Fibronectin (FN), a critical component of the extracellular matrix, is assembled into fibrils through a cell-mediated process. Heparan sulfate (HS) binds to the III module of FN and fibroblasts lacking this glycosaminoglycan exhibit reduced FN fibril assembly. To determine if HS depends on III to control FN assembly, we deleted both III alleles in NIH 3T3 cells using the CRISPR-Cas9 system. ΔIII cells assembled fewer FN matrix fibrils and less DOC-insoluble FN matrix than wild type cells. Little if any mutant FN matrix was assembled when purified ΔIII FN was provided to Chinese hamster ovary (CHO) cells, showing that lack of III caused the deficiency in assembly by ΔIII cells. Addition of heparin promoted the assembly of wild type FN by CHO cells, but it had no effect on the assembly of ΔIII FN. Furthermore, heparin binding stabilized the folded conformation of III and prevented it from self-associating with increasing temperature suggesting that stabilization by HS/heparin binding might regulate interactions between III and other FN modules. This effect would be particularly important at matrix assembly sites where our data show that ΔIII cells require both exogenous wild type FN and heparin in the culture medium to maximize assembly site formation. Our results show that heparin-promoted growth of fibril nucleation sites is dependent on III. We conclude that HS/heparin binds to III to promote and control the nucleation and development of FN fibrils.

The composition of the extracellular matrix (ECM) in nervous tissue plays an important role in controlling neuronal outgrowth and synapse development. Changes in both protein and glycosaminoglycan components of the ECM occur with tissue injury and may affect neuron growth. To investigate neuron responses to alterations in fibronectin (FN), a major component of the wound ECM, we grew cortical neurons on cell-derived decellularized matrices composed of wild type FN (FN) or of a mutant form of FN (FN) from which the III heparin-binding site had been deleted by CRISPR-Cas 9 gene editing. The most significant effect of the mutant FN was a reduction in dendrite outgrowth. Not only were dendrites shorter on mutant FN-collagen (COL) matrix than on wild type (FN-COL) matrix, but the number of dendrites and dendritic spines per neuron and the spine densities were also dramatically reduced on FN-COL matrices. Mass spectrometry and immunostaining identified a reduction in tenascin-C (TN-C) levels in the mutant matrix. TN-C is an ECM protein that binds to the III site of FN and modulates cell-matrix interactions and has been linked to dendrite development. We propose that TN-C binding to FN in the wound matrix supports dendrite and spine development during repair of damaged neural tissue. Overall, these results show that changes in ECM composition can dramatically affect elaboration of neurites and support the idea that the ECM microenvironment controls neuron morphology and connectivity.

The spinal cord has a poor ability to regenerate after an injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treating a spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface on the other side. When the cells are cultured on OPF via chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to that of the multichannel scaffold control, which is likely due to the greater number of axons growing across it. The immune cell number (microglia or hemopoietic cells: 50-120 cells/mm in all conditions), scarring (5-10% in all conditions), and ECM deposits (Laminin or Fibronectin: approximately 10-20% in all conditions) were equal in all conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery. This study provides a hydrogel scaffold construct that can be used in vitro for cell characterization or in vivo for future neuroprosthetics, devices, or cell and ECM delivery.

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly.

Fibrosis is an accumulation of extracellular matrix (ECM) proteins and fibers in a disordered fashion, which compromises cell and tissue functions. High glucose-induced fibrosis, a major pathophysiological change of diabetic retinopathy (DR), severely affects vision by compromising the retinal vasculature and ultimately disrupting retinal tissue organization. The retina is a highly vascularized, stratified tissue with multiple cell types organized into distinct layers. Chronically high blood glucose stimulates certain retinal cells to increase production and assembly of ECM proteins resulting in excess ECM deposition primarily in the capillary walls on the basal side of the endothelium. This subendothelial fibrosis of the capillaries is the earliest histological change in the diabetic retina and has been linked to the vascular dysfunction that underlies DR. Proteins that are not normally abundant in the capillary basement membrane (BM) matrix, such as the ECM protein fibronectin, are assembled in significant quantities, disrupting the architecture of the BM and altering its properties. Cell culture models have identified multiple mechanisms through which elevated glucose can stimulate fibronectin matrix assembly, including intracellular signaling pathways, alternative splicing, and non-enzymatic glycation of the ECM. The fibrotic subendothelial matrix alters cell adhesion and supports further accumulation of other ECM proteins leading to disruption of endothelial cell-cell junctions. We review evidence supporting the notion that these molecular changes in the ECM contribute to the pathogenesis of DR, including vascular leakage, loss of endothelial cells and pericytes, changes in blood flow, and neovascularization. We propose that the accumulation of ECM, especially fibronectin matrix, first around the vasculature and later in extravascular locations, plays a critical role in DR and vision loss. Strategies for DR prevention and treatment should consider the ECM a potential therapeutic target.

