This Review is part of a thematic series on Integrins, which includes the following articles:
Integrins and the Myocardium
Functional Consequences of Integrin Gene Mutations in Mice
Integrin and Growth Factor Receptor Crosstalk
Integrins in Vascular Development
Signaling and the Response of Endothelial Cell Signals From the Extracellular Matrix
David Cheresh, Guest Editor
Functional Consequences of Integrin Gene
Mutations in Mice
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Daniel Bouvard,* Cord Brakebusch,* Erika Gustafsson, Attila Aszódi, Therese Bengtsson,
Alejandro Berna, Reinhard Fässler
Abstract—Integrins are cell-surface receptors responsible for cell attachment to extracellular matrices and to other cells.
The application of mouse genetics has significantly increased our understanding of integrin function in vivo. In this
review, we summarize the phenotypes of mice carrying mutant integrin genes and compare them with phenotypes of
mice lacking the integrin ligands. (Circ Res. 2001;89:211-223.)
Key Words: integrin 䡲 knockout 䡲 extracellular matrix
C
initiation of a signaling cascade that modulates cell behavior
and gene transcription.2,3
During the last two decades, most of the information about
integrin function has been derived from in vitro cell culture
systems. Gene targeting technology recently made it possible
to generate mice that lack specific integrins in a constitutive
or cell type–specific manner. Analyses of these mice demonstrate how integrin-mediated adhesion and signal transduction affect development and maintenance of tissues and
provide additional insight into integrin function in various
diseases. In this review, we summarize the phenotypes of
mice carrying mutant integrin genes and compare them with
the relevant genetic models bearing mutations in some
integrin ligands (Table).
ell-cell and cell-matrix interactions play essential roles
in development, tissue function, and repair. During
morphogenesis and tissue homeostasis, specific signals
emerge from the environment, cross the cell membrane, and
control many aspects of cell behavior, including migration,
proliferation, survival, and differentiation. Integrins are cell
adhesion molecules that can transmit such signals. They are
heterodimeric transmembrane glycoproteins, composed of
noncovalently-associated ␣ and ␤ subunits. To date, 24
integrin receptors assembled from various combinations of 18
␣ and 8 ␤ chains have been described (Figure 1). The
extracellular domain of integrins binds the cognate ligand,
whereas the cytoplasmic domain is linked to the actin
cytoskeleton. Integrins can bind extracellular matrix (ECM)
proteins, such as collagens, fibronectins, and laminins, and
cellular receptors, such as vascular cell adhesion molecule-1
(VCAM-1) and the intercellular cell adhesion molecule
(ICAM) family.1 Certain integrins share the same extracellular ligand, but on the other hand, several ligands are recognized by the same integrin. Ligand binding is followed by
recruitment of several signaling proteins to the integrin and
Early Mouse Development
Ablation of integrin genes leads to various phenotypes during
mouse development, ranging from apparently normal mice to
early lethality (Figure 2). For example, disruption of the
ubiquitously expressed ␤1 integrin gene leads to the loss of at
least 12 different integrin receptors and results in peri-
Original received March 6, 2001; revision received June 20, 2001; accepted June 20, 2001.
From the Department of Experimental Pathology, Lund University, Lund, Sweden.
*Both authors contributed equally to this work.
Correspondence to Daniel Bouvard or Cord Brakebusch, Lund University Hospital, Department of Experimental Pathology, 22185 Lund, Sweden.
E-mail [email protected] or [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
211
212
Circulation Research
August 3, 2001
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Figure 1. Vertebrate integrin family. ␤ Integrin subunits associate with ␣ subunits in a noncovalent fashion. So far, 24 heterodimers have been described that can bind extracellular
matrix proteins, other cell surface receptors, and bacterial and
viral proteins.
implantation lethality characterized by an inner cell mass
(ICM) failure.4,5 Fertilization of ␤1-null oocytes and the entire
preimplantation development is normal. This is most likely
because of the presence of maternal ␤1 integrin mRNA and
protein.6 A possible explanation of the ICM failure could be
the loss of ␤1-mediated survival signals. At peri-implantation
stage, the ICM cells facing the blastocyst cavity differentiate
into primitive endodermal cells and lay down a basal membrane (BM). This leads to polarization of adjacent ICM cells
and their differentiation into ectodermal cells. The inability of
the ectodermal cells to interact with BM components through
integrins could lead to their loss by apoptosis and arrest of
additional development.7 Another possibility could be that the
lack of ␤1 integrins leads to abnormal BM assembly. ␤1
integrin is crucial for normal expression and correct assembly
of BM components into a supramolecular structure.8 –12 The
complete absence of a BM is observed in mice carrying a null
mutation in the laminin ␥1 chain gene.13 Laminins are
heterotrimeric glycoproteins composed of ␣, ␤, and ␥ subunits. Laminin ␥1-null mice fail to develop a BM between
primitive endoderm and ICM, and, like the ␤1-null mice, they
die at the peri-implantation stage.13 A recent study demonstrated the role of the BM in regulating apoptosis and cavity
formation using embryoid bodies derived from laminin ␥1null embryonic stem (ES) cells.14 In laminin ␥1-null embryoid
bodies, the ectoderm never polarizes and the inner cells never
apoptose to form cavities. An identical phenotype is observed
in ␤1-null embryoid bodies (R. Fässler, unpublished data,
1995).
Deletion of the integrin ␣5 gene leads to an embryonic
lethality around embryonic day 9.5 (E9.5) and E10 with
variable degrees of embryonic and extra-embryonic defects
that partly depend on the genetic background.15,16 ␣5␤1 plays
a role in neural crest survival and maintenance of mesoderm
derivatives after the gastrulation stage but is dispensable for
initial commitment of mesodermic cells.17 Knockout of the
␣5␤1 ligand fibronectin (FN) results in a more severe phenotype, suggesting that other FN-binding integrins compensate
in the ␣5-null mouse. Fibronectin-null embryos implant and
initiate gastrulation normally. Shortly thereafter, they show
defects in neural tube closure, in the cardiovascular system,
and in somite formation and die at E8.5.18
␣4-Null embryos fail to fuse the allantois with the chorion
during placentation, show defects in the heart and vasculature, and display abnormalities in the cranial and facial
structure.19 ␣4-Null embryos die at two different stages during
development. The first wave is around E9.5 to E11.5 and is
caused by the failure of allantois-chorion fusion, and the
second is between E11.5 and E14 and is caused by massive
heart hemorrhage. ␣4␤1 Integrin can bind fibronectin and
VCAM-1. The mutant phenotype is mainly attributable to a
defect in VCAM-1 binding, because mice lacking VCAM-1
also fail to fuse the allantois with the chorion and have
cardiac defect similar to ␣4-null mice.20,21
␣v Integrins can associate with ␤1, ␤3, ␤5, ␤6, and ␤8
subunits. They bind to both fibronectin and vitronectin,
except for ␣v␤6 integrin, which binds to fibronectin, vitronectin, and tenascin-C, and ␣v␤8, which binds only to fibronectin.1 Several reports have shown that ␣v␤3 and ␣v␤5 integrins
are crucial for tumor angiogenesis.22 Therefore, it came by
surprise that ␣v-null mice, which lack both of these receptors,
have no defects in vasculogenesis and angiogenesis. Approximately 80% of the ␣v-null mice die between E10 and E12
because of a placenta defect characterized by a poorly
developed labyrinthine zone and reduced interdigitation of
fetal and maternal vessels. The remaining 20% die at birth
from massive hemorrhages and cleft palates.23 Because vitronectin, tenascin-C, and osteopontin-null mice have no obvious phenotype during the early mouse development,24 the
␣v-null phenotype is most likely attributable to a lack of
fibronectin binding.
