* Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; Department
of Dermatology and Cutaneous Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107; and § The Jackson
Laboratory, Bar Harbor, Maine 04609
In patients with pemphigus vulgaris (PV),
autoantibodies against desmoglein 3 (Dsg3) cause loss
of cell-cell adhesion of keratinocytes in the basal and
immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on
protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in
cell adhesion has not been shown. To test whether Dsg3
mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 /
mice had no DSG3 mRNA by RNase protection assay
and no Dsg3 protein by immunofluorescence (IF) and
immunoblots. These mice were normal at birth, but by
8-10 d weighed less than DSG3 +/
or +/+ littermates, and at around day 18 were grossly runted. We
speculated that oral lesions (typical in PV patients)
might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and "tombstoning" of basal cells. EM showed
separation of desmosomes. Traumatized skin also had
crusting and suprabasilar acantholysis. Runted mice
showed hair loss at weaning. The runting and hair loss
phenotype of DSG3
/
mice is identical to that of a
previously reported mouse mutant, balding (bal).
Breeding indicated that bal is coallelic with the targeted
mutation. We also showed that bal mice lack Dsg3 by
IF, have typical PV oral lesions, and have a DSG3 gene
mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous
epithelia and suggest that pemphigus autoantibodies
might interfere directly with such a function.
Desmosomes are cell-cell adhesion junctions that
are found in epithelia and a small number of other
tissues (for review see Schwarz et al., 1990 Two types of transmembrane proteins, desmogleins (Dsg)1
and desmocollins (Dsc), are constitutive components of
desmosomes. Both represent small families of type 1 transmembrane glycoproteins (Dsg1, Dsg2, Dsg3; Dsc1, Dsc2,
Dsc3, for nomenclature see Buxton et al., 1993 The members of the desmoglein and desmocollin subfamily of desmosomal cadherins show a tissue- and cell
type-specific expression pattern (e.g., Koch et al., 1992 In this study we focused on the biological function of
one member of the desmoglein family, Dsg3. This protein
has been shown to be the antigen recognized by autoantibodies from patients with the disease pemphigus vulgaris
(PV), and therefore is also referred to as PV antigen (PVA)
(Amagai et al., 1991 PV is a life-threatening, autoantibody-mediated, blistering disease of the skin and mucous membranes (Stanley,
1993a IgG autoantibodies against the cell surface of keratinocytes of stratified squamous epithelia are present in the
skin and sera of PV patients, as detected by direct and indirect immunofluorescence, respectively (Stanley, 1993a Screening of a keratinocyte It is unclear exactly how PV autoantibodies cause loss of
cell-cell adhesion. One possibility is that Dsg3 functions in
cell-cell adhesion in the deep stratified squamous epithelia, and that autoantibodies from PV patients might interfere with its adhesive function. However, several points do
not support such a hypothesis. First, it has been suggested
that PV autoantibodies do not directly cause loss of cell
adhesion, but function through stimulating release of proteases (specifically, plasminogen activator) from keratinocytes (Schiltz et al., 1978 Another approach to investigate whether Dsg3 is important for adhesion of keratinocytes would be to eliminate it
from desmosomes in tissue. Therefore, in this study we genetically engineered a mouse with a targeted disruption of
the desmoglein 3 gene (DSG3). We hypothesized that if
the PV autoantibodies directly interfere with an adhesion
function of this molecule then such a mouse might have a
phenotype resembling human disease. Our findings demonstrate that Dsg3 is critical for cell adhesion in the basal
and immediate suprabasilar keratinocytes, especially in
the oral mucous membrane, thereby showing a differentiation- and tissue-specific adhesion function of one of the
desmogleins. Furthermore, because the pathology of these
mice strikingly resembles that of humans with PV, our findings are consistent with the idea that PV autoantibodies directly interfere with this adhesive function.
Cloning and Characterization of 129/Sv DSG3 Gene
We have previously cloned partial cDNAs corresponding to mouse Dsg3
(Ishikawa et al., 1994 Construction of Targeting Vector
The plasmid pPNT (Tybulewicz et al., 1991
Targeting of Embryonic Stem Cells and Generation of
DSG3-deficient Mice
The embryonic stem (ES) cell line RW4 (Genome Systems, St. Louis,
MO) was cultured according to the recommendations of the supplier. In a
typical electroporation experiment, 30 µg of the NotI-linearized targeting
vector was mixed with 8 × 106 RW4 cells suspended in 10 mM Hepes in
PBS, pH 7.5, and electroporated with a single pulse (960 µF, 0.22 kV) using a BioRad Gene Pulser II (Hercules, CA). Electroporated cells were
grown on neomycin-resistant mouse embryonic feeder cells (MEF; Genome Systems) in selection medium containing 220 µg/ml G418 and 2 µM
gancyclovir (Mansour et al., 1988 Southern Blots
Labeled DNA probes were synthesized using a random primer labeling
kit with [32P]dCTP (Prime-It Rmt; Stratagene). The blots were hybridized
for 2 h at 65°C with labeled probes in "rapid hybridization buffer" (Amersham, Little Chalfont, UK). Blots were washed twice (15 min each time)
in 2× SSC, 0.1% SDS at room temperature and twice (15 min each time)
in 0.2× SSC, 0.1% SDS at 65°C, and then exposed to X-Omat films (Eastman Kodak Co., Rochester, NY) for autoradiography.
Genomic DNA was extracted from 1 cm of mouse tails by digesting the
tails overnight at 55°C in 700 µl tail buffer containing 50 mM Tris, pH 8.0, 100 mM EDTA, 100 mM NaCl, 0.4 mg/ml proteinase K, and 1% SDS. The
lysate was extracted by phenol, 1:1 phenol/choroform, and then chloroform, and DNA was precipitated with isopropanol. Genomic DNA (10-20
µg) was digested overnight with restriction enzymes and electrophoresed
in 0.8% agarose gels. DNA was transferred to Nytran membranes by the
alkaline transfer method according to Schleicher and Schuell (Keene,
NH) and was cross-linked with 120 mJ of UV light.
