Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
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Abstract |
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Two splice variants of the 6 integrin subunit,
6A and
6B, with different cytoplasmic domains,
have previously been described. While
6B is expressed throughout the development of the mouse, the
expression of
6A begins at 8.5 days post coitum and is
initially restricted to the myocardium. Later in ontogeny,
6A is found in various epithelia and in certain
cells of the immune system. In this study, we have investigated the function of
6A in vivo by generating
knockout mice deficient for this splice variant. The Cre-
loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of
6A in embryonic stem cells, and the deletion
resulted in the expression of
6B in all tissues that normally express
6A. We show that
6A
/
mice develop normally and are fertile. The substitution of
6A by
6B does not impair the development and function
of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells
differentiated normally in
6A
/
mice. However, the
substitution of
6A by
6B leads to a decrease in the
migration of lymphocytes through laminin-coated
Transwell filters and to a reduction of the number of T
cells isolated from the peripheral and mesenteric lymph
nodes. Lymphocyte homing to the lymph nodes, which
involves various types of integrin-ligand interactions, was not affected in the
6A knockout mice, indicating
that the reduced number of lymph node cells could not
be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of
6A might be necessary for optimal lymphocyte migration on laminin in
certain pathological conditions.
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Introduction |
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THE adhesion receptors of the integrin family play a
major role in various physiological and developmental processes by regulating cell adhesion, migration, differentiation, and proliferation (Hynes, 1992).
Integrins are transmembrane heterodimers formed by
noncovalently linked
and
subunits. While the extracellular domain of integrins mediates cell-cell or extracellular matrix-cell interactions, the cytoplasmic domain
provides a link with proteins of the cytoskeleton and is involved in the transmission of intracellular signals (Clark and Brugge, 1995
).
16 and 8
subunits have been identified so far, constituting a family of over 20 distinct receptors. A further
degree of diversity arises from alternative RNA splicing,
which produces extracellular and cytoplasmic variants of
some of these subunits. Splice variants of the extracellular
domain of
6 (Delwel et al., 1995
),
7 (Ziober et al., 1993
),
and
II
(Bray et al., 1990
) and of the cytoplasmic domain
of
3 (Takada et al., 1991
),
6 (Cooper et al., 1991
; Hogervorst et al., 1991
),
7 (Song et al., 1993
; Ziober et al.,
1993
),
1 (Altruda et al., 1990
; Languino and Ruoslahti,
1992
; van der Flier et al., 1995
; Zhidkova et al., 1995
),
3
(van Kuppevelt et al., 1989
; Kumar et al., 1997
), and
4
(Hogervorst et al., 1990
; Suzuki and Naitoh, 1990
; Tamura
et al., 1990
; Clarke et al., 1994
; van Leusden et al., 1997
)
subunits have been described.
The 6 subunit dimerizes with either the
1 or the
4
subunit to form receptors for various laminin isoforms
(Delwel and Sonnenberg, 1996
). Two splice variants of the
cytoplasmic domain of
6,
6A and
6B, have been identified (Cooper et al., 1991
; Hogervorst et al., 1991
), the cytoplasmic domains of which are encoded by separate exons and which present entirely different sequences with
the exception of the GFFKR motif, present in all
subunits. The
6A and
6B mRNA variants are generated by
pre-mRNA splicing in such a way that exon A sequences
are either retained in or removed from the primary transcript. In the
6A transcripts, a stop codon at the 3' end of
exon A prevents translation to continue further into exon
B. The cytoplasmic sequences of
6A and
6B are conserved in mammalian species, suggesting that the existence of the two forms may be functionally advantageous. This
hypothesis is further supported by the homologies existing
between the
6 splice variants and the variants of two
other laminin-binding subunits,
3 and
7.
Little is known about the functions of 6A and
6B.
Transfection experiments have shown that whether
6
1
or
6
4 contain
6A or
6B makes no difference in the
regulation of their binding activity or ligand specificity
(Delwel et al., 1993
; Shaw et al., 1993
a) or the transduction
of inside-out signals, although only the variant A is phosphorylated upon phorbolester treatment (Hogervorst et
al., 1993a
; Shaw and Mercurio, 1993
b). The subcellular localization of the two variants and their interaction with the
cytoskeleton appear to be cell type specific: while both
variants distribute to the focal contacts in many cell lines,
staining of
6B revealed a punctate pattern distinct from
focal adhesions in embryonic fibroblasts (Cattelino et al.,
1995
). Differential interactions with cytoskeleton proteins
were further suggested by the finding that
6A induced
the formation of pseudopodia in a macrophage cell line
and promoted cell migration on laminin-1 to a greater extent than
6B (Shaw et Mercurio, 1994). This might be
related to the quantitative differences in tyrosine phosphorylation of certain proteins, including paxillin, upon ligation of integrins containing either of the two variants in
these cells (Shaw et al., 1995
). This property of
6A to facilitate cell migration was also observed in embryonic stem
(ES)1 cells (Domanico et al., 1997
) and could be crucial
during development and tissue remodeling.
The hypothesis that 6A and
6B differentially regulate
cell behavior is further supported by their specific distribution patterns in both embryonic and adult tissues. While
omnipotent mouse ES cells only express the
6B variant
in vitro, their differentiation was found to be correlated
with the expression of
6A (Cooper et al., 1991
; Hierck et
al., 1993
). Similarly,
6B is found in the earliest stages of
embryonic development, whereas expression of
6A does
not start until 8.5 days post coitum (dpc) and is initially restricted to the myocardium (Collo et al., 1995
; Thorsteinsdóttir et al., 1995
). At this stage,
6A is present in the
heart in a gradient from strong expression in the atrium to
a weaker expression in the ventricle. By 12.5 dpc, this gradient has disappeared, and
6A is found in other tissues,
including the epidermis, the gonads, and the epithelium of
the digestive tract (Thorsteinsdóttir et al., 1995
). In the
adult mouse,
6A is expressed in the epidermis, in the epithelia of the mammary gland and the digestive tract, in the
mature gonads, and in Schwann cells (Hogervorst et al., 1993b
; Salanova et al., 1995
). However, it is no longer
present in the adult myocardium. From early development
to the adult stage,
6B is expressed in the kidney, in endothelia, in certain epithelia, and in the nervous system
(Hogervorst et al., 1993b
).
