the evolution of multi - cellular eukaryotic organisms from single - celled ancestors was one of the most significant transitions in the evolution of life on earth .
it enabled the emergence of larger and more complex eukaryotes that could resist predation , evolve specialized tissues and higher order biological capacities , and colonize new environments .
multi - cellularity evolved independently in several eukaryotic lineages and , in terms of the number of cell types per organism , animals ( the metazoa ) include the most complex multi - cellular eukaryotes ( rokas , 2008 ) . a key mediator of metazoan multi - cellularity is the extracellular matrix ( ecm ) , a multi - component , proteinaceous network that bridges between cells , contributes to their spatial arrangements by binding cell - surface adhesion receptors , and supports cell survival , differentiation , and tissue organization ( hynes , 2009 ) .
the advantages of increased organism size for more efficient use of nutrients and escape from predation might have acted as selection pressures for the evolution of ecm , and increases in ocean oxygen levels around 850 million years ago likely provided a favorable environment for these changes ( rokas , 2008 ) . here , we discuss the adhesome of basal metazoa , the modern animals that provide a window on the earliest - evolving ecm components , and consider the expansion of adhesome complexity that occurred in the deuterostome / chordate lineage .
the pathway by which ecm components are secreted from cells is highly conserved in eukaryotes and clearly predates the metazoa ( dacks et al . , 2009 ) .
many ecm proteins are oligomeric ; the most prevalent oligomerization domains are the gxy motifs of collagens that assemble three polypeptides into a supercoiled triple helix , and the -helical heptad repeats that assemble ecm oligomers from trimers to pentamers .
both the gxy motif and heptad repeat are found throughout the domains of life and in viruses ( odgren et al . , 1996 ,
, the vwf_a domain , thrombospondin type 1 domain , or egf domain  are also of premetazoan origin ( whittaker and hynes , 2002 , tucker , 2004 , wouters et al . , 2005 ) .
thus , many constituent domains of ecm proteins predate the origin of animals . with the benefit of recent genome sequences and expressed sequence tag databases it is possible to assess the representation of adhesome components within the metazoa ( figure 1 ; supplemental tables s1s3 ; supplemental file 1 ) . by inference ,
proteins represented throughout the metazoa were present in the last common metazoan ancestor and so these studies give us insight into the core molecular machinery of the ecm .
ecm components that are conserved from sponges to human include the three clades of fibrillar collagens and basement membrane collagen iv ( figure 1 ) . for a gxy triple helix to be stable and assemble into higher order fibrils , additional protein domains and accessory proteins
the fibrillar collagen polypeptides of sponges contain all the expected domains , namely , a triple - helical domain and n- and c - terminal propeptide domains that align three procollagen polypeptides to nucleate the triple helix ( exposito et al . , 2008 , 2010 ) . sponges also encode the n- and c - propeptidases that enable higher order assembly of triple helices into fibrils .
correspondingly , collagen fibrils have been identified as macromolecular structures in sponges ( garrone et al . , 1975 , exposito et al . , 2010 ) .
of the four taxonomic groups of sponges , the homoscleromorpha also encode collagen iv and contain a basement membrane structure ( boute et al . , 1996 ) .
other key components of basement membranes , laminin and perlecan , are also present in sponges and are conserved throughout the metazoa ( figure 1 , table s1 ) .
fibrillin , that forms a separate microfibril system , is also universal ( reber - mller et al . , 1995 , ramirez and sakai , 2010 ) .
thus , three major structural systems of the ecm appear to have originated in the metazoan ancestor .
lower panels illustrate the complexity of ecm in tissues from nematostella ( asterisk indicates collagen - like fibrils ) or mouse ( * indicates collagen fibrils in longitudinal section ; + indicates collagen fibrils in cross - section ) . mouse dermis micrograph courtesy of douglas keene . surprisingly , ecm components such as agrin , known for its roles in synapses and at neuromuscular junctions ( wu et al . ,
2010 ) , along with thrombospondin ( tsp ) and sparc / osteonectin that have specialized roles in vertebrates , are also conserved in sponges ( figure 1 and supplemental table s1 ; bentley and adams , 2010 ) .
the functions of these proteins in basal metazoa are unknown ; however , their strong conservation suggests wide roles in the ecm . in mammals ,
tsps or sparc do not self - associate into fibrils , yet they affect the packing of collagen fibrils ( bornstein et al . , 2004 , halsz et al .