RNase Protection
The following cDNAs were used to synthesize biotinylated RNA antisense and sense probes with the Biotinscript kit (Ambion, Austin, TX):
B9 (338 bp of mouse Dsg3 cDNA starting from 63 bp upstream of the
translation initiation site), Dsg2.mc580 (mouse desmoglein 2 cDNA, provided by Werner W. Franke, German Cancer Research Center, Heidelberg, Germany), mDsg1exon15A (mouse desmoglein 1 exon 15), and
pTRI- Antibodies
A rabbit antiserum was raised against a synthetic peptide, HQWGIEGAHPEDKEITNIC (single letter amino acid code) (amino acids 667-685
[Amagai et al., 1991
The anti-desmoglein 1 and 2 mouse mAb DG3.10 was generously provided by W.W. Franke. We also used mAbs against desmoplakin I + II
(Biodesign, Kennebunk, ME) and plakoglobin (15F11 generously provided by M. Wheelock, University of Toledo, OH).
Anti-rabbit IgG, anti-human IgG, and anti-mouse IgG antibodies coupled to fluorescent dyes (FITC, Texas red) or enzymes (HRP) were purchased from Biosource (Camarillo, CA), Bio Rad Laboratories, and Molecular Probes (Eugene, OR).
Immunofluorescence Microscopy
Cryosections were fixed for 10 min in acetone or a 1:1 mixture of acetone/
methanol at Western Blotting
Whole tissue extracts were prepared by homogenizing mouse tongue in lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 420 mM NaCl, 1% Triton X-100, 100 µg/ml leupeptin, 0.5 mM PMSF, 50 µg/ml aprotinin) on ice.
The lysates were centrifuged for 10 min (4°C 16,000 g). The pellet was suspended in Laemmli buffer (Bio Rad Laboratories), incubated for 5 min at
100°C, and then centrifuged for 5 min at 16,000 g. The proteins from these
supernatants were separated in 8% Tris-glycine gels (Novex) and then
transferred to nitrocellulose (Trans-Blot; Bio Rad Laboratories). The
membranes were incubated for 1 h in blocking buffer (5% fat-free milk
powder, 1% BSA, 0.1% Tween-20 in PBS).The first antibody (diluted in
blocking buffer) was incubated overnight at 4°C. The membranes were
washed three times, for 15 min each, in 0.1% Tween-20/PBS, and then incubated with the secondary antibody (goat anti-mouse-HRP or goat anti- rabbit-HRP, 1:1,000 dilution in PBS/0.1% Tween-20). The membranes were washed as described above. Binding of the secondary antibody was
detected with the ECL system (Amersham Corp., Arlington Heights, IL).
Histology and EM
Paraffin-embedded, microtome-sectioned tissues were stained with hematoxylin and eosin by routine methods (Lavker et al., 1991 Detection and Verification of a DSG3 Mutation
in bal Mice
Information on the intron-exon organization of the mouse DSG3 gene allowed us to develop a strategy to screen for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis (Ganguly
et al., 1993 For PCR amplification, 250 ng of genomic DNA was used as a template
in the amplification buffer containing 20 pmol of each primer, 100 nmol
MgCl2, 20 mmol of each nucleotide, and 2.5 U of Taq polymerase (GIBCO
BRL, Gaithersburg, MD), in a total vol of 50 µl. The amplification conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 55°C for
45 s, and 72°C for 45 s. Aliquots of 5 µl of the PCR products were analyzed on 2% agarose gel electrophoresis, and 10 µl of the sample was prepared for heteroduplex analysis. The PCR products demonstrating heteroduplex formation were sequenced using an ABI automated sequencer.
Since the mutation in the DSG3 gene did not create or abolish a restriction endonuclease site, its presence was verified by allele-specific oligonucleotide hybridization. The oligonucleotide probes used for hybridization
were, for the wild-type (WT) sequence, 5 Cloning of the 129/Sv Mouse DSG3 Gene
To obtain homologous DNA sequences to target the
DSG3 gene in embryonic stem cells derived from 129/Sv
mice, we cloned the gene from a 129/Sv Generation of Mice with a Targeted Disruption of the
DSG3 Gene
The strategy underlying the experiments described below
was to delete the coding sequence from the first exon of
the DSG3 gene, thereby functionally inactivating the gene.
We constructed a targeting vector to delete, by homologous recombination, about one-third of the 3 An ES clone with a targeted allele (Fig. 1 C) was injected into C57Bl/6J blastocysts and seven male chimeras
were generated, three of which showed germline transmission of the ES cell genome as determined by the agouti
coat color of offspring from chimera × C57Bl/6J breedings. As expected, Southern blot analysis revealed that
~50% of the agouti animals were heterozygous (+/ Taken together, these findings indicated that the targeted mutation did not significantly interfere with prenatal
development. Furthermore, so far one male and one female homozygous mutant mouse were able to breed, demonstrating fertility of the mutants.
Mice Homozygous for the Targeted Mutation Do Not
Synthesize Dsg3
To demonstrate that the gene targeting resulted in a functional null mutation, we studied the Dsg3 mRNA expression and protein synthesis in pups derived from intercrosses between +/ These data indicate that the targeted mutation indeed
represented a functional null mutation.
DSG3 Around 15-20 d after birth,
Runting of the pups consistently sorted with the targeted mutation and was never observed in animals that
were wild type or heterozygous for the mutant allele. A
closer analysis revealed that at birth all pups showed similar weights, but, at 8-10 d after birth, Since the most characteristic lesions in pemphigus vulgaris patients are painful oral erosions that may interfere
with eating, we speculated that the DSG3
Interestingly, the epidermis of these mice did not show
extensive spontaneous lesions. However, when a female
DSG3 Among the other stratified epithelia examined, only the
vaginal epithelium of a DSG3 EM of nonlesional epidermis and lingual mucosa in
DSG3
An additional, and unexpected, phenotype developed at
around the time of weaning (3-4 wk after birth) in DSG3
There were no phenotypic abnormalities of any kind observed in DSG3 +/ Balding (bal) Mice Have the Same Phenotype as DSG3
The phenotype of runting and hair loss around the time of
weaning seen in the DSG3 Histological analysis revealed the presence of suprabasilar blisters on the tongue of bal/bal mice, indistinguishable
from those found in our DSG3 We then determined whether the balding mutation and
our targeted mutation were coallelic. A mouse heterozygous for the targeted mutation (DSG3 +/ Further analysis of the balding mice by immunofluorescence microscopy on tongue epithelium from mice heterozygous (bal/+) and homozygous (bal/bal) for the balding mutation was done using antibodies against extracellular
and intracellular epitopes of Dsg3. Whereas bal/+ epithelium clearly stained with these antibodies, no staining was
observed in the bal/bal epithelium (Fig. 6 C). Control antibodies against desmoglein 1 and 2, desmoplakin and plakoglobin stained both bal/+ and bal/bal epithelia equally
well (data not shown). However, using an RNase protection assay, we were able to detect a transcript derived from
the DSG3 locus (Fig. 6 D). Given the fact that we did not
detect Dsg3 by immunofluorescence analysis, we hypothesized that a mutation within the DSG3 gene of the bal/bal
mutants leads to a transcript that is either not translated or
that directs the synthesis of a truncated polypeptide that is
rapidly degraded. We therefore screened the DSG3 gene for putative mutations by heteroduplex analysis. Band
shifting of PCR products derived from exon 14 of the obligate heterozygous bal/+ mouse suggested the presence of
a mutation in the DSG3 gene (Fig. 7 A). Sequence analysis
of this aberrant PCR fragment identified heterozygous insertion of a thymidine residue at a position that would correspond to nucleotide 2,418 in the human cDNA (Amagai et al., 1991
Both the absence of Dsg3 immunostaining and the presence of suprabasilar blisters indicate that the balding
mouse represents a functional DSG3 null mutation.