A recent study investigated ␣5⫺/⫺␣v⫺/⫺ double knockouts,
which lack several major fibronectin receptors. The mutants
die between E7.5 and E8 and display a severe gastrulation
defect, with a lack of anterior mesoderm.16 It is noteworthy
that this phenotype is even more severe than that observed in
the fibronectin-null mutants, suggesting the presence of
additional ligands for these two receptors at this developmental stage.
Hematopoiesis
Hematopoietic stem cells (HSCs) and endothelial cells are
derived from a common precursor cell called hemangioblast.
HSCs are generated outside the embryo proper, in the yolk
sac (YS), and within the embryo in the para-aortic
splanchnopleura/aorta-gonad mesonephros region. At around
E8.5, when circulation starts in mouse, the HSCs are present
in the fetal blood, and at E10 they start to colonize the fetal
liver. Later, hematopoietic progenitors seed the thymus,
spleen, other lymphoid organs, and bone marrow. In the adult
mouse, HSCs reside in the bone marrow, but differentiated
leukocytes circulate through the body and extravasate into
infected tissues. Because of the early embryonic lethality of
integrin ␤1-null mice, chimeric mice have been used to
analyze ␤1 function in the hematopoietic system. These mice
revealed that ␤1 integrin is not required for the generation of
progenitor cells in the para-aortic splanchnopleura/aorta-
Bouvard et al
Integrin Mutations in Mice
213
Ablation of Integrin Genes in Mice and Their Resulting Phenotypes In Vivo
Gene
Phenotype
References
␣1
V, F
Increased collagen synthesis, reduced tumor vascularization
75, 76, 143, 147
␣2
䡠䡠䡠
L, birth
䡠䡠䡠
Defects in kidneys, lungs, and cerebral cortex; skin blistering
Not reported
␣3
␣4
L, E11–E14
Defects in chorioallantois fusion and cardiac development
19
␣5
L, E10
Defects in extraembryonic and embryonic vascular development
15
␣6
L, birth
Defects in cerebral cortex and retina; skin blistering
137
␣7
V, F
Muscular dystrophy
116
␣8
L⫹V/F
Small or absent kidneys; inner ear defects
130, 133
␣9
L, perinatal
Bilateral chylothorax
85
␣10
V, F
No apparent phenotype
(T. Bengtsson and R.
Fässler, unpublished
data, 2001)
␣11
䡠䡠䡠
L, E12–birth
䡠䡠䡠
Defects in placenta and in CNS and GI blood vessels; cleft palate
Not reported
䡠䡠䡠
Impaired leukocyte recruitment and tumor rejection
Not reported
␣L
䡠䡠䡠
V, F
␣M
V, F
Impaired phagocytosis and PMN apoptosis; obesity; mast cell
development
54–57
␣X
䡠䡠䡠
V, F
䡠䡠䡠
Inflammatory skin lesions
Not reported
␣IIb
V, F
Defective platelet aggregation
62
␤1
L, E5.5
Inner cell mass deterioration
4, 5
␤2
V, F
Impaired leukocyte recruitment; skin infections
43, 44
␤3
V, F
Defective platelet aggregation; osteosclerosis
61, 125
␤4
L, perinatal
Skin blistering
78, 94
␤5
V, F
No apparent phenotype
85
␤6
V, F
Skin and lung inflammation and impaired lung fibrosis
99, 100
␤7
V, F
Abnormal Peyer’s patches; decreased No. of intraepithelial lymphocytes
30
␤8
L, E12–birth
Defects in placenta and in CNS and GI blood vessels; cleft palate
(Z. Jiangwen and L.
Reichardt, personal
communication, 2001)
␣v
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
␣D
␣E
74, 95, 135
23
50
32
V indicates viable; F, fertile; L, lethal; L⫹V/F, mutations that disrupt development but also allow survival in a fraction of mice; CNS,
central nervous system; GI, gastrointestinal; and PMN, polymorphonuclear neutrophil.
gonad mesonephros region and the YS but is essential for
them to colonize the fetal liver.25,26 Differentiation of ␤1-null
precursor cells in the presence of cytokines in vitro and in
reaggregated fetal organ cultures of liver and thymus is
normal, indicating that ␤1 integrins are crucial for the migration of HSCs into, but not for their development within,
hematopoietic organs. Also, the homing of adult HSCs to the
bone marrow is critically dependent on ␤1 integrin expression. Accumulation of transferred ␤1-null precursor cells in
the blood in vivo and reduced adhesion of other cells to
endothelioma cells in vitro suggests that ␤1 integrin is
essential for the adhesion of HSC to the vessel wall.26
Whether it plays an additional role at later processes (transmigration though the endothelium or extravascular migration)
is not clear. Mouse HSCs express ␣4␤1, ␣5␤1, and ␣6␤1
integrin. Because individual knockouts of these ␣ subunits do
not interfere with HSC homing,27–29 either a combination of
these receptors or another integrin of ␤1 family mediates HSC
migration to the bone marrow.