Interestingly, the expression of 6 splice variants is regulated during the development of thymic endothelial and
stromal cells and cells of the thymocyte/T cell lineage.
Thus, while
6B is the first variant detected in the thymus
at 10 dpc,
6A becomes expressed later during ontogeny,
in association with both
1 and
4 subunits (Ruiz et al.,
1995
). The expression of
6 on endothelial thymic cells appears to be important for proper maturation of the immune system since anti-
6 antibodies block homing of T
cell progenitors to the thymus (Ruiz et al., 1995
), where
differentiation into CD4+ or CD8+ cells occurs. In the T
cell lineage,
6A and
6B are present on immature thymocytes, but mature cells no longer express
6 (Ruiz et al.,
1995
). However, both splice variants are found on human
peripheral blood T lymphocytes (Chang et al., 1995
).
Although the spatial and temporal regulation of 6A
and
6B expression strongly suggests that the splice variants have specific functions during embryogenesis as well
as in various organs at the adult age, this has never been
studied in vivo. To answer this question and to test the tentative conclusions from results obtained in vitro, we have
generated exon-specific knockout mice in which the exon
encoding the cytoplasmic domain of
6A is deleted. As a
consequence, only exon B is inserted in the
6 mRNA,
which results in the replacement of
6A by
6B in all tissues that normally express only
6A. Classical ES cell
technology allows the generation of null mutations by simple ablation and replacement of the gene of interest by a
selection marker cassette through homologous recombination. This approach could not be used in the case of an
exon-specific knockout since the selection marker remaining in the gene after homologous recombination might affect the splicing of the remaining exons. Therefore, we
made use of the Cre-loxP system of the bacteriophage P1
(Sauer and Henderson, 1988
) to subsequently excise the
selection marker cassette in the ES cells after replacement
of exon A.
The analysis of the phenotype of the 6 exon A-specific
knockout mice confirmed the involvement of
6A in some
aspects of cell migration but also revealed a number of
surprising and unexpected results.
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Materials and Methods |
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Construction of the Targeting Vector
A 14-kb clone containing the exon A encoding the cytoplasmic domain of
6A and the exon B encoding the cytoplasmic domain of
6B was isolated
from the
FIX-II 129/Sv mouse genomic library (Stratagene, La Jolla,
CA) and used to construct the targeting vector. The exon A and the exon-
intron boundaries were replaced by a BamHI site and a XhoI site using
PCR in the 3-kb XbaI-XbaI DNA fragment (Fig. 1 A). Next, a gene cassette containing the neor gene and the HSV-tk gene flanked by two loxP
sites (a gift of Dr. R. Fässler, Lund University Hospital, Lund, Sweden)
was inserted into the introduced BamHI and XhoI sites. Each selectable
marker gene is under the control of a PGK promoter and contains PGK
poly-adenylation sequences. The final targeting construct contained 2.8 kb
of flanking genomic sequences further upstream from the exon A to the
BglII site, and a 5.5-kb fragment including the exon B downstream from the exon A to the NsiI site.
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Generation of 6A-deficient Mice
E14 ES cells were transfected with 80 µg of the targeting construct by
electroporation. The transfected cells were grown on gelatin and selected
in the presence of G418 (200 µg/ml). Homologous recombinant clones
were identified by Southern blot hybridization with a cDNA probe corresponding to exon B. To excise the neo-tk cassette, several homologous recombinant clones were transfected with 10 µg of Cre-encoding plasmid by
electroporation. Transfected cells were grown on irradiated mouse embryonic fibroblasts for 48 h. Cells were then trypsinized and seeded on a fibroblast monolayer at a density of 3 × 103 ES cells/cm2. Selection with 1.5 mM gancyclovir started 3 d later. Resistant clones were screened by PCR
for the Cre-mediated recombination events using oligonucleotides in the
intronic sequences adjacent to the exon A, 83 bp upstream of the exon A
(5'-ACGGCACAGTGACTGCTCGCT-3'), and 36 bp downstream of
the exon A (5'-GCCACCACAACCACAGCAGGT-3'). Two independent clones were isolated and expanded, and their karyotype was checked
before injection into C57BL6 blastocysts. Male chimeric mice were bred
with 129/OLA and FVB females to obtain heterozygous 6A+/
mice,
identified by PCR. Intercrossing of these mice produced offspring homozygous for the mutation.
Histological Analysis
Whole embryos of 10.5 or 12.5 dpc and tissues from adult mice were collected and fixed in 20% ethanol/5% acetic acid/5% formalin for 48 h, embedded in paraffin, cut into 7-µm sections, and stained with haematoxylin/ eosin.
Immunofluorescence
Whole embryos of 12.5 dpc and skin samples from adult mice were collected and fixed in Tissue-Tek OCT compound (Miles Inc., Elkhart, IN)
and frozen in liquid nitrogen. Cryosections (5 µm) were prepared and air
dried. After blocking with PBS, 2% bovine serum albumin for 1 h, unfixed
sections were incubated with primary antibodies for 1 h at 37°C. The following monoclonal antibodies were used: rat anti-integrin 6 (GoH3;
Sonnenberg et al., 1987
), mouse anti-
6A (1A10; Hogervorst et al.,
1993a
,b) and mouse anti-
6B (PB36; de Melker et al., 1997
), mouse anti-
3A (29A3; de Melker et al., 1997
), and rat anti-mouse
7 (CA5; Yao et
al., 1996
). After washing in PBS, sections were incubated with FITC-conjugated and TRITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 45 min at 37°C,
washed again in PBS, and mounted in Vectashield (Vector Laboratories, Inc., Burlingame CA).
Ultrastructural Analysis
Skin samples from adult mice were collected and fixed in 2.5% glutaraldehyde/0.1 M cacodylate buffer, pH 7.2, post-fixed in 1% OsO4/0.1 M cacodylate, stained en bloc with UO2Ac2, and embedded in a mixture of LX112 and Araldite. Thin sections were examined with an electron microscope (model CM10; Philips Electron Optics, Mahwah, NJ).
Wound Healing Experiments
Wounding by tail amputation was conducted as previously described
(Guo et al., 1996). A small section of the tail end was amputated from age-
and sex-matched wild-type and
6A
/
mice. 2 or 4 d after wounding, an
additional segment of the tail was amputated, fixed in 20% ethanol/5%
acetic acid/5% formalin for 48 h, embedded in paraffin, cut into 7-µm sections, and stained with haematoxylin/eosin.
