Adhesion and matrix in vertebrate development

Adhesion and matrix in vertebrate

development

Douglas W DeSimone University

of Virginia,

Charlottesville,

USA

The extracellular matrix supports the adhesion and migration of cells during morphogenesis and influences cell differentiation. Cell interactions with the extracellular matrix are mediated in large part by members of the integrin family of cell-surface receptors. Recent progress in this area has resulted in the identification of multiple integrins, many of which are expressed in position-specific patterns during vertebrate development. The contributions of these receptors to specific developmental events are now being investigated in a variety of systems using a combination of genetic, molecular and immunological approaches. Current

Opinion

in Cell Biology

1994, 6:747-751

Introduction

lntegrin

Among the most notable achievements in developmental biology during the past two decades has been the identification of genes responsible for controlling pattern and specifjring cell fate. Leading the way in these efforts are powerful genetic analyses made possible through studies of organisms such as fruit flies and nematodes. In addition, new genetic techniques and sophisticated molecular approaches have made it possible to investigate the developmentally significant contributions of specific genes in mammals and other vertebrates. Although many of these studies have helped advance the concept of development as an unfolding genetic program, there is still much to learn about the downstream molecular events involved in executing morphogenetic change at the cellular level. Adhesive interactions long have been considered to play essential roles in the morphogenetic movements that occur in early development. These interactions often involve multiple adhesive mechanisms [l], many of which are now being characterized at the molecular level. This review focuses specifically on adhesion mediated by integrins, arguably the most thoroughly understood and important receptors for extracellular matrix (ECM). A great deal has been learned about integrin structure and function in recent years and there is now growing interest in the roles of these receptors in embryogenesis [2*,3’,4]. I begin this review with a brief introduction to integrins, with an emphasis on the features of these receptors that are of developmental interest. Select examples of integrin expression in a number of vertebrate systems are discussed and a survey of recent functional approaches included. For a general overview of integrin structure, function and cell biology, the reader is referred to a number of recent reviews [5-Y].

Integrins are heterodimeric transmembrane glycoproteins composed of distinct a- and B-subunits. Different a/3 subunit combinations differ in their ligand-binding specificities. Among the vertebrates, eight different Bsubunits and 15 a-subunits have been reported thus far, and have been shown to combine to form more than two dozen distinct heterodimers. Most integrins are involved in binding to ECM molecules, although some integrins also bind to counter-receptors that include members of the immunoglobulin superfamily (e.g. I-CAM, VCAM). Some heterodimers recognize only one ligand (e.g. a& binds fibronectin) whereas others can bind several (e.g. a& binds laminin, collagen IV and fibronectin). Ligand specificity may also differ, depending upon the cell type in which a given integrin is expressed [lo]. Because most cells express multiple integrins, there is considerable potential for functional redundancy. Integrins form associations with cytoskeletal elements [ 111 and thereby function as transmembrane links between extracellular and intracellular compartments. Integrins have also been shown to participate in a variety of contact-dependent signalling events that influence cellular physiology and gene expression [2*,6,7].

ECM-extracellular

matrix; I-CAM-intercellular 0 Current

expression

These functionally diverse properties of integrins have attracted the attention of developmental biologists interested in morphogenesis and pattern formation. Because the adhesive behaviors of cells expressing distinct integrins often differ from one another, it is reasonable to suggest that localized expression of these receptors is important for proper patterning of early morphogenetic events. Ample evidence is now available to support this view, as discussed below.

Abbreviations adhesion molecule; V-CAM-vascular Biology

in development

Ltd ISSN 0955-0674

cell adhesion molecule. 747

748

Cell-to-cell

contact

and extracellular

lntegrins as position-specific morphogenesis?

