Genetic analyses of integrin function in mice

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Genetic analyses of integrin function in mice Reinhard Fissler*, Elisabeth Georges-Labouesset and Emilio Hirsch*

Recent mutations of most integrin genes in the mouse have provided new exciting insights into the role of these integrins in cell-extracellular matrix interactions during development. The embryonic lethal phenotypes obtained by ablating integrins which are predominantly expressed in the mesenchyme confirmed the essential function of those integrins in morphogenesis. In contrast, null alleles for several epithelial integrins which bind components of basement membranes showed milder phenotypes, suggesting the presence of novel and unexpected redundant and compensatory mechanisms.

Addresses *Max Planck Institutefor Biochemie, Abteilung Proteinchemie,Am Klopferspitz 18A, D-82152 Martinsried,MiJnchen, Germany tlnstitut de G6n6tique et de Biologie Mol~culaire et Cellulaire, 1 Rue Laurent Fries, 67404 IIIkirch,C.U. de Strasbourg, France

Null mutations integrins

in f i b r o n e c t i n

receptor

T h e ablation of the ot5 integrin gene was the first integrin knockout published [1]. T h e o~5131 integrin is one of at least eight integrins which bind fibronectin (FN). Null mutations in either the ct5 integrin gene or the FN gene [3] lead to embryonic lethality. T h e resulting phenotypes, however, are different. FN-null embryos develop abnormalities shortly after gastrulation. Defects are found in mesodermal derivatives all along the anterior-posterior axis and in the yolk sac vasculature. Mutant embryos have neither a notochord nor somites along the entire axis, although precursor cells for those tissues are present [4]. Interestingly, a similar phenotype has been observed in mice lacking the focal adhesion kinase (FAK) [5], which can bind to and phosphorylate the cytoplasmic domain of [51 integrin.

Current Opinion in Cell Biology 1996, 8:641-646 © Current Biology Ltd ISSN 0955-0674 Abbreviations ECM extracellularmatrix FN fibronectin ES embryonicstem ICM inner cell mass VCAM-1 vascularendothelialcell adhesion molecule-1

Introduction

T h e first report of a targeted mutation of an integrin subunit gene was published only three years ago [1]. To date, most of the known integrin genes have been mutated in mice. T h e rapid improvements in the gene-targeting technology have made this progress possible and brought a wealth of information. In this review, we will discuss the recent genetic analyses of integrin-null mice. Mammals contain more than 20 integrins, which are composed of an ~ and a 13 subunit [2]. Many of the different ot and [5 subunits come in alternatively spliced forms which are expressed in a tissue-specific manner. Each cell expresses multiple integrins, but each cell type expresses a distinct set of integrins. Integrins specifically bind to one or more ligands, and one ligand can be bound by multiple integrins. This overlap in distribution and binding specificity indicates complex functional relationships among these receptors. Therefore, it was a surprise that most of the integrin mutations produce distinct phenotypes (see Table 1), suggesting a fine-tuned specialization of these molecules.

In contrast, embryos with null mutations in the (:(5 integrin gene [2] gastrulate and form a normal notochord and somites in their anterior half. Failure to form a notochord and somites is seen only in the posterior region, distal to somite 10, of a5-null embryos (see Fig. 1). Different integrins could thus be implicated in the development of anterior versus posterior structures. Similar defects in the development of posterior structures have also been reported in three other mouse strains carrying mutations in genes encoding Brachyury (a transcription factor) [6], Wnt-3A (an extracellular signalling molecule) [7] and Csk (a tyrosine kinase belonging to the Src family) [8,9]. Whether the three signalling cascades are involved in regulating the expression of integrins and extracellular matrix (ECM) molecules awaits further investigations. A very surprising finding was that cells derived from ot5-null embryos still polymerize FN even though, from in vitro findings, ot5l~1 was thought to be the receptor required for FN assembly [2,10]. Recent evidence from studies of [51-null cells and transfected cells show that ~k,[53 integrin [11 °] and an activated form of ~ilbl~3 integrin [12"] are able to assemble FN. Other FN receptors, which are widely expressed, belong either to the [51 integrin family (i.e. o~31~!,o~4~1 and ~8131)or to the av integrin family (i.e. CCv[51,O~v[53, Ctv~5, ~v[56, O~v[57 and Otv~8). Null mutations in the ct4 integrin gene [13"] also lead to embryonic lethality, due either to a defect in placentation characterized by a faulty fusion of the allantois with the chorion (see Fig. 1), or to an abnormal development of the epicardium and coronary vessels