Primary Keratinocyte Culture and Immunoprecipitations
Keratinocytes were isolated and cultured as previously described (Hennings, 1994) with some modifications. In brief, newborn mice were killed
by decapitation, and the tail and limbs were removed. Skin was removed
from the base of the tail to the head and rinsed in PBS containing penicillin/streptomycin, and fat tissue underlying the dermis was discarded. Skins
were floated, dermis side down, on a 0.25% trypsin solution overnight at
4°C. Dermis was then removed with forceps and discarded. Epidermis was
minced, and keratinocytes were mechanically isolated by stirring at 4°C
for 30 min. The cell suspension was then filtered through a 70-µm nylon
filter, and keratinocytes were centrifuged and seeded on Matrigel (Collaborative Biomedical Products, Becton Dickinson Labware, Mountain
View, CA) in SFM keratinocyte medium (GIBCO BRL, Paisley, UK).
From the first passage, cells were grown on tissue culture-treated plastic
dishes.
Immunoprecipitation from lysates of 125I surface-labeled cells was performed as previously described (Hogervorst et al., 1993b) using the following antibodies: anti-
6 (GoH3), anti-
6A (1A10), anti-
6B (PB36),
anti-
3A (29A3), anti-mouse
4 (346-11A; Kennel et al., 1986
), and rat
anti-mouse
1 (MB1.2; von Ballestrem et al., 1996
).
Keratinocyte Adhesion and Migration
For adhesion assays, subconfluent keratinocytes were trypsinized, washed twice in SFM medium, seeded in 96-well plates (105 cells/well) previously coated with laminin-1 (Collaborative Biomedical Products), laminin-5 (a gift from Dr. P. Rousselle, Institut de Biologie et Chimie des Protéines, Lyon, France), or fibronectin (Sigma Chemical Co., St. Louis, MO), and blocked with 1% BSA. After 50 min incubation at 37°C, cells were washed with PBS, fixed with 1% glutaraldehyde, and stained with crystal violet for 30 min. Cells were then thoroughly washed and solubilized in 0.2% Triton X-100. Adhesion was quantified as a measure for the OD at 540 nm.
For migration assays, 105 keratinocytes were seeded in the upper compartment of 8-µm-pore Transwell filters (Costar, Cambridge, MA) previously coated with 10 µg/ml of laminin-1, laminin-5, or fibronectin on the lower face of the filter only. After 6 or 18 h of migration, cells in the upper chamber of the filter were removed, and keratinocytes on the lower side of the filter were fixed in methanol and stained with crystal violet. The number of cells under the microscope was assessed by counting a minimum of five fields/filter.
Flow Cytometry
Total cell suspensions from the various lymphoid organs were isolated,
and red blood cells were lysed by routine techniques. Cells were washed
twice in PBS, incubated with purified anti-mouse CD32/CD16 (2.4G2;
PharMingen, San Diego, CA) to block Fc receptors for 30 min at 4°C,
and stained for flow cytometry with the following antibodies from PharMingen for 30 min at 4°C: PE-conjugated anti-mouse CD3
(145-2C11),
FITC-conjugated anti-mouse CD4 (L3T4, RM4-5), PE-conjugated anti-
mouse CD8b.2 (Ly-3.2), FITC-conjugated anti-mouse IgM (R6-60.2), and
biotin-conjugated anti-mouse B220/CD45R. The samples were washed
three times in PBS and analyzed in a FACScan® using CellQuest software
(Becton Dickinson Labware).
T Cell Purification and Proliferation
Axillary, brachial, inguinal, and mesenteric lymph nodes from 2-mo-old
wild-type and 6A
/
mice were collected, and cells were extracted in Iscove's medium (GIBCO BRL) supplemented with 5% heat inactivated
FCS, penicillin, streptomycin, and 30 µM
-mercaptoethanol. After lysis
of red cells, cell suspensions were first depleted from large adhering cells
by passing them through a nylonwool column (Polysciences, Inc., Warrington, PA) for 45 min at 37°C. Unbound cells (containing T lymphocytes) were subjected to further purification on anti-MHCII-coupled
beads (anti-mouse I-Ad/I-Ed antibody 2G9 from PharMingen; beads from
PerSeptive Biosystems, Inc., Framingham, MA). Purity was checked by
FACS® analysis using a PE-conjugated anti-mouse CD3
antibody, and cell preparations containing less than 97% of CD3-positive cells were discarded.
For proliferation assays, 96-well plates were first coated overnight at
4°C with anti-CD3 followed by coating with mouse laminin-1 for 4 h at
37°C. T lymphocytes (105 cells/well) were cultured in 5% FCS Iscove's
medium for 48, 72, or 96 h, pulsed with [3H]thymidine (0.5 µCi/well) for
the last 12 h, and harvested onto glass fiber filter paper. Radioactivity was
determined by liquid scintillation counting. In some cases, anti-CD28 antibody (1 µg/ml) was added to the wells.
In Vitro T Cell Migration
5-µm-pore Transwells (Costar) were coated overnight at 4°C with laminin-1 or fibronectin at the indicated concentrations on both sides of the filter and subsequently washed in PBS. SDF-1 or MCP-1 was added to 0.5% BSA Iscove's medium (GIBCO BRL) in the lower compartment of the Transwell only. 105 purified lymph node T lymphocytes (isolated from the axillary, brachial, inguinal, and mesenteric lymph nodes) were seeded in the upper compartment of the Transwell in 0.5% BSA Iscove's medium and allowed to migrate for 2 h. Migration was terminated by removal of the filter and counting of the cells collected on the bottom of the well.
In Vivo Homing Assay
Single cell suspensions were prepared from peripheral (axillary, brachial,
and inguinal) and mesenteric lymph nodes from age- and sex-matched
wild-type and 6A
/
mice and used as lymph node cells. Cells were
washed twice in 0.5% Iscove's medium (GIBCO BRL) and labeled with
either PKH2 or PKH26 fluorescent cell linkers (Sigma Chemical Co., St.