Histology of the bald area in DSG3
It has proven difficult, using in vitro methods, to demonstrate whether desmogleins mediate cell adhesion because
they normally are organized in the desmosome with multiple other proteins that may affect their function. A direct
way to analyze their function in vivo is to determine what
happens to cell adhesion if one desmoglein is eliminated.
We therefore genetically engineered a mouse with a targeted disruption of DSG3.
The phenotype of this mouse resembled in many, but
not all, ways that of patients with PV who have autoantibodies directed against DSG3. Painful oral mucous membrane erosions resulting from suprabasilar acantholysis
are the most characteristic lesions of PV (Lever, 1965 Unlike some patients, these mice did not develop extensive spontaneous skin lesions, but did develop some
crusted lesions around the eyes, on the snout, and on the
nipples of a nursing mother, resulting presumably from
trauma. The skin and mucocutaneous junction of the eyelids also showed histologically typical pemphigus lesions as
did some biopsies near the edges where skin was traumatized in cutting. An analogous phenomenon, called the Nikolski sign, occurs in patients with active PV in whom rubbing on normal skin may cause erosions with resultant
crusting (Lever, 1965 The fact that skin of mice is very different from that of
humans, with many more hair follicles, may account for
the finding that most skin areas in mice do not develop extensive erosions. Furthermore, mice may not develop skin
erosions as extensive as those sometimes seen in humans
because, as discussed in the Introduction, autoantibody
binding to human skin might stimulate release of proteases that could amplify blister formation. In addition,
the hair phenotype of these mice may be a reflection of the
difference in hair in rodents and humans, as humans with
PV usually do not develop a balding phenotype. Preliminary analysis of the hair phenotype in these mice shows a
normal first hair cycle but loss of hair after this cycle. In
bald areas, histology of the skin reveals dilated telogen follicles lacking a hair shaft. Previous analysis of balding mice
has shown necrosis immediately above the hair matrix
leading to some scattered generalized follicular necrosis
(Sundberg, 1994 Moreover, considering the major difference in mouse
and human stratified squamous epithelia and adnexal
structures, it is remarkable how similar the phenotype of
DSG3 The phenotype of Dsg3 The phenotype of DSG3 knockout mice underscores the
importance of Dsg3 for cell adhesion and mechanical stability in the deepest layers of stratified epithelia. The other
transmembrane adhesion molecules present, such as Dsg2,
desmocollins, E-cadherin, and P-cadherin, apparently cannot compensate for the loss of Dsg3. In addition, Dsg3
seems to be particularly important for adhesion in the oral
mucous membrane, where lesions always occurred in DSG3 Finally, the phenotype of the Dsg3 knockout mice not
only demonstrates the importance of Dsg3 for cell adhesion in the deep stratified squamous epithelia, but also is
consistent with the idea that, at least in part, PV autoantibodies cause loss of cell adhesion by directly interfering
with the adhesive function of Dsg3.
). These
junctions have two basic functions: in addition to mediating cell-cell coupling, they provide anchorage for intermediate filaments via their cytoplasmic plaque and therefore function as organizational centers for part of the cytoskeleton. Given these structural features and the abundance of
desmosomes in certain tissues, especially in stratified squamous epithelia, it is assumed that desmosomes are critical
in providing mechanical stability to these tissues.
) that are
sequence related to the previously characterized family of
calcium-dependent cell adhesion molecules, the cadherins (Koch et al., 1990
, 1991a,b, 1992; Goodwin et al., 1990
;
Amagai et al., 1991
; Collins et al., 1991
; Mechanic et al.,
1991
; Nilles et al., 1991
; Parker et al., 1991
; Wheeler et al.,
1991
; King et al., 1993
; Theis et al., 1993
; Troyanovsky et
al., 1993
; Schäfer et al., 1994
; for reviews see Koch and
Franke, 1994
; Schmidt et al., 1994
).
;
Arnemann et al., 1993
; Theis et al., 1993
; Schäfer et al.,
1994
, 1996; Schmidt et al., 1994
; Nuber et al., 1995
, 1996
;
Amagai et al., 1996
; North et al., 1996
). Some tissues, e.g.,
simple epithelia, express Dsg2 and Dsc2 only. In stratified
squamous epithelia, however, all three desmoglein and
desmocollin isoforms are present, although the expression of some of these proteins is restricted to certain strata. In human skin, for example, Dsg1 is expressed in suprabasal
cell layers, Dsg2 in the basal cell layer only, and Dsg3 in
the basal as well as the immediate suprabasal cell layer
(Amagai et al., 1996
; Schäfer et al., 1996
).
).
). Blisters in these patients result from loss of keratinocyte cell-to-cell adhesion in the basal and immediate suprabasal level of stratified squamous epithelia. The typical
histology of an early lesion in PV shows detachment of the
epithelium just above the basal cells, usually with a few
acantholytic (detached and rounded-up) cells in the blister
cavity. There is no, or minimal, inflammation in an early
lesion. In addition, the basal cells may detach slightly from
one another while maintaining their attachment to the
basement membrane, a histologic pattern referred to as a
"row of tombstones" (Lever, 1965
; also shown schematically in Stanley, 1993b
). Besides the resemblance of the individual basal cells to tombstones, this designation of the
histologic appearance also reflected the dismal prognosis
of the disease which, before the advent of corticosteroid therapy, was almost uniformly fatal. Without therapy, patients died because these blisters rapidly lost the superficial epithelia, resulting in large areas of erosions on mucous membranes and skin. The mucous membrane lesions
prevented adequate food and fluid intake, while erosions
on the skin resulted in protein and electrolyte loss as well
as infection. Histology of older lesions shows erosions with
inflammation (which is characteristically seen in mucous membrane or skin eroded from any cause, probably secondary to infection, colonization with microbes, and/or irritation due to loss of the barrier function) and attempts at
reepithelialization.