Deletion of the ␤7 integrin subunit, which can associate
with ␣4 and ␣E, results in defective migration of T cells to
Peyer’s patches and a reduced number of lamina propria and
intraepithelial lymphocytes.30 Migration of lymphocytes to
the Peyer’s patches is mediated by ␣4␤7 binding to the
endothelial MadCAM-1 receptors on the high endothelial
venules in the gut. This was confirmed with mice lacking
MadCAM-1 because of ablation of the transcription factor
NKX2.3 gene, which controls MadCAM-1 expression. These
mice have very small Peyer’s patches only visible after
microscopic inspection.31 Targeted disruption of the ␣E gene
leads to a reduction of T cells in lamina propria and gut
epithelium but not in Peyer’s patches.32 Mice lacking both ␤7
integrin and L-selectin suffer from a nearly complete impairment of lymphocyte migration to mesenterial lymph nodes.33
Because of the early embryonic lethality of ␣4-null mice,
the function of ␣4 integrin in the hematopoietic system was
studied in ␣4-null chimeric mice. The ␣4 subunit can associate
with ␤1 and ␤7 integrin and is important for the differentiation
214
Circulation Research
August 3, 2001
Figure 2. Integrin function during mouse
development. Diagram showing the
embryonic and perinatal lethal phenotype
of single and double integrin–null mutant
mice. The sizes of the embryos from fertilization (E0) to birth (P0) are not drawn
to scale. Fertilization occurs in all integrin
mutants, possibly because of the presence of maternal mRNAs. The earliest
lethal phenotype has been observed in
␤1-deficient mice. Studies of double
␣5/␣v-null and ␣3/␣6-null mice revealed a
redundant and/or compensatory function
for ␣5/␣v during early mouse development and for ␣3/␣6 in several organs and
tissues, ie, brain, lung, and AER.
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of erythroid, myeloid, and B cell progenitors during embryogenesis.34 Because in vitro differentiation in colony assays is
normal, the defects observed in vivo are quite likely attributable to impaired interaction of the ␣4-null progenitors with
the microenvironment in the hematopoietic organs. Furthermore, no ␣4-null erythrocytes, almost no B lymphoid cells,
and only a few myeloid cells are present in adult chimeric
mice, indicating an important role of ␣4 integrins in the
postnatal hematopoietic development.34 The underlying
mechanism proposed for this severe phenotype is an important role for the ␣4 integrin for transmigration of progenitors
through the stroma. In addition, proliferation of ␣4-null
precursor cells might be decreased, because ␣4-deficient preB
cells that did transmigrate in vitro showed a poor mitotic
activity.34 Similar to ␤7-deficient mice, the contribution of
␣4-null T cells to Peyer’s patches is impaired.27 ␣4-Null
chimeras that are older than 1 month after birth lack ␣4deficient thymocytes in the thymus, although T cell precursors are present in the bone marrow.27 The few peripheral
␣4-null T cells present show a reduced migration into the
inflamed peritoneum.29 Because mice with a disrupted
VCAM-1 gene have normal hematopoiesis, the ␣4-null phenotype is most likely attributable to impaired interactions of
␣4␤1 with fibronectin.35 Mice with a conditional inactivation
of the VCAM-1 gene show reduced number of mature B cells
in the BM attributable to a defective migration of lymphocyte
to the BM.36,37 In addition, T cell– dependent immune response is impaired.36
Integrins play a central role in leukocyte adhesion and
migration. Leukocytes express several integrins in a lowaffinity state, ensuring that they do not make inappropriate
interactions with ligands normally present in blood or on
endothelial cells. To leave the blood, leukocytes attach
loosely to activated endothelium via selectins and begin to
roll. Chemokines released by the endothelium or the surrounding tissue shift the leukocyte integrin into a highaffinity state that allows binding to endothelial cell adhesion
molecules such as ICAMs and VCAM-1, resulting in firm
adhesion of the blood cells to the vessel wall. Finally, the
leukocytes cross the endothelial cell layer and underlying BM
and adhere and migrate within the extravascular tissue.
␤2 integrin is specifically expressed on leukocytes and
associates with ␣L (lymphocyte function–associated antigen-1
[LFA-1]), ␣M (Mac-1), ␣X, and ␣D subunits. Important
counter-receptors of ␤2 integrins are members of the ICAM
family.1 However, Mac-1, ␣X␤2, and ␣D␤2 also bind to other
proteins: Mac-1 to inactivated complement factor (iC3b),
fibrinogen, factor X, ␣X␤2 to iC3b and type I collagen,38 and
␣D␤2 to VCAM-1.39 In addition, Mac-1 and ␣X␤2 can interact
specifically with various polysaccharides, such as ␤-glucan,
mannose, N-acetyl-D-glucosamine, and glucose, which mediate attachment to bacteria.40 – 42
Humans with a mutation in the ␤2 gene develop leukocyte
adhesion deficiency type I. These patients have a massive
leukocytosis, recurrent bacterial infections, impaired wound
healing, and severe gingivitis. Almost no neutrophils extravasate into infected tissues. Mice lacking ␤2 integrin display a
very similar phenotype.43,44 The degree of neutrophil extravasation, however, is variable and depends on the model of
inflammation.43– 45 Some neutrophil extravasation processes
like the croton oil-induced migration into skin are completely
abolished, whereas others, like the sterile peritonitis, still
occur. These migrations are perhaps mediated by ␤1 integrins.
In chronic inflammations, ␣4␤1 integrin seems to play an
important role.46 Another candidate is ␣9␤1, which is highly
expressed on human neutrophils and involved in transendothelial migration in vitro.47 ␤2 integrin also plays an important
role in T cell proliferation44 and contributes to the accumulation of dendritic cells in the lung.48
LFA-1 and, to a lesser degree, Mac-1 contribute to neutrophil adhesion to endothelium. In a tumor necrosis factor
(TNF)-induced air pouch inflammation model, neutrophil
migration was equally reduced in ␤2-deficient and LFA-1–
deficient mice, whereas mice lacking Mac-1 showed a
marked increase of neutrophil extravasation.49 In the absence
of LFA-1, immune responses against systemic viral infections
are normal, but alloantigen triggered T cell proliferation and
cytotoxicity are severely impaired, leading to defective hostversus-graft reaction and abrogated tumor rejection.50 –52
LFA-1 is also involved in the homing of lymphocytes to
peripheral lymph nodes and, to a lesser degree, to mesenteric
lymph nodes and Peyer’s patches.53 Mac-1–null neutrophils
have an impaired phagocytosis and degranulation, leading to
Bouvard et al
a delayed neutrophil apoptosis.54,55 Furthermore, mast cells
were reduced in some but not all locations of Mac-1–
deficient mice.56 In an acute glomerulonephritis model, Mac1–null neutrophils extravasated normally but did not stay in
the tissue, apparently because of an absent cytoskeletal
rearrangement mediated by Mac-1.57 Unexpectedly, mice
lacking Mac-1 or the Mac-1 ligand ICAM-1 are obese,
suggesting a role of Mac-1 in the regulation of fat metabolism.58 No knockouts have been reported so far for ␣X and ␣D
integrin.
