Embracing the diversity of model systems to deconstruct the basis of regeneration and tissue repair.

The eighth EMBO conference in the series 'The Molecular and Cellular Basis of Regeneration and Tissue Repair' took place in Barcelona (Spain) in September 2022. A total of 173 researchers from across the globe shared their latest advances in deciphering the molecular and cellular basis of wound healing, tissue repair and regeneration, as well as their implications for future clinical applications. The conference showcased an ever-expanding diversity of model organisms used to identify mechanisms that promote regeneration. Over 25 species were discussed, ranging from invertebrates to humans. Here, we provide an overview of the exciting topics presented at the conference, highlighting novel discoveries in regeneration and perspectives for regenerative medicine.

Reactive oligodendrocyte progenitor cells (re-)myelinate the regenerating zebrafish spinal cord.

Spinal cord injury (SCI) results in loss of neurons, oligodendrocytes and myelin sheaths, all of which are not efficiently restored. The scarcity of oligodendrocytes in the lesion site impairs re-myelination of spared fibres, which leaves axons denuded, impedes signal transduction and contributes to permanent functional deficits. In contrast to mammals, zebrafish can functionally regenerate the spinal cord. Yet, little is known about oligodendroglial lineage biology and re-myelination capacity after SCI in a regeneration-permissive context. Here, we report that, in adult zebrafish, SCI results in axonal, oligodendrocyte and myelin sheath loss. We find that OPCs, the oligodendrocyte progenitor cells, survive the injury, enter a reactive state, proliferate and differentiate into oligodendrocytes. Concomitantly, the oligodendrocyte population is re-established to pre-injury levels within 2 weeks. Transcriptional profiling revealed that reactive OPCs upregulate the expression of several myelination-related genes. Interestingly, global reduction of axonal tracts and partial re-myelination, relative to pre-injury levels, persist at later stages of regeneration, yet are sufficient for functional recovery. Taken together, these findings imply that, in the zebrafish spinal cord, OPCs replace lost oligodendrocytes and, thus, re-establish myelination during regeneration.

Acoustic signals in air and water generated by very shallow marine seismic sources: An experimental study.

When a marine seismic source, like an airgun, is fired close to the water surface the oscillating bubble interacts with the water-air interface. The main interest for seismic applications is how this effect impacts the acoustic signal propagating into the water. It is known that the sound transmission into air is abnormally strong when the sound source is very close to the sea surface relative to the emitted wavelength. Detailed insight into how the acoustic signal changes when the source depth is changed is useful in seismic data analysis and processing. Two experiments are conducted in a water tank with two different types of seismic sources. In experiment A the source is a small cavity that is sufficiently far away from the water-air interface so that it can be assumed that no interaction between the cavity and water surface occurs. In experiment B the source is a larger air bubble that is very close to the water-air interface, and hence interaction between the bubble and water surface occurs. The effects on the water surface, oscillating bubble, and emitted acoustic pressure into air are discussed. It is demonstrated that the moving surface contributes significantly to the acoustic signal measured in air.

Spinal motor neurons are regenerated after mechanical lesion and genetic ablation in larval zebrafish.

In adult zebrafish, relatively quiescent progenitor cells show lesion-induced generation of motor neurons. Developmental motor neuron generation from the spinal motor neuron progenitor domain (pMN) sharply declines at 48 hours post-fertilisation (hpf). After that, mostly oligodendrocytes are generated from the same domain. We demonstrate here that within 48 h of a spinal lesion or specific genetic ablation of motor neurons at 72 hpf, the pMN domain reverts to motor neuron generation at the expense of oligodendrogenesis. By contrast, generation of dorsal Pax2-positive interneurons was not altered. Larval motor neuron regeneration can be boosted by dopaminergic drugs, similar to adult regeneration. We use larval lesions to show that pharmacological suppression of the cellular response of the innate immune system inhibits motor neuron regeneration. Hence, we have established a rapid larval regeneration paradigm. Either mechanical lesions or motor neuron ablation is sufficient to reveal a high degree of developmental flexibility of pMN progenitor cells. In addition, we show an important influence of the immune system on motor neuron regeneration from these progenitor cells.