).
These antibodies are pathogenic, i.e., they cause blister
formation, as proven by several lines of evidence (for review see Stanley, 1990
). In general, autoantibody titer, as
determined by indirect immunofluorescence, correlates with disease activity. That is, the higher the antibody titer, the more severe the disease. Furthermore, neonatal PV
has been reported in infants born to mothers with active
PV. As the passively transferred maternal IgG is catabolized, the infant recovers. Similarly, PV IgG passively
transferred to neonatal mice causes clinically and histologically typical blisters. Finally, normal skin in organ culture
incubated with PV IgG develops typical blisters, without
the addition of complement or inflammatory cells.
gt11 expression library
with patient sera was used to isolate cDNA encoding PVA
(Amagai et al., 1991
). Analysis of the predicted amino acid
sequence defined PVA as Dsg3 (Buxton et al., 1993
). Antibodies raised in rabbits to recombinant Dsg3, as well as
patient sera, localized it, like the other desmogleins, to
desmosomes, in particular to their extracellular face (Akiyama et al., 1991
; Karpati et al., 1993
). Recent studies have
shown that antibodies against the extracellular domain of Dsg3 can cause suprabasilar blisters in neonatal mice
(Amagai et al., 1992
) and that the extracellular domain of
Dsg3, expressed by baculovirus in Sf9 insect cells, can adsorb out all pathogenic antibodies from PV sera (Amagai
et al., 1994a
; Memar et al., 1996
). Therefore, the antibodies against Dsg3 in patient sera are pathogenic. Finally, it
has recently been shown that Dsg3 mRNA and antibodies to Dsg3 in patients' sera localize in epidermis to the basal
and immediate suprabasal layer, exactly where blisters occur in PV (Arnemann et al., 1993
; Shimizu et al., 1995
;
Amagai et al., 1996
).
, 1979
; Farb et al., 1978
; Hashimoto et al., 1983
, 1989; Morioka et al., 1987
; Naito et al.,
1989
). Furthermore, although the classical cadherins (e.g.,
E-cadherin, N-cadherin, P-cadherin) have been directly
shown to be calcium-dependent cell adhesion molecules,
the desmogleins have not. Expression of classical cadherins in transfected L cells (mouse fibroblasts) mediates calcium-dependent aggregation (Nagafuchi et al., 1987
).
However, experiments with desmogleins do not show similar aggregation (Kowalczyk et al., 1996
) nor do studies using desmocollins (Chidgey et al., 1996
). Even a chimeric
molecule of the extracellular domain of Dsg3 with the cytoplasmic domain of E-cadherin, which allows for proper
interaction with the actin cytoskeleton through catenins (an interaction shown to be critical for E-cadherin function), mediates only weak aggregation when expressed in
L cells (Amagai et al., 1994b
). These studies suggest that
Dsg3 might mediate adhesion but only does so effectively
when organized with other desmosomal molecules.
Materials and Methods
). To initiate the cloning of the mouse DSG3 gene,
three different cDNA probes corresponding to mouse cDNA sequences
were used for screening of a mouse 129/Sv genomic
FixII library (Stratagene, La Jolla, CA) (Sambrook et al., 1989
). These three cDNAs, 413, 405, and 404 bp in size, respectively, corresponded to the amino-terminal, central, and carboxyl-terminal regions of the coding sequences. (These sequence data are available from EMBL/Genbank/DDBJ under accession
number U86016.) A total of nine unique genomic clones were isolated and
characterized by restriction enzyme digestions and Southern analysis with
synthetic oligonucleotides based on exon sequences derived from the
cDNA. The endonuclease digestion products were fractionated by electrophoresis on 1% agarose gels, and the sizes were estimated by comparison with standard DNA markers (New England Biolabs, Beverly, MA). Subcloning and sequencing of the genomic DNA, in comparison with cDNA sequences, allowed identification of the intron-exon borders. The
sizes of the introns in the genomic DNA were determined by direct nucleotide sequencing, estimated by generating PCR products using synthetic
nucleotide primers placed on the flanking exons, or by Southern blot analysis. (These sequence data [for exon 1/intron 1/exon 2] are available from
EMBL/Genbank/DDBJ under accession number U86015.)
), which contains a neomycin
resistance (neo) and a herpes simplex virus thymidine kinase (HSV-tk)
minigene (both under the control of the phosphoglycerate kinase promoter), was used to construct the targeting vector (Fig. 1 B). DSG3 gene
fragments were derived from two overlapping
FixII clones, both of which
contained portions of exon 1. In the targeting vector the neo-cassette is
flanked by a 2.1-kb DNA fragment (5
arm, derived from ~400 bp of the
5
end of exon 1 and ~1.7 kb of sequences upstream of exon 1) inserted
into the BamHI and EcoRI sites of pPNT and a 5-kb DNA fragment (3
arm, derived from intron 1) inserted into the XhoI and NotI sites of
pPNT. This targeting vector, through homologous recombination with the
DSG3 gene locus, is predicted to delete exon 1 coding sequences for
amino acids 1-16, 164 bp upstream of this coding sequence, and ~500 bp
of intron 1.
Fig. 1.
Targeting strategy and Southern blot confirmation of
targeted alleles. (A) Intron-exon organization of the mouse DSG3
gene. The exons (vertical blocks) and introns (horizontal lines) are drawn to scale, with the exception of intron 1 that is >6 kb in size.
(B) Targeting strategy. Vertical box indicates exon 1 (lighter shading indicates part of exon 1 that is deleted in targeting strategy). neo, neomycin-resistance cassette; tk, herpes thymidine kinase
cassette; B, BamHI sites; NotI, restriction site used to linearize
vector; (bold horizontal lines) portions of the DSG3 gene that were
used in targeting vector; (thickest horizontal line) pUC vector sequences. Probe indicated was used in Southern blots described
below. (C) Southern blot of DNA from wild type (+/+) and targeted (+/) ES cell clones. DNA was digested with BamHI. Targeted allele shows a 2.7-kb band; wild-type allele shows a 9-kb
band. (D) Southern blot of tail DNA from pups of DSG3 +/
× DSG3 +/
mating.
