NRF2 Shortage in Human Skin Fibroblasts Dysregulates Matrisome Gene Expression and Affects Collagen Fibrillogenesis.

NRF2 is a master regulator of the antioxidative response that was recently proposed as a potential regulator of extracellular matrix (ECM) gene expression. Fibroblasts are major ECM producers in all connective tissues, including the dermis. A better understanding of NRF2-mediated ECM regulation in skin fibroblasts is thus of great interest for skin homeostasis maintenance and aging protection. In this study, we investigate the impact of NRF2 downregulation on matrisome gene expression and ECM deposits in human primary dermal fibroblasts. RNA-sequencing‒based transcriptome analysis of NRF2 silenced dermal fibroblasts shows that ECM genes are the most regulated gene sets, highlighting the relevance of the NRF2-mediated matrisome program in these cells. Using complementary light and electron microscopy methods, we show that NRF2 deprivation in dermal fibroblasts results in reduced collagen I biosynthesis and impacts collagen fibril deposition. Moreover, we identify ZNF469, a putative transcriptional regulator of collagen biosynthesis, as a target of NRF2. Both ZNF469 silenced fibroblasts and fibroblasts derived from Brittle Corneal Syndrome patients carrying variants in ZNF469 gene show reduced collagen I gene expression. Our study shows that NRF2 orchestrates matrisome expression in human skin fibroblasts through direct or indirect transcriptional mechanisms that could be prioritized to target dermal ECM homeostasis in health and disease.

Lack of the myotendinous junction marker col22a1 results in posture and locomotion disabilities in zebrafish.

The myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

A dynamic and mosaic basement membrane controls cell intercalation in Drosophila ovaries.

Basement membranes (BM) are extracellular matrices assembled into complex and highly organized networks essential for organ morphogenesis and function. However, little is known about the tissue origin of BM components and their dynamics in vivo Here, we unravel the assembly and role of the BM main component, Collagen type IV (ColIV), in Drosophila ovarian stalk morphogenesis. Stalks are short strings of cells assembled through cell intercalation that link adjacent follicles and maintain ovarian integrity. We show that stalk ColIV has multiple origins and is assembled following a regulated pattern leading to a unique BM organisation. Absence of ColIV leads to follicle fusion, as observed upon ablation of stalk cells. ColIV and integrins are both required to trigger cell intercalation and maintain mechanically strong cell-cell attachment within the stalk. These results show how the dynamic assembly of a mosaic BM controls complex tissue morphogenesis and integrity.

Stiffness measurement is a biomarker of skin ageing in vivo.

Human skin is particularly vulnerable to age-related deterioration and undergoes profound structural and functional changes, reflected in the external skin appearance. Skin ageing is characterized by features such as wrinkling or loss of elasticity. Even if research advances have been done concerning the molecular mechanisms that underlie these changes, very few studies have been conducted concerning the structure stiffness of the skin organ as a whole. In this study, we showed, thanks to human skin reconstructs and the Japanese Medaka fish model, that biomechanics is a new biomarker of skin ageing. We revealed that global stiffness measurement by Atomic Force Microscopy, since modulated through ageing in these models, can be a new biomarker of skin ageing, and reflects the profound reorganization of the dermis extracellular matrix, as shown by Transmission Electron Microscopy. Moreover, our data unveiled that the Japanese Medaka fish could represent a highly relevant integrated model to study skin ageing in vivo.

Scavenger Receptor Cysteine-Rich domains of Lysyl Oxidase-Like2 regulate endothelial ECM and angiogenesis through non-catalytic scaffolding mechanisms.

Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic cross-linking activity.

Gene profile of zebrafish fin regeneration offers clues to kinetics, organization and biomechanics of basement membrane.

