role of the extracellular matrix in morphogenesis

Role of the extracellular matrix in morphogenesisHynda K Kleinman�, Deborah Philp and Matthew P Hoffman

The extracellular matrix is a complex, dynamic and critical

component of all tissues. It functions as a scaffold for tissue

morphogenesis, provides cues for cell proliferation and

differentiation, promotes the maintenance of differentiated

tissues and enhances the repair response after injury. Various

amounts and types of collagens, adhesion molecules,

proteoglycans, growth factors and cytokines or chemokines are

present in the tissue- and temporal-specific extracellular

matrices. Tissue morphogenesis is mediated by multiple

extracellular matrix components and by multiple active sites on

some of these components. Biologically active extracellular

matrix components may have use in tissue repair, regeneration

and engineering, and in programming stem cells for tissue

replacement.

AddressesCell Biology Section, CDBRB, National Institute of Dental and

Craniofacial Research, NIH, 30 Convent Drive, MSC 4370, Bethesda,

MD 20892, USA�e-mail: [email protected]

Current Opinion in Biotechnology 2003, 14:526–532

This review comes from a themed issue on

Tissue and cell engineering

Edited by Jeffrey Hubbell

0958-1669/$ – see front matter

� 2003 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2003.08.002

AbbreviationsECM extracellular matrixFGF fibroblast growth factor

HGF hepatocyte growth factor

VEGF vascular endothelial growth factor

IntroductionMost cells in multicellular organisms are in contact with

an intricate meshwork of interacting extracellular col-

lagens, proteoglycans and adhesion proteins, as well as

growth factors, chemokines and cytokines. Together,

these components constitute the extracellular matrix

(ECM) [1,2]. Many ECM proteins form large families

with some 30 genes identified for collagens and 12

identified for laminins. Additional events, such as alter-

native splicing, proteolytic processing and glycosylation,

increase the number of unique structures and expand the

functions of these large, multifunctional molecules.

Major recent advances have been made in the identifica-

tion of new ECM molecules and in the determination

of the domain structures and molecular organization of

many components. The role of these components in

development has also been defined and progress has

been made in the identification of structurally and bio-

logically active sites. The amount and type of these

components vary considerably in different tissues and

usually differ within the same tissue depending on the

developmental stage.

ECMs not only provide support, tensile strength and

scaffolding for tissues and cells, but also serve as three-

dimensional substructures for cell adhesion and move-

ment, as a storage depot for growth factors, chemokines

and cytokines, and as signals for morphogenesis and

differentiation (Box 1). Cartilage ECM, which is highly

enriched in large proteoglycans and collagen II, has an

additional unique function in resisting compression. By

contrast, basement membrane matrices, which are

enriched in the glycoproteins laminin and entactin/nido-

gen and collagen IV with lesser amounts of proteoglycans

and growth factors, regulate cell polarity, separate differ-

ent tissue types, and have the specialized function of

acting as a molecular filter in the kidney.

With the identification of many new ECM family mem-

bers and elucidation of the atomic structures by X-ray

crystallography and nuclear magnetic resonance (NMR)

spectroscopy, advances have been made in defining the

domain structure and higher order architecture of extra-

cellular components and matrices [3��]. Such approaches

have begun to specify the intermolecular interactions

important in ECM assembly and biological activity.

For example, agrin was found to be important in acetyl-

choline receptor clustering in the neuromuscular junc-

tion and requires laminin-binding to localize to the

synaptic basement membrane [4]. The surface-exposed

residues on the g1 laminin chain directly interact with an

N-terminal agrin domain.

In general, the biological response involves multiple

cellular interactions with individual ECM molecules

and with multiple sites within the same molecule; the

response is also influenced by the biomechanical proper-

ties of the ECM. Multiple ECM signals transmitted via

diverse surface receptors are integrated by intracellular

signaling pathways to affect the cellular response. The

biological activity of the ECM has been studied in vitrousing either complex three-dimensional matrices (either

cell- or tissue-derived), gels of individual components

(collagen or laminin), proteolytic or recombinant frag-

ments of ECM components or peptides. Three-

dimensional matrices appear to mimic the in vivofunctions of the ECM, and the function of individual

components has been further studied with synthetic

526

Current Opinion in Biotechnology 2003, 14:526–532 www.current-opinion.com

peptides and recombinant fragments to identify targets

for tissue engineering and repair [5��,6�,7].