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration.

Fibronectin (Fn1) fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live-imaging and single-molecule localization microscopy data are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, in which nanodomains are spaced with an average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. Treatment with FUD, a peptide that binds the Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocked the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis. This article has an associated First Person interview with the first author of the paper.

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the function of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned, meshwork matrix formed matrix assembly sites around the cell periphery but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independently of cell elongation. We conclude the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites which form during the initial stages of cell-FN interactions.

BACKGROUND AND OBJECTIVE: Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment repair failure. However, the molecular pathogenesis remains incompletely understood. Determining the proteome of PVR will help to identify novel therapeutic targets. MATERIALS AND METHODS: Preretinal tissue samples, delaminated during surgery from six PVR cases and one idiopathic epiretinal membrane (ERM) were analyzed by mass spectrometry. Tandem mass spectra were extracted using the UniProt database, generating a list of 896 proteins, which were subjected to pathway set and fold-change (ERM vs PVR) analyses. RESULTS: Two pathways were enriched in PVR: extracellular matrix (ECM) organization and extracellular structure organization. A fold-change analysis comparing mean total spectral counts from PVR to an ERM control identified fibronectin, the ECM glycoprotein, as the protein most significantly elevated in PVR compared to ERM. CONCLUSION: These data identify pathwayskey to PVR progression, including thoseinvolved in cell-mediated ECM assembly and thus tractional force generation at the cellular level. .

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration.

Spinal cord injury (SCI) results in cell death, demyelination, and axonal loss. The spinal cord has a limited ability to regenerate, and current clinical therapies for SCI are not effective in helping promote neurologic recovery. We have developed a novel scaffold biomaterial that is fabricated from the biodegradable hydrogel oligo(poly(ethylene glycol)fumarate) (OPF). We have previously shown that positively charged OPF scaffolds (OPF+) in an open spaced, multichannel design can be loaded with Schwann cells to support axonal generation and functional recovery following SCI. We have now developed a hybrid OPF+ biomaterial that increases the surface area available for cell attachment and that contains an aligned microarchitecture and extracellular matrix (ECM) proteins to better support axonal regeneration. OPF+ was fabricated as 0.08 mm thick sheets containing 100 μm high polymer ridges that self-assemble into a spiral shape when hydrated. Laminin, fibronectin, or collagen I coating promoted neuron attachment and axonal outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and neurites aligned in the direction of the ridge. Schwann cells seeded on laminin coated OPF+ sheets aligned along the ridges over a 6-day period and could myelinate dorsal root ganglion neurons over 4 weeks. This novel scaffold design, with closer spaced ridges and Schwann cells, is a novel biomaterial construct to promote regeneration after SCI.

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here we describe the creation of a novel multi-layered scaffold with distinct fiber organizations - aligned to unaligned and dense to porous - to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations. This article is protected by copyright. All rights reserved.

Mesangial cells are the major extracellular matrix (ECM)-producing cells in the kidney glomerulus and, when exposed to elevated glucose levels, they up-regulate assembly of fibronectin (FN) and other ECM proteins. Increases in glucose concentration are known to alter gene expression; here we investigated the connection between increased ECM production and changes in gene expression in mesangial cells. Comparison of mesangial cells grown in normal or high glucose conditions by RNA-sequencing showed significant expression changes in over 6000 genes and, when grouped by KEGG pathway analysis, identified the ECM-receptor interaction and focal adhesion pathways among the top 5 upregulated pathways. Of note was the significant increase in expression of tenascin-C (TN-C), a known regulator of FN matrix assembly. Mouse TN-C has multiple isoforms due to alternative splicing of 6 FNIII repeat exons. In addition to the transcriptional increase with high glucose, exon inclusion via alternative splicing was also changed resulting in production of higher molecular weight isoforms of TN-C. Mesangial cells grown in normal glucose secreted small isoforms with 1-2 variable repeats included whereas in high glucose large isoforms estimated to include 5 repeats were secreted. Unlike the smaller isoforms, the larger TN-C was not detected in the FN matrix. This change in TN-C isoforms may affect the regulation of FN matrix assembly and in this way may contribute to increased ECM accumulation under high glucose conditions.