matrix

participahts

in

Integrins initially were described as “position-specific” (PS). antigens in Drosophila embryos on the basis of their characteristic patterns of expression [12] (see also Gotwals et al., this issue, pp 734-739). The Drosophila studies provided early support for the general hypothesis that integrins confer ‘adhesive identities’ to cells in the embryo, and thereby play a role in specifjling positional information during morphogenesis. Investigations of integrin expression in vertebrate embryos have been hampered by a lack of subunit-specific antibodies and cDNAs suitable for use in non-human models. The situation is rapidly changing and a number of reagents have now been reported for rodent, avian and amphibian species. A complete survey of integrin expression in these systems is beyond the scope of the current discussion, but is the subject of several recent reviews [3*,4,13]. Most of the current literature on integrin involvement in development focuses on the b1 family, which is widely expressed in embryonic and adult tissues (see, for example, [14-171) and includes the majority of the known receptors for ECM [5]. The pt subunit is expressed as maternal mRNA and protein in amphibian and mouse oocytes and early cleavage stage embryos [l&20]. What functions might integrins have at these early stages? One possibility is that an integrin serves as a ligand for a sperm receptor at fertilization. Indirect support for this idea comes from studies of the sperm surface protein PH30, recently shown to be a trammembrane heterodimer with homology both to viral proteins involved in membrane fusion and to a family of potent integrin ligands found in snake venoms, termed ‘disintegrins’ [21]. Interestingly, although present at the plasma membrane throughout oogenesis, B1 integrins are internalized and stored following oocyte maturation in Xcnopus [22*] and are subsequently re-inserted into all newly formed plasma membranes during embryo cleavage [16]. This makes B1 integrins improbable participants in Xenopus fertilization; however, it is possible that other integrins (e.g. 83 and Bh) known to be present at these times [17] may be available at the oolemma surface during sperm-egg binding. The developmental regulation of integrin expression in mouse trophoblast cells suggest that these receptors also participate in embryo implantation [20]. lntegrin involvement in this process is further supported by observations that trophoblast attachment and outgrowth on ECM can be blocked with antibodies directed against integrins [23]. Although the current emphasis is on the roles of p, integrins in early development, in most cases the specific ab heterodimers involved have not been identified. Even less information is available concerning other b-subunits in development, but initial studies suggest a more limited pattern of expression for these integrins [17,20,24]. Given that 10 different a-subunits are known to associate with the & subunit and that g, expression is ubiquitous,

the identification of a-subunits is a necessary step in understanding integrin function in the embryo. Several recent studies confirm that a-subunit expression in vertebrates is ofien developmentally regulated and localized to specific tissues or embryonic regions (reviewed in [3*,13]). For example, at is expressed in developing smooth and striated muscle, and in the central and peripheral nervous systems in avian embryos [25]. lntegrins a8 and av are also expressed throughout the chick nervous system [26,27] along with a6 [28,29], which is alternatively spliced to yield two isofotms with distinct spatial patterns of expression in the developing retina [30]. The distribution of a4 in chick embryos is widespread and includes expression in the somitic tissues, heart, gut, CNS and neural crest [31]. In Xenopus, a2, ag, a4, a5 and elf, subunit mRNAs are each expressed by gastrulation [32*]. The integrin a3 mRNA is localized to the mesoderm of the dorsal lip at gastrulation and to the developing notochotd [32*]. The c+, subunit is first expressed in the dorsal ectodetm and later in the neural plate, developing nervous system and pronephros (TE Lallier, CA Whittaker, DW DeSimone, unpublished data). Perhaps the most abundant and widely expressed integrins in early amphibian development are av (D Alfandari, CA Whittaker, DW DeSimone, T Darribere, unpublished data) and a5 [32*], the latter of which is located in both the somitic mesoderm and presumptive neural crest (TO Joos, F Meng, CA Whittaker, DW DeSimone, P Hausen, unpublished data). Similarly, a5 is highly expressed in early chick embryos but is downregulated at later stages of development

[331The distributions of amphibian a-subunit mRNAs at very early embryonic stages are generally non-overlapping. This raises the question of how these expression patterns are generated. One intriguing possibility is that integrin-gene expression is responsive to gradients of embryonic inducing factors or morphogens that have been shown to be important in axis formation and patterning [34]. Initial studies indicate that integrin a3, a4 and elf, mRNAs are upregulated in response to induction with activin-A, a member of the transforming growth factor-p family of growth factors [32*]. Additional studies will be needed to determine the extent to which morphogenic gradients influence integrin expression and/or functional activation in vim. In summary, integrin-subunit expression during development is dynamic and often position-specific. The descriptive data provide a good starting point for integrin functional analyses, which have recently begun in earnest in several systems.

lntegrin

function

in vertebrate

development

Although detailed information on integrin function in vertebrate embryos is limited, two general conclusions can be drawn from the studies published thus far. The

Adhesion

first is that integrins appear to play a number of important roles in morphogenesis and development [2*,3*,13]. The second is that studies of integrin function are complicated by the presence of redundant adhesive mechanisms (both integrin-dependent and independent) operating together in the embryo.