/
, both DSG3 alleles are targeted, resulting in only a 2.7-kb band. +/
animals show both a recombinant
(2.7 kb) and wild-type (9 kb) allele, whereas +/+ animals only
show a wild-type allele. Lane C shows control ES cell DNA.
[View Larger Version of this Image (19K GIF file)]
) for 9 d. With procedures previously described (Ramirez-Solis et al., 1992
, 1993), G418/gancyclovir-resistant ES
cell clones were cultured in duplicate; one set of cultures was frozen while the duplicate set was screened by Southern analysis. In the initial Southern blot screening, BamHI-digested DNA was hybridized to the probe
shown in Fig. 1 B. Out of 628 clones tested, one clone had undergone homologous recombination (Fig. 1 C). Additional Southern blot analysis
with additional restriction enzymes and a probe located in the DSG3 gene
just 3
to the homologous sequences used in the targeting vector confirmed the targeting event in this cell line (data not shown.). A neo-derived
probe indicated the presence of a single copy of the neo-minigene in the
recombinant ES cell clone (data not shown). The targeted ES cells were
found to possess a normal male karyotype (Hogan et al., 1994
) and had no
detectable mycoplasma infection (Mycoplasma PCR Primer Set; Stratagene). The recombinant ES cells were injected into C57Bl/6J blastocysts.
Chimeric offspring were crossed with C57Bl/6J mice. DNA from agouti
offspring was prepared from tail or, if mice were killed, other organs
(Hogan et al., 1994
) and tested for the presence of the targeted allele by
Southern blot hybridization. Mice heterozygous for the targeted mutation
were intercrossed to obtain homozygous DSG3 mutants.
-actin (mouse
-actin; Ambion). Tissue lysates from the skin of
2-4-d-old pups or the tongue of adult animals were prepared (Direct Protect; Ambion). Products of the protection assay were separated in 6%
Tris-borate-EDTA-urea gels (Novex, San Diego, CA), and then transferred to nylon membranes (BrightStar Plus; Ambion). Biotin-labeled
RNA fragments were detected with a chemiluminescent detection system
(BrightStar Biodetect; Ambion).
]), from the "intracellular anchor" domain of Dsg3,
coupled to keyhole limpet hemocyanin (Imject Activated Immunogen
Conjugation Kit; Pierce Chemical Co., Rockford, IL) (Tanaka et al.,
1990
). The rabbit antiserum was affinity purified on the peptide (ImmunoPure Ag/Ab Immobilization Kit 2; Pierce Chemical Co.) and concentrated to 0.3 mg/ml. On immunoblots, the resulting antibody identified the
130-kD Dsg3, but not the 160-kD Dsg1 or Dsg2, extracted from mouse
(Fig. 2 C) or human (not shown) stratified squamous epithelia. In addition, two sera from PV patients, Nos. 1172 and 1409, were used to identify
Dsg3 by immunofluorescence of mouse tissue.
Fig. 2.
Lack of Dsg3 RNA and protein in homozygous targeted mice. (A) RNase protection assay of tongue lysates from
wild type (+/+) and targeted (/
) mice. (B) Immunofluorescence of tongue from wild-type and targeted mice shows absence
of Dsg3 from
/
mice, but presence of Dsg 1 and 2 (Dsg1/2,
identified by mAb DG3.10), and plakoglobin. The submucosa of
/
mice shows increased nonspecific fluorescence probably as a
result of inflammation. (C) Western blot of tongue lysates. Dsg3
is absent in
/
mice (arrowhead), but Dsg1 and 2 are present
(arrow) to the same degree as in +/+ mice. Bar, 25 µm.
[View Larger Version of this Image (93K GIF file)]
20°C, and then washed in PBS. In some experiments, tissue
samples were washed for 5 min in PBS with 1% Triton X-100 after fixation, followed by several washes in PBS. The tissue samples were incubated with the first antibody at the appropriate dilution in 1% BSA in
PBS for 1 h. After washing three times for 5 min each in PBS, samples
were incubated with a fluorescent dye-coupled antibody diluted in 1%
BSA/PBS for 30 min and washed as described above. Stained sections
were examined and photographed with a BX60 photomicroscope (Olympus Corp., Lake Success, NY).
). We used routine, previously described methods to examine the ultrastructure of lesional mouse skin by transmission EM (Lavker et al., 1991
).
). For heteroduplex analysis, total DNA isolated from homozygous bal/bal, heterozygous bal/+, or wild-type mice (+/+) was used as
template for amplification of exons within DSG3. Oligonucleotide primers spanning each of the 15 exons were synthesized on the basis of intronic
sequences and used to generate PCR products spanning the exons. Specifically, to amplify exon 14 containing the mutation, the following primers were used: sense, 5
-GCCATAGCATGAACTGTTAG-3
; antisense, 5
GTTGGCTTGTCTTGTGAGTT-3
.
-TTGAAGGACTATGCTGCGC-3
, and, for the mutant (M) allele, 5
-TGAAGGACTTATGCTGCGC-3
. These oligomers were end labeled with [
-32P]ATP and hybridized to PCR-amplified DNAs that had been immobilized to Zetabind
nylon membrane. Hybridizations were carried out in 5× SSPE, 0.5% SDS,
0.1% BSA, 0.1% polyvinyl pyrrolidone/Ficoll, at 37°C for 1 h, followed by
washing in 2× SSPE, 0.1% SDS at 58°C. Radioactive oligomer DNA hybrids were visualized by autoradiography on exposure to x-ray film.
Results
FixII genomic library and determined the intron-exon structure, which revealed 15 exons spanning ~25 kb of DNA (Fig. 1 A). The
smallest exon (13) was 64 bp in size, while the last exon
(15) consisted of 3,677 bp, including a segment corresponding to the 3
untranslated sequence ending at the
polyadenylation signal. Exon 1 contained the putative
translation initiation codon, ATG, as well as an upstream
5
untranslated region.
end of exon
1 from one DSG3 allele in mouse 129/Sv ES cells (Fig. 1 B)
(Horie et al., 1994
; Thomas et al., 1992
; Zhang et al., 1994
).