Diabetic nephropathy, a devastating consequence of diabetes mellitus, is characterized by the accumulation of extracellular matrix (ECM) that disrupts the kidney's filtration apparatus. Elevated glucose levels increase the deposition of a fibronectin (FN) matrix by mesangial cells, the primary matrix-producing cells of the kidney, and also increase acetyl-CoA leading to higher levels of lysine acetylation. Here, we investigated the connection between acetylation and the ECM and show that treatment of mesangial cells with deacetylase inhibitors increases both acetylation and FN matrix assembly compared to untreated cells. The matrix effects were linked to lysine 794 (K794) in the β1 integrin cytoplasmic domain based on studies of cells expressing acetylated (K794Q) and non-acetylated (K794R) mimetics. β1(K794Q) cells assembled significantly more FN matrix than wildtype β1 cells, while the non-acetylated β1(K794R) form was inactive. We show that mutation of K794 affects FN assembly by stimulating integrin-FN binding activity and cell contractility. Wildtype and β1(K794Q) cells but not β1(K794R) cells further increased their FN matrix when stimulated with deacetylase inhibitors indicating that increased acetylation on other proteins is required for maximum FN assembly. Thus, lysine acetylation provides a mechanism for glucose-induced fibrosis by up-regulation of FN matrix assembly.

A two-step synthesis is described for activating the surface of a fully hydrated hydrogel that is of interest as a possible scaffold for neural regeneration devices. The first step exploits the water content of the hydrogel and the hydrophobicity of the reaction solvent to create a thin oxide layer on the hydrogel surface using a common titanium or zirconium alkoxide. This layer serves as a reactive interface that enables rapid transformation of the hydrophilic, cell-nonadhesive hydrogel into either a highly hydrophobic surface by reaction with an alkylphosphonic acid, or into a cell-adhesive one using a (α,ω-diphosphono)alkane. Physically imprinting a mask ("debossing") into the hydrogel, followed by a two-step surface modification with a phosphonate, allows for patterning its surface to create spatially defined, cell-adhesive regions.

The extracellular matrix (ECM) proteins fibronectin (FN) and type I collagen (collagen I) are codistributed in many tissues, and collagens have been shown to depend on an FN matrix for fibrillogenesis. Microscopic analysis of a fibroblast ECM showed colocalization of procollagen I with FN fibrils, and proteolytic cleavage of procollagen to initiate fibril formation was significantly reduced with inhibition of FN matrix assembly. We examined the role of FN matrix in procollagen processing by the C-propeptide proteinase bone morphogenetic protein 1 (BMP-1). We found that BMP-1 binds to a cell-assembled ECM in a dose-dependent manner and that, like procollagen, BMP-1 colocalizes with FN fibrils in the matrix microenvironment. Binding studies with FN fragments identified a binding site in FN's primary heparin-binding domain. In solution, BMP-1-FN interactions and BMP-1 cleavage of procollagen I were both enhanced by the presence of heparin, suggesting a role for heparin in complex formation during proteolysis. Indeed, addition of heparin enhanced the rate of procollagen cleavage by matrix-bound BMP-1. Our results show that matrix localization of this proteinase facilitates the initiation of collagen assembly and suggest a model in which FN matrix and associated heparan sulfate act as a scaffold to organize enzyme and substrate for procollagen processing.

Spondylometaphyseal dysplasia (SMD) is characterized by developmental changes in long bones and vertebrae. It has large phenotypic diversity and multiple genetic causes, including a recent link to novel variants in the extracellular matrix (ECM) protein fibronectin (FN), a regulator of ECM assembly and key link between the ECM and proper cell function. We identified a patient with a unique SMD, similar to SMD with corner fractures. The patient has been followed over 19 years and presents with short stature, genu varum, kyphoscoliosis, and pectus carinatum. Radiography shows metaphyseal changes that resolved over time, vertebral changes, and capitular avascular necrosis. Whole exome sequencing identified a novel heterozygous FN1 variant (p.Cys97Trp). Using mass spectroscopy, mutant FN was detected in plasma and in culture medium of primary dermal fibroblasts isolated from the patient, but mutant protein was much less abundant than wild-type FN. Immunofluorescence and immunoblotting analyses show that mutant fibroblasts assemble significantly lower amounts of FN matrix than wild-type cells, and mutant FN was preferentially retained within the endoplasmic reticulum. This work highlights the importance of FN in skeletal development, and its potential role in the pathogenesis of a subtype of SMD.