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Table 1 Effects of mutations in integrin genes on mouse development.

Integrin subunit

Phenotype associated with mutations in integrin genes

al (x2 c¢3 (i 4 ~5 c(6 c~7 c~s (x9 ck, o~E (7.L (1M (xx (111b

The only mutated integrin subunit so far without a corresponding phenotype. No knockout mouse available. Perinatal lethality; displays abnormalities in kidneys. Embryonic lethality; shows abnormal formation of placenta or heart. Embryonic lethality; displays mesodermal abnormalities. Perinatal lethality; displays skin blistering. Viable; displays muscular dystrophy. Perinatal lethality; shows malformation of kidneys, Mice die within the first two weeks after birth. Perinatal (and maybe embryonic) lethality; some vessels are abnormal. No knockout mouse available. No knockout mouse available. No knockout mouse available. No knockout mouse available. No knockout mouse available. Peri-implantation lethality; shows ICM failure. Knockout mouse available with reduced expression of ~2 integrin; viable with impairment of white blood cell function. No knockout mouse available. Perinatal lethality; shows skin blistering. No knockout mouse available. Viable with macrophage infiltration in skin and lung. Lack of lymphocytes in gut-associated tissues. No knockout mouse available.

131 ~2 I]3

~4 135 J~s ~7 ~8

Reference [21] [16] [13"] [1] [23 °] * t :~ §

[29"°,30 °°] [47]

[24 °] [20] [45]

*U Meyer et eL, unpublished data. tU MiJller, L Reichardt, personal communication. SD Sheppard, XZ Huang, personal communication. §B Bader, R Hynes, personal communication.

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fusion © 1996 Current Opinion in Cell Biology

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Diagram showing the embryonic and perinatal lethal phenotypes of integrin-null mutant mice. The sizes of the embryos from fertilization (E0) to birth (PO) are not drawn to scale. Fertilization occurs in all integrin mutants due to the presence of maternal mRNAs. The earliest lethal phenotype has been observed in J]l integrin deficient mice. Studies of the (x4-null mice revealed new functions for c(4 integrin, such as regulation of chorion-allantois fusion and development of the heart. Unexpectedly mild phenotypes are found in o¢6-null and (Xv-null mice, which show skin detachment and brain bleeding, respectively, but do not die until birth.

(see Fig. 1). Similar defects in chorioallantoic fusion and heart development also occur in vascular endothelial cell adhesion molecule-1 (VCAM-1) knockout mice [14°,15°].

VCAM-1 is a known counter-receptor for cc413t and o~4~7 integrins; it belongs to the Ig family of cell adhesion molecules and mediates adhesion of leukocytes and tumor

Genetic analyses of integrin function in mice F~ssler,Georges-Labouesseand Hirsch