Louis, MO) according to the manufacturer's instructions. In brief, cells
were resuspended in diluent C, after which they were immediately added
to an equal volume of a 4 µM PKH2 or PKH26 solution in diluent C. The
final cell concentration was 107 cells/ml. The cells were then incubated at
room temperature for 2 min, after which the staining reaction was stopped
by the addition of an equal volume of FCS. After 1 min, an equal volume
of Iscove's medium supplemented with 10% FCS was added to the cells.
Cells were then washed four times in the same medium. Labeled
6A+/+ and
6A
/
cells were mixed in equal number and immediately injected into the tail vein of wild-type recipient animals (3 × 107 cells for each cell
type). The percentage of PKH2- and PKH26-labeled cells in the cell mixture was checked by FACS® analysis. After 2 or 20 h, mice were killed,
and the spleen, peripheral, and mesenteric lymph nodes were isolated.
Cell suspensions were extracted from each organ, and the percentages of
labeled wild-type (
6A+/+) and
6A
/
cells were determined by
FACS® analysis.
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Results |
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Generation of 6A Integrin-deficient Mice
The two cytoplasmic variants 6A and
6B result from alternative splicing of the
6 pre-mRNA (Cooper et al.,
1991
; Hogervorst et al., 1991
). In the case of the variant A,
exon A is inserted in the
6 mRNA, and the presence of a
stop codon at the 3' end of exon A prevents translation to
continue further into exon B. In the case of
6B, an
6
RNA transcript is produced that lacks exon A sequences.
To define the function of the two 6 variants in the development of the mouse and to determine whether
6B
could functionally replace
6A, we specifically deleted the
exon A. To this end, we have used a gene targeting approach combining classical methods of gene inactivation
by homologous recombination and the use of the Cre-loxP
system (Fig. 1 A). This technique has been successfully used to generate knockout mice in which the exon coding
for a splice variant of the
1 integrin subunit was selectively deleted (Baudoin et al., 1998
). The Cre recombinase
of the bacteriophage P1 excises DNA residing between repeats of 34 bp termed loxP sites, leaving one loxP site in
the gene locus (Sauer and Henderson, 1988
). We made use
of this property to delete the selection marker cassette after inactivation of exon A. We predicted that the removal of the 4.8-kb cassette would allow normal splicing between
the exon coding for the transmembrane domain of the integrin subunit and exon B (Fig. 1 B), and thus only
6B
was expected to be expressed in all tissues that normally
express
6A.
In the first step, a FIX-II 129/Sv mouse genomic library
was screened with a cDNA probe corresponding to the
exon coding for the cytoplasmic domain of
6A. One of
the resulting clones, m
6A
1, was characterized further,
and its restriction map is shown in Fig. 1 A. The position of
exon A and exon B was determined by PCR using oligonucleotides specific for these exons. The vector was designed to replace exon A and the exon-intron boundaries by a selection marker cassette containing the neor gene for
positive selection and the HSV-thymidine kinase gene for
negative selection.
The linearized targeting vector was transfected in E14
ES cells by electroporation, and cells were subjected to
positive selection (G418). Cell clones containing a recombined 6 allele were identified by Southern blot analysis
after digestion of genomic DNA with BglII and hybridization with a cDNA probe corresponding to exon B. Targeted clones yielded a 7.5-kb band in addition to the 10-kb band corresponding to the wild-type allele (Fig. 2 A). 30%
of the resistant clones were positive for homologous recombination.
|
Clones that had undergone homologous recombination
were transfected with a Cre-encoding plasmid and subjected to negative selection (gancyclovir). As the size of
the loxP site remaining in the gene is smaller than that of
exon A, PCR was used to identify the resistant colonies in
which Cre-loxP-mediated recombination had occurred
(Fig. 2 B), and the results were confirmed by Southern blot analysis (not shown). 90% of the resistant clones
scored positive for Cre-loxP-mediated recombination.
Two independent 6A+/
clones were used to generate
chimeric males, which transmitted the mutated allele to
their progeny. Mice heterozygous for the mutation in the
6 integrin gene were identified by PCR analysis on tail
DNA (Fig. 2 C).
Deletion of Exon A Causes Replacement of 6A by
6B
in Tissues That Normally Express
6A
Intercrossing heterozygous animals produced offspring
homozygous for the deletion, which occurred in the expected mendelian frequency. Such animals developed apparently normally and were fertile. These surprising results indicate that 6B can fully functionally replace
6A
in mouse development. We first verified that the deletion
of exon A caused
6A to be replaced by
6B in the embryonic myocardium by using antibodies directed against the cytoplasmic domain of
6A or
6B on tissue sections
of wild-type (
6A+/+) and
6A
/
embryos collected at
12.5 dpc (Fig. 3). As previously described (Collo et al.,
1995
; Thorsteinsdóttir et al., 1995
), the
6A variant is expressed in the myocardium of the atria and the ventricles
of 12.5 dpc
6A+/+ embryos, and although a reaction was
seen throughout the ventricle wall, the protruding cells at
the inner face of the ventricular myocardium reacted more
intensely than the peripheral cells. No staining for
6B could be detected in the myocardium. A weak staining of
the blood vessels feeding the heart was observed with anti-
6B antibodies and probably corresponded to the expression of this variant in endothelial cells (Thorsteinsdóttir et
al., 1995
). This was in agreement with finding
6B mRNA
by reverse transcription PCR in heart preparations (Thorsteinsdóttir et al., 1995
). In contrast, the somites, the kidneys, and the head of the embryo strongly reacted with the
anti-
6B antibody (not shown).
|
As predicted, no 6A was detected in the myocardium
of
6A
/
embryos of 12.5 dpc. In contrast,
6B was
found to have replaced
6A and was now present in the
atria and ventricles in a gradient, the expression being
strongest at the inner face of the ventricular myocardium
and weakening towards the peripheral cells, similar to the
gradient of expression of
6A in control mice.
These data demonstrate that the specific removal of
exon A using homologous recombination and the Cre-
loxP system resulted in the replacement of 6A by
6B in
a tissue that normally expresses
6A.