BACKGROUND: Despite the widespread practice of using biologic scaffolds for soft tissue reinforcement over prosthetic implants, the impact of acellular dermal matrix (ADM) on surgical wound fluid biomarkers over the initial postoperative period after prosthetic breast reconstruction remains poorly understood. METHODS: Patients undergoing prosthetic breast reconstruction surgery where ADM was likely to be used were consented to have fluid samples collected from surgical drains after surgery. Sample collections occurred at an "Early" time point at 24 to 48 hours after surgery and then a "Late" time point approximately 1 to 2 weeks after surgery. All procedures were performed by a single surgeon. Acellular dermal matrix was placed when prosthetic coverage with autologous tissue could not be achieved. Laboratory analyses were performed in blinded fashion without the knowledge of whether the samples came from the ADM "Present" or "Not Present" group. RESULTS: Twenty-one patients were in the ADM Present group and 18 patients were in the Not Present group. Both groups showed similar demographics based on age and body mass index. Analyses for cell concentration, protein concentration, extracellular matrix protein levels, cell proliferation activity, and matrix metalloproteinase activity showed no significant differences between wound fluid samples from the 2 groups. CONCLUSIONS: The presence of ADM does not appear to significantly impact wound biomarkers in prosthetic breast reconstruction. The current study provides useful data regarding the impact of ADM on surgical wound fluid during the initial postoperative period, laying important groundwork for more extensive future studies on the impact of biologic scaffolds on wound biology.

A major challenge of tissue engineering is to generate materials that combine bioactivity with stability in a form that captures the robust nature of native tissues. Here we describe a procedure to fabricate a novel hybrid extracellular matrix (ECM)-synthetic scaffold biomaterial by cell-mediated deposition of ECM within an electrospun fiber mat. Synthetic polymer fiber mats were fabricated using poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) co-spun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. PEG removal increased the overall mat porosity and produced a mat with a layered structure that could be peeled into separate sheets of about 50 μm in thickness. Individual layers had pore sizes and wettability that facilitated cell infiltration over the depth of the scaffold. Confocal microscopy showed the formation of a highly interpenetrated network of cells, fibronectin fibrils, and synthetic fibers mimicking a complex ECM as observed within tissues. Decellularization did not perturb the structure of the matrix or the fiber mat. The resulting hybrid ECM-scaffold promoted cell adhesion and spreading and stimulated new ECM assembly by stem cells and tumor cells. These results identify a new technique for fabricating highly porous synthetic fibrous scaffolds and an approach to supplement them with natural biomimetic cues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2017.

Retinal fibrosis, characterized by dysregulation of extracellular matrix (ECM) protein deposition by retinal endothelial cells, pigment epithelial cells, and other resident cell-types, is a unifying feature of several common retinal diseases. Fibronectin is an early constituent of newly deposited ECM and serves as a template for assembly of other ECM proteins, including collagens. Under physiologic conditions, fibronectin is found in all layers of Bruch's membrane. Proliferative vitreoretinopathy (PVR), a complication of retinal surgery, is characterized by ECM accumulation. Among the earliest histologic manifestations of diabetic retinopathy (DR) is capillary basement membrane thickening, which occurs due to perturbations in ECM homeostasis. Neovascularization, the hallmark of late stage DR as well as exudative age-related macular degeneration (AMD), involves ECM assembly as a scaffold for the aberrant new vessel architecture. Rodent models of retinal injury demonstrate a key role for fibronectin in complications characteristic of PVR, including retinal detachment. In mouse models of DR, reducing fibronectin gene expression has been shown to arrest the accumulation of ECM in the capillary basement membrane. Alterations in matrix metalloproteinase activity thought to be important in the pathogenesis of AMD impact the turnover of fibronectin matrix as well as collagens. Growth factors involved in PVR, AMD, and DR, such as PDGF and TGFβ, are known to stimulate fibronectin matrix assembly. A deeper understanding of how pathologic ECM deposition contributes to disease progression may help to identify novel targets for therapeutic intervention.

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM.