This part of exon 1 was replaced by a neo-cassette in the
recombinant locus. The deleted part of exon 1 encodes the
first 16 amino acids (aa) of the Dsg3 signal peptide and 164 bp upstream of this coding sequence (see above and Ishikawa et al., 1994
). The next methionine in the aa sequence is located at position 177 (encoded by exon 6) in
the second extracellular domain (EC2) of Dsg3 (Ishikawa
et al., 1994
; Amagai et al., 1991
). Therefore, even if the
neo-cassette would be spliced out of a primary transcript
generated at a targeted gene locus, the resulting mRNA
would encode a truncated polypeptide lacking signal sequences necessary for insertion into the cell membrane.
Furthermore, the first extracellular domain of Dsg3 (EC1;
Amagai et al., 1991
) would be missing in this polypeptide. Although the specific function of EC1 in desmogleins is
unknown, it has been shown for other cadherins (e.g.,
E-cadherin) that this domain of the protein is crucial for
homophilic interactions (Nose et al., 1990
; Blaschuk et al.,
1990
).
) for
the targeted mutation. The DSG3+/
animals were
healthy and indistinguishable from wild-type (+/+) littermates. Heterozygous mice (F1 generation) were then intercrossed to produce offspring homozygous (
/
) for the
targeted allele. 132 pups derived from these intercrosses were genotyped (example in Fig. 1 D). Of those, 23% were
+/+, 54% +/
, and 23%
/
, indicating inheritance of
the targeted mutation according to Mendel's laws with no
indication of significant embryonic lethality of the homozygous state. The litter size in the F2 generation was
normal, and, at birth, +/+, +/
, or
/
animals could not be distinguished by visual inspection.
animals. Dsg3 mRNA was absent in
/
animals, whereas the mRNA was detected in +/+
and +/
animals, as determined by an RNase protection
assay. (Fig. 2 A). The levels of Dsg1 and Dsg2 mRNA expression were not affected by the targeted mutation and
were similar in +/+, +/
, and
/
mice (data not shown).
Immunofluorescence microscopy on tongue epithelium using antibodies against extracellular or cytoplasmic epitopes
of Dsg3 indicated the absence of the protein in
/
mutants (Fig. 2 B). Control antibodies against other desmogleins (e.g., mAb 3.10 that recognizes Dsg1 and Dsg2)
(Fig. 2 B), as well as antibodies against the desmosomal
plaque proteins plakoglobin (Fig. 2 B) or desmoplakin
(not shown), showed no difference in the staining patterns
in samples derived from +/+, +/
, and
/
mice. Furthermore, Western blot analysis of tongue extracts from
/
mutants demonstrated the absence of the Dsg3 polypeptide but the presence of Dsg1 and Dsg2, as indicated
by mAb 3.10 (Fig. 2 C).
/
Mice Show Loss of Keratinocyte Cell
Adhesion Resulting in a Phenotype That Resembles
That of Patients with PV
/
animals differed in size
from +/
and +/+ animals. The
/
mutants were much
smaller than their littermates (Fig. 3 A). Autopsies revealed a dramatic reduction in body fat in the
/
animals
that resembles what is seen in starvation.
Fig. 3.
DSG3 /
mice
are runts and have skin erosions and eye lesions. (A)
DSG3
/
mice are runts.
Upper mouse is DSG3
/
;
lower mouse is a +/+ littermate. (B) Weight graph
shows that
/
mice (open
circles), compared with +/+
and +/
littermates (filled circles), are born with equal
weight but by about day 8-10
are lagging in weight gain.
Weight loss is seen about day
20, approximately the time of
weaning and start of solid
food. (C) Nipple erosions in
a DSG3
/
nursing mother.
(D) Snout erosion and conjunctivitis in a DSG3
/
mouse.
[View Larger Versions of these Images (65 + 22K GIF file)]
/
animals showed
reduced bodyweight (Fig. 3 B). In the following days, the
mutants grew at a much slower rate than +/
and +/+
mice. Between days 18 and 25, the growth of
/
animals
slowed down even more with most mutants losing weight and a few dying (Fig. 3 B). However, >80% of mutants
survived and again started to gain weight, but they were
still clearly smaller than their littermates. No significant
weight difference between +/
and +/+ mice was observed.
/
animals
might have similar lesions preventing them from feeding
sufficiently that would, in turn, result in runting. Indeed,
histological examination of the oral mucosa in DSG3
/
mice showed a full spectrum of the types of lesions typical
of PV. The most common lesion was an inflammatory erosion, sometimes seen with reepithelialization (Fig. 4 A).
This is typical of a late PV blister after the superficial epidermis is lost and the resulting irritation and/or colonization of the erosion results in acute inflammation and loss
of the basal cells (Fig. 4 B). Further examination showed
intermediate lesions with the superficial epidermis lost,
leaving the basal cells still attached to the basement membrane, but slightly detached from each other (Fig. 4 C).
This appearance has been called the "row of tombstones"
in patients with PV (Fig. 4 D). Finally, we could also detect
the earliest lesion of PV in the DSG3
/
mice, namely a
suprabasilar split in the epithelium, with minimal inflammation (Fig. 4 E). Oropharyngeal biopsies of essentially
all DSG3
/
mice, examined at ages 3 d-5 mo, showed
these changes, but DSG3 +/
and +/+ mice never
showed them. We speculate from this data that suckling
resulted in the trauma necessary to cause these lesions initially, with the beginning of solid food at 16-20 d exacerbating them. The resulting lesions presumably decreased food intake enough to result in the runting of these mice.
Fig. 4.
Histology of oral mucous membranes and skin in DSG3 /
mice and human PV patients. (A) DSG3
/
: inflammatory oral
erosion of the tongue. (B) Human PV: inflammatory oral erosion. (C) DSG3
/
: oral lesion shows basal cells are separated from each
other and the suprabasilar epithelium is lost. This is a characteristic histology called a "row of tombstones." (D) Human PV: oral lesion shows a "row of tombstones." (E) DSG3
/
: early oral lesion shows suprabasilar acantholysis with intact suprabasilar epithelium separated from basal cells. (F) DSG3
/
: early skin lesion on dorsum of foot near where skin was traumatized by cutting. (G) Human PV:
skin lesion shows typical suprabasilar acantholysis. Bar: (A and B) 160 µm; (C-G) 40 µm.
[View Larger Version of this Image (134K GIF file)]
/
mouse delivered pups, their suckling caused
erosions around the nipples (Fig. 3 C) and some mice
showed crusting on the skin around the eyes and snout
(Fig. 3 D), areas that are normally traumatized by scratching. Histology of traumatized skin also showed typical suprabasilar blisters (Fig. 4 F), similar to the histology of skin lesions in pemphigus vulgaris patients (Fig. 4 G).