Signaling networks organizing regenerative growth of the zebrafish fin.

In contrast to mammals, adult salamanders and fish can completely regenerate their appendages after amputation. The cellular and molecular mechanisms underlying this fascinating phenomenon are beginning to emerge, including substantial progress in the identification of signals that control regenerative growth of the zebrafish caudal fin. Despite the fairly simple architecture of the fin, the regulation of its regeneration is complex. Many signals, including fibroblast growth factor (FGF), Wnt, Hedgehog (Hh), retinoic acid (RA), Notch, bone morphogenic protein (BMP), activin, and insulin-like growth factor (IGF), are required for regeneration. Much work needs to be done to dissect tissue-specific functions of these pathways and how they interact, but Wnt/ß-catenin signaling is already emerging as a central player. Surprisingly, Wnt/ß-catenin signaling appears to largely indirectly control epidermal patterning, progenitor cell proliferation, and osteoblast maturation via regulation of a multitude of secondary signals.

Mature osteoblasts dedifferentiate in response to traumatic bone injury in the zebrafish fin and skull.

Zebrafish have an unlimited capacity to regenerate bone after fin amputation. In this process, mature osteoblasts dedifferentiate to osteogenic precursor cells and thus represent an important source of newly forming bone. By contrast, differentiated osteoblasts do not appear to contribute to repair of bone injuries in mammals; rather, osteoblasts form anew from mesenchymal stem cells. This raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, a special feature of the lepidotrichia bone of the fish fin, or a process found more generally in fish bone. Here, we show that dedifferentiation of mature osteoblasts is not restricted to fin regeneration after amputation, but also occurs during repair of zebrafish fin fractures and skull injuries. In both models, mature osteoblasts surrounding the injury downregulate the expression of differentiation markers, upregulate markers of the pre-osteoblast state and become proliferative. Making use of photoconvertible Kaede protein as well as Cre-driven genetic fate mapping, we show that osteoblasts migrate to the site of injury to replace damaged tissue. Our findings suggest a fundamental role for osteoblast dedifferentiation in reparative bone formation in fish and indicate that adult fish osteoblasts display elevated cellular plasticity compared with mammalian bone-forming cells.

Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration.

Zebrafish can completely regenerate amputated fins via formation of a blastema, a proliferative mass of undifferentiated precursor cells. During regenerative growth, blastema proliferation must be tightly coordinated with cellular differentiation, but little is known about how this is achieved. Here, we show that Notch signaling is essential for maintenance of blastema cells in a proliferative undifferentiated state. We found that the Notch pathway is activated in response to fin amputation in the highly proliferative region of the blastema. Chemical interference with Notch signaling resulted in a complete block of regeneration. Notch signaling was not required for the earliest known cellular processes during blastema formation, i.e. dedifferentiation and migration of osteoblasts, but specifically interfered with proliferation of blastema cells. Interestingly, overactivation of the pathway via misexpression of the intracellular domain of the Notch receptor (NICD) likewise inhibited regenerative outgrowth. In NICD-overexpressing fins, overall blastemal cell proliferation was not enhanced, but expanded into proximal regions where cellular differentiation normally occurs. Similarly, blastemal and epidermal gene expression territories invaded proximal regions upon sustained Notch activation. Concomitantly, NICD overexpression suppressed differentiation of osteoblasts and caused an expansion of the undifferentiated blastema. Together, these data suggest that Notch signaling activity maintains blastemal cells in a proliferative state and thus coordinates proliferation with differentiation during regenerative growth.

Patient and Health-Care Provider Interpretation of do not Resuscitate and do not Intubate.