6B Takes Over the Function of
6A in
Heart Development
The viability of the 6A
/
mice and the normal morphology of atria and ventricles of 12.5-dpc
6A
/
embryos suggested that cardiogenesis was not affected by the
replacement of
6A by
6B. Careful histological analysis
of the heart was conducted on 10.5- and 12.5-dpc embryos
to investigate whether there were any subtle morphological defects due to the mutation, but no aberrations in the
morphology of the heart and cardiomyocyte organization were observed at these stages (not shown). We tested the
possibility that the normal development of the heart in the
6A
/
mice could be the result of compensatory mechanisms involving other laminin-binding integrins such as
3
1 and
7
1. These integrins were found to be absent in
the myocardium of both control and knockout 12.5-dpc embryos (not shown), suggesting that
6B alone can take
over the function of
6A in the formation of the heart.
Replacement of 6A by
6B Does Not Impair
the Differentiation of the Epidermis, Hemidesmosome
Formation, or Wound Closure
Although 6B is present in the epidermis of the embryo
(Thorsteinsdóttir et al., 1995
), only
6A is expressed there
after birth and in the adult, in association with the
4 integrin subunit. However, no obvious abnormalities were detected in histological sections of the epidermis of
6A
/
mice (not shown). The substitution of
6A by
6B in the
epidermis of the knockout mice was first confirmed with
antibodies directed against
6A or
6B (Fig. 4 A). In control animals, the staining for
6A was restricted to the
basal keratinocyte layer, and a weak staining of the blood vessels in the dermis corresponding to that of
6B in endothelial cells was observed. As expected,
6A was not expressed in the epidermis of the knockout mice and was replaced by
6B. The absence of
6A in skin was confirmed
by Northern blot analysis (not shown).
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Ultrastructural analysis revealed that hemidesmosomes
were normal in number and morphology in the skin of
knockout animals (Fig. 4 B), indicating that 6B in association with
4 supports hemidesmosome formation. Since
the
6
4 integrin is redistributed from the hemidesmosomes to a more even distribution over the membrane during keratinocyte migration (Kurpakus et al., 1991
), we
investigated whether the replacement of
6A by
6B
would lead to the disassembly of hemidesmosomes and, as
a consequence, would affect keratinocyte migration during
wound healing. Therefore, a wound closure experiment by
tail amputation was conducted on
6A+/+ and
6A
/
mice. Keratinocyte migration was assessed at day 2 and 4 after amputation of the tail by hematoxylin/eosin staining.
No apparent irregularities were detected in the epidermis
of the reepithelializing skin, and wounds healed as quickly
in the
6A
/
mice as in the control mice (Fig. 4 C).
Together, these results show that the differentiation of
the epidermis, hemidesmosome formation, and reepithelialization after wound healing in mice expressing only the
variant 6B are normal.
Keratinocyte Adhesion and Motility Are Not Altered in
6A
/
Mice
To further confirm the replacement of 6A by
6B in the
epidermis and to study the adhesion of the
6A
/
keratinocytes and their migration properties, we isolated keratinocytes from newborn mice and cultured them in vitro.
At none of the passages was the morphology of
6A
/
cells different from that of control keratinocytes (not
shown). The pattern of expression of laminin-binding integrins on both cell types was analyzed by immunoprecipitation from 125I-labeled cells (Fig. 5 A). The
6A variant but
not
6B was precipitated from
6A+/+ keratinocytes. On
the contrary,
6A was not expressed at the surface of
6A
/
keratinocytes. It had been replaced by
6B. As
expected, the expression of the
3A,
4, and
1 chains was
not altered by the deletion. The absence of a band corresponding to the
6 polypeptide in the
1 immunoprecipitates indicated that mouse keratinocytes, like those of humans, do not express
6
1.
|
Data obtained by Tennenbaum et al. (1995) revealed
that the introduction of the
6B variant in a papilloma cell
line induced an increase of the adhesion of these cells to
laminin-1, which is a ligand for
6
4 (Lee et al., 1992
;
Niessen et al., 1994
). We have assessed the adhesion of keratinocytes to laminin-1, which interacts with
6
4, to
laminin-5, involving both
3
1 and
6
4, and to fibronectin, mediated by
5
1 and
v integrins. As shown in Fig. 5
B, the adhesion properties of
6A+/+ and
6A
/
keratinocytes on all three substrates are similar.
Finally, because in vivo wound healing involves both cell
migration and proliferation, we investigated the migratory
properties of 6A
/
keratinocytes in vitro using Transwell filters coated with laminin-1, laminin-5, or fibronectin
on the lower side. We could not observe any significant
difference in the motility of the two cell types after 6 or 18 h
of migration (not shown). These results indicate that substitution of
6A by
6B does not modify keratinocyte adhesion and motility.
Replacement of 6A by
6B Does Not Affect
Lymphocyte Differentiation and Proliferation but
Reduces the Motility of These Cells
In the thymus during development, the expression of 6A
and
6B is regulated in both the thymocytes and the endothelial cells (Chang et al., 1995
; Ruiz et al., 1995
). In addition, it has been previously shown that the homing of T
cell precursors into the thymus is blocked by anti-
6 antibodies (Ruiz et al., 1995
). Therefore, it was expected that
T lymphocyte differentiation might be impaired in our
6A
/
mice. Furthermore, laminin has been shown to
promote the proliferation of thymocytes and T cells plated
on anti-CD3 antibody (Shimizu et al., 1990a
; Chang et al.,
1995
). Finally, lymphocytes encounter different laminin
isoforms as they recirculate and transmigrate through the
basement membranes underlying the endothelial cells in
secondary lymphoid organs and at sites of inflammation.
The receptor for laminin on T cells is
6
1 (Shimizu et al.,
1990b
), and because the cytoplasmic domain of
6A might give a specific signal to lymphocytes upon interaction with
laminin, we investigated the ability of
6B to sustain T cell
differentiation, proliferation, and migration.