Hemostasis
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Platelets express several integrins on their surface, including
␣2␤1, ␣5␤1, ␣6␤1, ␣v␤3, and ␣IIb␤3, which play different roles
during blood coagulation. ␣IIb␤3 is crucially important for the
aggregation and adhesive spreading of platelets during hemostasis.59 On resting platelets, ␣IIb␤3 is present in a low-affinity
form. Inside-out signaling triggered by various agonists,
including thrombin, ADP, and collagen induces a shift to a
high affinity conformation. Activated ␣IIb␤3 then binds to
fibrinogen, which crosslinks platelets, leading to thrombus
formation and finally sealing of wounds. In addition,
outside-in signaling induced by ligand binding to ␣IIb␤3
results in calcium mobilization, tyrosine phosphorylation of
several proteins, including ␤3 integrin itself, and additional
cytoskeletal rearrangement, which is especially important for
stable clot formation.59 This was confirmed in mice with a
selective loss of outside-in signaling caused by replacement
of the two cytoplasmic tyrosines of the ␤3 subunit by
phenylalanine residues. These mice are impaired in the late
phase of platelet aggregation and in clot retraction in vitro
and show an increased rebleeding tendency in vivo.60 Patients
having no or only a mutated ␣IIb␤3 integrin on platelets suffer
from Glanzmann thrombasthenia characterized by impaired
platelet aggregation and prolonged bleeding time. The same
phenotype was replicated in mice lacking the ␤3 or the ␣IIb
integrin gene.61,62
When platelets get in contact with injured or diseased
blood vessel walls, they tether to the subendothelial matrix
through interactions with von Willebrand factor. Integrin
␣2␤1 was thought to be essential for the subsequent platelet
adhesion to collagens, facilitating interactions with the activating platelet collagen receptor glycoprotein VI (GPVI).
This hypothesis was based on observations from patients with
reduced expression of ␣2␤1 integrin on platelets who suffered
from excessive posttraumatic bleeding and menorrhagia.63,64
In addition, their platelets display profound defects in
collagen-induced adhesion/aggregation.65 However, it is presently unclear whether these patients also carried other defects
contributing to the pathological condition. A few ␣2␤1 integrin function– blocking antibodies have been shown to interfere with collagen-induced platelet aggregation.66 – 68 Surprisingly, however, mice lacking ␤1 integrin on megakaryocytes
show no significant changes in platelet counts or bleeding
time.69 Aggregation of ␤1-null platelets on collagen is delayed
but not reduced. On the other hand, platelets lacking GPVI
cannot activate integrins and consequently fail to adhere to
and aggregate on collagen.69 Therefore, GPVI plays the
central role in platelet-collagen interactions by activating
Integrin Mutations in Mice
215
different adhesive receptors, including ␣2␤1 integrin. ␣2␤1
Strengthens adhesion but is not essential for it.
Vasculogenesis and Angiogenesis
Studies of mice carrying integrin-null mutations support the
concept that endothelial integrins are involved in the formation and maintenance of the vascular network. Endothelial
cells express at least 11 different integrins, including ␣1␤1,
␣2␤1, ␣3␤1, ␣4␤1, ␣5␤1, ␣6␤1, ␣v␤1, ␣v␤3, ␣v␤5, ␣v␤8, and ␣6␤4.
The most severe vascular defect is present in mice lacking
the ␣5 subunit. They have impaired development of the
extraembryonic (YS) and embryonic (heart and aorta) vasculature.15 Blood islands form, and hematopoiesis occurs in the
YS. Subsequently, the mesoderm and endoderm layers are
separated, and the primitive blood cells lie in sacs between
the two germ layers and leak into the exocoelomic space. This
splitting of the germ layers could be explained by defective
cell-matrix interaction or mesoderm defects. All ␣5-null
embryos form heart and blood vessels that contain a few
primitive blood cells. The vessels, however, are distended and
leak. A very similar but more severe vascular phenotype is
present in mice lacking fibronectin,18,70 the major ligand of
␣5␤1. The fibronectin mutants display variable cardiovascular
defects depending on the genetic background.70 On the
C57BL/6 background, the defects are less severe, whereas on
the 129/Sv background, no hearts have properly formed
vessels. In C57BL/6 mutants that form hearts, the myocardial
and endocardial cells are disorganized, resulting in a bulbous
heart tube. The aorta contains endothelial cells that lack
contact with the surrounding mesenchyme, whereas the yolk
sac is completely devoid of vessels. On a 129/Sv background,
endothelial cells differentiate but fail to assemble into vessels.
These findings clearly suggest that ␣5␤1 is not required for
initial vasculogenesis but is necessary for the proper formation and maintenance of blood vessels. Fibronectin, on the
other hand, seems necessary for vasculogenesis, suggesting
that in the absence of ␣5, other fibronectin receptors can
compensate. Similar to the ␣5- and fibronectin-null mice,
mice lacking vascular endothelial growth factor (VEGF)-R1
(flt-1) have normal hematopoietic progenitors but show
impaired blood vessel formation.71 Mice lacking the tie-2
receptor72 or its ligand angiopoetin-173 show defects in the
interactions of endothelial cells with the surrounding matrix
and mesenchyme. These abnormalities are reminiscent of
those observed in ␣5- and fibronectin-deficient mice, suggesting that these signaling systems cooperate in blood vessel
assembly.
Null mutations of other ␣ subunits associated with ␤1 show
milder or no obvious vascular defects. Mice lacking integrin
␣4 die during embryonic development because of defects in
heart and placenta.19 In addition, these mutants lack coronary
vessels.19 Because epicardial coronary vessels connect with
those in the myocardium, blood from the myocardial vessels
leaks, resulting in cardiac hemorrhage in the ␣4-deficient
mice. It is not clear, however, if this is a direct consequence
of the loss of ␣4 or an indirect secondary defect attributable to
a loss of the epicardium. A vascular abnormality is also
present in ␣3-null mice. Their glomerular capillaries show
reduced branching and a distended lumen.74 Glomerular
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August 3, 2001
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endothelial cells express ␣3␤1, and, therefore, the defect could
result from an abnormal formation of capillary structure of
the glomerulus. It is also possible that the altered capillaries
are caused by the failure of podocytes to provide adequate
scaffolding, within which the newly formed capillaries would
form. Mice lacking the ␣1 subunit show normal development
of the blood vessel system75 but have a reduced tumor
angiogenesis.76 This reduction in tumor capillary numbers is
caused by overexpression of metalloproteinase 7 (MMP-7)
and MMP-9 in the mutants leading to elevation of angiostatin
that has been shown to inhibit endothelial cell proliferation in
tumors but not during embryogenesis. Isolated ␣1-null endothelial cells from adult lung, however, show a reduced
proliferation on both ␣1-dependent and -independent substrata, indicating that this response is not restricted to tumor
endothelial cells.76 The formation of a normal embryonic
vasculature suggests that there are phenotypic differences
between embryonic versus adult and tumor endothelial cells.
