueous OsO4
and stained en bloc with 0.2% uranyl acetate. After
dehydration, embedding in epon, sectioning, and staining
with uranyl acetate and lead citrate, the tissues were
examined with a Phillips CM12 transmission electron
microscope.
Oncogene
Type II transmembrane serine protease in development
K List et al
3778
Scanning electron microscopy
Tissues were ®xed in 4% formaldehyde/4% glutaraldehyde
on 0.1 M cacodylate bu€er, pH 7.4, for 3 days, rinsed in
cacodylate bu€er and incubated overnight in 2% glycine,
overnight in 2% tannic acid pH 4.0, and 6 h in 2% OsO4.
The ®xed tissues were dehydrated in graded ethanol
solutions, put under vacuum for 2 h, mounted onto stubs
with Leit-C adhesive (Electron Microscopy Sciences, Fort
Washington, PA, USA) and were examined with a Hitachi S3500N variable pressure scanning electron microscope.
Immunohistochemistry
For visualization of epidermal structural antigens, sections of
newborn skin were mounted on cardboard and ®xed for 24 h
in 90% ethanol, processed into paran, and sectioned.
Antigens were retrieved by the boiling of sections for 20 min
in DAKO retrieval bu€er (DAKO, Carpinteria, CA, USA),
blocked with 2% bovine serum albumin, and incubated
overnight at 48C with rabbit antibodies to mouse keratin 1, 5,
10, 14, ®llagrin, and loricrin (Covance, Richmond, CA,
USA). Bound antibodies were visualized with a Vectastain
ABC peroxidase kit (vector Laboratories, Burlingame, CA,
USA) using diaminobenzidine as chromogenic substrate.
Apoptosis staining was performed on parasagittal sections
of whole newborn pups ®xed for 24 h in 4% PFA using TdT
incorporation of digoxigenin-dUTP, and visualization of
incorporated digoxigenin-dUTP by horseradish peroxidaselabeled anti-digoxigenin antibodies and new fuchin chromogen (DAKO). Cell proliferation was visualized by staining of
PFA-®xed tissues with monoclonal proliferation cell nuclear
antigen antibodies, and visualization of bound antibodies
with a Vectastain ABC peroxidase kit and diaminobenzidine.
Skin permeability assay
Newborn mice were euthanized and subjected to methanol
dehydration and subsequent rehydration as described (Koch
et al., 2000). The pups were then washed in PBS for 1 min
and stained overnight at room temperature in 0.1% Toluidine
Blue/PBS (Fisher Scienti®c, Pittsburgh, PA, USA), destained
for 15 min in PBS at room temperature and examined with a
dissection microscope for epidermal due penetration.
Transepidermal fluid loss assay
Newborn pups from Matriptase/MT-SP1+/7 parents were
separated from their mother to prevent ¯uid intake and
placed in a 378C incubator. The rate of epithelial ¯uid loss
was estimated by measuring the reduction of body weight of
the individual pups as a function of time over a period of 5 h.
Flow cytometry analysis
Cell staining for surface markers was performed essentially as
described (Chen et al., 2001). Brie¯y, thymocytes from
newborn pups were isolated, stained simultaneously with
phycoerythrin-conjugated CD4 antibodies, ¯uorescein-conjugated CD8 antibodies (both from BD/PharMingen, San
Diego, CA, USA), and 7-amino-actinomycin D (Calbiochem-Novabiochem, San Diego, CA, USA). Cell subsets were
gated and 7-amino-actinomycin D staining on ¯uorescence
channel-3 versus forward scatter channel was displayed.
Acknowledgments
We thank the NIDCR gene targeting core for blastocyst
injections, Drs Henning Birkedal-Hansen, Silvio Gukind,
Kenn Holmbeck, Keld Danù, and Mary Jo Danton for
critically reading the manuscript, Dr Panomwat Amornphimoltam for advice on keratin immunostaining, and Dr Ulrike
Lichti for advice on hair canal formation. We also thank
Yamei Gao, Elizabeth Smith, Hannah Aaronson, and David
Mitola for their excellent technical assistance. K List was
supported by fellowships from the Danish Cancer Society
and Svend Cole Frederiksen and Hustrus Foundation.
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