Laminin has been the most vigorously studied ECM

molecule using this approach and has been analyzed with

much success; more than 40 active sites for specific

functions have been defined. In addition, laminin and

other ECM molecules contain cryptic active sites that are

released or become available after proteolytic processing.

For example, cleavage of laminin during mammary gland

involution releases a fragment that binds to the epidermal

growth factor (EGF) receptor and increases cell migra-

tion. Similarly, the anti-angiogenic molecules endostatin

and tumstatin are degraded products of collagen XVIII

and collagen IV, respectively [8�,9]. These cryptic sites,

as well as the active sites, relate to in vivo functions in

development and during remodeling of tissues, as ev-

idenced by the mammary gland.

The biological responses to the ECM are regulated by

specific cell-surface receptors [10�]. Many different

receptors have been identified that transduce signals

to the cytoskeleton and nucleus. These include members

of the heterodimeric integrins, receptor tyrosine kinases

and phosphatases, immunogloblulin superfamily recep-

tors, dystroglycan, and cell-surface proteoglycans. It is

important to note that most of the biologically active

ECM molecules, including laminins, collagens, throm-

bospondin and fibronectin, contain multiple active sites,

often for different activities, and interact with different

receptors. For example, some 40 active sites have been

identified on laminin-1 and 20 different receptors have

been characterized.

The importance of ECM molecules in development has

been proven by in vivo studies using gene targeting

(Table 1) [11]. Mice lacking certain ECM component

genes die before birth, whereas others survive and exhibit

unique tissue phenotypes. The basement membrane, a

critical ECM component during early development, is

composed of the trimeric glycoprotein laminin, collagen

IV, perlecan, entactin/nidogen, and various growth factors

[12–14]. At the two-cell stage, the first ECM molecule,

the laminin g1 chain, is synthesized and serves as an

initial matrix scaffolding (organizer of the ECM). Mice

that lack the gene encoding laminin g1 fail to organize a

basement membrane matrix and die at embryonic day 5.5

and stem cells that lack this gene cannot form an epiblast

[11,15�]. It is thought that laminin self-polymerization

preceeds and is required for proper basement membrane

formation. Mice lacking the laminin a2 chain have a

muscular-dystrophy-like phenotype. More than half of

the human congenital muscular dystrophies are asso-

ciated with mutations or loss of the laminin a2 chain.

Mice lacking the laminin a3 chain have skin blisters

similar to certain bullous diseases. The comparison of

perlecan knockout mouse cartilage defects with those of

the human genetic disorders Schwartz–Jampel syndrome

and Dyssegmental dysplasia (Silverman–Handmaker

type), led to the discovery that these disorders were

caused by mutations in perlecan [16�]. It is likely that

additional genetic diseases will be found to result from

mutations in ECM molecules.

The large number of ECM molecules and their interac-

tions with each other define unique biological matrices

important in morphogenesis [17]. There is considerable

variation in the amount and type of specific components

present in ECMs in different tissues and at different

stages of development [1,2]. For example, the basement

membrane of the kidney glomerulus has different pro-

portions and types of collagens and laminins from that of

the skin basement membrane. Also, the specific laminin

isoform in each of these tissues varies during develop-

ment. In addition, there are different growth factors in

Box 1 Functions of the extracellular matrix.

StructuralScaffold

Tensile strength

Cushioning (cartilage)

Molecular filter (kidney)

Boundary between different tissue types

Storage depot (for growth factors, cytokines and chemokines)

Conformational blocking of cryptic sites

BiologicalCell polarity

Cell adhesionMorphogenesis/differentiation

Migration

Proliferation

Prevention of apoptosis

Table 1

Selected examples of extracellular matrix knockout micephenotypes.

