Role of the extracellular matrix in epithelial morphogenesis

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

Biochemical and mechanical extracellular matrix properties dictate mammary epithelial cell motility and assembly

Biochemical and mechanical cues of the extracellular matrix have been shown to play important roles in cell-matrix and cell-cell interactions. We have experimentally tested the combined influence of these cues to better understand cell motility, force generation, cell-cell interaction, and assembly in an in vitro breast cancer model. MCF-10A non-tumorigenic mammary epithelial cells were observed on surfaces with varying fibronectin ligand concentration and polyacrylamide gel rigidity. Our data show that cell velocity is biphasic in both matrix rigidity and adhesiveness. The maximum cell migration velocity occurs only at specific combination of substrate stiffness and ligand density. We found cell-cell interactions reduce migration velocity. However, the traction forces cells exert onto the substrate increase linearly with both cues, with cells in pairs exerting higher maximum tractions observed over single cells. A relationship between force and motility shows a maximum in single cell velocity not observed in cell pairs. Cell-cell adhesion becomes strongly favored on softer gels with elasticity ≤ 1250 Pascals (Pa), implying the existence of a compliance threshold that promotes cell-cell over cell-matrix adhesion. Finally on gels with stiffness similar to pre-malignant breast tissue, 400 Pa, cells undergo multicellular assembly and division into 3D spherical aggregates on a 2D surface.     

Local, three-dimensional strain measurements within largely deformed extracellular matrix constructs

The ability to create extracellular matrix (ECM) constructs that are mechanically and biochemically similar to those found in vivo and to understand how their properties affect cellular responses will drive the next generation of tissue engineering strategies. To date, many mechanisms by which cells biochemically communicate with the ECM are known. However the mechanisms by which mechanical information is transmitted between cells and their ECM remain to be elucidated. "Self-assembled" collagen matrices provide an in vitro-model system to study the mechanical behavior of ECM. To begin to understand how the ECM and the cells interact mechanically, the three-dimensional (3D) mechanical properties of the ECM must be quantified at the micro-(local) level in addition to information measured at the macro-(global) level. Here we describe an incremental digital volume correlation (IDVC) algorithm to quantify large (>0.05) 3D mechanical strains in the microstructure of 3D collagen matrices in response to applied mechanical loads. Strain measurements from the IDVC algorithm rely on 3D confocal images acquired from collagen matrices under applied mechanical loads. The accuracy and the precision of the IDVC algorithm was verified by comparing both image volumes collected in succession when no deformation was applied to the ECM (zero strain) and image volumes to which simulated deformations were applied in both ID and 3D (simulated strains). Results indicate that the IDVC algorithm can accurately and precisely determine the 3D strain state inside largely deformed collagen ECMs. Finally, the usefulness of the algorithm was demonstrated by measuring the microlevel 3D strain response of a collagen ECM loaded in tension.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

Role of the extracellular matrix in epithelial morphogenesis: A view from C. elegans

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

MT1-MMP-dependent neovessel formation within the confines of the three-dimensional extracellular matrix

During angiogenesis, endothelial cells initiate a tissue-invasive program within an interstitial matrix comprised largely of type I collagen. Extracellular matrix-degradative enzymes, including the matrix metalloproteinases (MMPs) MMP-2 and MMP-9, are thought to play key roles in angiogenesis by binding to docking sites on the cell surface after activation by plasmin- and/or membrane-type (MT) 1-MMP-dependent processes. To identify proteinases critical to neovessel formation, an ex vivo model of angiogenesis has been established wherein tissue explants from gene-targeted mice are embedded within a three-dimensional, type I collagen matrix. Unexpectedly, neither MMP-2, MMP-9, their cognate cell-surface receptors (i.e., beta3 integrin and CD44), nor plasminogen are essential for collagenolytic activity, endothelial cell invasion, or neovessel formation. Instead, the membrane-anchored MMP, MT1-MMP, confers endothelial cells with the ability to express invasive and tubulogenic activity in a collagen-rich milieu, in vitro or in vivo, where it plays an indispensable role in driving neovessel formation.     

Three-dimensional modeling of mechanical forces in the extracellular matrix during epithelial lumen formation

Mechanical interactions between cells and extracellular matrix (ECM) mediate epithelial cyst formation. This work relies on the combination of numerical modeling with live cell imaging, to piece together a novel nonintrusive method for determining three-dimensional (3D) mechanical forces caused by shape changes of a multicellular aggregate at the early stages of epithelial cyst formation. We analyzed the evolution of Madin-Darby canine kidney cells in 3D cultures using time-lapse microscopy, with type I collagen gel forming the ECM. The evolving 3D interface between the ECM and the cell aggregate was obtained from microscopy images, and the stress on the surface of a proliferating aggregate and in the surrounding ECM was calculated using the finite element method. The viscoelastic properties of the ECM (a needed input for the finite element method solver) were obtained through oscillatory shear flow experiments on a rheometer. For validation purpose, the forces exerted by an aggregate on a force-sensor array were measured and compared against the computational results.     

Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila

Many epithelia produce apical extracellular matrices (aECM) that are crucial for organ morphogenesis or physiology. Apical ECM formation relies on coordinated synthesis and modification of constituting components, to enable their subcellular targeting and extracellular assembly into functional matrices. The exoskeleton of Drosophila, the cuticle, is a stratified aECM containing ordered chitin polysaccharide lamellae and proteinaceous layers, and is suited for studies of molecular functions needed for aECM assembly. Here, we show that Drosophila mummy (mmy) mutants display defects in epithelial organisation in conjunction with aberrant deposition of the cuticle and an apical matrix needed for tracheal tubulogenesis. We find that mmy encodes the UDP-N-acetylglucosamine pyrophosphorylase, which catalyses the production of UDP-N-acetylglucosamine, an obligate substrate for chitin synthases as well as for protein glycosylation and GPI-anchor formation. Consequently, in mmy mutants GlcNAc-groups including chitin are severely reduced and modification and subcellular localisation of proteins designated for extracellular space is defective. Moreover, mmy expression is selectively upregulated in epithelia at the time they actively deposit aECM, and is altered by the moulting hormone 20-Hydroxyecdysone, suggesting that mmy is part of a developmental genetic programme to promote aECM formation.     

Micropatterned cell sheets with defined cell and extracellular matrix orientation exhibit anisotropic mechanical properties

For an arterial replacement graft to be effective, it must possess the appropriate strength in order to withstand long-term hemodynamic stress without failure, yet be compliant enough that the mismatch between the stiffness of the graft and the native vessel wall is minimized. The native vessel wall is a structurally complex tissue characterized by circumferentially oriented collagen fibers/cells and lamellar elastin. Besides the biochemical composition, the functional properties of the wall, including stiffness, depend critically on the structural organization. Therefore, it will be crucial to develop methods of producing tissues with defined structures in order to more closely mimic the properties of a native vessel. To this end, we sought to generate cell sheets that have specific ECM/cell organization using micropatterned polydimethylsiloxane (PDMS) substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges. Adult bovine aortic smooth muscle cells cultured on these substrates in the presence of ascorbic acid produced ECM-rich sheets several cell layers thick in which both the cells and ECM exhibited strong alignment in the direction of the micropattern. Moreover, mechanical testing revealed that the sheets exhibited mechanical anisotropy similar to that of native vessels with both the stiffness and strength being significantly larger in the direction of alignment, demonstrating that the microscale control of ECM organization results in functional changes in macroscale material behavior.     

Experimental prostate epithelial morphogenesis in response to stroma and three-dimensional matrigel culture.

To reproduce the structural and functional differentiation of human prostatic acini in vivo, prostatic epithelial and stromal cells derived from human primary cultures were cocultured in Matrigel. In the absence of stroma and serum, epithelial spheroids composed of solid masses of stratified and cuboidal cells formed. Outer cells of the spheroid expressed cytokeratins 1, 5, 10, and 14, whereas the inner cells expressed cytokeratin 18. The addition of 2% serum induced formation of a lumen surrounded by a layer of one or two cuboidal and columnar epithelial cells. The further addition of stromal cultures, dihydrotestosterone, and estrogen induced polarization of the epithelium and increased spheroid-forming efficiency. Epithelium expressed either cytokeratin 18 alone or additionally cytokeratins 1, 5, 14, and 10. All spheroid epithelium expressed prostate-specific antigen and prostate-specific membrane antigen. Androgen receptor was only detected in the presence of stroma, serum, and hormones. Thus, development of a functional and morphologically correct prostate gland in vitro is dependent on extracellular matrix, steroid hormones, and factors from stromal cells and serum.     

Molecular assembly and mechanical properties of the extracellular matrix: A fibrous protein perspective

The extracellular matrix is an integral and dynamic component of all tissues. Macromolecular compositions and structural architectures of the matrix are tissue-specific and typically are strongly influenced by the magnitude and direction of biomechanical forces experienced as part of normal tissue function. Fibrous extracellular networks of collagen and elastin provide the dominant response to tissue mechanical forces. These matrix proteins enable tissues to withstand high tensile and repetitive stresses without plastic deformation or rupture. Here we provide an overview of the hierarchical molecular and supramolecular assembly of collagens and elastic fibers, and review their capacity for mechanical behavior in response to force. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Expression of lacunin, a large multidomain extracellular matrix protein, accompanies morphogenesis of epithelial monolayers in Manduca sexta.

