Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

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.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells.

BACKGROUND: Angiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression. RESULTS: By combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients. CONCLUSIONS: We demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

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.

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.

Hepatitis C virus infection propagates through interactions between Syndecan-1 and CD81 and impacts the hepatocyte glycocalyx.

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.

Slow Muscle Precursors Lay Down a Collagen XV Matrix Fingerprint to Guide Motor Axon Navigation.

The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development. SIGNIFICANCE STATEMENT: In addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an unexpected role of the extracellular matrix collagen XV in motor axon pathfinding. We show that the synthesis of collagen XV-B by slow muscle precursors and its deposition in the common motor path are dependent on a novel two-step mechanism that determines axon decisions at a choice point during motor axonogenesis. Zebrafish and humans use common molecular cues and regulatory mechanisms for the neuromuscular system development. And as such, our study reveals COL15A1 as a candidate gene for orphan neuromuscular disorders.

Transcriptomic analysis of mouse limb tendon cells during development.

The molecular signals driving tendon development are not fully identified. We have undertaken a transcriptome analysis of mouse limb tendon cells that were isolated at different stages of development based on scleraxis (Scx) expression. Microarray comparisons allowed us to establish a list of genes regulated in tendon cells during mouse limb development. Bioinformatics analysis of the tendon transcriptome showed that the two most strongly modified signalling pathways were TGF-ß and MAPK. TGF-ß/SMAD2/3 gain- and loss-of-function experiments in mouse limb explants and mesenchymal stem cells showed that TGF-ß signalling was sufficient and required via SMAD2/3 to drive mouse mesodermal stem cells towards the tendon lineage ex vivo and in vitro. TGF-ß was also sufficient for tendon gene expression in late limb explants during tendon differentiation. FGF does not have a tenogenic effect and the inhibition of the ERK MAPK signalling pathway was sufficient to activate Scx in mouse limb mesodermal progenitors and mesenchymal stem cells.

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.

Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish.

Zebrafish fin regeneration involves initial formation of the wound epidermis and the blastema, followed by tissue morphogenesis. The mechanisms coordinating differentiation of distinct tissues of the regenerate are poorly understood. Here, we applied pharmacologic and transgenic approaches to address the role of bone morphogenetic protein (BMP) signaling during fin restoration. To map the BMP transcriptional activity, we analyzed the expression of the evolutionarily conserved direct phospho-Smad1 target gene, id1, and its homologs id2a and id3. This analysis revealed the BMP activity in the distal blastema, wound epidermis, osteoblasts, and blood vessels of the regenerate. Blocking the BMP function with a selective chemical inhibitor of BMP type I receptors, DMH1, suppressed id1 and id3 expression and arrested regeneration after blastema formation. We identified several previously uncharacterized functions of BMP during fin regeneration. Specifically, BMP signaling is required for remodeling of plexus into structured blood vessels in the rapidly growing regenerate. It organizes the wound epithelium by triggering wnt5b expression and promoting Collagen XIV-A deposition into the basement membrane. BMP represents the first known signaling that induces actinotrichia formation in the regenerate. Our data reveal a multifaceted role of BMP for coordinated morphogenesis of distinct tissues during regeneration of a complex vertebrate appendage.

Tinkering signaling pathways by gain and loss of protein isoforms: the case of the EDA pathway regulator EDARADD.

BACKGROUND: Only a handful of signaling pathways are major actors of development and responsible for both the conservation and the diversification of animal morphologies. To explain this twofold nature, gene duplication and enhancer evolution were predominantly put forth as tinkering mechanisms whereas the evolution of alternative isoforms has been, so far, overlooked. We investigate here the role of gain and loss of isoforms using Edaradd, a gene of the Ecodysplasin pathway, implicated in morphological evolution. A previous study had suggested a scenario of isoform gain and loss with an alternative isoform (A) newly gained in mammals but secondarily lost in mouse lineage. RESULTS: For a comprehensive view of A and B Edaradd isoforms history during mammal evolution, we obtained sequences for both isoforms in representative mammals and performed in vitro translations to support functional predictions. We showed that the ancestral B isoform is well conserved, whereas the mammal-specific A isoform was lost at least 7 times independently in terminal lineages throughout mammal phylogeny. Then, to gain insights into the functional relevance of this evolutionary pattern, we compared the biological function of these isoforms: i) In cellulo promoter assays showed that they are transcribed from two alternative promoters, only B exhibiting feedback regulation. ii) RT-PCR in various tissues and ENCODE data suggested that B isoform is systematically expressed whereas A isoform showed a more tissue-specific expression. iii) Both isoforms activated the NF-κB pathway in an in cellulo reporter assay, albeit at different levels and with different dynamics since A isoform exhibited feedback regulation at the protein level. Finally, only B isoform could rescue a zebrafish edaradd knockdown. CONCLUSIONS: These results suggest that the newly evolved A isoform enables modulating EDA signaling in specific conditions and with different dynamics. We speculate that during mammal diversification, A isoform regulation may have evolved rapidly, accompanying and possibly supporting the diversity of ectodermal appendages, while B isoform may have ensured essential roles. This study makes the case to pay greater attention to mosaic loss of evolutionarily speaking "young" isoforms as an important mechanism underlying phenotypic diversity and not simply as a manifestation of neutral evolution.

Collagen XXII binds to collagen-binding integrins via the novel motifs GLQGER and GFKGER.

Collagen XXII, a FACIT (fibril-associated collagen with interrupted triple helices), is expressed at the myotendinous junction and the articular surface of joint cartilage. Cellular receptors like collagen-binding integrins are known to bind collagens with distinct binding motifs following the sequence GXOGER. In the present study, we demonstrate the sequences GLQGER and GFKGER as novel binding motifs between collagen XXII and collagen-binding integrins, especially α2ß1 integrin. Solid-phase assays and surface plasmon resonance spectroscopy revealed a direct interaction between α2ß1 integrin and the motif GFKGER. In addition, immunohistochemical analysis demonstrated partial co-localization of collagen XXII, α2ß1 integrin and α11ß1 integrin at the myotendinous junction. Furthermore, computational modelling of the motifs GLQGER and GFKGER showed perfect fitting of the sequences into the binding pocket of collagen-binding integrins. Taken together, we demonstrated that collagen XXII interacts with collagen-binding integrins via the new motifs GLQGER and GFKGER.

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.

Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin-dependent trafficking.

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti-HCV action of silymarin-derived compounds at the molecular level. By using live-cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV-infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin-coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin-coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well-known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin-mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad-spectrum antiviral therapy.

Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a K(i) of 1.5 ± 0.5 nM, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

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.

Quantitative Image Analysis of Axonal Morphology in In Vivo Model.

Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.

Cell differentiation and matrix organization are differentially affected during bone formation in osteogenesis imperfecta zebrafish models with different genetic defects impacting collagen type I structure.

Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1-/-, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1-/- was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1-/- by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.