cells to the endothelium. Thus, the predominant function of 0~4131 integrin in early embryogenesis, as revealed by this genetic approach, is to mediate VCAM-1, rather than FN, functions. Both oc3-null and O~v-null embryos die on the first day after birth (see Fig. 1). The ct3-knockout mice have abnormal kidneys characterized by dilated tubules in the medullary regions, and podocytes with a decreased number of foot processes along the glomerular basement membrane [16]. T h e ~tv-null mutation results in perinatal (and probably also embryonic) lethality. These animals show vascular hemorrhage, although many blood vessels appear to develop normally (B Bader, R Hynes, personal communication). This relatively mild phenotype is astonishing, as etv integrin associates with five different integrin 13 subunits which are widely expressed and bind multiple ligands. T h e ligands implicated here are not identified. T h e observation of abnormal blood vessel development correlates with in vivo observations that ligand binding of Otv133 and ~v115 integrins is required for cytokine-induced formation and survival of blood vessels [17-19]. A knockout of the ~8 integrin gene results in neonatal lethality (see Fig. 1). Mutant mice are born without kidneys or with kidney rudiments, suggesting an important role for cx8 during the early steps in kidney morphogenesis (U Mialler, L Reichardt, personal communi~ation). Mice lacking the I]6 gene [20] are viable and fertile but have juvenile baldness associated with infiltration of macrophages into the skin. T h e y also accumulate activated lymphocytes around conductive airways of the lung. Thus far, none of the phenotypes obtained with FN receptor knockouts recapitulates the full range of defects observed in the FN-null embryos. Null m u t a t i o n s in l a m i n i n r e c e p t o r i n t e g r i n s Laminins are major components of basement membranes. T h e importance of laminin-integrin interactions is impressively documented by mice lacking the laminin ~1 chain which leads to a loss of laminin-1. Although details of the phenotype have not been published yet, this laminin-l-null mutation results in the formation of normal blastocysts which die around the time of implantation (N Smith, D Edgar, personal communication).

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[21]. T h e o~2 integrin gene has not yet been ablated in mice. In humans, a single patient has been described with reduced expression of a 2 integrin on platelets, which led to a bleeding disorder in which platelets failed to bind to collagen [22]. Unfortunately, it is not known whether aZ integrin expression is also reduced or completely absent in other tissues of this patient. T h e phenotype of the a3 integrin subunit mutation was described above. T h e o~6 subunit can associate with the 131 and 134 subunits [2]. All cells express 131 integrin, but only stratified squamous and transitional epithelia and a few other tissues express 134 integrin [2]. Considering the distinct distribution of these 13 subunits, it is surprising that embryos with null mutations in the o~6 [23 °] and 134 [24 °] genes are born with the same phenotype (see Fig. 1). Both mutant mouse strains suffer from severe blistering of the skin and detachment of other epithelia, for example in tongue, nasal cavities and esophagus. Both mouse strains fail to develop hemidesmosomes, which are dense cytoplasmic plaques, found at the basal side of basal keratinocytes, that link the intracellular keratin filaments with the extracellular basement membrane. This phenotype resembles a human disease known as epidermolysis bullosa, which can be caused by loss of, or mutations in, the I]4 integrin gene [25°]. T h e presence of subtle defects could not have been completely excluded in the first set of experiments published (more sophisticated experiments may reveal defects which have so far escaped detection), but already the published results raise the unexpected possibility that the function of c~61]1 integrin is either unnecessary or redundant or can be compensated for in many tissues such as heart or kidney. T h e first possibility is very unlikely, as a number of studies using specific antibodies revealed roles of 0~6131 integrin in adhesion, migration, morphogenesis and differentiation [26]. Functional redundancy might operate in ~6-null mice, as many cells express multiple laminin-binding integrins. T h e most likely scenario, however, is functional compensation. In some c~6-null mice the 134 subunit is still expressed on basal keratinocytes [23°], raising the possibility that I]4 integrin is associated with another subunit. Although this aberrant integrin clearly results in defective formation of hemidesmosomes, it might still fulfill some c~6131integrin function during development.

So far, it is not known which integrin mediates laminin function during the peri-implantation stage. At least seven integrins (all]l, ~2131, (3(3131,(3(6131,o~7131,°c9131and o~6134)can bind to laminins. In addition to binding laminins, some of these seven receptors (namely oc1131, ~2131 and ~31]1) bind collagens while others (namely tz9131)bind tenascinC. With the exception of ~t2, all integrin subunits have been ablated in mice.