During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12-14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Tissue formation and cell differentiation depend on a properly assembled extracellular matrix (ECM). Fibronectin is a key constituent of the pericellular ECM, forming essential connections between cell surface integrin receptors and structural components of the ECM. Recent studies using vertebrate models, conditional gene knockouts, tissue explants, and cell culture systems have identified developmental processes that depend on fibronectin and its receptor α5β1 integrin. We describe requirements for fibronectin matrix in the cardiovascular system, somite and precartilage development, and epithelial-mesenchymal transition. Information about molecular mechanisms shows the importance of fibronectin and integrins during tissue morphogenesis and cell differentiation, as well as their cooperation with growth factors to mediate changes in cell behaviors.

The G protein-coupled estrogen receptor-1, GPER-1, coordinates fibronectin (FN) matrix assembly and release of heparan-bound epidermal growth factor (HB-EGF). This mechanism of action results in the recruitment of FN-engaged integrin α5β1 to fibrillar adhesions and the formation of integrin α5β1-Shc adaptor protein complexes. Here, we show that GPER-1 stimulation of murine 4 T1 or human SKBR3 breast cancer cells with 17β-estradiol (E2β) promotes the formation of focal adhesions and actin stress fibers and results in increased cellular adhesion and haptotaxis on FN, but not collagen. These actions are also induced by the xenoestrogen, bisphenol A, and the estrogen receptor (ER) antagonist, ICI 182, 780, but not the inactive stereoisomer, 17α-estradiol (E2α). In addition, we show that GPER-1 stimulation of breast cancer cells allows for FN-dependent, anchorage-independent growth and FN fibril formation in "hanging drop" assays, indicating that these GPER-1-mediated actions occur independently of adhesion to solid substrata. Stable expression of Shc mutant Y317F lacking its primary tyrosyl phosphorylation site disrupts E2β-induced focal adhesion and actin stress fiber formation and abolishes E2β-enhanced haptotaxis on FN and anchorage-dependent growth. Collectively, these data demonstrate that E2β action via GPER-1 enhances cellular adhesivity and FN matrix assembly and allows for anchorage-independent growth, cellular events that may allow for cellular survival, and tumor progression.

The filtration unit of the kidney is the glomerulus, a capillary network supported by mesangial cells and extracellular matrix (ECM). Glomerular function is compromised in diabetic nephropathy (DN) by uncontrolled buildup of ECM, especially type IV collagen, which progressively occludes the capillaries. Increased levels of the ECM protein fibronectin (FN) are also present; however, its role in DN is unknown. Mesangial cells cultured under high glucose conditions provide a model system for studying the effect of elevated glucose on deposition of FN and collagen IV. Imaging of mesangial cell cultures and analysis of detergent-insoluble matrix show that, under high glucose conditions, mesangial cells assembled significantly more FN matrix, independent of FN protein levels. High glucose conditions induced protein kinase C-dependent β1 integrin activation, and FN assembly in normal glucose was increased by stimulation of integrin activity with Mn(2+). Collagen IV incorporation into the matrix was also increased under high glucose conditions and colocalized with FN fibrils. An inhibitor of FN matrix assembly prevented collagen IV deposition, demonstrating dependence of collagen IV on FN matrix. We conclude that high glucose induces FN assembly, which contributes to collagen IV accumulation. Enhanced assembly of FN might facilitate dysregulated ECM accumulation in DN.

In the mammary gland, the stromal extracellular matrix (ECM) undergoes dramatic changes during development and in tumorigenesis. For example, normal adult breast tissue is largely devoid of the ECM protein fibronectin (FN) whereas high FN levels have been detected in the stroma of breast tumors. FN is an established marker for epithelial-mesenchymal transition (EMT), which occurs during development and has been linked to cancer. During EMT, epithelial cell adhesion switches from cell-cell contacts to mainly cell-ECM interactions, raising the possibility that FN may have a role in promoting this transition. Using MCF-10A mammary epithelial cells, we show that exposure to exogenous FN induces an EMT response including upregulation of the EMT markers FN, Snail, N-cadherin, vimentin, the matrix metalloprotease MMP2, α-smooth muscle actin and phospho-Smad2, as well as acquisition of cell migratory behavior. FN-induced EMT depends on Src kinase and extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase signaling but not on the immediate early gene EGR-1. FN initiates EMT under serum-free conditions; this response is partially reversed by a transforming growth factor (TGF)β-neutralizing antibody, suggesting that FN enhances the effect of endogenous TGFβ. EMT marker expression is upregulated in cells on a fragment of FN containing the integrin-binding domain but not other domains. Differences in gene expression between FN and Matrigel are maintained with addition of a subthreshold level of TGFβ1. Together, these results show that cells interacting with FN are primed to respond to TGFβ. The ability of FN to induce EMT shows an active role for the stromal ECM in this process and supports the notion that the increased levels of FN observed in breast tumors facilitate tumorigenesis.