Antibody

blocking

and antisense

experiments

Most studies have utilized function-blocking antibodies to investigate the roles of integrins in developmental processes.For example, antibodies directed against the avian bt complex block myotube adhesion, fusion and muscle differentiation in vitro [35,36] and can be used to perturb neural crest cell migration [37], somitogenesis and vasculogenesis irt vivo [38,39]. The differentiation of murine myoblasts can be blocked by antibodies specific for integrin a4 or its counter-receptor, V-CAM-l [40]. A number of reports indicate that integrin-ECM interactions are also involved in neurite outgrowth [1,3*,27,41,42]. In a different approach, antisense experiments have been used to analyze integrin function in development. Retroviral expression of antisense Bt mRNAs in chick embryos inhibits neuroblast migration into the optic tectum and indicates a possible requirement for integrin-ECM interactions in maintaining the viability of these cells [43]. Lallier and Bronner-Fraser [44] used antisense oligodeoxynucleotides to block the synthesis of pt and various a-subunits in neural crest cells to show the involvement of at least three distinct receptors in the adhesion of these cells in vitro.

Cell-matrix

adhesion

in amphibian

gastrulation

Some of the most compelling in vivo functional analyses of cell-matrix interactions have been accomplished using amphibian embryos [13]. Boucaut and colleagues [45,46] used RGD peptide (single-letter code for amino acids) and anti-fibronectin antibody injections to demonstrate that mesodermal cell migration on fibronectin is required for gastrulation in the salamander Pleurodeles wu/t/. Similar results were obtained when antibodies directed against bt integrins were injected into these embryos [47]. Recently, it has been shown that antibodies directed against Xenopus fibronectin or B, integrins also block gastrulation in Xenopus [48]. The adhesion and migration of Xenopus mesodermal cells on fibronectin is a rapid and early response to mesoderm induction [49,50]. Prospective ectodermal cells cultured in the presence of activin-A, a potent mesoderm inducer, spread and migrate on fibronectin, whereas cells that have not been induced attach but remain rounded. Interestingly, this change in adhesive behavior on fibronectin is not accompanied by an increase in the synthesis of Bt integrins [49] or additional receptor at the cell surface (JW Ramos, DW DeSimone, unpublished data). These studies suggest that functional activation of pre-existing integrins may be responsible for the observed changes in

and matrix

in vertebrate

develoDment

DeSimone

adhesive behavior. Furthermore, the presence of a given integrin during development does not necessarily imply functional activity; therefore, ‘inside-out’ signalling (e.g. via second-messenger pathways) may play an important role in regulating integrin function in the embryo. Integrins also appear to be participants in the formation of fibronectin fibrils in the ECM that lines the roof of the blastocoel before gastrulation in amphibians. Intracellular injection of antibodies directed against the cytoplasmic domain of the pt subunit prevent fibronectin matrix assembly [51]. These experiments suggest that normal integrin-cytoskeletal interactions are required for fibril formation. This interpretation is supported by recent dominant-negative experiments in Xenopus. Overexpression of a chimera constructed from the ectodomain of viral hemagglutinin and the cytoplasmic domain of integrin pt blocks matrix assembly, presumably by competing with endogenous integrins for cytoskeletal components (DG Ransom, S Kateeshock. DW DeSimone, unpublished data).