6A was only present in the T cells of lymph nodes from
control mice and not in those from knockout mice, while
6B was expressed in
6A
/
T cells as determined by
reverse transcription PCR (not shown). The levels of expression of the
6 subunit were the same on both cell
types as determined by FACS® analysis using the GoH3
antibody (not shown). Differentiation of the T cell-lineage
was assessed by reactivity of total cell suspensions isolated
from the thymus of age- and sex-matched control and
knockout mice with anti-CD3 (Fig. 6 A). A normal percentage of strongly CD3-positive T cells was found in the
thymus of
6A
/
mice. Moreover, CD4/CD8 ratios
were similar in knockout and control mice, indicating that
the development of various T cell subsets proceeds normally in the presence of
6B alone. As the presence of
6
integrin on B cells has been reported in one study (Ohguro and Tsubota, 1996
), we also studied B cell maturation using antibodies directed against B220/CD45R and IgM on
spleen cells. Fig. 6 B shows that their differentiation is not
affected in
6A
/
mice.
|
To determine whether the substitution of 6A by
6B
affected the proliferative response triggered by laminin-1,
we conducted costimulation experiments by incubating
purified lymph node T cells on plate-bound laminin-1 and
anti-CD3 antibody for 48 h (not shown), 72 h (Fig. 7), or
96 h (not shown). While the incorporation of [3H]thymidine was low in cells stimulated by anti-CD3 antibody
alone, costimulation by soluble anti-CD28 antibody (Gross
et al., 1992
) showed that
6A+/+ and
6A
/
T cells displayed the same ability to proliferate. More importantly,
incorporation of [3H]thymidine was found to be increased
by binding to laminin-1 to the same extent in T cells isolated from knockout mice and control animals, indicating
that the proliferative response to laminin-1, although
6
1
mediated (Shimizu et al., 1990a
), is not dependent on the
nature of the cytoplasmic domain of
6.
|
Finally, the ability of 6B to support migration of cells
of the immune system was studied. Although
6A has previously been reported to increase motility of cells of a
macrophage cell line on laminin-1 (Shaw and Mercurio,
1994
), we could not investigate the effects of the absence
of
6A in cells of this type because laminin-1 failed to support attachment and migration of peritoneal macrophages
isolated from either our control or knockout mice (not
shown). Migratory properties of T lymphocytes were first studied in vitro using Transwell filters coated with either
laminin-1 or fibronectin, and in the presence of the
chemoattractant SDF-1, in the lower compartment of the
chamber. Interestingly, we have found that the motility of
6A
/
T cells was reduced by 46% compared with that
of control cells on laminin-1-coated filters, whereas migration through fibronectin-coated filters was not affected (Fig. 8 A). Similar results were obtained with another
chemoattractant, MCP-1 (not shown). Importantly, the
decrease in cell migration was not due to differences in the
activation state of T cells from both normal and knockout
mice, as verified by the expression of the activation
marker CD69 (not shown). Finally, a decrease in cell motility was observed in cells originating from two independent ES cell clones. This indicates that optimal migration
of T lymphocytes is dependent on the expression of the cytoplasmic domain of
6A.
|
After T cell purification, we consistently observed a
strong decrease (30-40%) in the absolute number of cells
isolated from the peripheral and mesenteric lymph nodes
of 6A
/
mice (Fig. 8 B). To test the hypothesis that this
could be due to alterations in the migration of T cells to
the lymph nodes, we have conducted in vivo lymphocyte
homing experiments using PKH2 and PKH26 fluorescent cell linkers. Lymph node cells from
6A+/+ and
6A
/
cells were labeled with one of the fluorescent cell linkers,
mixed in equal number, and injected into the tail vein of
wild-type animals. The percentage of each cell type in the
cell mixture was checked by FACS® analysis and was close
to 50% (not shown). Fluorescent-labeled cells homing into
spleen, peripheral, and mesenteric lymph nodes was determined by FACS® analysis 2 or 20 h after the injection.
Two series of experiments were performed with the injection of either PKH2-labeled
6A+/+ and PKH26-labeled
6A
/
cells or PKH2-labeled
6A
/
and PKH26-labeled
6A+/+ cells. Results obtained with both combinations were similar and are presented in Table I. The
frequency of
6A
/
cells in spleen, peripheral, and mesenteric lymph nodes was comparable to that of the
6A+/+ lymphocytes 2 h after injection. A higher percentage of cells were found in the lymph nodes after 20 h
of homing, but no significant difference could be observed
between the two cell types.
|
Together, these data suggest that, although the expression of 6A is necessary for the optimal migration of T
cells on laminin-1 in vitro, it is dispensable for in vivo lymphocyte homing to the secondary lymphoid organs.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Development Is Not Impaired in Mice Expressing Only
the 6B Variant
Classical ES cell technology combined with the Cre-loxP
system of the bacteriophage P1 was used to create exon-specific knockout mice that did not express the A variant
of the 6 integrin subunit. The deletion resulted in the replacement of
6A by
6B in all tissues that normally express
6A, such as the developing heart, in which
6A is
associated with the
1 subunit, and the adult epidermis, in
which
6A
4 is expressed in the basal keratinocyte layer.
Previous studies have indicated that
6 is apparently not essential for proper prenatal development (Georges-Labouesse et al., 1996
). However, the importance of
6A
for the function of the epidermis, mature gonads, Schwann
cells, and the immune system could not be addressed since
6
/
pups die shortly after birth because of detachment
of the epidermis. Moreover, it was not possible to investigate the consequences of the lack of
6A during cardiogenesis on the function of the adult heart. We show here
that
6B can sustain normal mouse development until maturity and that
6A
/
animals are fertile. We did not detect any abnormalities in either the morphology or the histology of various organs and tissues, and we argue that the
presence of exon A in
6 mRNA is not essential for the
proper control of cell migration and proliferation during development. These findings were surprising in the light of
the recently published work by Domanico et al. (1997)
,
who have shown that the ectopic expression of
6A in undifferentiated ES cells conferred a highly migratory phenotype to these cells as compared with their normal counterparts, which express only
6B. Importantly, these
stimulatory effects on cell migration were found to be independent from adhesion to laminin-1, suggesting that the
cytoplasmic domain of
6A was sufficient for promoting
motility. However, our own results unequivocally show
that the cytoplasmic tail of
6B can support cell migration
during development.
Heart Development and Function Are Normal in
6A
/
Mice
Most surprising was the absence of an apparently abnormal phenotype during heart ontogeny in 6A
/
mice.