in addition , two major groups of ecm proteases , the matrix metalloproteinases ( mmp ) and the disintegrin and metalloproteinase with thrombospondin repeats proteases ( adamts ) , are both also conserved throughout the metazoa ( nicholson et al . , 2005 , fanjul - fernndez et al . , 2010 ;
thus , the core adhesome apparently includes multiple inputs for regulation or dynamic turnover of the higher order organization of collagen fibrils .
this is an important consideration for the design of synthetic , ecm - mimetic , three - dimensional environments .
in contrast , with the exceptions of perlecan and agrin , basal metazoa do not encode secreted proteoglycans equivalent to those of vertebrates ( observations from blast searches ) .
nevertheless , carbohydrate - based interactions contribute to tissue organization , as in the cell  cell cohesion of sponges ( bucior et al . , 2004 ) .
these functions might be conserved ; for example , a nonsulfated chondroitin of hydra stabilizes membrane tubulation during assembly of the stinging organelle of nematocytes .
this activity is similar to the function of chondroitin sulfate in perineuronal ecm nets of the mammalian cns ( yamada et al .
. further investigation of proteoglycan core proteins of basal metazoa by nongenomic approaches ( e.g. , targeted proteomics of glycosaminoglycan - substituted proteins ) is needed to assess the significance of proteoglycans in the core adhesome .
the attachment of ecm components to cell surfaces is also essential for ecm function and transmission of mechanical forces .
certain adhesion receptor families : integrins , syndecan , and glypican membrane proteoglycans and cd36 , are present in sponges and throughout the metazoa ( figure 1 and supplemental table s2 ) ( brower et al . , 1997 , hughes , 2001 , mueller et al . , 2004 ,
others [ e.g. , dystroglycan and discoidin domain receptor ( ddr ) , a receptor tyrosine kinase that binds collagens at the same site as sparc ( carafoli et al . , 2009 ) ] originated later within the metazoa ( supplemental table s2 ) .
the high conservation of genes encoding core ecm and adhesion receptors is especially striking for trichoplax adhaerens , a basal metazoan with four cell types in which a morphologically recognizable intercellular ecm has not been detected by transmission electron microscopy ( srivastava et al .
the high conservation of core adhesome components has raised the fascinating issue that their evolutionary origins need to be sought outside the metazoa ( king , 2004 , rokas , 2008 ) . on present data , ecm proteins and most of the associated adhesome arose through a major wave of innovation that occurred in conjunction with the origin of the metazoa ( figure 1 ) .
choanoflagellates are considered the closest outgroup to the metazoa ( baldauf , 2003 , king , 2004 ) and genome sequencing for the unicellular choanoflagellate monosiga brevicollis and the colonial salpingoeca rosetta has enabled the question of metazoan ecm origins to be pursued in more depth .
m. brevicollis and s. rosetta each encode two proteins that contain repeated gxy motifs and several other proteins that contain collagen c - propeptide  like domains ( king et al .
vital accessory proteins for collagen fibril assembly , collagen propeptidases and lysyl oxidase , are not encoded .
thus , a current model is that fibrillar collagens originated at the dawn of the metazoa by domain shuffling ( exposito et al . , 2008 ) .
similarly , although some of the individual domains are present ( e.g. , the laminin g domain ) , none of the core basement membrane components are encoded in the choanoflagellates examined to date ( king et al . , 2008 ) .
intriguingly , both choanoflagellates encode a fibrillin - like protein ( supplemental file 1 ) .
it will be of great interest to learn whether this protein has fibril - forming capacity .
a second intriguing molecule is usherin , which in humans has specialized roles in the retina as an ecm component at photoreceptor cell synapses and in the inner ear as a transmembrane component of the usher protein network ( reiners et al . , 2006 ) .
the conservation of usherin in cnidaria ( tucker , 2010 ) and the existence of homologues in choanoflagellates ( supplemental file 1 ) suggests that a fundamental role in cell interactions remains to be uncovered .
turning to the adhesion receptors , ddr and membrane - bound proteoglycans are not apparent outside the metazoa ( supplemental file 1 ) .
a cd36-like protein is evident in m. brevicollis , the opisthokont capsaspora owczarzaki and the slime mold dictyostelium discoideum ( supplemental file 1 ) .
mammalian cd36 binds collagen and thrombospondin-1 and also has roles in the internalization of long chain fatty acids , oxidized phospholipids , and lipid microparticles ( silverstein and febbriao , 2009 ) . in the sponge , cd36 contributes to the assembly of aquiferous channels that form a primitive circulation system ( mueller et al . , 2004 ) .