Mesenchymal cell condensation is the initiating event in endochondral bone formation. Cell condensation is followed by differentiation into chondrocytes, which is accompanied by induction of chondrogenic gene expression. Gene mutations involved in chondrogenesis cause chondrodysplasias and other skeletal defects. Using mesenchymal stem cells (MSCs) in an in vitro chondrogenesis assay, we found that knockdown of the diastrophic dysplasia (DTD) sulfate transporter (DTDST, also known as SLC26A2), which is required for normal cartilage development, blocked cell condensation and caused a significant reduction in fibronectin matrix. Knockdown of fibronectin with small interfering RNAs (siRNAs) also blocked condensation. Fibrillar fibronectin matrix was detected prior to cell condensation, and its levels increased during and after condensation. Inhibition of fibronectin matrix assembly by use of the functional upstream domain (FUD) of adhesin F1 from Streptococcus pyogenes prevented cell condensation by MSCs and also by the chondrogenic cell line ATDC5. Our data show that cell condensation and induction of chondrogenesis depend on fibronectin matrix assembly and DTDST, and indicate that this transporter is required earlier in chondrogenesis than previously appreciated. They also raise the possibility that certain of the skeletal defects in DTD patients might derive from the link between DTDST, fibronectin matrix and condensation.

BACKGROUND: The mechanisms that govern directional changes in cell migration are poorly understood. The migratory paths of two distal tip cells (DTC) determine the U-shape of the C. elegans hermaphroditic gonad. The morphogenesis of this organ provides a model system to identify genes necessary for the DTCs to execute two stereotyped turns. RESULTS: Using candidate genes for RNAi knockdown in a DTC-specific strain, we identified two transcriptional regulators required for DTC turning: cbp-1, the CBP/p300 transcriptional coactivator homologue, and let-607, a CREBH transcription factor homologue. Further screening of potential target genes uncovered a network of integrin adhesion-related genes that have roles in turning and are dependent on cbp-1 and let-607 for expression. These genes include src-1/Src kinase, tln-1/talin, pat-2/α integrin and nmy-2, a nonmuscle myosin heavy chain. CONCLUSIONS: Transcriptional regulation by means of cbp-1 and let-607 is crucial for determining directional changes during DTC migration. These regulators coordinate a gene network that is necessary for integrin-mediated adhesion. Overall, these results suggest that directional changes in cell migration rely on the precise gene regulation of adhesion.

Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn(2+) treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn(2+) treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.

Templating of cell spreading and proliferation is described that yields confluent layers of cells aligned across an entire two-dimensional surface. The template is a reactive, two-component interface that is synthesized in three steps in nanometer thick, micron-scaled patterns on silicon and on several biomaterial polymers. In this method, a volatile zirconium alkoxide complex is first deposited at reduced pressure onto a surface pattern that is prepared by photolithography; the substrate is then heated to thermolyze the organic ligands to form surface-bound zirconium oxide patterns. The thickness of this oxide layer ranges from 10 to 70 nanometers, which is controlled by alkoxide complex deposition time. The oxide layer is treated with 1,4-butanediphosphonic acid to give a monolayer pattern whose composition and spatial conformity to the photolithographic mask are determined spectroscopically. NIH 3T3 fibroblasts and human bone marrow-derived mesenchymal stem cells attach and spread in alignment with the pattern without constraint by physical means or by arrays of cytophilic and cytophobic molecules. Cell alignment with the pattern is maintained as cells grow to form a confluent monolayer across the entire substrate surface.

