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.

The roles of inter-tissue adhesion in development and morphological evolution.

The study of how neighboring tissues physically interact with each other, inter-tissue adhesion, is an emerging field at the interface of cell biology, biophysics and developmental biology. Inter-tissue adhesion can be mediated by either cell-extracellular matrix adhesion or cell-cell adhesion, and both the mechanisms and consequences of inter-tissue adhesion have been studied in vivo in numerous vertebrate and invertebrate species. In this Review, we discuss recent progress in understanding the many functions of inter-tissue adhesion in development and evolution. Inter-tissue adhesion can couple the motion of adjacent tissues, be the source of mechanical resistance that constrains morphogenesis, and transmit tension required for normal development. Tissue-tissue adhesion can also create mechanical instability that leads to tissue folding or looping. Transient inter-tissue adhesion can facilitate tissue invasion, and weak tissue adhesion can generate friction that shapes and positions tissues within the embryo. Lastly, we review studies that reveal how inter-tissue adhesion contributes to the diversification of animal morphologies.

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.

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.

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.

Assessment of the Commitments and Performance of the European Food Industry to Improve Population Nutrition.

Objectives: Food companies could play an important role in improving population diets, but often escape accountability through unspecific commitments. This study evaluated nutrition-related commitments and estimated performance of the largest packaged food and non-alcoholic beverage manufacturers, supermarkets and quick-service restaurants (QSR) in Europe. Methods: To quantitatively assess companies' publicly available commitments in 2020, the "Business Impact Assessment on Obesity and Population Nutrition" was applied. The proportion of sales from ultra-processed and "unhealthy" food categories (product categories not-permitted to be marketed to children) and over time changes in the number of QSR transactions and QSR and supermarket outlets were calculated. Results: Company commitments fell short of best practice recommendations (median overall score of 21%, range: 1%-62%). Food and beverage companies generated 82% (15%-100%) and 58% (1%-100%) sales from ultra-processed and "unhealthy" products, respectively. The number of QSR outlets and transactions substantially increased in Europe since 2011, while QSR commitments to improve population nutrition remained limited. Conclusion: Whilst most companies made some nutrition-related commitments, they did not comply with best practice recommendations. A large proportion of sales was generated from ultra-processed/unhealthy products and QSR outlets increased. Government regulations are urgently needed.

Integrin intra-heterodimer affinity inversely correlates with integrin activatability.

Integrins are heterodimeric cell surface receptors composed of an α and ß subunit that mediate cell adhesion to extracellular matrix proteins such as fibronectin. We previously studied integrin α5ß1 activation during zebrafish somitogenesis, and in the present study, we characterize the integrin αV fibronectin receptors. Integrins are activated via a conformational change, and we perform single-molecule biophysical measurements of both integrin activation via fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) and integrin intra-heterodimer stability via fluorescence cross-correlation spectroscopy (FCCS) in living embryos. We find that integrin heterodimers that exhibit robust cell surface expression, including αVß3, αVß5, and αVß6, are never activated in this in vivo context, even in the presence of fibronectin matrix. In contrast, activatable integrins, such as integrin αVß1, and alleles of αVß3, αVß5, αVß6 that are biased to the active conformation exhibit poor cell surface expression and have a higher intra-heterodimer dissociation constant (KD). These observations suggest that a weak integrin intra-heterodimer affinity decreases integrin cell surface stability and increases integrin activatability.

Collagen XV, a multifaceted multiplexin present across tissues and species.

Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years.

Fibronectin is a smart adhesive that both influences and responds to the mechanics of early spinal column development.

An extracellular matrix of Fibronectin adheres the neural tube to the two flanking columns of paraxial mesoderm and is required for normal vertebrate development. Here, we find that the bilaterally symmetric interfaces between the zebrafish neural tube and paraxial mesoderm function as optimally engineered adhesive lap joints with rounded edges, graded Fibronectin 'adhesive' and an arced adhesive spew filet. Fibronectin is a 'smart adhesive' that remodels to the lateral edges of the neural tube-paraxial mesoderm interfaces where shear stress is highest. Fibronectin remodeling is mechanically responsive to contralateral variation morphogenesis, and Fibronectin-mediated inter-tissue adhesion is required for bilaterally symmetric morphogenesis of the paraxial mesoderm. Strikingly, however, perturbation of the Fibronectin matrix rescues the neural tube convergence defect of cadherin 2 mutants. Therefore, Fibronectin-mediated inter-tissue adhesion dynamically coordinates bilaterally symmetric morphogenesis of the vertebrate trunk but predisposes the neural tube to convergence defects that lead to spina bifida.