Gene Phenotype

Laminin a2 Lethal, muscular dystrophy

Laminin a3 Lethal, skin blistering

Osteonectin/SPARC Cataracts, osteopenia

Vitronectin No phenotype

Fibronectin Lethal, mesodermal and cardiovascular

defects

Tenascin C CNS and hematopoietic defects

Thrombospondin-1 Increased vascularity, defects in lung,

pancreas

Entactin/nidogen Neurological defects

Collagen a1 (I) Lethal, vascular defects

Collagen a3 (IV) Lethal, renal failure, progressive

glomerulonephritis

Perlecan Lethal, cartilage defects

Biglycan Osteoporosis

CNS, central nervous system; SPARC, secreted protein acidic and

rich in cysteine.

The extracellular matrix in morphogenesis Kleinman, Philp and Hoffman 527

www.current-opinion.com Current Opinion in Biotechnology 2003, 14:526–532

each of these tissue ECMs. This review will focus on the

role of the ECM in a few organ systems, but it should be

noted that all cells are influenced by the ECM. Emphasis

will be placed on the role of the basement membrane and

its components, as this is an area of active discovery.

Stem cell differentiationEmbryonic stem cells have the capacity to proliferate and

to differentiate. In vivo, these cells are contacted by

various soluble and insoluble ECM components that

influence their differentiation [17]. In vitro studies have

shown that ECM components and growth factors regulate

the differentiation of stem cells. For example, a feeder

layer of fibroblasts is not required if a basement mem-

brane ECM (MatrigelTM, containing laminin-1, collagen

IV, perlecan, entactin and growth factors) and fibroblast-

conditioned medium are used to culture human embryo-

nic stem cells [18]. When human embryonic stem cells are

injected into mice they form all three germ layers. When

bone marrow-derived adult stem cells are cultured on

basement membrane MatrigelTM with fibroblast growth

factor 4 (FGF-4) and hepatocyte growth factor (HGF),

they have morphological, functional, and phenotypical

characteristics of hepatocytes [19]. Finally, monkey blas-

tocyst stem cells will differentiate in vitro into immature

gland-like structures on polymerized MatrigelTM or in the

presence of soluble MatrigelTM (Figures 1a,b) (D Philp,

unpublished results), but will differentiate in vivo into

cartilage when cultured with an extract of cartilage matrix

(Figure 1c). Tissue-specific matrices may therefore influ-

ence developmental lineage and may further promote

organ differentiation. Being able to ‘pre-program’ plur-

ipotent stem cells before injection into patients might

help to alleviate the need for isolation of committed cells

from tissues.

Vascular morphogenesisThe ECM is important in both angiogenesis (blood vessel

formation from pre-existing vessels) and vasculogenesis

(blood vessel formation de novo). During vasculogenesis,

endothelial cells interact with ECM components to

migrate, proliferate and to form three-dimensional tubu-

lar structures [6�,20,21,22�]. This tubular morphogenesis

requires integrin receptors for the ECM components,

signaling processes that regulate cell shape through

changes in the cytoskeleton, and cell–cell interactions that

control the shape of the tubules [23,24]. Surprisingly,

various ECMs will support endothelial cell tubule forma-

tion. In vitro, both collagen I gels and basement membrane

MatrigelTM induce capillary-like formation of endothelial

cells. The cells attach to both three-dimensional gels,

migrate, align to form tubules and generate lumens. The

process is slower, however, on collagen I than on

MatrigelTM, requiring several days, and the capillaries have

their basement membrane deposited intraluminally. Also,

on collagen I, changes in the cytoskeleton through

suppression of cyclic AMP and protein kinase A are

required for cell alignment and tubule formation [25].

Laminin is also important for capillary morphogenesis and

can act as a pro-angiogenic/vasculogenic molecule. Active

sites on laminin regulate capillary morphogenesis on

MatrigelTM either by direct activation or by competition

with the laminin in the MatrigelTM [26,27]. Various sites

have been identified in the N- and C-terminal globular

domains of laminin as well as in the coiled-coil region.