BACKGROUND: Advance directives and end of life care are difficult discussions for both patients and health-care providers (HCPs). A HCP requires an accurate understanding of advanced directives to educate patients and their family members to allow them to make an appropriate decision. Misinterpretations of the do not resuscitate (DNR), do not intubate (DNI), and the Physicians Orders for Life-Sustaining Treatment (POLST) form result in ineffective communication and confusion between patients, family members, and HCPs. METHODOLOGY: An anonymous, multiple choice online and paper survey was distributed to patients, family members of patients (PFMs), and HCPs from December 12, 2012 to March 6, 2013. Data regarding demographics, the accuracy of determining the correct definition of DNR and DNI, the familiarity of the POLST form and if a primary care physician had discussed advanced directives with the participants were collected. RESULTS: A total of 687 respondents participated in the survey. Patients and PFMs could not distinguish the definition of DNR (95% confidence interval [CI] [1.453-2.804]) or DNI (95% CI (1.216-2.334)) 52% of the time while HCPs 35% and 39% of the time (P < 0.0005). Regarding the POLST form, 86% of patients and PFMs and 50% of HCPs were not familiar with the POLST form. Sixty-nine percent of patients and family members reported that their primary care physician had not discussed advance directives with them. Twenty-four percent of patients and family members reported that they had previous health-care experience and this was associated with increased knowledge of the POLST form (P < 0.0005). An association was also seen between the type of HCP taking the survey and the ability to correctly identify the correct definition of DNR (P < 0.0005). CONCLUSION: Discussion of end of life care is difficult for patients and their family members. Often times multiple discussions are required in order to effectively communicate the definition of DNR, DNI, and the POLST form. Education of patients, family members, and HCPs is required to bridge the knowledge gap of advance directives.

Estimation of the mass density of biological matter from refractive index measurements.

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms. One of the basic biophysical properties is the mass density (MD). It affects the dynamics in sub-cellular compartments and plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge, as it requires detailed knowledge of the biochemical composition of the sample. Here we propose a methodology on how to account for assumptions about the biochemical composition of the sample and respective RI measurements. To this aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations and Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well-characterized complex mixtures given by bovine milk and intralipid emulsion and employ it to estimate the MD of living zebrafish (Danio rerio) larvae trunk tissue. Our results illustrate the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

Bringing Light into the Dark-Overview of Environmental Impacts of Carbon Fiber Production and Potential Levers for Reduction.

Carbon fibers (CFs) are a crucial material for lightweight structures with advanced mechanical performance. However, there is still a paucity of detailed understanding regarding the environmental impacts of production. Previously, mostly singled-out scenarios for CF production have been assessed, often based on scarce transparent inventory data. To expand the current knowledge and create a robust database for future evaluation, a life cycle assessment (LCA) was carried out. To this end, a detailed industry-approved LCI is published, which also proved plausible against the literature. Subsequently, based on a global scenario representing the market averages for precursor and CF production, the most relevant contributors to climate change (EF3.1 climate change, total) and the depletion of fossil energy carriers (EF3.1 resource use, fossil) were identified. The energy consumption in CF manufacturing was found to be responsible for 59% of the climate change and 48% of the fossil resource use. To enable a differentiated discussion of manufacturing locations and process energy consumption, 24 distinct scenarios were assessed. The findings demonstrate the significant dependence of the results on the scenarios' boundary conditions: climate change ranges from 13.0 to 34.1 kg CO2 eq./kg CF and resource use from 262.3 to 497.9 MJ/kg CF. Through the investigated scenarios, the relevant reduction potentials were identified. The presented results help close an existing data gap for high-quality, regionalized, and technology-specific LCA results for the production of CF.

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

An exception to the rule? Regeneration of the injured spinal cord in the spiny mouse.

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury in the adult has long been thought to be a unique feature of certain non-mammalian vertebrates. However, in this issue of Developmental Cell, Nogueira-Rodrigues et al. report an astonishingly high regenerative ability in the spiny mouse.

Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing.

Zebrafish regenerate their spinal cord after injury, both at larval and adult stages. Larval zebrafish have emerged as a powerful model system to study spinal cord injury and regeneration due to their high optical transparency for in vivo imaging, amenability to high-throughput analysis, and rapid regeneration time. Here, we describe a protocol for the mechanical transection of the larval zebrafish spinal cord, followed by whole-mount tissue processing for in situ hybridization and immunohistochemistry to elucidate principles of regeneration. For complete details on the use and execution of this protocol, please refer to Wehner et al. (2017) and Tsata et al. (2021).