Some DSG3
/
mice also showed suppurative conjunctivitis (Fig. 3 D) with suprabasilar blisters of eyelids and
mucocutaneous conjunctiva, similar to what has been reported in patients (Hodak et al., 1990
).
/
mouse showed suprabasilar blistering. Histology of the esophagus, the cardiac
portion of the stomach, and the thymus (for Dsg3 expression in thymus see Schäfer et al., 1994
) revealed no abnormalities.
/
mice showed normal differentiation. Desmosomes were present, and individual desmosomes appeared
indistinguishable from those of unaffected littermates:
both had well-defined membrane lipid bilayers, intracellular dense plaques anchored to aggregated intermediate filaments, and extracellular domains with typical central,
electron-dense disks formed by flocculent material (Fig. 5,
D and E). The extracellular domains were consistently 20-
25 nm thick in both control and affected animals. Our general impression was that the number of desmosomes in
DSG3
/
mice may have been diminished focally along the lateral and apical surfaces of basal cells, but, as a result of the focal nature of this finding, we could not quantitate
it convincingly. EM of lesions in DSG3
/
mice showed
cellular detachment primarily at the apical and lateral surfaces of the basal cells (Fig. 5, A and B). Accordingly, the
acantholytic cleft was formed as a result of separation of
basal cells from suprabasal cells and from each other at the
cell membranes. Hemidesmosomes were structurally normal (Fig. 5, F and G). Separated acantholytic cells retained
"half" desmosomes, containing the intracytoplasmic dense
plaque with attached intermediate filaments, along the
plasma membrane that abutted the cleavage plane (Fig. 5
C). These half desmosomes contained a finely flocculent
material (i.e., desmoglea) on their extracellular surface.
These residual half desmosomes were particularly prominent along the apical surfaces of basal cells and tended to
aggregate and coalesce. Individual intact desmosomes directly adjacent to acantholytic areas appeared normal.
Fig. 5.
Ultrastructure of lesional posterior lingual epithelium in DSG3 /
mice. (A) An edge of a blister cavity, denoted by "C" in a
DSG3
/
mouse. The base is formed by a single layer of basal keratinocytes with a characteristic "tombstone" cytoarchitecture. The
roof consists of intact suprabasal epithelium with occasional associated acantholytic keratinocytes (*). (B) Blister in a DSG3
/
mouse
shows separation of desmosomes which form half-desmosomes (double arrows) with tonofilaments still attached. (C) Higher magnification of a half-desmosome shows an intact intracytoplasmic dense plaque and its associated tonofilaments. There is residual flocculent
material (arrowheads) along the cell membrane. (D) Desmosome in DSG3 +/+ littermate. (E) Desmosome in DSG3
/
mouse has
normal appearance. (F) Hemidesmosome in DSG3 +/+ littermate. (G) Hemidesmosome in DSG3
/
mouse shows normal appearance. Bars: (A) 10 µm; (B) 0.5 µm; (C-G) 50 nm.
[View Larger Version of this Image (162K GIF file)]
/
mice. They started to lose their hair and developed
completely bald areas, first on the forehead, and then proceeding onto the entire back (Fig. 6 A). This phenomenon
was observed whether the mutants were housed in groups
or alone. The ventral coat at this age seemed thin but basically intact. New hair regrowth started from the head to
the back. These cycles of hair loss and regrowth occurred repeatedly, but after two to three cycles they lost the head
to tail synchronization and occurred as bald patches with
regrowth, involving both the ventral and dorsal coats. As
hair loss is not a major finding in PV patients, this phenotype either is specific for a genetic loss of Dsg3 or is peculiar to mice as opposed to humans.
Fig. 6.
Analysis of balding mice compared with DSG3 /
mice. (A) Balding phenotype of a 1-mo-old DSG3
/
mouse
compared with a normal littermate (above). (B) Histology of an
oral mucosal blister in a bal/bal mouse shows suprabasilar acantholysis. (C) Immunofluorescence of bal/bal tongue shows no
Dsg3 compared with control littermate. (D) RNase protection assay of tongue lysates demonstrates that bal/bal mice synthesize
DSG3 mRNA, as compared with DSG3
/
mice that do not.
Note that DSG3 +/
mice demonstrate about half of the mRNA
of wild-type mice.
[View Larger Versions of these Images (120 + 122 + 41K GIF file)]
mice.
/
Mice and Have a Null Mutation in the DSG3 Gene
/
mutants has been described in mice with a spontaneous mutation termed bal
(Sundberg, 1994
). This recessive mutation has recently
been mapped to a position close to the DSG3 locus on
mouse chromosome 18 (Davisson et al., 1994
). We therefore speculated that bal might be a DSG3 null mutation.
/
mice (Fig. 6 B). These
lesions were never found in bal/+ or +/+ littermates.
) was bred to a
mouse heterozygous for the bal mutation (bal/+). Two of
the seven pups obtained from these crosses were runts.
Furthermore, histological analysis demonstrated the presence of multiple lesions in the oropharynx of the runts
(but not the normal size littermates) with the typical PVlike morphology (data not shown). By Southern blot analysis, we showed that one runt possessed a targeted DSG3
allele, and mutation analysis (see below) demonstrated the
presence of the heterozygous balding mutation as well
(data not shown). Additionally, at the time of weaning, these runts lost their hair in a pattern identical to that described above for the mice with the targeted DSG3 mutation. We conclude that the runt had inherited the targeted
allele from one parent and the bal mutation from the
other, and developed the same phenotype as DSG3
/
and bal/bal mice. These data demonstrate that the targeted mutation and the balding mutation are coallelic.
), as determined by the nucleotide and aa homology in this region with the mouse sequence (Fig. 7 B).
This insertion causes a frame-shift resulting in a premature
stop codon 78 bp downstream from the site of insertion.
The bal/bal mouse was homozygous for the insertion (Fig.
7 B). The presence of the insertion mutation was verified
by allele-specific oligonucleotide hybridization (Fig. 7 C).
By homology to the reported human sequence, the mutated transcript would encode a polypeptide that lacks
most of the intracellular domain of Dsg3 (after aa 778 [Amagai et al., 1991
]), in particular the amino acid sequence to which plakoglobin binds (Mathur et al., 1994
;
Troyanovsky et al., 1994
; Roh and Stanley, 1995
; Chitaev et al., 1996
; Kowalczyk et al., 1996
). The fact that the Dsg3 antibodies used in this study (e.g., those that bind extracellular epitopes and an intracellular epitope encoded upstream of the frame-shift mutation) did not stain bal/bal
epithelium strongly suggests that a truncated Dsg3 polypeptide, if synthesized in these mice, is not inserted into
the plasma membrane and/or is rapidly degraded.
