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.

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.

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.