Matrix stiffness mechanosensing modulates the expression and distribution of transcription factors in Schwann cells.

After peripheral nerve injury, mature Schwann cells (SCs) de-differentiate and undergo cell reprogramming to convert into a specialized cell repair phenotype that promotes nerve regeneration. Reprogramming of SCs into the repair phenotype is tightly controlled at the genome level and includes downregulation of pro-myelinating genes and activation of nerve repair-associated genes. Nerve injuries induce not only biochemical but also mechanical changes in the tissue architecture which impact SCs. Recently, we showed that SCs mechanically sense the stiffness of the extracellular matrix and that SC mechanosensitivity modulates their morphology and migratory behavior. Here, we explore the expression levels of key transcription factors and myelin-associated genes in SCs, and the outgrowth of primary dorsal root ganglion (DRG) neurites, in response to changes in the stiffness of generated matrices. The selected stiffness range matches the physiological conditions of both utilized cell types as determined in our previous investigations. We find that stiffer matrices induce upregulation of the expression of transcription factors Sox2, Oct6, and Krox20, and concomitantly reduce the expression of the repair-associated transcription factor c-Jun, suggesting a link between SC substrate mechanosensing and gene expression regulation. Likewise, DRG neurite outgrowth correlates with substrate stiffness. The remarkable intrinsic physiological plasticity of SCs, and the mechanosensitivity of SCs and neurites, may be exploited in the design of bioengineered scaffolds that promote nerve regeneration upon injury.

Know How to Regrow-Axon Regeneration in the Zebrafish Spinal Cord.

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury is poor in mammals but remarkable in some vertebrates, including fish and salamanders. The cellular and molecular basis of this interspecies difference is beginning to emerge. This includes the identification of target cells that react to the injury and the cues directing their pro-regenerative responses. Among existing models of successful spinal cord regeneration, the zebrafish is arguably the most understood at a mechanistic level to date. Here, we review the spinal cord injury paradigms used in zebrafish, and summarize the breadth of neuron-intrinsic and -extrinsic factors that have been identified to play pivotal roles in the ability of zebrafish to regenerate central nervous system axons and recover function.

A switch in pdgfrb+ cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord.

In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM.

A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord.

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant" synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first" scenario of motor axon growth in zebrafish.

Restoration of anatomical continuity after spinal cord transection depends on Wnt/ß-catenin signaling in larval zebrafish.

This data article contains descriptive and experimental data on spinal cord regeneration in larval zebrafish and its dependence on Wnt/ß-catenin signaling. Analyzing spread of intraspinally injected fluorescent dextran showed that anatomical continuity is rapidly restored after complete spinal cord transection. Pharmacological interference with Wnt/ß-catenin signaling (IWR-1) impaired restoration of spinal continuity. For further details and experimental findings please refer to the research article by Wehner et al. Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish (Wehner et al., 2017) [1].

Dynamic control of proinflammatory cytokines Il-1ß and Tnf-α by macrophages in zebrafish spinal cord regeneration.

Spinal cord injury leads to a massive response of innate immune cells in non-regenerating mammals, but also in successfully regenerating zebrafish. However, the role of the immune response in successful regeneration is poorly defined. Here we show that inhibiting inflammation reduces and promoting it accelerates axonal regeneration in spinal-lesioned zebrafish larvae. Mutant analyses show that peripheral macrophages, but not neutrophils or microglia, are necessary for repair. Macrophage-less irf8 mutants show prolonged inflammation with elevated levels of Tnf-α and Il-1ß. Inhibiting Tnf-α does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-α. In contrast, decreasing Il-1ß levels or number of Il-1ß+ neutrophils rescue functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1ß function impairs regeneration in irf8 and wildtype animals. Hence, inflammation is dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.