Differentiation methods often rely exclusively on growth factors to direct mouse embryonic stem cell (ESC) fate, but the niche also contains fibrillar extracellular matrix (ECM) proteins, including fibronectin (FN) and laminin, which could also direct cell fate. Soluble differentiation factors are known to increase ECM expression, yet ECM's ability to direct ESC fate is not well understood. To address the extent to which these proteins regulate differentiation when assembled into a matrix, we examined mouse ESC embryoid bodies (EBs) and found that their ability to maintain pluripotency marker expression was impaired by soluble serum FN. EBs also showed a spatiotemporal correlation between expression of FN and GATA4, a marker of definitive endoderm (DE), and an inverse correlation between FN and Nanog, a pluripotency marker. Maintenance of mouse ESC pluripotency prevented fibrillar matrix production, but induction medium created lineage-specific ECM containing varying amounts of FN and laminin. Mouse ESC-derived matrix was unlike conventional fibroblast-derived matrix, which did not contain laminin. Naïve mouse ESCs plated onto ESC- and fibroblast-derived matrix exhibited composition-specific differentiation. With exogenously added laminin, fibroblast-derived matrix is more similar in composition to mouse ESC-derived matrix and lacks residual growth factors that mouse ESC matrix may contain. Naïve mouse ESCs in DE induction medium exhibited dose-dependent DE differentiation as a function of the amount of exogenous laminin in the matrix in an α3 integrin-dependent mechanism. These data imply that fibrillar FN is necessary for loss of pluripotency and that laminin within a FN matrix improves DE differentiation.

We report a robust strategy for conjugating mixtures of two or more protein domains to nonfouling polyurethane surfaces. In our strategy, the carbamate groups of polyurethane are reacted with zirconium alkoxide from the vapor phase to give a surface-bound oxide that serves as a chemical layer that can be used to bond organics to the polymer substrate. A hydroxyalkylphosphonate monolayer was synthesized on this layer, which was then used to covalently bind primary amine groups in protein domains using chloroformate-derived cross-linking. The effectiveness of this synthesis strategy was gauged by using an ELISA to measure competitive, covalent bonding of cell-binding (III(9-10)) and fibronectin-binding (III(1-2)) domains of the cell adhesion protein fibronectin. Cell adhesion, spreading, and fibronectin matrix assembly were examined on surfaces conjugated with single domains, a 1:1 surface mixture of III(1-2) and III(9-10), and a recombinant protein "duplex" containing both domains in one fusion protein. The mixture performed as well as or better than the other surfaces in these assays. Our surface activation strategy is amenable to a wide range of polymer substrates and free amino group-containing protein fragments. As such, this technique may be used to create biologically specific materials through the immobilization of specific protein groups or mixtures thereof on a substrate surface.

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.

In Caenorhabditis elegans gonad morphogenesis, the final U-shapes of the two hermaphrodite gonad arms are determined by migration of the distal tip cells (DTCs). These somatic cells migrate in opposite directions on the ventral basement membrane until specific extracellular cues induce turning from ventral to dorsal and then centripetally toward the midbody region on the dorsal basement membrane. To dissect the mechanism of DTC turning, we examined the role of a novel gene, F40F11.2/mig-38, whose depletion by RNAi results in failure of DTC turning so that DTCs continue their migration away from the midbody region. mig-38 is expressed in the gonad primordium, and expression continues throughout DTC migration where it acts cell-autonomously to control DTC turning. RNAi depletion of both mig-38 and ina-1, which encodes an integrin adhesion receptor, enhanced the loss of turning phenotype indicating a genetic interaction between these genes. Furthermore, the integrin-associated protein MIG-15/Nck-interacting kinase (NIK) works with MIG-38 to direct DTC turning as shown by mig-38 RNAi with the mig-15(rh80) hypomorph. These results indicate that MIG-38 enhances the role of MIG-15 in integrin-dependent DTC turning. Knockdown of talin, a protein that is important for integrin activation, causes the DTCs to stop migration prematurely. When both talin and MIG-38 were depleted by RNAi treatment, the premature stop phenotype was suppressed. This suppression effect was reversed upon additional depletion of MIG-15 or its binding partner NCK-1. These results suggest that both talin and the MIG-15/NCK-1 complex promote DTC motility and that MIG-38 may act as a negative regulator of the complex. We propose a model to explain the dual role of MIG-38 in motility and turning.

The extracellular matrix (ECM) is an intricate network of proteins that surrounds cells and has a central role in establishing an environment that is conducive to tissue-specific cell functions. In the case of stem cells, this environment is the stem cell niche, where ECM signals participate in cell fate decisions. In this Commentary, we describe how changes in ECM composition and mechanical properties can affect cell shape and stem cell differentiation. Using chondrogenic differentiation as a model, we examine the changes in the ECM that occur before and during mesenchymal stem cell differentiation. In particular, we focus on the main ECM protein fibronectin, its temporal expression pattern during chondrogenic differentiation, its potential effects on functions of differentiating chondrocytes, and how its interactions with other ECM components might affect cartilage development. Finally, we discuss data that support the possibility that the fibronectin matrix has an instructive role in directing cells through the condensation, proliferation and/or differentiation stages of cartilage formation.