Several integrins have been identified as the cellular

receptors for these active sites, including a5b1, avb3,

a3b1, a6b1 and a2b1 [28]. Furthermore, on MatrigelTM,

laminin mediates the anti-angiogenic activity of endosta-

tin, a fragment of collagen XVIII, by binding directly to

Figure 1

In vitro and in vivo rhesus monkey embryonic stem-cell studies on MatrigelTM and cartilage extract (Cartrigel) substrates. (a) Embryonic stem cells

grown on MatrigelTM-coated culture dishes for 4 days. (b) Hematoxylin (H) and eosin (E) section of a rotating wall vessel (microgravity) stem-cell

culture in the presence of MatrigelTM for 21 days. (c) H and E section of tumor tissue formed 15 weeks post-injection of embryonic stem cells grown

with Cartrigel before injection.

528 Tissue and cell engineering


endostatin [29]. Thus, laminin has multiple and potent

active sites for regulating directly and indirectly capillary

morphogenesis.

Growth factor signaling pathways act in concert with

signals for the ECM and are important in capillary mor-

phogenesis. For example, sonic hedgehog, a morphogen

in many tissues, promotes capillary morphogenesis of

endothelial cells on MatrigelTM via phosphoinositide

3-kinase [30]. The Rho GTPases Cdc42 and Rac1 are

required for vacuole coalescence leading to lumen for-

mation, and RhoA stabilizes the capillary tube networks

[22�]. Vascular endothelial growth factor A (VEGF A) is

important in vascular branching [31], as mice engineered

to express VEGF A minus the heparin-binding domain

have fewer branched vessels. VEGF also increases the

expression of stromal-derived factor-1, which functions to

promote endothelial cell tube formation on MatrigelTM

[32]. Antibodies to stromal-derived factor-1 and to its

receptor, CXCR4, block tube formation in vitro and

new vessel formation in vivo. These are a few of the

many examples of growth factor–ECM interactions reg-

ulating vasculogenesis.

Organ morphogenesisMorphogenesis of the salivary gland and other organs,

including lung, breast, prostate, pancreas and kidney, is

dependent on the multiple activities of the ECM as well

as on soluble factors [33�,34��]. The embryonic salivary

gland is a classic model to study organ morphogenesis exvivo (Figure 2). The signals from the ECM to the salivary

gland epithelium are integrated with signals from growth

Figure 2

Morphogenesis of the salivary gland in ex vivo culture is dependent on the multiple activities of the ECM as well as on growth factors. Salivary

gland epithelial cell proliferation and morphogenesis are decreased with SU5402 treatment, an FGF receptor signaling inhibitor. Light micrographs of

(a) control and (b) SU5402-treated salivary glands cultured for 24 h show a decrease in the size of the epithelial buds and the overall amount ofbranching morphogenesis. In the two lower panels, proliferating cells are labeled with bromodeoxyuridine (BrdU) incorporation at 24 h and detected

using fluorescein isothiocyanate-labeled BrdU. The proliferating cell nuclei appear as green punctate spots. Peanut lectin-rhodamine stains the

epithelium red and antiperlecan (a heparan sulfate proteoglycan) stains mesenchyme and the basement membrane blue. (c) The control gland shows

BrdU labeling concentrated on the epithelial buds and at the periphery of the mesenchyme. (d) SU5402 treatment results in less BrdU labeling on

the epithelial buds. Proliferating cells are still apparent in the mesenchyme. The images in (c) and (d) are compressed stacks of optical sections

through the entire gland.



factors, particularly FGFs. Inhibition of FGF signaling

decreases epithelial cell proliferation and branching mor-

phogenesis (Figure 2). Gene profiling of mouse salivary

glands at different stages of development demonstrated

that certain ECM molecules are highly expressed early in

morphogenesis (fibronectin, fibrillin-1, etc.), whereas

others show constant expression at all stages of develop-

ment (e.g. laminin g1 and collagen a6(I)) [34��]. The

coordinated clefting and proliferation result in branching

morphogenesis. Additionally, fibronectin is important in

early cleft formation during branching morphogenesis in

the salivary gland and in other organs, including lung and

kidney [35]. Fibrillar fibronectin can induce cell–matrix

interactions in culture, suggesting that in vivo it converts

cell–cell to cell–matrix adhesion to initiate clefting of

the epithelium.

Salivary gland epithelium organ culture on MatrigelTM

mimics in vivo growth and branching morphogenesis;

growth factor signaling via phosphoinositide 3-kinase

has an important role in this process [36]. Specific anti-

bodies to laminin-1 will inhibit branching in the salivary

gland and in lung, breast and pancreas, while antibodies

that block the laminin–entactin/nidogen interaction inhi-

bit salivary and kidney branching morphogenesis [37].