Morphogenesis is a complex process operating at several levels of organization--organism, tissues, cells, and molecules. Complex interactions occur between and within these levels. Many of the molecules that mediate these interactions are predictably turning out to be large multidomain proteins. Here we describe one such novel protein associated with remodeling of epithelial monolayers in embryos and developing wings of the moth Manduca sexta. On the basis of its sequence and its expression pattern along lacunae of developing wings, we propose the name lacunin for this extracellular matrix protein that contains nine different types of domains, most of which are present in multiple copies. These include domains of various types: Kunitz proteinase inhibitors, thrombospondin type I, immunoglobulin-like, and several newly defined domains of unknown function (PAL, PLAC, and lagrin domains). This rich patchwork of distinct domains probably exerts multiple effects on a variety of cell behaviors associated with the complex phenomenon of epithelial morphogenesis.     

Laminin-rich extracellular matrix association with mammary epithelial cells suppresses Brca1 expression

Brca1 mRNA was detectable in female mouse mammary gland tissue from adult virgins, during pregnancy and early lactation. It was associated with phases of mammary epithelial cell proliferation and differentiation but the pattern of Brca1 expression was dissociable from that of a true differentiation marker, beta-casein, by virtue of its significant expression in the virgin gland and termination of its expression in early lactation. In a primary cell culture model, association of a laminin-rich extracellular matrix (ECM) with mammary epithelial cells was required for cell survival and cell differentiation and suppressed Brca1 expression in these cells. ECM-association may significantly contribute to the final restriction in Brca1 expression in the lactating gland in vivo. Interestingly, our results suggest that mammary epithelial cells undergo apoptosis both when expressing and when not expressing Brca1, depending on whether the dying cell populations had been terminally differentiated or not.     

Influence of tissue- and cell-scale extracellular matrix distribution on the mechanical properties of tissue-engineered cartilage

The insufficient load-bearing capacity of today’s tissue- engineered (TE) cartilage limits its clinical application. Generally, cartilage TE studies aim to increase the extracellular matrix (ECM) content, as this is thought to determine the load-bearing properties of the cartilage. However, there are apparent inconsistencies in the literature regarding the correlation between ECM content and mechanical properties of TE constructs. In addition to the amount of ECM, the spatial inhomogeneities in ECM distribution at the tissue scale as well as at the cell scale may affect the mechanical properties of TE cartilage. The relative importance of such structural inhomogeneities on mechanical behavior of TE cartilage is unknown. The aim of the present study was, therefore, to theoretically elucidate the influence of these inhomogeneities on the mechanical behavior of chondrocyte-agarose TE constructs. A validated non-linear fiber-reinforced poro-elastic swelling cartilage model that can accommodate for effects of collagen reinforcement and swelling by proteoglycans was used. At the tissue scale, ECM was gradually varied from predominantly localized in the periphery of the TE construct toward an ECM-rich inner core. The effect of these inhomogeneities in relation to the total amount of ECM was also evaluated. At the cell scale, ECM was gradually varied from localized in the pericellular area, toward equally distributed throughout the interterritorial area. Results from the tissue-scale model indicated that localization of ECM in either the construct periphery or in the inner core may reduce construct stiffness compared with that of constructs with homogeneous ECM. Such effects are more significant at high ECM amounts. At the cell scale, localization of ECM around the cells significantly reduced the overall stiffness, even at low ECM amounts. The compressive stiffness gradually increased when ECM distribution became more homogeneous and the osmotic swelling pressure in the interterritorial area increased. We conclude that for the same amount of ECM content in TE cartilage constructs, superior mechanical properties can be achieved with more homogeneous ECM distribution at both tissue and cell scale. Inhomogeneities at the cell scale are more important than those at the tissue scale.     

Adhesion between cells and extracellular matrix with special reference to hepatic stellate cell adhesion to three-dimensional collagen fibers

Hepatic stellate cells are located in the perisinusoidal space (space of Disse), and extend their dendritic, thin membranous processes and fine fibrillar processes into this space. The stellate cells coexist with a three-dimensional extracellular matrix (ECM) in the perisinusoidal space. In turn the three-dimensional structure of the ECM regulates the proliferation, morphology, and functions of the stellate cell. In this review, the morphology of sites of adhesion between hepatic stellate cells and extracellular matrix is described. Hepatic stellate cells cultured in polystyrene dishes spread well, whereas the cells cultured on or in type I collagen gel become slender and elongate their long cellular processes which adhere directly to the collagen fibers. Cells in type I collagen gel form a large number of adhesive structures, each adhesive area forming a face but not a point. Adhesion molecules, integrins, for the ECM are localized on the cell surface. Elongation of the cellular processes occurs via integrin-binding to type I collagen fibers. The signal transduction mechanism, including protein and phosphatidylinositol phosphorylation, is critical to induce and sustain the cellular processes. Information on the three-dimensional structures of ECM is transmitted via three-dimensional adhesive structures containing the integrins.