Cell migration and morphogenesis are key events in tissue development and organogenesis. In Caenorhabditis elegans, the migratory path of the distal tip cells determines the morphology of the hermaphroditic gonad. The distal tip cells undergo a series of migratory phases interspersed with turns to form the gonad. A wide variety of genes have been identified as crucial to this process, from genes that encode components and modifiers of the extracellular matrix to signaling proteins and transcriptional regulators. The connections between extracellular and transmembrane protein functions and intracellular pathways are essential for distal tip cell migration, and the integration of this information governs gonad morphogenesis and determines gonad size and shape.

Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.

Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN-FN and cell-FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.

Traditional tissue regeneration approaches to activate cell behaviors on biomaterials rely on the use of extracellular-matrix-based or soluble growth-factor cues. In this article, a novel approach is highlighted to dynamically steer cellular phenomena such as cell motility based on nanoscale substratum features of biological ligands. Albumin-derived nanocarriers (ANCs) with variable nanoscale-size features are functionalized with fibronectin III9-10 matrix ligands, and their effects on primary human keratinocyte activation are investigated. The presentation of fibronectin fragments from ANCs significantly enhances cell migration as compared to free ligands at equivalent concentrations. Notably, cell migration is influenced by the size of the underlying ANCs even for variably sized ANCs covered in comparable levels of fibronectin fragment. For equivalent ligand concentrations, cell migration on the smaller-sized ANCs (30 and 50 nm) is significantly enhanced as compared to that on larger-sized ANCs (75 and 100 nm). In contrast, the enhancement of cell migration on nanocarriers is abolished by the use of immobilized, biofunctionalized ANCs, indicating that "dynamic" nanocarrier internalization events underlie the role of nanocarrier geometry on the differential regulation of cell migration kinetics. Uptake studies using fluorescent ANCs indicate that larger-sized ANCs cause delayed endocytic kinetics and hence could present barriers for internalization during the cell adhesion and motility processes. Motile cells exhibit diminished migration upon exposure to clathrin inhibitors, but not caveolin inhibitors, suggesting the role of clathrin-mediated endocytosis in facilitating cell migratory responsiveness to the nanocarriers. Overall, a monotonic relationship is found between the nanocarrier cytointernalization rate and the cell migration rate, suggesting the possibility of designing biointerfacial features for the dynamic control of cell migration. Thus, the functionalization of a mobile nanocarrier by a biorelevant ligand can be used to sensitize cellular motility activation to the adhesion ligands, and such nanocarrier interfaces can dynamically attune cell migration kinetics by modulating the uptake of the ligand-nanocarrier complex via nanocarrier size.

Cells within tissues are surrounded by fibrillar extracellular matrix (ECM) that supports cell adhesion via integrin receptors. The strength of cell interactions with fibrillar matrix and the effects of force on these interactions have not been quantified. To this end, we used a spinning disc device to apply radially increasing shear to human HT1080 fibrosarcoma cells attached to a cell-derived fibrillar fibronectin (FN) matrix. The shear required to detach 50% of HT1080 cells was eight times greater on a FN-coated, rigid glass substrate than on fibrillar FN matrix. Covalent crosslinking of the FN matrix increased its stiffness tenfold and produced a modest increase in shear detachment force for these cells. On FN-coated surfaces, cells detach by releasing interactions between alpha5beta1 integrin and FN. By contrast, cell detachment from fibrillar matrix occurred through a novel mechanism of fibril breakage, which left holes in the matrix visible by fluorescence microscopy. These results show that cells require less force to detach from fibrillar matrix than from FN adsorbed on glass and that detachment occurs through breaking fibrils instead of by release of integrin-matrix bonds. Thus, ECM fibril breakage is another molecular feature to consider when understanding cell and tissue homeostasis.

Fibronectin extracellular matrix is assembled and remodeled throughout embryogenesis and plays key roles in early vertebrate development. In this issue of Developmental Cell, Dzamba et al. reveal, through their studies of Xenopus embryos, a novel mechanism for regulating fibronectin matrix assembly through Wnt signaling and cadherin-mediated cell-cell adhesion.