How some animals regenerate missing body parts is not well understood. Taking advantage of the zebrafish caudal fin model, we performed a global unbiased time-course transcriptomic analysis of fin regeneration. Biostatistics analyses identified extracellular matrix (ECM) as the most enriched gene sets. Basement membranes (BMs) are specialized ECM structures that provide tissues with structural cohesion and serve as a major extracellular signaling platform. While the embryonic formation of BM has been extensively investigated, its regeneration in adults remains poorly studied. We therefore focused on BM gene expression kinetics and showed that it recapitulates many aspects of development. As such, the re-expression of the embryonic col14a1a gene indicated that col14a1a is part of the regeneration-specific program. We showed that laminins and col14a1a genes display similar kinetics and that the corresponding proteins are spatially and temporally controlled during regeneration. Analysis of our CRISPR/Cas9-mediated col14a1a knockout fish showed that collagen XIV-A contributes to timely deposition of laminins. As changes in ECM organization can affect tissue mechanical properties, we analyzed the biomechanics of col14a1a-/- regenerative BM using atomic force microscopy (AFM). Our data revealed a thinner BM accompanied by a substantial increase of the stiffness when compared to controls. Further AFM 3D-reconstructions showed that BM is organized as a checkerboard made of alternation of soft and rigid regions that is compromised in mutants leading to a more compact structure. We conclude that collagen XIV-A transiently acts as a molecular spacer responsible for BM structure and biomechanics possibly by helping laminins integration within regenerative BM.

Fishing for collagen function: About development, regeneration and disease.

Collagens are the most abundant vertebrate extracellular matrix proteins. They form a superfamily of 28 members that show a remarkable diversity in molecular and supramolecular organization, tissue distribution and function and mutations in collagen genes result in a wide range of inherited connective tissue diseases. In the recent years, unexpected and very diverse regulatory and mechanical collagen functions have been reported. But the structural and functional landscape of the collagen superfamily is still far from being complete. Zebrafish has emerged over the last decades as a powerful model to interrogate gene function and there are numerous advantages of using zebrafish for collagen research, including recent advances in genome editing technologies and the characterization of the zebrafish matrisome. One can confidently predict that zebrafish will rapidly become a popular vertebrate model to investigate the role of collagens in development, disease and regeneration as discussed in this chapter.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17ß-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

Upregulation of bone morphogenetic protein-1/mammalian tolloid and procollagen C-proteinase enhancer-1 in corneal scarring.

PURPOSE: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. METHODS: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. RESULTS: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. CONCLUSIONS: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction.

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7ß1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

CCM1-ICAP-1 complex controls ß1 integrin-dependent endothelial contractility and fibronectin remodeling.

The endothelial CCM complex regulates blood vessel stability and permeability. Loss-of-function mutations in CCM genes are responsible for human cerebral cavernous malformations (CCMs), which are characterized by clusters of hemorrhagic dilated capillaries composed of endothelium lacking mural cells and altered sub-endothelial extracellular matrix (ECM). Association of the CCM1/2 complex with ICAP-1, an inhibitor of ß1 integrin, prompted us to investigate whether the CCM complex interferes with integrin signaling. We demonstrate that CCM1/2 loss resulted in ICAP-1 destabilization, which increased ß1 integrin activation and led to increased RhoA-dependent contractility. The resulting abnormal distribution of forces led to aberrant ECM remodeling around lesions of CCM1- and CCM2-deficient mice. ICAP-1-deficient vessels displayed similar defects. We demonstrate that a positive feedback loop between the aberrant ECM and internal cellular tension led to decreased endothelial barrier function. Our data support that up-regulation of ß1 integrin activation participates in the progression of CCM lesions by destabilizing intercellular junctions through increased cell contractility and aberrant ECM remodeling.

Alteration of cartilage mechanical properties in absence of ß1 integrins revealed by rheometry and FRAP analyses.