T h e ~x7 and ~9 integrin genes have been knocked out, but results are not yet published. Null mutations in the ~7 gene lead to muscular dystrophy without any signs of paralysis (U Meyer et al., unpublished data). This phenotype is in agreement with the expression of ~7 in skeletal muscle and myocardium. T h e c~9-null embryos complete embryonic development and die within the first two to three weeks after birth (D Sheppard, XZ Huang, personal communication).

Null mutations in the ~1 integrin gene produce no phenotype either during embryogenesis or in adulthood

As is the case for FN (see above), none of the mutations in laminin-binding integrins produce the laminin-l-null

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Cell-to-cellcontactand extracellularmatrix

phenotype. Therefore, it will be important to create the a2-null mutation and to test for synergy in double mutant strains.

Null mutation in the 131 integrin gene One could easily predict from the early embryonic lethality of several integrin-null mice that a lack of 131 integrin (which is found in as many as ten integrin heterodimers) would have massive consequences early in mouse development or would even result in a ceil-lethal phenotype. The latter possibility was ruled out with the establishment of 131-null F9 cells [27] and of 131-null embryonic stem (ES) cells [28] which display alterations in adhesion, migration and morphogenesis, but clearly still proliferate. A complete knockout of the 131 integrin gene in mice resulted in peri-implantation lethality (see Fig. 1) and revealed a number of surprises [29",30"]. Fertilization of 131-null oocytes is normal, even though the binding between oocyte and sperm is dependent on the interaction between ot6131 integrin and fertilin, which are expressed on the surface of the oocyte and the sperm, respectively [31"']. This normal fertilization of 131-null eggs supports earlier data showing that oocytes contain maternal 131 integrin mRNA [32]. Preimplantation development is normal, although 131 integrin ligands such as basement membrane components (laminin, collagen IV and nidogen) and FN are already expressed and deposited during this period [33]. In line with these results, normal blastocysts are also produced without laminin-1 or FN expression. This might indicate that integrin-ECM interactions do not play an important role for the blastomere development, or that those interactions might be possible through maternally derived products. Hatching and implantation of 131-deficient blastocysts occurs; however, embryos die shortly thereafter. T h e most fascinating result is that the 131-null trophoblast is able to invade the uterine stroma and that the 131-null inner cell mass (ICM) deteriorates before the trophoblast does, hence indicating a requirement for 131 integrins in the survival of ICM cells but not in the survival of trophoblast cells. The rapid deterioration of the 131-null ICM, both in vivo [29"'] and in cultured blastocysts [30"], points to an essential role of 131 integrins in ICM survival. Integrin-ECM interactions are important for the formation of the proamniotic cavity; this process occurs in the ICM shortly after implantation [34"']. This cavitation results in a two-layered egg cylinder, consisting of an outer layer of endodermal cells and an inner layer of ectodermal cells separated by a basement membrane. Antibody perturbation experiments show that the survival of cell layers depends on the proper interaction of ceils with the basement membrane. T h e role of integrins in cell survival is well established for endothelial cells and for a number of epithelial cells [35-37].

In contrast, when 131-null ES cells are introduced into wild-type blastocysts, mutant cells survive and contribute at low levels to many differentiated tissues in viable chimeric mice [29"']. The most surprising observation was that neural crest cells can migrate both short and long distances in the absence of 131 integrin. This was evident by the presence of mutant cells in neural crest derivatives such as dorsal root ganglia (representing a short distance to travel) and the medulla of the adrenal gland (representing a long distance to travel). Formation of neural crest derived tissues was also observed in the ot4-null mice [13"']. These findings are in contrast with earlier results based on the microinjection of RGD (Arg-Gly-Asp)-containing peptides or antibodies against ct4 or 131 integrin into amphibian and chicken embryos which results in a block in migration of neural crest cells [38,39]. Similarly, ~l-null neurons are present in many regions of the central nervous tissue [29"'] although retroviraily transduced 131 integrin antisense RNA blocks neuroblast migration in vivo [40]. One explanation for these conflicting results is the existence of a compensation mechanism whereby other integrin subfamilies or nonintegrin cell surface receptors may substitute for 131 integrins. Another possibility is that mutant cells interact with normal cells via cell adhesion molecules such as cadherins. In this model, normal cells of the chimeric embryo bind to 131-null cells and carry them around in a rucksack-like manner. A conditional mutation in the 131integrin gene will allow us to distinguish between these two possibilities. If neurons migrate in a situation in which 131 integrin expression is abolished in all neurons, active migration is occurring and is probably mediated by different integrins or other adhesion molecules. On the other hand, if neurons do not migrate in such a situation, it would indicate that the distribution of 131-null neurons observed in the chimeric mice is due to interactions of 131-null and migrating wild-type cells via cell-cell contacts.