In embryos, the spinal cord starts out as a flat sheet of cells that curls up to form a closed cylinder called the neural tube. The folding tube is attached to the surrounding tissues through an extracellular matrix of proteins and sugars. Overlapping strands of a protein from the extracellular matrix called Fibronectin connect the neural tube to adjacent tissues, like a kind of biological glue. However, it remained unclear what effect this attachment had on the embryonic development of the spinal cord. Connecting two overlapping objects with glue to form what is known as an 'adhesive lap joint' is common in fields such as woodworking and aeronautical engineering. The glue in these joints comes under shearing stress whenever the two objects it connects try to pull apart. But, thanks to work in engineering, it is possible to predict how different joints will perform under tension. Now, Guillon et al. have deployed these engineering principles to shed light on neural tube development. Using zebrafish embryos and computational models, Guillon et al. investigated what happens when the strength of the adhesive lap joints in the developing spine changes. This revealed that Fibronectin works like a smart adhesive: rather than staying in one place like a conventional glue, it moves around. As the neural tube closes, cells remodel the Fibronectin, concentrating it on the areas under the highest stress. This seemed to both help and hinder neural tube development. On the one hand, by anchoring the tube equally to the left and right sides of the embryo, the Fibronectin glue helped the spine to develop symmetrically. On the other hand, the strength of the adhesive lap joints made it harder for the neural tube to curl up and close. If the neural tube fails to close properly, it can lead to birth defects like spina bifida. One of the best-known causes of these birth defects in humans is a lack of a vitamin known as folic acid. Cell culture experiments suggest that this might have something to do with the mechanics of the cells during development. It may be that faulty neural tubes could close more easily if they were able to unglue themselves from the surrounding tissues. Further use of engineering principles could shed more light on this idea in the future.

Organization of Embryonic Morphogenesis via Mechanical Information.

Embryonic organizers establish gradients of diffusible signaling molecules to pattern the surrounding cells. Here, we elucidate an additional mechanism of embryonic organizers that is a secondary consequence of morphogen signaling. Using pharmacological and localized transgenic perturbations, 4D imaging of the zebrafish embryo, systematic analysis of cell motion, and computational modeling, we find that the vertebrate tail organizer orchestrates morphogenesis over distances beyond the range of morphogen signaling. The organizer regulates the rate and coherence of cell motion in the elongating embryo using mechanical information that is transmitted via relay between neighboring cells. This mechanism is similar to a pressure front in granular media and other jammed systems, but in the embryo the mechanical information emerges from self-propelled cell movement and not force transfer between cells. The propagation likely relies upon local biochemical signaling that affects cell contractility, cell adhesion, and/or cell polarity but is independent of transcription and translation.

Characterization of spatial and temporal expression pattern of Col15a1b during zebrafish development.

In mammals, collagen XV is primarily expressed in skeletal and cardiac muscles, and loss of its expression in mice results in a mild skeletal myopathy. We recently identified Col15a1a, a zebrafish ortholog of the human collagen XV gene which expression was restricted to notochord in embryos. Col15a1a knockdown led to defects in muscle maintenance via Shh signaling. Here we report that zebrafish express a second ortholog Col15a1b. The identification of its complete primary sequence showed that the overall structure of collagen XV is well conserved between vertebrates. Whole mount in situ hybridization and RT-PCR analysis revealed that at 12hpf Col15a1b is mainly expressed in slow muscle cell lineage and in nervous tissues, and, at later stages transcripts are detected in eyes, otic placodes and aortic arches. Based on the expression pattern of col15a1b, sequence alignments and synteny comparisons, we conclude that, contrary to collagen XVa, the zebrafish collagen XVb likely displays the same or similar function than the mammalian orthologs.