These data demonstrate that ␣1 integrins regulate MMP
expression and that a tightly controlled MMP expression is
crucial for angiogenesis. Although MMP synthesis is essential for new blood vessel formation by degrading the ECM
and thus facilitating remodelling and invasion, an increased
MMP expression can also lead to the release of factors that
inhibits cell growth, such as angiostatin.
Endothelial cells express both ␣6␤1 and ␣6␤4 integrins.
Mice lacking ␣6 or ␤4 integrin show no obvious vascular
defects.77,78 Other members of the integrin family or
␣-dystroglycan may compensate for the loss of ␣6 function.
Mice lacking ␣-dystroglycan die before the first vessels
form,79 and, therefore, only tissue-specific gene inactivation
of the ␣-dystroglycan gene will reveal whether it has a
function during vessel formation. Targeted null mutation of
the ␤1 integrin gene leads to embryonic lethality before the
vascular system begins to form. With the use of ␤1-null ES
cells, however, it is possible to demonstrate an important role
for the ␤1 subfamily during blood vessel formation. Chimeric
mice derived from ␤1-null ES cells lack ␤1-null endothelial
cells in liver,4 indicating that endothelial cells either fail to
form or fail to participate in blood vessel formation. In vitro
differentiation of ␤1-null ES cells into embryoid bodies show
that endothelial cells differentiate but initiation of vasculogenesis is significantly delayed.80 In addition, the responsiveness to VEGF is impaired,80 suggesting that VEGF action is
upstream of ␤1 integrin function. Finally, in ␤1-null teratomas, the ␤1 mutant ES cells fail to participate in tumor vessel
formation, which is in sharp contrast to wild-type ES cells.80
Endothelial cells express at least 4 members of the ␣vintegrin subfamily, ␣v␤1, ␣v␤3, ␣v␤5, and ␣v␤8. Numerous
studies using blocking antibodies suggested that this family
of integrins is of crucial importance for angiogenesis, as
shown in chorioallantoic membrane assays,81 tumor models,82
and neovascularization studies of the mouse retina.83,84 The
generation and analyses of ␣v-null mice, however, revealed
that 20% of the mutants are born alive with no defects in
vasculogenesis and angiogenesis.23 Basic endothelial processes of proliferation, migration, tube formation, branching,
and basement membrane assembly occur in the mutants. The
intracerebral and intestinal vasculature, however, becomes
distended and eventually ruptures, resulting in severe hemorrhage. Interestingly, ␤8-null mutant mice share many similarities to those of the ␣v mutants, with early defects in placental
development and later deficits in formation and stabilization
of the vascularization of the central nervous system (Z.
Jiangwen and L. Reichardt, personal communication, 2001).
Mice lacking ␤3 or ␤5, two of the ␣v-associated ␤ subunits, are
viable and show no obvious defects in vascular development.61,85 The ␤3-null mutants were additionally investigated
for defects in postnatal retinal angiogenesis, but no abnormalities were observed.61 In addition, null mutants of many
␣v ligands, including vitronectin,86 tenascin-C,87,88 osteopontin,89 fibrinogen,90 perlecan,91 and von Willebrand factor,92
show no defects in vessel formation.
Skin
The skin is composed of an epidermal and a dermal layer,
separated by a basement membrane. The epidermis is made of
keratinocytes at different stages of differentiation. The proliferating basal keratinocytes adhere to the basement membrane via specialized adhesion junctions called hemidesmosomes, containing ␣6␤4 integrin, and via other ECM
receptors, including ␣3␤1 integrin and ␣-dystroglycan. Both
␣6␤4 and ␣3␤1 integrins bind laminin 5 (␣3␤3␥1) of the BM.
During differentiation, the keratinocytes lose their integrin
expression, detach from the basement membrane, move to the
suprabasal layers, and terminally differentiate to form the
stratum corneum. Mutations in the ␣6 or the ␤4 gene result in
a reduction of hemidesmosomes, causing the epidermolysis
bullosa in humans, a severe blistering disease leading in most
cases to early postnatal lethality.93 Deletion of the ␣6 or the ␤4
gene in mice leads to a complete absence of hemidesmosomes, a severe detachment of the epidermis and death
shortly after birth.77,78,94 Interestingly, the basal lamina is not
affected by the loss of ␣6␤4 integrin.
␣3-Null mice die shortly after birth because of defects in
kidney and lung organogenesis.74 These mice also display mild
blisters caused by disruption of the basement membrane organization.95 Keratinocyte-restricted disruption of the ␤1 integrin
gene during early development causes a similar but more severe
phenotype with death shortly after birth, suggesting that ␣2␤1 or
␣5␤1, which both are expressed in skin, play an additional role in
basement membrane formation and skin homeostasis.96 Skinspecific ablation of the ␤1 integrin gene at around birth demonstrated a role of ␤1 integrin in the processing of the BM
components and in the growth and maintenance of hair follicles.
␤1-deficient basal keratinocytes have an aberrant morphology
and a reduced proliferation rate, but are still able to terminally
differentiate.10 Interestingly, in the absence of both ␣3 and ␣6 or
␤4 integrin, basal keratinocytes can initially attach to the basal
lamina and proliferate, indicating additional adhesion mechanisms.97 A candidate receptor for maintaining proliferation could
be ␣-dystroglycan.
The critical importance of laminin-5 as a ligand for ␣6␤4
integrin is demonstrated by patients with mutations in the
laminin subchains ␣3, ␤3, or ␥2, which develop lethal junctional epidermolysis bullosa.93 Similarly, mice lacking a
functional laminin ␣3 gene have severe blistering and abnormal hemidesmosomes but an ultrastructurally normal basal
Bouvard et al
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lamina.98 Adhesion experiments with laminin-5– deficient
basement membrane suggested that ␣3␤1 also interacts with
other basement membrane components, in contrast to ␣6␤4
integrin.