CONTEXT: Mechanical properties are essential for biological functions of the hyaline cartilage such as energy dissipation and diffusion of solutes. Mechanical properties are primarily dependent on the hierarchical organization of the two major extracellular matrix (ECM) macromolecular components of the cartilage: the fibrillar collagen network and the glycosaminoglycan (GAG)-substituted proteoglycan, mainly aggrecan, aggregates. Interaction of chondrocytes, the only cell type in the tissue, with the ECM through adhesion receptors is involved in establishing mechanical stability via bidirectional transduction of both mechanical forces and chemical signals. In this study, we aimed to determine the role of the transmembrane ß1 integrin adhesion receptors in cartilage biomechanical properties by the use of genetic modification in mice. METHODS: Costal cartilages of wild type and mutant mice lacking ß1 integrins in chondrocytes were investigated. Cartilage compressive properties and solute diffusion were characterized by rheometric analysis and Fluorescence Recovery After Photobleaching (FRAP), respectively. Cartilage tissue sections were analyzed by histology, immunohistochemistry and transmission electron microscopy (TEM). RESULTS: At the histological level, the mutant costal cartilage was characterized by chondrocyte rounding and loss of tissue polarity. Immunohistochemistry and safranin orange staining demonstrated apparently normal aggrecan and GAG levels, respectively. Antibody staining for collagen II and TEM showed comparable expression and organization of the collagen fibrils between mutant and control cartilages. Despite the lack of gross histological and ultrastructural abnormalities, rheological measurements revealed that the peak elastic modulus in compression of mutant cartilage was 1.6-fold higher than the peak elastic modulus of wild-type sample. Interestingly, the diffusion coefficient within the mutant cartilage tissue was found to be 1.2-fold lower in the extracellular space and 14-fold lower in the pericellular (PCM) space compared to control. CONCLUSION: The results demonstrate that the absence of ß1 integrins on the surface of chondrocytes increases the stiffness and modifies the diffusion properties of costal cartilage. Our data imply that ß1 integrins-mediated chondrocyte-matrix interactions directly affect cartilage biomechanics probably by modifying physical properties of individual cells. This study thus highlights the crucial role of ß1 integrins in the cartilage function.

Control of collagen production in mouse chondrocytes by using a combination of bone morphogenetic protein-2 and small interfering RNA targeting Col1a1 for hydrogel-based tissue-engineered cartilage.

Because articular cartilage does not self-repair, tissue-engineering strategies should be considered to regenerate this tissue. Autologous chondrocyte implantation is already used for treatment of focal damage of articular cartilage. Unfortunately, this technique includes a step of cell amplification, which results in dedifferentiation of chondrocytes, with expression of type I collagen, a protein characteristic of fibrotic tissues. Therefore, the risk of producing a fibrocartilage exists. The aim of this study was to propose a new strategy for authorizing the recovery of the differentiated status of the chondrocytes after their amplification on plastic. Because the bone morphogenetic protein (BMP)-2 and the transforming growth factor (TGF)-ß1 are cytokines both proposed as stimulants for cartilage repair, we undertook a detailed comparative analysis of their biological effects on chondrocytes. As a cellular model, we used mouse chondrocytes after their expansion on plastic and we tested the capability of BMP-2 or TGF-ß1 to drive their redifferentiation, with special attention given to the nature of the proteins synthesized by the cells. To prevent any fibrotic character of the newly synthesized extracellular matrix, we silenced type I collagen by transfecting small interfering RNA (siRNA) into the chondrocytes, before their exposure to BMP-2 or TGF-ß1. Our results showed that addition of siRNA targeting the mRNA encoded by the Col1a1 gene (Col1a1 siRNA) and BMP-2 represents the most efficient combination to control the production of cartilage-characteristic collagen proteins. To go one step further toward scaffold-based cartilage engineering, Col1a1 siRNA-transfected chondrocytes were encapsulated in agarose hydrogel and cultured in vitro for 1 week. The analysis of the chondrocyte-agarose constructs by using real-time polymerase chain reaction, Western-blotting, immunohistochemistry, and electron microscopy techniques demonstrated that the BMP-2/Col1a1 siRNA combination is effective in reinitializing correct production and assembly of the cartilage-characteristic matrix in agarose hydrogel, without production of type I collagen. Because agarose is known to favor long-term expression of the chondrocyte phenotype and agarose-based hydrogels are approved for clinical trials, this strategy appears very promising to repair hyaline cartilage.

Zebrafish collagen XIV is transiently expressed in epithelia and is required for proper function of certain basement membranes.