While
6B is expressed throughout development, the expression of
6A begins at 8.5 dpc and is initially restricted
to the developing myocardium (Collo et al., 1995
; Thorsteinsdóttir et al., 1995
). The onset of
6A expression immediately precedes the initiation of the looping process,
which converts anterior-posterior patterning of the heart tube into left-right asymmetry (Fishman and Olson, 1997
),
strongly suggesting a critical role of this integrin subunit in
cardiogenesis. Although
6
/
mice did not present any
abnormalities of the heart (Georges-Labouesse et al.,
1996
), we assumed that the replacement of
6A by
6B,
while not preventing adhesion to laminin substrata, could
result in the transmission of inappropriate molecular signals into the cardiomyocytes and thus could have more
profound effects on heart formation than the complete absence of
6. Surprisingly, histological analysis and immunofluorescence data revealed no overt abnormal phenotype
of the heart, and the pattern of expression of
6B was similar to that of
6A in control mice, the inner cells of the
myocardium being more intensely stained than the peripheral cells. Because an abnormal phenotype is more likely
to be detected in a simplified, in vitro differentiation
model (Bagutti et al., 1996
), double knockout ES cells
were generated and aggregated into embryoid bodies. We
found that
6A
/
ES cells displayed the same ability to
differentiate into cardiac contracting cells as their wild-type counterparts (Gimond, C., unpublished results), further supporting the finding that the substitution of
6A by
6B is compatible with normal cardiogenesis. Finally, this
substitution does not seem to impair the function of the
adult heart either, since the level of the atrial natriuretic
peptide, a marker of heart dysfunction (Edwards et al.,
1988
), was normal in the ventricles of our
6A knockout
mice (our unpublished results).
Although the simplest explanation for the lack of an
overt abnormal phenotype in the heart is that 6B, in spite
of its different cytoplasmic domain, can substitute for
6A
during cardiomyocyte differentiation and reorganization,
more complex compensatory mechanisms, involving other
laminin-binding receptors, might have occurred during development. However, if compensation occurred, our results show that this is not due to the induction of the expression of two other laminin-binding integrins,
3A
1
and
7
1.
Keratinocytes Expressing 6B Assemble
Hemidesmosomes and Display Normal Adhesive and
Migratory Properties
In contrast to the embryonic heart, in which 6A is complexed with the
1 subunit, in developing epidermis both
6A and
6B are expressed in association with the
4 subunit (Thorsteinsdóttir et al., 1995
). Only
6A remains after birth, and although the reasons for the loss of expression of
6B in this tissue is not clear, the expression
patterns of the two variants might correspond to key
stages in skin development or to specific physiological conditions. However, the epidermis apparently differentiated normally in
6A
/
mice, and more importantly, it
contains hemidesmosomes in normal number and of normal morphology. This is interesting because phosphorylation of
6A was thought to be required for the nucleation of hemidesmosome assembly (Baker et al., 1997
). Thus, although
6B was never found to become phosphorylated, it is capable, in association with
4, of supporting
hemidesmosome formation. This suggests that, for this
function of
6, only binding to laminin, which does not depend on the cytoplasmic domain of the subunit, is required. Alternatively, the cytoplasmic domain of
6B may
render conformation of
6
4 permissive for hemidesmosome formation. Nevertheless, the substitution of
6A by
6B might have subtle effects on the regulation of the assembly or the disruption of hemidesmosomes, in which
4
is involved (Dowling et al., 1996
; van der Neut et al., 1996
),
and although the migration of keratinocyte per se was
found to be independent of
6
4 (Kurpakus et al., 1991
), the cytoplasmic domain of this integrin might play a role in
hemidesmosome disassembly and the onset of cell movement. However, given our results both in vivo and in vitro,
it appears that the expression of only
6B does not perturb keratinocyte migration and wound closure.
Previous work by Tennenbaum et al. (1995) has shown
that overexpression of
6B in a papilloma cell line resulted in increased binding to laminin-1, whereas overexpression of
6A had no effect on adhesion. In contrast to
those results, we show that when using an in vivo approach
in which the expression level of
6 is not modified, adhesion properties of keratinocytes expressing either the one
or the other splice variant are not different.
Lymphocyte Motility Is Reduced in 6A
/
Mice
During maturation of the immune system, recirculation,
and inflammation, lymphocytes encounter different laminin isoforms as they cross the basement membrane underlining various blood vessels (Springer, 1994). In addition,
laminin-1, -3, and -5, together with other extracellular matrix proteins, are present in the stroma of various lymphoid organs such as the thymus and lymph nodes and in
the bone marrow (Chang et al., 1993
; Jaspars et al., 1996
),
in which lymphocytes proliferate and differentiate. The
production of laminins by these tissues is mirrored by the stronger expression of
6 integrins on immature than on
mature T cells (Ruiz et al., 1995
), suggesting a critical role
of laminin-integrin interactions in the differentiation process. Moreover,
6 expressed by thymic endothelial cells
was shown to participate in the homing of T cell progenitors to this organ (Ruiz et al., 1995
). A differential role for
the cytoplasmic domains of the
5 and
6 subunits in mediating signals for proliferation and differentiation has recently been described in myoblasts (Sastry et al., 1996
),
and because of their entirely different cytoplasmic domain, we expected the ability of
6A and
6B to regulate
these cellular responses to be different. It was therefore
surprising to find that T and B cells differentiated normally in
6A
/
mice and that the
6-dependent costimulatory effect of laminin-1 on T cell proliferation (Shimizu et al., 1990a
; Chang et al., 1995
) can be transmitted as efficiently by
6B as by
6A. This finding might be explained
by the critical function of the cytoplasmic tail of the
subunit in cell growth (Merredith and Schwartz, 1997
). Alternatively, the adhesion-dependent activation of integrin-associated protein could be involved in the induction of
proliferative signals in T cells regardless of which integrin
is ligated (Reinhold et al., 1997
). Finally, adhesion of lymphocytes to extracellular matrix proteins in the bone marrow or in the thymus might be sufficient to allow their sustained interaction with stromal cells and stimulation by
cytokines, which are the conditions for proper selection
and differentiation (Anderson et al., 1996
). In that case,
substitution of
6A by
6B is not expected to affect maturation, since the ligand-binding activities of the two splice
variants are identical.