Gene targeting

experiments

in mice

Gene targeting by homologous recombination has recently been used to disrupt both fibronectin [52”] and the a& receptor for fibronectin in mice [53**]. Surprisingly, although both null mutants show defects in mesodermally derived structures, gastrulation proceeds without obvious phenotypic defects. Comparisons of the fibronectin and a5 knockout phenotypes also provide a convincing demonstration of redundant adhesive mechanisms likely to be operating in the embryo. Although both conditions are embryonic lethal, a5 null mutants progress further than fibronectin deficient embryos (i.e. embryonic stage 10-l 1 versus 9-10). This indicates that receptors other than as& must be both present and sufficient to support at least some fibronectin-dependent adhesive functions. Can the mouse data be reconciled with the amphibian studies, which suggest an important role for fibronectins and integrins in gastrulation? One formal possibility is that amphibian and mammalian embryos gastrulate using fundamentally different molecular mechanisms. It is important to note, however, that the presence of mesodermal derivatives in an embryo (e.g. in mice lacking a5 or fibronectin) does not necessarily imply evidence of normal gastrulation, because although proper positioning of mesodermal structures is dependent on gastrulation, induction of mesodermal tissue is not. In fact, mice and frogs may share more similarities than differences in this regard, given that perturbation of gastrulation in Xenopus with anti-fibronectin antibodies does not prevent the formation of mesoderm as defined by the expression of the brarhyury gene, the presence of extensive mesodermal-cell movements, or the subsequent appearance of at least some mesodermally derived tissues (JW Ramos. DW DeSimone. unpublished data). Clearly, however, mesoderm moves through the primitive streak

749

750

Cell-to-cek

contact

and extracellular

matrix

in fibronectin-minus mice [51], indicatini that there is no absolute requirement for fibronectin in this process. Whether the extent, rate or ‘accuracy’ of this migration is perturbed in the absence of fibronectin is unclear, but such subtle effects may help explain many of the defects observed following gastrulation in these mice.

Extracellular Matrix. Edited by Cheresh DA, Mecham York: Academic Press; 1994:ll l-l 40. This review contains a compilation of the published literature integrin expression in a variety of developmental systems. 4.

Clukhova

5.

Hynes

Cell 6.

Conclusions

7.

Although the past several years have witnessed remarkable advances in our understanding of the ‘master switches’ and molecular mechanisms that are likely to control development at the level of the gene, subsequent cellular events involved in mediating morphogenetic change remain poorly understood. In one sense, integrin-dependent adhesive interactions may be viewed as ‘end points’ in a series of developmental instructions required to execute a given morphogenetic event. On the other hand, outside-in signalling via integrins may serve as an important mechanism whereby gene expression and differentiation are influenced by changes in the composition of embryonic ECMs. Although compelling patterns of integrin expression are now being described for a variety of vertebrate embryos, a central challenge in the years ahead will be to sort out the contributions of individual receptors and their ligands to the complex adhesive interactions that drive morphogenesis. Future advances undoubtedly will benefit l?om the combination of approaches and developmental systems currently under study.

Acknowledgements

1.

Hynes RO, Lander AD: Contact and Adhesive Specificities in the Associations, Migrations, and Targeting of Cells and Axons.

2. .

period

117:1183-1198.

Lallier ment.

TE, Hens In lntegrins:

MD,

DeSimone

Molecular

and

DW:

l&grins

Biological

in Develop Response

Curr

Y: Integrins: 1994.

Opin

Kirchhofer D, Languino LR, aa& lntegrins from Different ing Specificities.) Biol Chem

Cell

rhe Biological

to the

to

in Devel-

and Signaling by Integrins.

2: Signal Transduction Matrix: Cooperative

10.

in

Trends

by lntegrin Recep Processing of Extra-

Biol

1992,

Problems.

4:772-781.

Boca

Raton:

CRC

Mecham RP: Integrins: Molecular and Biologilo rhe Extracellular Matrix. New York: Academic

Ruoslahti

E, Pierschbacher

Cell Types 1990,

Show

MD:

Different

Bind-

265:615-618.

11.

Sastry SK, Horwitz AF: lntegrin Cytoplasmic Domains: Mediators of Cytoskeletal linkages and Extra- and Intracellular Transmembrane Signalling. Curr Opin Cell Biol 1993, 5:819-831.

12.

Wilcox

M,

Brown

N,

Piovant

M,

Smith

RJ, White

RA:

The

Drosophila Position-Specific Antigens Are a Family of Cell Surface Clycoprotein Complexes. FMBO / 1984, 3:2307-2313. 13.