The integrin ␣v␤6, which interacts with fibronectin, vitronectin, and tenascin-C, is only expressed on epithelial cells
during local injury or inflammation. Mice deficient for ␤6
integrin show normal embryonic development and wound
healing.99 Surprisingly, however, mutant mice exhibit juvenile baldness attributable to macrophage infiltration of the
dermis of the affected areas. Furthermore, ␤6-null mice
demonstrate an increased airway responsiveness, a hallmark
of asthma, and an infiltration of activated B and T cells into
the lung. This phenotype was explained by the finding that
␣v␤6 can bind to a latent form of the transforming growth
factor-␤1 (TGF-␤1), inducing a conformational change in the
ligand, which allows its interaction with TGF-␤ receptors.
Therefore, loss of ␣v␤6 reduces the amount of active TGF-␤1
in epithelia, which at least partially mimics a local TGF-␤1–
null phenotype.100 Expression of ␣v␤6 in injury and inflammation, therefore, leads to increased local activation of
TGF-␤1, which, among other effects, modulates inflammatory
cell function.
Skeletal Muscle
The ␤1 subunit is expressed throughout skeletal muscle
development. Initially, muscle precursor cells and fetal muscle express the cytoplasmic ␤1A splice variant. After birth, the
␤1A splice variant becomes substituted by the ␤1D variant.101
During early muscle development, ␤1A is expressed with ␣1,
␣3, ␣4, ␣5, ␣6, ␣7, ␣9, and ␣v. With the exception of the ␣7
integrin, the other ␣ subunits are downregulated around birth.
In vitro studies using Drosophila cells revealed an important
role for integrins in generating the sarcomeric cytoarchitecture. Normal Drosophila embryo cells fuse and develop
sarcomeres in which integrins and actins concentrate at
Z-bands. In contrast, integrin-deficient fly cells fail to form
sarcomeres.102 In mice, antibody perturbation experiments
suggested an important role of ␤1 integrin for migration of
muscle precursor cells from the dermomyotome to the limbs,
fusion of myoblasts into primary and secondary myotubes,
and assembly of the contractile proteins/apparatus.103–105
Interaction between ␣4␤1 on the primary myotubes and
VCAM-1 on the secondary myoblasts was suggested to be
essential for the formation of the secondary myotubes.105
Such a function for ␣4 integrin, however, was neither confirmed in ␣4-null chimeric mice nor in ␣4-null embryoid
bodies.34 In addition, no muscle phenotype was found in the
few VCAM-1 null mice that survive the embryonic
defects.20,21
␣5␤1 Integrin is expressed in adhesion plaque–like structures along the myotube during development106 but is downregulated in adult muscle. ␣5-Null mice die during early
development before muscle is formed. ␣5-Null chimeras with
a high contribution of ␣5-deficient cells to skeletal muscles
develop typical alterations resembling muscular dystrophy,
including giant muscle fibers, vacuoles, and centrally located
nuclei.28 The cause of the muscle degeneration is reduced
adhesion and survival of ␣5-null myoblasts.28
Integrin Mutations in Mice
217
The most abundant integrin in skeletal muscle is ␣7␤1,
which is expressed during all stages of muscle development107,108 and interacts with laminin 2 (␣2␤1␥1) and laminin
4 (␣2␤2␥1) in skeletal muscle.109 –111 ␣7 Exists in two extracellular (X1 and X2) and three cytoplasmic (A, B, and C)
splice variants, which display a tissue-specific and developmentally regulated expression pattern.112–115 Skeletal muscles
express ␣7A, ␣7B, ␣7C, and ␣7X2, whereas ␣7B, ␣7X1, and ␣7X2 are
found in the heart. After birth, ␣7A and ␣7B concentrate at
neuromuscular and myotendinous junctions, whereas ␣7C is
the predominant extrajunctional splice variant.107,113 Mice
with a targeted deletion of the ␣7 integrin develop a progressive muscular dystrophy after birth. The major defect is
severe disruption of the myotendinous junctions.116 Mutations in the ␣7 gene have also been identified in patients
suffering from congenital myopathies characterized by delayed motor milestones.117 The genetic findings from mouse
and human suggest no role for ␣7␤1 in myogenesis but a
maintenance function on the mature muscle by firmly linking
the muscle fibers to the extracellular matrix. Muscle expresses laminin 4 that binds the ␣7␤1 integrin. Naturally
occurring mutations in the laminin ␣2 gene are found in
human and mouse (dy/dy mouse strain) and lead to a
congenital muscular dystrophy118 resembling the phenotype
produced by loss of ␣7 integrin expression.
These data demonstrate an important function of ␣5␤1A and
␣7␤1D integrins in the maintenance of muscles and show that
they cannot compensate each other. However, in ␤1-null
chimeric mice, ␤1-deficient myoblasts migrate, form myotubes, and develop a normal sarcomeric cytoarchitecture in
␤1-null chimeric mice.119 The formation of myotubes in
␤1-null embryoid bodies is delayed but otherwise normal.119,120 The absence of any obvious muscular dystrophy in
the ␤1-null chimeric mice could be explained by both a low
contribution of ␤1-null muscle cells and the presence of
wild-type cells, which can functionally compensate. Replacement of the muscle-specific splice variant ␤1D by ␤1A had no
effect on the formation of skeletal muscles.121 No muscle
phenotype has been reported from knockouts of ␣1, ␣3, ␣4, ␣6,
␣9, and ␣v integrins, respectively.
Heart
Cardiomyocytes express several integrin subunits during
development: ␤1A, ␤1D, ␣1, ␣3, ␣5, ␣6, ␣7, and ␣v. Like in the
skeletal muscle, after birth, adult cardiac muscle expressed
the ␤1D variant. Expression of ␣1, ␣5, and ␣6 is downregulated
at birth. On the basis of in vitro analysis, integrin-mediated
attachment to the ECM has been suggested to be important
for controlling growth and differentiation of cardiomyocyte.122 Furthermore, integrins were proposed to function as
mechanoreceptors that transform mechanical stimuli into
biochemical signals that affect cellular function.122 Several
genetic mouse models demonstrate an important function of
␤1 integrin in cardiac muscle in vivo. Mice expressing ␤1A
instead of ␤1D in heart develop, without exposure to mechanical stress, mild cardiac defects characterized by an increase
in atrial natriuretic peptide (ANP) synthesis. ANP is a
vasorelaxant diminishing volume overload and hypertension.
Increased levels of ANP have been correlated to, among other
218
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August 3, 2001
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things, reduced ventricular function and induction of ventricular hypertrophy. The increased expression of ANP suggests
that the ␤1A subunit, at least in the heart, is not fully
compensating for ␤1D.121 Even more severe defects occur
when both ␤1A and ␤1D are absent or functionally inactivated.