Integrin function during migration and differentiation of blood cells T h e members of the 131 integrin subfamily were originally identified on activated lymphocytes and thus called 'very late activating antigens' [41]. Much later, it was realized that this subfamily is ubiquitously expressed and binds to ECM proteins. Recent experiments with chimeric mice made from 131-null cells, however, have reconfirmed the role of 131 integrins in migration and differentiation of blood cells. T h e lack of the entire 131integrin subfamily does not affect the differentiation of hematopoietic stem cells in the yolk sac but it does block their migration into the fetal liver [42"']. As a consequence, no 131-null cells are present in the hematopoietic organs of adult 131-null chimeric mice. T h e ability of 131-null hematopoietic stem cells from the yolk sac tissue and from the fetal blood to form blood cell colonies containing erythroid and myeloid blood cells

Genetic analyses of integrin function in mice F~issler, Georges-Labouesse and Hirsch

suggests that blood cell differentiation can occur in the absence of 131 integrin. A lack of or4 integrin leads to a defect in B-cell and T-cell development [43"]. In (x4-null chimeric mice, B-cell differentiation is blocked before the pro-B-cell stage. In contrast to these findings, 131-null ES cells can differentiate into B cells which have rearranged their Ig heavy and light chain genes [42"']. As 131-null ES cells lack (x4131 but have ot4137, one of the two (x4-containing integrins could be sufficient for normal B-cell differentiation. Alternatively, embryonic hematopoietic precursor cells (developing in the yolk sac or in the fetal liver) may be able to compensate for the lack of oc4 integrin whereas bone marrow derived precursor cells might no longer have the plasticity needed for a functional compensation. In young cx4-null chimeric mice, T cells, which in the fetus originate from yolk sac or fetal liver [44], are normal in number and phenotype in blood and many secondary lymphatic organs, except in Peyer's patches. T h e lack of T cells in Peyer's patches could be a consequence of the loss of ot4137 as 137-null mice display the same homing defect [45]. In (x4-null chimeric mice that are older than one month, however, the number of T cells decreases and the thymic tissue involutes. As, after birth, T-cell precursors originate exclusively from bone marrow, this suggests that the differentiation of the T-cell precursors in the bone marrow is defective. T h e nature of the T-cell defect is not known.

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T h e next generation of mutations will include subtle mutations in integrin domains or conditional knockouts. As the lack of a gene product might be more easily compensated for than an altered gene product, we should prepare ourselves for an exciting future with many new and surprising functions of integrins being revealed.

Acknowledgements We thank all the people who provided preprints and allowed citation of unpublished data and Antonio Iglesias, Hanny Mayer and Mikael Wendel for critically reading the manuscript. R F~issler is supported by the Hermann and Lilly Schilling Stiftung. E Hirsch is on leave of absence from the Dipartimento di Genetica, Bilogia e Chimica Medica, University of Torino, Italy.

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Conclusions and perspectives

9.

A wealth of new information has been obtained by analyzing mice carrying null mutations in integrin genes. This new information has confirmed many earlier results and refuted others. T h e big surprises are the few overlapping, and the many distinct, phenotypes obtained with each integrin mutation.