Specific ablation of the entactin/nidogen-binding site on

the laminin g1 chain in mice results in lack of kidney

formation and impaired lung development, whereas abla-

tion of the entire entactin/nidogen molecule results in

viable animals with neurological abnormalities [38,39]. In

related studies, the interaction of another basement

membrane component, dystroglycan, also appears to be

important for epithelial morphogenesis in the salivary

gland and in other organs (lung and kidney), as antibodies

against dystroglycan that block binding to laminin inhibit

branching morphogenesis in culture [40]. Furthermore,

antibodies to laminin that are specific for the dystroglycan-

binding site also block branching. Ablation of the laminin

a5 chain results in normal lung branching morphogenesis

but abnormal lobular septation, demonstrating that

branching and septation are not linked and have different

laminin isoform requirements [41]. These studies illus-

trate the importance of laminin and its interactions with

other proteins in branching morphogenesis.

Cell lines derived from many organs have all been shown

to undergo tubulogenesis in the presence of growth

factors and a three-dimensional collagen-rich matrix.

On MatrigelTM, cultured mammary cells form gland-like

structures with polarized epithelia, tissue-specific gene

expression and reduced apoptosis [42]. For salivary gland

cells, the adhesion molecule laminin is very important

and certain peptides alone can mimic the morphological

differentiation of cultured cells into acinar-like structures

[43]. Such peptides will also block salivary gland organ

differentiation on MatrigelTM by competing with the

laminin in the matrix [37].

Conclusions and future directionsThe ECM has profound structural and biological effects

on developmental processes. Studies with in vitro cell

and organ culture models include the use of ECM pro-

teins, simple gels (laminin and collagen), complex gels

(MatrigelTM), and complex three-dimensional matrices

(matrices laid down by cells in culture or derived from

tissues). Based on the success of the collagen I and base-

ment membrane matrices in vitro and in vivo, it is likely that

additional ECMs will be developed that are tissue and

development stage specific. For example, endothelial cell-

specific basement membrane may be more potent than

MatrigelTM for promoting and maintaining capillary-like

structures. Likewise, an organ-specific matrix may pro-

mote functional organ development. Defining the ECM

interactions for a particular tissue or developmental stage

will allow for the development of specific biomimetics.

The identification of biologically active sites on mole-

cules will be important in tissue engineering for tissue

replacement and repair [44]. Already, laminin and fibro-

nectin peptides have been coupled to biodegradable

scaffolds and shown to be functionally active in vitro,but these have not been tested in vivo [45,46��,47].

Mimetics with more potent activity are likely to be

developed in the future. Because different ECM mole-

cules are important at different stages of development,

controlled release and spatial orientation are likely to be

important future considerations. The new field of stem-

cell biology will continue to be an active area of inves-

tigation with ECM components. Specific potential

benefits of understanding stem-cell–matrix interactions

are prolonged stem-cell survival, proliferation and dif-

ferentiation in vitro, as well as improved targeting,

survival and differentiation in vivo.

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� of special interest��of outstanding interest

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45. Patel N, Padera R, Sanders GHW, Gannizzaro SM, Davies MC,Langer R, Roberts CJ, Tendler SJB, Williams PM, Shakeesheff KM:Spatially controlled cell engineering on biodegradable polymersurfaces. FASEB J 1998, 12:1447-1454.

46.��

Mochizuki M, Kadoya Y, Wakabayasgi Y, Kato K,Okazaki I, Yamada M, Sato T, Sakairi N, Nishi N, Nomizu M:Laminin-1 peptide-conjugated chitosan membranes asa novel approach for cell engineering. FASEB J 2003:in press.

This paper shows the potent biological activity of several laminin peptidescoupled to a biodegradable scaffold.

47. Mann BK, West JL: Cell adhesion peptides alter smooth musclecell adhesion, proliferation, migration, and matrix proteinsynthesis on modified surface and in polymer scaffolds.J Biomed Mater Res 2002, 60:86-93.



role of the extracellular matrix in morphogenesis

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