DeSimone

opment:

DW: In&grin Function Perspectives from Studies

/n&grins: press. 14.

the Biological

Duband

JL, Rocher

in Early Vertebrate Develof Amphibian Embryos. In

Problems,

S, Chen

Edited

Wl,

by Takada

Yamada

KM,

Y. 1994:in

Thiery

JP: Cell

Adhesion and Migration in the Early Vertebrate Embryo: Location and Possible Role of the Putative Fibronectin Receptor Complex. I Cell Biol 1986, 102:160-l 78. 15.

Krotoski DM, Domingo C, Bronner-Fraser M: Distribution of a Putative Cell Surface Receptor for Fibronectin and Laminin in the Avian Embryo. 1 Cell Biol 1986, 103:1061-l 071.

16.

Cawantka

V, Ellinger-Ziegelbauer

P: &-lntegrin

H, Hausen

Ransom

DC,

Hens

Formed

Is a Plasma

Development

MD, DeSimone DW: lntegrin Expression in Embryos: cDNA Cloning and CharacterizaBl, b2, 83 and @6 Subunits. Dev Biol 1993,

Tarone G, Russo MA, Hirsch E, Odorisio L, Siracusa C: Expression of pl lntegrin

T, Altruda

Surface of Unfertilized 117:1369-l 375.

Development

Mouse

Oocvte.

F, Sileneo

.Complexes

on

t’;e

1993,

19.

DeSimone DW, Hynes RO: Xenopus faevis Integrins. Structural Conservation and Evolutionary Divergence of lntegrin fJ Subunits. J Biol Chem 1988, 263:5333-5340.

20.

Sutherland

ulation Mouse 21.

of Development and DifMatrix. Developmenr 1993,

Interesting and comprehensive review that concentrates on the roles of the extracellular matrix and integrins in regulating differentiation and development. 3. .

Werb

Cheresh DA, cal Response Press; 1994.

of

68:303-322.

Adams JC, Watt FM: Regulation ferentiation by the Extracellular

CH,

Extracellular Information.

Early Amphibian tion of Xenopus 160~265-275.

of particular interest, have been highlighted of special interest of outstanding interest

1992,

the annual

Damsky

tars for cellular

Signalling

9.

reading within

MA: Transmembrane 1992, 2:304-306.

Schwartz Cell Biol

Takada Press;

17.

Papers review, . ..

Cell

published as:

Integrins: Versatility, Modulation, Cell 1992, 69:l l-25.

0.

18.

and recommended

RO:

Adhesion.

related

l&grins

Maternal Protein that Is inserted into all Newly Membranes during Early Xenopus Embryogenesis. 1992, 115:595405.

Research in the author’s laboratory is supported by grants from the USYHS (HD3640Z). the American Cancer Society (CB93) and by a Pew Scholars Award in the Biomedical Sciences. The author also acknowledges the generous support of a Junior Faculty Research Award fi-om the American Cancer Society.

References

Thiery JP: Fibronectin and Cancer Biol 1993,4:241-249.

MA, Semin

opment.

RP. New

AE, Calarco

PC,

Damsky

of lntegrin Expression Embryo. Development

Blobel White

CP, Wolf&a JM: A PotenthI

Domain

in a Protein

TG,

Turck

1993, CW,

Developmental of Implantation 119: 1175-l 186. CH:

at Time

Mvles

DC,

Regin the

Primakoff

P,

Fusion Peptide a;ld an lntegrin Ligand Active in Sperm-Egg Fusion. Nature 1992,

3561248-252. 22. .

Muller

AH,

Gawantka

V, Ding

P: Maturation Inby Xenopus Oocyies and Pool. Mechanisms of De-

X, Hausen

duced Internalization of pl-In&grin Formation of the Maternal lntegrin

velopment 1993, 42177-88. . This report and I161 provide evidence for post- translational regulation of integrin expression at the egg cell surface. & integrin complexes, in-

Adhesion ternalized following the progesterone induced maturation of oocytes are stored in vesicles and re-inserted into newly formed membranes following fertilization. Although the Bt subunit continues to be synthesized throughout cleavage and blastula stages, the integrins appearing at the cell surface arise from the maternally stored complexes. 23.

24.

25.