We found that zebrafish has two differentially expressed col14a1 paralogs. col14a1a expression peaked between 18-somite stage and 24 hours postfertilization (hpf), whereas col14a1b was first expressed at 32 hpf. To uncover functions of collagen XIV (COLXIV) during early embryogenesis, we focused our study on col14a1a. We characterized the α1 (XIV-A) chain as a collagenase-sensitive 200-kDa protein that formed dimer that could be reduced at high pH. As observed for the transcript, COLXIV-A protein expression peaked between 24 and 48 hpf. Using antisense probes and polyclonal antibodies, we show that col14a1a and its protein product COLXIV-A are transiently expressed in several epithelia, including epithelia undergoing shape changes, such as the fin folds. In contrast, anti-COLXII antibodies stained only connective tissues. COLXIV-A was also detected in the basement membrane (BM), where it co-localized with COLXII. At later developmental stages, COLXIV-A was not expressed in epithelia anymore but persisted in the BM. Morpholino knockdown of COLXIV-A provoked a skin detachment phenotype. Electron microscopy analysis revealed that morpholino-injected embryos lacked a lamina densa and lamina lucida at 24 hpf, and BM defects, such as gaps in the adepidermal granules, were still detected at 48 hpf. These BM defects were accompanied by a rupture of the dermis and detachment of the epidermis. Taken together, these data suggest an unexpected role of COLXIV-A in undifferentiated epithelia and in the formation of embryonic basement membranes.

In vivo evidence for a bridging role of a collagen V subtype at the epidermis-dermis interface.

Collagen V is the defective product in most cases of classical Ehlers-Danlos syndrome (EDS), a connective tissue disorder typically characterized by skin fragility and abnormal wound healing. Collagen V assembles into diverse molecular forms. The predominant α1(V)(2)α2(V) heterotrimer controls fibrillogenesis in skin and other tissues. The α1(V)(3) minor form is thought to occur in skin, but its function is unknown. To elucidate its role, we generated transgenic mice that overexpress the human α1(V)(3) homotrimer in the epidermis. The transgene-derived product is deposited as thin unstriated fibrillar material in the basement membrane zone of embryonic and perinatal epidermis and hair follicles. Accumulation of α1(V)(3)-containing fibrils leads to ultrastructural modifications at the epidermis-dermis interface and provokes changes in biomechanical properties, although not statistically significant. Using superparamagnetic immunobeads to isolate authentic suprastructures and protein-binding assays, we demonstrate that the homotrimer is part of a protein network containing collagen IV, laminin-111, and the dermal collagen VI. Our data show that the homotrimer serves as a bridging molecule that contributes to the stabilization of the epidermal-dermal interface. This finding strongly suggests that collagen V may be expressed in skin as different subtypes with important but distinct roles in matrix organization and stability.

Development of the zebrafish myoseptum with emphasis on the myotendinous junction.

Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.

Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.

Use of magnetically oriented orthogonal collagen scaffolds for hemi-corneal reconstruction and regeneration.

We recently showed that the highly organized architecture of the corneal stroma could be reproduced using scaffolds consisting of orthogonally aligned multilayers of collagen fibrils prepared using a high magnetic field. Here we show that such scaffolds permit the reconstruction in vitro of human hemi-corneas (stroma + epithelium), using primary human keratocytes and limbal stem cell derived human keratinocytes. On the surface of these hemi-corneas, a well-differentiated epithelium was formed, as determined both histologically and ultrastructurally and by the expression of characteristic markers. Within the stroma, the keratocytes aligned with the directions of the fibrils in the scaffold and synthesized a new extracellular matrix with typical collagen markers and small, uniform diameter fibrils. Finally, in vivo experiments using a rabbit model showed that these orthogonally oriented multi-layer scaffolds could be used to repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization.

Collagen XV, a novel factor in zebrafish notochord differentiation and muscle development.

Muscle cells are surrounded by extracellular matrix, the components of which play an important role in signalling mechanisms involved in their development. In mice, loss of collagen XV, a component of basement membranes expressed primarily in skeletal muscles, results in a mild skeletal myopathy. We have determined the complete zebrafish collagen XV primary sequence and analysed its expression and function in embryogenesis. During the segmentation period, expression of the Col15a1 gene is mainly found in the notochord and its protein product is deposited exclusively in the peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1 causes defects in notochord differentiation and in fast and slow muscle formation as shown by persistence of axial mesodermal marker gene expression, disorganization of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome. In addition, the number of medial fast-twitch muscle fibers was substantially increased, suggesting that the signalling by notochord derived Hh proteins is enhanced by loss of collagen XV. Consistent with this, there is a concomitant expansion of patched-1 expression in the myotome of morphant embryos. Together, these results indicate that collagen XV is required for notochord differentiation and muscle development in the zebrafish embryo and that it interplays with Shh signalling.