we speculate that cd36 evolved originally for nutritional functions , as in the uptake of prey or lipid materials , and later acquired ecm - binding capacities .
remarkably , thecamonas trahens ( a protist ) and c. owczarzaki both encode integrin  and  subunits as well as key components of intracellular integrin signaling ( seb - pedrs et al . , 2010 ) .
these data place the evolutionary debut of integrin heterodimers before the metazoan / fungal divergence ( considerably earlier than previous estimates ) and indicate lineage - specific losses of integrins in the fungi and choanoflagellates examined to date ( king et al . , 2008 , shalchian - tabrizi et al . , 2008 ,
the sib ( similar to integrin beta ) proteins of dictyostelium discoideum are intriguing for their similar function in substratum attachment , yet in structure these proteins are only similar to integrin beta by the presence of an extracellular vwf_a domain and npxy - dependent talin - binding capacity of the cytoplasmic domain ( cornillon et al . , 2006 , 2008 ) .
present data do not distinguish whether sibs and integrin  share a common ancestor or are of independent evolutionary origin .
metazoan integrins function in phagocytosis and cell - cell interactions as well as ecm attachment , and it is plausible that integrins in single - celled organisms have  pre - ecm  roles in signaling cytoskeletal reorganizations during prey capture and uptake .
if so , the protist integrins could be of great interest for studies of integrin structure and signaling activation mechanisms .
deuterostomes in general and vertebrates in particular have many elaborations and specializations of tissue ecm distinct from those of basal metazoa and protostomes .
these include novel splice variants ( e.g. , agrin ) , gene family expansions ( e.g. , the laminin , thrombospondin , integrin and adamts protease families ; tzu and marinkovich , 2008 , huhtala et al . , 2005 , nicholson et al . , 2005 , mckenzie et al . , 2006 ) , and the evolutionary origin of many novel ecm components [ e.g. , tenascin , fibronectin ( fn ) , ccn ( cyr61 , ctgf , nov ) proteins , and facit collagens ( fibril associated collagens with interrupted triple helices ) , ( tucker et al . , 2006 , huxley - jones et al . , 2007 , tucker and chiquet - ehrismann , 2009a , katsube et al . , 2009 ;
these innovations exemplify shuffling of preexisting domains into novel combinations ( e.g. , tenascin , ccn ) , or inclusion of novel domains ( e.g. , in fibronectin the fn - i domain is deuterostome - specific and the fn - ii domain is chordate - specific ) ( kawashima et al . , 2009 )
many adhesome components represented by single genes in invertebrate deuterostomes ( sea urchin , acorn worm , or sea squirt ) form a gene family in vertebrates , most likely due to the two rounds of genome - wide duplication that took place early in the vertebrate lineage ( dehal and boore 2005 ; huxley - jones et al . , 2007 ) ( figure 1 and supplemental file 1 ) .
frequently , as exemplified by syndecan and tenascin , even larger gene families are present in bony fish that underwent a third , fish - specific genome duplication ( meyer and van de peer , 2005 , chakravarti and adams , 2006 , tucker et al . , 2006 )
the subsequent evolution of the duplicated genes has involved subfunctionalization , such that the modern paralogues are expressed under control of different regulatory pathways in distinct cell - types or tissues ( e.g. , sun et al . , 2005 , tucker and chiquet - ehrismann , 2009b ) .
collectively , these molecular innovations and gene family expansions have resulted in many changes and additions to ecm network systems ( figure 1 ) .
collagen fibrils are of increased size and molecular complexity , due to presence of facit collagens and the repertoire of collagen - binding proteoglycans ( exposito et al . , 2010 ) .
the evolution of hyaluronan synthase has resulted in the presence of large ( 225 micron long ) , water - retaining hyaluronan / aggrecan polymers in the cartilage and brain ecm of vertebrates ( weigel and deangelis , 2007 , yoneda et al . ,
the predicted fn - like protein of ciona savignyi contains all three canonical domains ( fn - i , fn - ii , fn - iii ) but is unusual in having only one fn - i domain , lacking an rgd motif exposed for integrin - binding , and having immunoglobulin - like domains interspersed with the fn - iii domains ( tucker and chiquet - ehrismann , 2009a ) . for vertebrate fn ,
fibrillogenesis is a cell - dependent process , requiring integrin - binding and cell contractility in addition to intermolecular interactions between multiple fn - i domains in the n - terminal region ( mao and schwarzbauer , 2005 ) .