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T h e 132 integrins are exclusively expressed on white blood cells and play a role during extravasation and normal function of blood cells. A lack of 132 integrin can occur in humans and is characterized by severe infections [46]. Mice lacking the 132 integrin are not yet available. Mice with a reduced expression of 132, however, have been reported [47].

Future work will show whether some of the phenotypes, even if derived from a single integrin loss, are additionally influenced by altered expression of other integrins and/or cell adhesion molecules. Cross-talk between integrins is indeed observed for the ot5 and 131 subunits in ES cells, in which a reduction or a lack of 131 reduces the expression of ot5 [28]. Similar cross-talks might operate in a number of other integrin knockouts.

11. •

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Aim°ida EA, Huovila AP, Sutherland AE, Stephens LE, Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A, Primakoff P, Myles DG, White JM: Mouse egg integrin ocs~1 functions as a sperm receptor. Cell 1995, 81:1095-1104. Elegant experiments identify ec6J31integrin as the long sought sperm receptor on oocytes.

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Coucouvanis E, Martin GR: Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Ce//1995, 83:279-287. Integrin-basement membrane interactions confer survival to cells, which then line cavities that are being formed in the developing embryo. This integrinmediated survival might be important for all cavitation processes throughout development. 35.

Meredith JE Jr, Fazeli B, Schwartz MA: The exb'acellular matrix as a cell survival factor. Mol Biol Cell 1993, 4:953-961.

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Georges-Labouesse E, Messadeq N, Yehia G, Cadalbert L, Dierich A, Le Meur M: Absence of the (xs integrin leads to epidermolysis bullosa and neonatal death in mice. Nat Genet 1996, 13:370-373. The finding of this paper suggests that the function of the early and ubiquitously expressed (x6~1 integrin is redundant or can be efficiently compensated for.

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22.

Nieuwenhuis HK, Akkerman JW, Houdijk WP, Sixma JJ: Human blood platelets showing no response to collagen fail to express surface glycoprotein la. Nature 1985, 318:470-472.

23. •

Van der Neut R, Krimpenfort P, Calafat J, Niessen C, Sonnenberg A: Epithelial detachment due to absence of hemidesmosomes in integrin J34null mice. Nat Genet 1996, 13:366-369. See annotation [25"]. 25. •

Vidal F, Aberdam D, Miquel C, Christiano AM, Pulkkinen L, Uitto J, Ortonne JP, Meneguzzi G: Integrin 134 mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nat Genet 1995, 10:229-234. The paper and [24 °] demonstrate common but also different roles of ~4 integrin in humans and mice: a lack of [[34 integrin is associated with the absence of hemidesmosomes in mice but not in man. A common function for [34 integrin is to anchor the basal keratinocytes to the basement membrane. 26.

Kadoya Y, Kadoya K, Durbeej M, Holmvall K, Sorokin L, Ekblom P: Antibodies against domain E3 of laminin-1 and integrin alpha 6 subunit perturb branching epithelial morphogenesis of submandiular gland, but by different modes. J Cell Biol 1995, 129:521-534.

42. o•

Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, F~LsslerR: Impaired migration but not differentiation of haematopoietic stem cells in the absence of 131 integrins. Nature 1996, 380:171-175. Whereas most cells are capable of migrating in the absence of ~1 integrins (see [29"]), hematopoietic precursor cells need 131 integrins to migrate into and/or seed the fetal liver. 43. ••

Arroyo AG, Yang JT, Rayburn H, Hynes RO: Differential requirements for o.4 integrins during fetal and adult hematopoiesis. Cell 1996, 85:997-1008. This paper identifies an important role for C(4~1 integrin in the migration of T-cell and B-cell precursors from the bone marrow into secondary lymphoid tissues. 44.

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27.

Stephens LE, Sonne JE, Fitzgerald ML, Damsky CH: Targeted deletion of ~1 integrins in F9 embryonal carcinoma cells affects morphological differentiation but not tissue-specific gene expression. J Cell Biol 1993, 1 2 3 : 1 6 0 7 - 1 6 2 0 .

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