The areas with ␤1-null cardiac muscle cells in the heart of
␤1-null chimeras become smaller with time and show signs of
degeneration. Ultrastructural analysis revealed alterations in
the sarcomeric architecture. In addition, transgenic mice
expressing a dominant-negative form of ␤1 integrin, in which
the extracellular and transmembrane domain of CD4 is fused
to cytoplasmic domain of ␤1 integrin, show hypertrophic
changes in the heart. Mice that express high levels of the
transgene die around birth and display a replacement fibrosis.123 Overexpression of a constitutively active form of ␣5
integrin in the heart results in electrocardiographic abnormalities, cardiomyopathy, and death within one month after
birth.124
Skeleton
The vertebrate skeleton develops via two distinct ways: by
replacing cartilaginous precursors (endochondral ossification) or by direct development within a mesenchymal tissue
(intramembranous ossification). The major cell types of the
skeleton, the chondrocytes, the bone depositing osteoblasts,
and the bone resorbing osteoclasts, express various integrin
receptors at their surfaces. Despite numerous in vitro studies,
their function in vivo during skeletogenesis is not well
understood. Mice deficient in ␤3 integrin produce dysfunctional osteoclasts, leading to osteosclerosis characterized by
increased bone mass.125 The mutant osteoclasts fail to form
the proper ruffled membrane important for their resorptive
function, leading to hypocalcemia in vivo and decreased
resorption of whale dentin in vitro. Furthermore, ␤3-deficient
osteoclasts do not spread in culture and lack the characteristic
actin rings, suggesting that ␣v␤3-mediated signals are important for the organization of cytoskeleton in these cells.
The most severe skeletal abnormalities have been identified in ␣3⫺/⫺␣6⫺/⫺ double-knockout mice.126 They display
exencephaly attributable to the failure of neural tube closure,
kinked tail, and limb anomalies characterized by shortening,
abnormal shape, lack of digit separation, and fusion of
phalanges between digits 2 and 3. Detailed analyses revealed
discontinuity of the surface ectoderm of the distal limb and
defective formation of a specialized limb bud epithelial
structure, called apical ectodermal ridge (AER). AER is
located at the tip of the bud and is involved in the control of
limb outgrowth and differentiation. Both ␣3 and ␣6 integrin
chains are expressed in this region, and a lack of both
integrins results in structural disorganization and reduced
proliferation of the ridge cells. Because single ␣3 or ␣6
mutants do not show such defects, the observations suggest
that the presence of both integrin chains is crucial for the
organization and proper function of AER and reveal the
synergistic actions of these integrins in limb morphogenesis.
Similar abnormalities of the distal limbs are present in the
laminin ␣5 knockout mice.127 Altogether, these observations
indicate that ␣3 and ␣6 integrins are the major cell-surface
receptors for laminins containing ␣5 chains in the distal limb
ectoderm.
Transgenic mice expressing a dominant-negative ␤1 integrin subunit under the control of the osteoblast-specific
osteocalcin promoter show impaired membranous bone formation.128 The phenotype is characterized by a decreased rate
of cortical bone formation and reduced bone mass of cortical
and flat bones in young animals, indicating the importance of
␤1 integrin in osteoblast function. The defect of flat bones was
normalized in older males but not in females, suggesting
sex-specific differences in adult animals.
Kidney
So far, two integrins, ␣3␤1 and ␣8␤1, have been shown to be
crucial for kidney development. In ␣3⫺/⫺ mutant mice, the
glomerular basement membrane of the kidney is disorganized
and glomerular podocytes are unable to form mature foot
processes, suggesting that ␣3 integrin might be important for
basal membrane organization. In addition, these mutant mice
have a lung defect with reduced branching of the conducting
airway74 and show an alteration of the basal membrane
organization of the submandibular gland.129
Inactivation of the ␣8 integrin leads to a failure in kidney
development, with different levels of severity ranging from a
reduction of kidney size to a complete loss of kidneys.130 The
kidney defect is characterized by abnormal growth of the
ureteric bud and abnormal branching and formation of the
ureteric epithelium. Fibronectin, tenascin-C, and vitronectin
bind to ␣8␤1. However, they seem not to be involved in the
phenotype, because they are not detectably expressed during
this stage of kidney development. Osteopontin can also bind
␣8␤1 and has been proposed to mediate this interaction.
Osteopontin-null mice, however, are normal and develop no
apparent phenotype in the kidney.131,132
Most integrin ␣8-null mice die soon after birth, but a few
survive until adulthood. These mice are deaf and have
balance defects attributable to a structural inner-ear defect,
indicating a role of ␣8␤1 integrin and its ligand fibronectin in
hair-cell differentiation and function.133
Brain
Evidence coming from fly and worm suggest a critical role
for the integrin family during brain development and maintaining brain function.134 This was also confirmed in mice.
␣3-Null mice display a defect in neuron migration and a
disorganized layering of the cerebral cortex, suggesting that
this integrin is involved in radial neuronal cell migration.135 A
very similar phenotype is observed in reeler mice, which lack
the extracellular matrix protein reelin. In these mutant mice,
migration of Cajal-Retzius cells is impaired, leading to an
abnormal lamination of the cerebral cortex. A recent study
showed that ␣3␤1 integrin can bind reelin.136 This interaction
may provide a stop signal and arrest neuronal migration.
␣6-Null mice have abnormalities in the laminar organization of the developing cerebral cortex and retina, ectopic
neuroblastic outgrowth on the brain surface and in the eye,
and an abnormal laminin deposition.137 In the ␣3⫺/⫺␣6⫺/⫺
double-knockout mice, the cortical disorganization appears
earlier and is more severe, likely because of an additive effect
Bouvard et al
of the two mutations. Ablation of the laminin ␣5 gene leads
also to a brain phenotype similar to the ␣3⫺/⫺␣6⫺/⫺ double
knockouts. In addition, the ␣3⫺/⫺␣6⫺/⫺ mutants have several
other abnormalities, including exencephaly, syndactyly, and
kidney defects.126,127,138
Axotomy leads to an increased expression of ␣7␤1 integrin
on axons and growth cones during peripheral nerve regeneration. An impaired axonal regeneration has been reported in
␣7-null mutants, suggesting that this integrin plays a role in
this process.139
Signaling
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Integrin-mediated adhesion results in the activation of various
signaling cascades inside the cell, including activation of
tyrosine kinases like focal adhesion kinase (FAK) and Src,
serine/threonine kinases like mitogen activated protein kinase
(MAPK), and small G proteins.3,140 Direct evidence for
signaling defects in integrin-deficient mice is scarce. In
␣7-null mice, a constitutive activation of c-Raf-1 kinase was
observed in muscle, suggesting that ␣7␤1 integrin may be
involved in the negative control of this pathway.141 However,
this finding could also be caused indirectly by the muscle
degeneration observed in these mice. Analysis of the inner ear
development in ␣8-null mice revealed a defect in stereocilia
formation correlating with a loss of FAK at the cell
membrane.133
Cell cultures studies revealed that integrin-mediated signals are involved in cell proliferation, cell survival, prevention of apoptosis (also called anoikis), and cell differentiation.142 Presently, it is not clear to what degree the integrin
signaling observed in vitro also has a role in vivo. The
observations collected so far suggest an involvement of
integrins in regulating cell proliferation and programmed cell
death in vivo.