we hypothesize that ciona fn - like protein does not form a distinct fibril system , but would bind onto other ecm component such as fibrillin or tenascin , or cell - surface heparan sulfate proteoglycans .
although information on fn in the lamprey and shark is currently missing , it is clear that the fns of bony fish have all the expected molecular features for fibril assembly ( sun et al . , 2005 ) .
the early embryonic lethality of fn gene knockout mice demonstrates the vital importance of this fibril system in vertebrates ( george et al . , 1993 ) .
deuterostomes also exemplify many innovations or neofunctions of cell - ecm adhesion receptors ( figure 1 ; supplemental table s3 and supplemental file 1 ) . whereas rgd- and laminin - binding integrins exist in both protostomes and deuterostomes , major lineage - specific novelties in chordates include the collagen - binding i domain integrin  subunits and the  subunit clades ( hughes , 2001 , ewan et al .
although i domain  containing integrins are present in ciona intestinalis , their structural and functional characteristics indicate an independent evolutionary lineage ( ewan et al .
, 2005 , huhtala et al . , 2005 , tulla et al . , 2007 ) .
the major rgd - dependent , fn - binding integrin of vertebrates , 51 , appeared after the divergence of tunicates and before the divergence of bony fish ( ewan et al .
the hyaluronan receptor , cd44 , first appears in bony fish ( supplemental table s3 ) .
these examples illustrate how innovations , domain shuffling , and coevolutionary events expanded the possibilities for tissue specificity , microenvironmental complexity and subtlety of ecm - dependent intracellular signaling and gene expression .
these new potentialities contributed to major tissue and bodyplan innovations , including development of the notochord , the pharyngeal arches and neural crest cells ( zhang and cohn , 2006 , jeffery et al .
the origination of cartilaginous tissue based on collagen ii provided the framework for the development of endochondrial ossification in vertebrates ( wada , 2010 ) .
this is not to overlook the vast diversity of ecm that supports distinctive tissue- or species - specific functions .
elastic proteins have evolved in multiple lineages as counterparts to the relatively inflexible collagen fibers ( gosline et al . , 2002 ) .
the production of mineralized bio - composites with remarkable mechanical properties that are based on collagen fibrils occurs in many phyla ; the 3-m - long silica spicules of glass sponges and the byssus material of mussels are fascinating examples .
such forms of ecm are of increasing interest to bioengineers ( harrington et al . , 2010 , mller et al . , 2009 ) .
contrastingly , lineage - specific ecm components can fulfill major tissue functions , as exemplified by the role of lamprin in lamprey cartilage ( robson et al . , 1993 ) .
given the vast number of arthropod and mollusk species and the relatively low representation of these phyla from sequenced genomes , it is likely that the full extent of ecm diversity remains to be uncovered .
the evolution of metazoa can not be separated from the evolution of their ecm . ecm representation in modern
this viewpoint has been enabled by comparative genomics , but functional investigations in basal metazoa and choanoflagellates are now needed , complemented by proteomics to identify lineage - specific components .
knowledge of the networks and hierarchies by which tissue ecms are built is relevant to stem cell biology , tissue engineering , and many clinical conditions .
understanding the evolutionary history of the ecm is an important approach to deciphering these systems .
 we present a perspective on the molecular evolution of the extracellular matrix ( ecm ) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries . 
 ecm components do not function in isolation , and the biological ecm system or  
 adhesome  also depends on posttranslational processing enzymes , cell surface receptors , and extracellular proteases . 
 we focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage . 
 the analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa . 
 integrin , cd36 , and certain domains predate the metazoa , and some ecm - related proteins are identified in choanoflagellates as predicted sequences . 
 modern deuterostomes and vertebrates have many novelties and elaborations of ecm as a result of domain shuffling , domain innovations and gene family expansions . 
 knowledge of the evolution of metazoan ecm is important for understanding how it is built as a system , its roles in normal tissues and disease processes , and has relevance for tissue engineering , the development of artificial organs , and the goals of synthetic biology . 
INTRODUCTION
EXTRACELLULAR MATRIX ORIGINS
DEUTEROSTOME/CHORDATE/VERTEBRATE INNOVATIONS
VARIATION AND INNOVATION WITHIN THE ECM FRAMEWORK
CONCLUSION
Supplementary Material