Sutherland AE, Calarco PC, Damsky CH: tion of Cell Surface Extracellular Matrix Blastocyst Attachment and Outgrowth. 186:1331-1348.

Expression Receptors / Cell

and Funcin Mouse Biol 1988,

Fettri ML, Scherer SS, Nemni R, Kamholz J, Voeelbacher H, Scott MO, Canal N, Quaranta V, Wrabetz L: g4”lntegrin Exoression in Mvelinatinr Schwann Cells Is Polarized, Developmentally Regulated and Axonally Dependent. Dev&opmenr 1994, 120:12871301. Duband JL, pression of Myogenesis ment 1992,

Belkin AM, Syfrig J, Thiery JP, Koteliansky VE: Exal Integrin, a Laminin-Collagen Receptor, during and Neurogenesis in the Avian Embryo. Develop116:585-600.

26.

Neugebauer KM, Emmett CJ, Venstrom ronectin and Thrombospondin Promote growth: Developmental Regulation and ron 1991, 6:345-358.

KA, Reichardt LF: VitRetinal Neurite OutRole of Integrins. Neu-

27.

Boss+ B, Bossv-Wetzel E, Reichardt LF: Characterization lntegrin a8 Subunit: a New lntegrin beta l-Associated which Is Prominently Expressed on Axons and on Contact with Basal Laminae in Chick Embryos. EM60 182375-2385.

of the Subunit Cells in 1 1991,

20.

DeCurtis I, Ouaranta V, Tamura RN, Reichardt LF: Laminin Receptors in. the Retina: Sequence Analysis of the Chick lntegrin a6 Subunit. Evidence for Transcriptional and Posttranslational Regulation. 1 Cell Eio/ 1991, 113:405-416.

29.

Bronner-Fraser M, Artinger M, Muschler J, Horwitz velopmentally Regulated Expression of a6 lntegrin Embryos. Developmenf 1992, 115:197-211.

30.

DeCurtis I, Reichardt LF: Function and Spatial Distribution Developing Chick Retina of the Laminin Receptor a6Bl Its Isoforms. Developmenf 1993, 118:377-388.

31.

Stepp MA, Urry LA, Hynes RO: Expression mRNA and Protein and Fibronectin in the bryo. Cell Adhes Commun 1994, in press.

Whittaker CA, DeSimone DW: lntegrin a Subunit mRNAs Are Differentially Expressed in Early Xenopus Embryos. Developmenr 1993, 117:1239-1249. . a--, * Degenerate oligodeoxynucleotrde primers and IXK metnods were used to identify and characterize the temporal expression of six a-subunits, five of which are expressed by gastrulation in Xenopus. In situ hybridization reveals that the a3 subunit is expressed in the dorsal lip region of Spemann’s organizer. Evidence is also presented to show that the expression of at least some a-subunits is regulated during mesoderm induction. 33.

Muschler JL, Horwitz AF: Down-Regulation gl In&grin Fibronectin Receptor during opmenf 1991, 113:327-337.

34.

Green JB, New HV, Smith JC: Responses of Embryonic Xenopus Cells to Activin and FCF Are Separated by Multiple Dose Thresholds and Correspond to Distinct Axes of the Mesoderm. Cell 1992, 71:731-739.

35.

. 36.

of the Chicken a5 Development. Devel-

Neff NT, Lowrey C, Decker C, Tovar A, Damsky C, Buck C, Horwitz AF: A Monoclonal Antibody Detaches Embryonic Skeletal Muscle from Extracellular Matrices. 1 Cell Biol 1982, 95:654666. Menko AS, Boettiger D: Occupation of the Extracellular Receptor, Integrin, Is a Control Point for Myogenic tiation. Cell 1987, 5151-57.

37.

Bronner-Fraser M: An Antibody to a Receptor and Laminin Perturbs Cranial Neural Crest Wvo. Dev Eiol 1986, 117:528-536.

30.

Drake CJ, Davis LA, gl tntegrin-Mediated Shape and Behavior.

Hungerford Adhesions Dev Biol

Matrix Differen-

develoDment

DeSimone

751

Drake CJ, Davis LA, Little CD: Antibodies to gl-lntegrins Cause Alterations of Aortic Vasculogenesis, in viva Dev Dynamics 1992, 193:83-91.