Several integrin-null mice show reduced proliferation of
certain cell populations. Mice lacking ␣3 and ␣6 integrin have
a reduction of cell proliferation in the AER,126 and mice
lacking ␤1 integrin in keratinocytes lose proliferating hair
matrix cells and display a reduced proliferation of basal
keratinocytes.10 Deletion of the ␣1 gene results in decreased
proliferation of dermal fibroblasts.143 In vitro culture of these
fibroblasts demonstrated a failure to recruit and activate the
adaptor protein Shc, which triggers the MAPK activation
cascade leading to cell proliferation. In a dominant-negative
approach, the cytoplasmic and transmembrane domain of ␤1
integrin was fused to the extracellular domain of CD4, which
interferes with endogenous ␤1 integrin function in vitro.
Expression of such a chimeric protein in the mammary gland
of transgenic mice results in decreased proliferation, increased apoptosis, and impaired differentiation of the mammary epithelial cells.144 Additional biochemical analysis of
these mice revealed no change in FAK activation in the
mammary gland but a significant decrease in the phosphorylation of Shc, Grb2, Akt, c-Jun N-terminal kinase, and
extracellular signal–regulated kinase.145 Lack of the ␤4 integrin cytoplasmic domain has been shown to reduce proliferation in the skin and intestine,146 which agrees with in vitro
data showing that ␤4 integrin is able to bind and activate Shc.
Integrin Mutations in Mice
219
Cell survival defects have been reported in some integrinnull mice. For instance, ␣5 integrin was shown to be important for the survival of neural crest cells in vivo,17 and
␣3⫺/⫺␣6⫺/⫺ mice have syndactyly resulting from an impaired
cell death of interdigital cells.126 On the other hand, loss of ␤1
integrin–mediated adhesion of basal keratinocytes does not
result in cell death, in contrast to expectations from cell
culture experiments.10
Concerning differentiation processes, so far gene ablation
approaches revealed only a minor role for integrins. Absence
of ␤1 integrin does not interfere with differentiation of
neurons and neural crest– derived cells,4 fetal hematopoietic
cells,26 or keratinocytes.10 Double knockouts for ␣3 and ␣6
integrin show normal keratinocyte differentiation, despite the
absence of the major integrin receptors ␣3␤1 and ␣6␤4.126 In
vitro myoblast differentiation and fusion occurs independently of ␤1 integrin but is delayed.119 A similar delay was
found in blood vessel formation in ␤1-null embryoid bodies.80
Such a delay could be attributable to the absence of ␤1
integrin or the experimental design that does not reflect the in
vivo conditions, eg, growth factors that are absent in fetal calf
serum but not in vivo or absence of complex matrices in vitro.
On the other hand, teratomas using ␤1-null cells never give
rise to an endothelial cell population, suggesting that ␤1
integrin could be involved during their differentiation or
survival.80
Future
Analysis of integrin function by gene-targeted mice often
confirmed but sometimes also contradicted previous results
obtained by antibody or peptide inhibition studies. For
instance, blocking ␣v␤3 function by inhibitory antibodies
suggested an essential role of ␣v␤3 integrin in angiogenesis.
However, both ␣v and ␤3 knockout mice show blood vessel
formation. Similarly, a few ␣2 blocking antibodies prevent
collagen-induced platelet aggregation, whereas deletion of ␤1
integrin on platelets revealed only a minor and clearly
dispensable role for ␣2␤1 during this process. One obvious
disadvantage of antibodies is that they do not completely
block protein function, whereas a targeted gene disruption
leads to a complete loss of the protein. In addition, antibodies
might have unexpected side effects by steric hindrance of
other ligand-receptor interactions or only partial inhibition of
the integrin signaling. However, constitutive gene knockout
also has disadvantages. The mutated mouse might react to the
loss of a protein by upregulating the activity or expression of
other molecules, thus compensating the defect. Because
totipotent embryonic cells, at least theoretically, have a high
capacity for compensation, this risk is real in constitutive
knockouts, and compensation has already been demonstrated
on several occasions. A way to reduce the chance of compensation is to induce the knockout in more differentiated
cells, which are less able to change their expression pattern.
This is possible using the cre/loxP system generating mice
with tissue restricted or inducible gene deletions.10,26,69,96 The
generation of mice carrying floxed integrin genes to induce
cell type–specific deletions, but also the introduction of subtle
mutations into the extracellular and intracellular domain of
integrin subunits and the interbreeding of integrin-null mice
220
Circulation Research
August 3, 2001
to create double and triple mutant animals, will additionally
advance our understanding of how integrins function in vivo.
In addition, the mutant mice will allow the establishment of
cell lines that can be used to study in vivo observations at a
molecular level.
More information about the role of integrins in signaling in
vivo will come from the analysis of mutant tissues with
antibodies specific for activated forms of signaling molecules, from knockin mice carrying subtle mutations of the
integrin molecules interfering specifically with certain in
vitro signaling functions, from knockouts of integrinassociated proteins, and from the rescue of integrin-null mice
with transgenes encoding activated forms of signal transduction molecules suspected to act downstream of the respective
integrins.
Acknowledgments
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This work was supported by the Craaford Foundation (E.G.) and the
Swedish National Research Foundation (R.F. and C.B.). D.B. is a
Wenner-Gren fellow. We are grateful to Drs Z. Jiangwen and L.
Reichardt for sharing information before publication. We thank Drs
T. Sakai, Kamin Johnson, and Michael Dictor for critical reading the
manuscript.
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Functional Consequences of Integrin Gene Mutations in Mice
Daniel Bouvard, Cord Brakebusch, Erika Gustafsson, Attila Aszódi, Therese Bengtsson,
Alejandro Berna and Reinhard Fässler
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Circ Res. 2001;89:211-223
doi: 10.1161/hh1501.094874
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