Interestingly, the motility of 6A
/
T cells through
laminin-1-coated filters was markedly decreased. A promoting effect of
6A on cell migration has already been
reported (Shaw and Mercurio, 1994
; Domanico et al.,
1997
), but in previous work, the effects of ectopic and
overexpression of either one or the variants in two different cell lines was analyzed. In the present paper, we show,
for the first time, that the endogenous expression of
6A more efficiently supports the migration of primary T lymphocytes in vitro. When overexpressed in ES cells,
6A
was reported to enhance migration not only on laminin-1
but also on other extracellular matrix proteins that do not
use
6
1 as a receptor (Domanico et al., 1997
). Yet, we
show here that the effects of the absence of
6A on T cell
migration only occur on laminin-1. We have also observed
that the number of T cells isolated from the lymph nodes was consistently lower in the
6A
/
than in wild-type
mice. Since lymphocytes must cross basement membranes
containing laminins when recirculating through the organism, one hypothesis for this difference is that impaired migration of
6A
/
cells could result in a less efficient
homing to the peripheral and mesenteric lymph nodes,
causing the number of cells in these organs to be lower. In
this regard, it is interesting to note that antilaminin antibodies were found to inhibit lymphocyte trafficking and
homing to the peripheral lymph nodes (Kupiec-Weglinski
and De Sousa, 1991
). However, we could not demonstrate
any defects in lymphocyte homing in our
6A
/
mice,
which indicates that the reduced cell number in lymph
nodes has other causes, e.g., defects in cell proliferation. Although we have shown that the substitution of
6A by
6B does not affect T cell proliferation in vitro in conditions where both CD3 and
6
1 are ligated, the lack of
6A in lymphocytes or in lymph node stromal cells might
lead to a decrease in lymphocyte proliferation in vivo.
The altered cell motility on laminin-1 that we observed
in vitro does not seem to have consequences for lymphocyte homing. This might be due to the ability of lymphocytes to use several other integrins from their repertoire to
migrate on the various extracellular matrix proteins
present in basement membranes, and the lack of 6A is
likely to be compensated by other types of integrin-matrix
interactions. Such compensatory mechanisms cannot be
used by lymphocytes migrating on a laminin-1 matrix, and
therefore, defects resulting of the absence of
6A are
more likely to be detected in vitro. Redundancy and compensatory mechanisms could also explain the absence of
effects of the lack of
6A on the development of the
mouse. Nevertheless, we cannot rule out that the promoting role of
6A on cell motility might be important for the
fast recruitment of lymphocytes to sites of inflammation or
in other pathological conditions.
The molecular basis for the promoting effect of 6A on
cell migration has not yet been elucidated, but it might involve the different ability of the two splice variants to trigger the phosphorylation of certain cytoskeletal proteins
(such as paxillin; Shaw et al., 1995
) that are involved in cell
migration (Aznavoorian et al., 1996
; Tourkin et al., 1996
).
In agreement with this notion is the induction of filopodia
in cells of both ES and macrophage cell lines by
6A
(Shaw and Mercurio, 1994
; Domanico et al., 1997
), which
reflects cytoskeleton rearrangements. Since the only structural differences between
6A and
6B reside in their cytoplasmic domains, it is clear that these unique sequences
are responsible for the transmission of distinct signals into
the cell. This is not restricted to the
6 subunit since Chan
et al. (1992)
, using chimeric integrin molecules, have previously shown that the unique intracellular domains of the
2 and
4 integrin subunits triggered either collagen gel
contraction or cell migration, respectively. Whether the
cytoplasmic domain of the
chain itself is able to transduce signals has not yet been elucidated. Alternatively, it
could regulate interactions of the cytoplasmic tail of
1
with intracellular proteins involved in transduction. Regulation of the phosphorylation state of
6A, for example by
chemoattractants, might also play a role in cell motility.
Such a posttranslational regulation has never been reported for
6B and may account partly for their distinct
functional properties. Finally, internalization of
6
1,
which is an important aspect of cell migration, could be
regulated by the cytoplasmic domains of
6A and
6B in a
different manner, although previous reports argue against
this suggestion (Gaietta et al., 1994
).
In conclusion, the specific removal of exon A from the
6 gene allowed us to study in detail the role of endogenously expressed
6A in vivo and to determine whether
6B could functionally replace it. Our results revealed
that, in contrast to previous assumptions and despite its remarkable up-regulation at the stage of the heart-looping process,
6A is not essential for proper cardiogenesis and
cell migration during development. However, we have
demonstrated that the two splice variants
6A and
6B
are not equivalent in supporting lymphocyte migration on
laminin-1, and thus, although its absence does not impair
lymphocyte homing into the lymph nodes,
6A might contribute to some aspects of host defense. The
6A
/
mice
will provide a useful tool for future studies aiming at fully
understanding the molecular mechanisms activated by the
two cytoplasmic variants of
6.
![]() |
Footnotes |
---|
Received for publication 20 April 1998 and in revised form 21 July 1998.
All of the ES cell work was carried out in the division of Molecular Genetics (Head Prof. Dr. A. Berns) at the Netherlands Cancer Institute. This
work was supported by a fellowship from the European Commission
(ERB4050PL930847) to C. Gimond, a Marie Curie Research training
grant from the European Commission (ERBFMBICT961823) to C. Baudoin, and grants from the Netherlands Heart Foundation (NHS 96.006) and
the Netherlands Organization for Scientific Research (NWO 900-511-043).
Address all correspondence to Arnoud Sonnenberg, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: (31) 20 512 1942. Fax: (31) 20 512 1944. E-mail: asonn{at}nki.nl
Ronald van der Neut's present address is INSERM U434, 27 rue Juliette Dodu, 75010 Paris, France.
We thank A.J. Schrauwers for mice husbandry; M. van der Valk, D. Hoogervorst, J. Bulthuis, and K. de Goeij for their help with histological
analysis of the mice; L. Oomen for confocal laser scanning microscopy; H. Janssen for electronic microscopy; N. Ong for artwork; and R. Soede for
his helpful comments on lymphocyte migration. A special thank you goes
to D. Amsen for his valuable advice on experiments on lymphocyte differentiation and critical reading of the manuscript, and to P. Rousselle for
providing us with laminin-5 and for her helpful advice on keratinocyte culture. We also thank B.M.C. Chang for the rat monoclonal antibody to murine 1; S.J. Kennel for the antibody to murine
4; R. Kramer for the rat
antibody to murine
7; and R. Fässler for providing us with the loxP-neo-tk-loxP cassette and for useful suggestions.
![]() |
Abbreviations used in this paper |
---|
dpc, days post coitum; ES, embryonic stem.
![]() |
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