40.

Rosen CD, Sanes JR, LaChance R, Cunningham JM, Roman J, Dean DC: Roles for the In&grin WA-4 and Its Counter Receptor VCAM-1 in Myogenesis. Cell 1992, 691107-l 119.

41.

Letourneau PC, Pech IV, Furcht LT: Growth Cone trix Components Depends Clioma Cells Does Not. J

42.

Tomaselli KJ, Neugebauer KM, Bixby JL, Lilien J, Reichardt LF: N-Cadherin and Integrins: Two Receptor Systems that Mediate Neuronal Process Outgrowth on Astrocyte Surfaces. Neuron 1988, 1:33-43.

43.

Galileo DS, Majors J, Horwitz AF, Sanes JR: Retrovirally Introduced Antisense lntegrin RNA Inhibits Neuroblast Migration in viva. Neuron 1992, 9~117-1131.

44.

Lallier T, Bronner-Fraser Attachment by lntegrin 1993, 259:692-695.

45.

Boucaut JC, Darribere T, Boulekbache H, Thiery JP: Prevention of Castrulation but Not Neurulation by Antibodies to Fibronectin in Amphibian Embryos. Nafure 1984, 307364-367.

46.

Boucaut JC, Darribere T, Poole TJ, Aoyama H, Yamada KM, Thiery JP: Biolo&allv Active Synthetic Peptides as Probes of Embryonic Dev
47.

Darribere T, Yamada KM, Johnson KE, Boucaut JC: The 148 kDa Fibronectin ReceptorComplex ts Required for Mesodermal Cell Adhesion during Gastndation in the Amphibian Pleurodeles waltlii. Dev Biof 1988, 126:182-194.

48.

Howard Xenopus

49.

Smith JC, Symes K, Hynes RO, DeSimone D: Mesoderm Induction and the Control of Gastrulation in Xenopus laevk the Roles of Fibronectin and Integrins. Development 1990, 108:229-238.

50.

Howard JE, Smith Types of Gastrulation MesodennInducing 1993, 43t37-48.

51.

Darribere T, Guida K. Lariava H. Johnson KE, Yamada KM, Thiery JP, Boucaut JC:‘/n v;‘vo Analyses of l&grin gl Subunit Function in Fibronectin Matrix Assembly. I Cell Biol 1990, 110:1813-1823.

of a4 integrin Early Chick Em-

32. .

in vertebrate

39.

AF: Dein Avian in and

and matrix

Rogers SL, Palm SL, McCarthy JB, Migration across Extracellular Maon Integrin, but Migration across Neurosci Res 1988, 21:28&297.

M: Inhibition of Neural Antisense Oligonucleotides.

JE, Hirst EM, Smith JC: Are gl In&grins Gastrulation?. Mech Dev 1992, 38109-l

JC:

Analysis Movement Factors in

Crest Cell Science

involved 19.

in

of Gastrulation: Different Are Induced by Different Xempus hevis. Mech Dev

52. ..

George EL, George+Labouesse EN, Patel-King RS, Rayburn H,Hynes RO: Defects in Mesoderm, Neural Tube and Vascular Development in Mouse Embryos Lacking Fibronectin. Development 1993, 11%X1079-1091. .-~ --. See J53”J. 53. ..

Yang JT, Rayburn H, Hynes RO: Embryonic Mesodermal Defects in a5 Integrin-Decicient Mice. Development 1993, 119:1093-1105. The authors of [52**] and this paper report the use of Rene targeting by homologous recombination to produce’homozygous mutant r&e l&kinR fibronectin [52**J and the integrin as subunit [53**]. They provide aninteresting example of the potential power of knockout approaches for studying adhesive interactions during development. They also point out many of the difficulties encountered in interpreting the contributions of single molecules to complex morphogenetic events that are likely to involve redundant adhesive mechanisms.

for Fibronectin Development in

JE, Little CD: Perturbation Results in Altered Somite 1992, 149:327-338.

of Cell

1IW olobv, USA.

I>eSimone, Uox

439,

University School

of Virginia. of Medicine,

I>epartment Charlottesville,

of VA

Cell

I%-

33908,