Fig. 7.
Analysis of the DSG3 mutation in balding mice. (A)
Heteroduplex analysis of a 400-bp segment of PCR-amplified
mouse DSG3, containing exon 14 and flanking intronic sequences, revealed heteroduplex bands in case of a heterozygous
bal/+ mouse (lane 2), while control mouse (lane 1) and a homozygous bal/bal (lane 3) showed a homoduplex band only. (B)
Direct nucleotide sequencing of DNA shown on lane 2 in A revealed a heterozygous 1-bp insertion, 2275insT, which resulted in
frame-shift (upper panel), in comparison with normal sequence in
DNA obtained from a control mouse shown on lane 1 in A (lower
panel). Sequencing of DNA on lane 3 revealed that bal/bal mouse
was homozygous for the 2275insT mutation (middle panel). (C)
Allele-specific oligonucleotide hybridization (ASO) was used to
verify the presence of the mutation in balding mice. Specifically,
PCR-amplified DNA from the control mouse hybridized with the
wild-type oligomer (WT) only (lane 1), while homozygous bal/bal
mouse hybridized with the mutant (M) probe only (lane 3). DNA
from bal/+ mouse hybridized with both the WT and M probes, indicating that these mice were heterozygous for the mutation (lane 2).
[View Larger Versions of these Images (25 + 27K GIF file)]
/
mice shows
slightly cystic telogen hair follicles that lack a hair shaft
(Fig. 8). Further detailed analysis will be needed to determine the exact cause of this hair loss.
Fig. 8.
Histology of bald back skin from a 24-d-old DSG3 /
mouse. Note dilated telogen hair follicle containing a clump of
pigment but no hair shaft. Bar, 35 µm.
[View Larger Version of this Image (92K GIF file)]
Discussion
). Patients often present with oral mucous membrane lesions,
and these may persist, without skin lesions, for months. Some patients only have oral lesions. Patients may lose
weight and become dehydrated because these painful lesions often interfere with normal food and fluid intake.
Similarly, the major lesions in DSG3
/
mice were oral
mucous membrane erosions. As in PV patients, early lesions showed suprabasilar acantholysis with later lesions
showing inflammatory erosions. We presume that these lesions interfered with oral intake, as these mice, which were born with normal weights, became runts once they relied
on oral intake for food. We noticed individual variations in
the severity of the mutant phenotype; i.e., a few animals
died whereas most reached adulthood. These variations
were not unexpected and might be, at least in part, due to
the presence of "modifier genes" in this outbred line. Finally, electron microscopic analysis of a blister in DSG3
/
mice reveals very similar findings to those of patients' lesions.
In both, separating desmosomes are seen, with acantholytic
cells showing single attachment plaques to which tonofilaments insert (Lever, 1979
).
). The findings of conjunctivitis and
PV-like lesions in the eyes of DSG3
/
mice have also
been reported in patients (Hodak et al., 1990
).
). Additional analysis will be required to
better define the pathophysiology of hair loss in these
DSG3
/
and balding mice.
/
mice is to PV patients.
/
mice is quite different from
that of a recently described transgenic mouse that expresses an amino-terminal deleted Dsg3 under the control
of the K14 promoter (Allen et al., 1996
). The transgene
used consisted of the cytoplasmic and transmembrane domain as well as part of the extracellular domain of Dsg3
and was expressed mainly in the lower layers of the epidermis. The transgenic mice, thought to express a dominant negative effect of the truncated Dsg3, showed swelling of paws and digits, focal flakiness of the skin, and
necrotic changes on the tips of the tails that ultimately resulted in tail degeneration. Furthermore, histological analysis revealed epidermal thickening and widening of the intercellular space between keratinocytes but did not show loss of cell-cell adhesion (i.e., acantholysis). At the ultrastructural level, a reduction in the number of desmosomes
and the occurrence of aberrant desmosomes were reported. Although these mice were noted to have a wet and
matted hair coat (probably because of excess grooming),
they did not show an obvious loss of hair. Our DSG3
/
mice clearly had a different phenotype and distinct histologic and ultrastructural abnormalities. DSG3
/
mice
did not show any tail abnormality and did not have flaky
skin, but they developed crusted erosions in areas of
trauma. They also had a striking balding phenotype. Histologically these mice showed obvious acantholysis. Finally, ultrastructurally, the DSG3
/
mice showed normally appearing desmosomes in intact skin and "half"-desmosomes
where cells were acantholytic. We conclude from these differences between the transgenic and DSG3
/
mice that
the truncated Dsg3 did not act in a dominant negative
fashion to totally inactivate DSG3 function.
/
mice, and less so in other stratified squamous
epithelia such as esophagus where lesions were never seen.
The importance of Dsg3 in skin seems intermediate because lesions were mainly seen secondary to trauma.
These studies, then, show that specific desmogleins may
have tissue- and differentiation-specific adhesion functions.
Received for publication 3 February 1997 and in revised form 20 March 1997.
Please address all correspondence to John R. Stanley, Department of Dermatology, University of Pennsylvania School of Medicine, 211 CRB, 415 Curie Blvd., Philadelphia, PA 19104. Tel.: (215) 898-3240. Fax: (215) 573-2033. e-mail: jrstan{at}mail.med.upenn.eduWe thank Dr. Chin Howe for valuable discussions and advice regarding knockout technology; Dr. Victor Tybulewicz for providing the targeting vector; Daniela Simon who did karyotyping; Dr. Jean Richa, from the University of Pennsylvania Transgenic Facility, who performed the ES cell injections; Qi Tian, Drs. Stephan Schäfer, and Werner Franke for providing Dsg2 cDNA and antibodies; Dr. Margaret Wheelock for plakoglobin antibodies; Dr. Kehua Li for assistance in cloning the mouse DSG3 gene; and Dr. Sarolta Karpati for helping with anti-Dsg3 antibody preparation.
This work was supported by National Institutes of Health grants 1RO1AR43776, PO1AR38923, and CA20408. P. Koch was supported by The Thyssen Foundation and a research fellowship from the Dermatology Foundation.
aa, amino acid; Dsc, desmocollin; Dsg, desmoglein; ES, embryonic stem; neo, neomycin resistance; PV, pemphigus vulgaris; PVA, PV antigen.