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

Julia Kolb, Vasiliki Tsata, Nora John, Kyoohyun Kim, Conrad Möckel, Gonzalo Rosso, Veronika Kurbel, Asha Parmar, Gargi Sharma, Kristina Karandasheva, Shada Abuhattum Hofemeier, Olga Lyraki, Timon Beck, Paul Müller, Raimund Schlüßler, Renato Frischknecht, Anja Wehner, Nicole Krombholz, Barbara Steigenberger, Dimitris Beis, Aya Takeoka, Ingmar Blümcke, Stephanie Möllmert, Kanwarpal Singh, Jochen Guck, Katja Kobow, Daniel Wehner

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.

Highly Nonlinear Dynamics of In Vivo Deep-Tissue Interaction with Femtosecond Laser Pulses at 1030 nm

Soyeon Jun, Andreas Herbst, Kilian Scheffter, Nora John, Julia Kolb, Daniel Wehner, Hanieh Fattahi

We report on the highly nonlinear behavior observed in the central nervous system tissue of zebrafish (Danio rerio) when exposed to femtosecond pulses at 1030 nm. At this irradiation wavelength, photo damage becomes detectable only after exceeding a specific peak intensity threshold, which is independent of the photon flux and irradiation time, distinguishing it from irradiation at shorter wavelengths. Furthermore, we investigate and quantify the role of excessive heat in reducing the damage threshold, particularly during high-repetition-rate operations, which are desirable for label-free and multi-dimensional microscopy techniques. To verify our findings, we examined cellular responses to tissue damage, including apoptosis and the recruitment of macrophages and fibroblasts at different time points post-irradiation. These findings substantially contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing deep-tissue, precise and localized injuries using near-infrared femtosecond laser pulses.

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

Aldine Amiel, Stephanie Tsai, Daniel Wehner

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.

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

Daniel Wehner, Catherina G. Becker

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

Nora John, Julia Kolb, Daniel Wehner

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.

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

Gonzalo Rosso, Daniel Wehner, Christine Schweitzer, Stephanie Möllmert, Elisabeth Sock, Jochen Guck, Victor Shahin

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.

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

Leonardo Cavone, Tess McCann, Louisa K. Drake, Erika A. Aguzzi, Ana-Maria Oprisoreanu, Elisa Pedersen, Soe Sandi, Jathurshan Selvarajah, Themistoklis M. Tsarouchas, Daniel Wehner, Marcus Keatinge, Karolina S. Mysiak, Beth E. P. Henderson, Ross Dobie, Neil C. Henderson, Thomas Becker, Catherina G. Becker

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.

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

Vasiliki Tsata, Daniel Wehner

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

Vasiliki Tsata, Stephanie Möllmert, Christine Schweitzer, Julia Kolb, Conrad Möckel, Benjamin Böhm, Gonzalo Rosso, Christian Lange, Mathias Lesche, Juliane Hammer, Gokul Kesavan, Dimitris Beis, Jochen Guck, Michael Brand, Daniel Wehner

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.

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

Vasiliki Tsata, Volker Kroehne, Daniel Wehner, Fabian Rost, Christian Lange, Cornelia Hoppe, Thomas Kurth, Susanne Reinhardt, Andreas Petzold, Andreas Dahl, Markus Loeffler, Michell M. Reimer, Michael Brand

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 reestablished 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.

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

Ana-Maria Oprisoreanu, Hannah L. Smith, Sukrat Arya, Richard Webster, Zhen Zhong, Daniel Wehner, Marcos J. Cardozo, Thomas Becker, Kevin Talbot, Catherina G. Becker

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.

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

Themistoklis M. Tsarouchas, Daniel Wehner, Leonardo Cavone, Tahimina Munir, Marcus Keatinge, Marvin Lambertus, Anna Underhill, Thomas Barrett, Elias Kassapis, Nikolay Ogryzko, Yi Feng, Tjakko J. van Ham, Thomas Becker, Catherina G. Becker

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-alpha and Il-1 beta. Inhibiting Tnf-a does not rescue axonal growth in irf8 mutants, but impairs it in wildtype animals, indicating a pro-regenerative role of Tnf-a. In contrast, decreasing Il-1 beta levels or number of Il-1 beta+ neutrophils rescue functional regeneration in irf8 mutants. However, during early regeneration, interference with Il-1 beta function impairs regeneration in irf8 and wildtype animals. Hence, inflammation is dynamically controlled by macrophages to promote functional spinal cord regeneration in zebrafish.

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

Daniel Wehner, Thomas Becker, Catherina G. Becker

This data article contains descriptive and experimental data on spinal cord regeneration in larval zebrafish and its dependence on Wnt/beta-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/beta-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]. (C) 2017 The Authors. Published by Elsevier Inc.

Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish

Daniel Wehner, Themistoklis M. Tsarouchas, Andria Michael, Christa Haase, Gilbert Weidinger, Michell M. Reimer, Thomas Becker, Catherina G. Becker

The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/beta-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/beta-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/beta-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/beta-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/beta-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.

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

Jochen Ohnmacht, Yujie Yang, Gianna W. Maurer, Anton Barreiro-Iglesias, Themistoklis M. Tsarouchas, Daniel Wehner, Dirk Sieger, Catherina G. Becker, Thomas Becker

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

Daniel Wehner, Gilbert Weidinger

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/beta-catenin signaling is already emerging as a central player. Surprisingly, Wnt/beta-catenin signaling appears to largely indirectly control epidermal patterning, progenitor cell proliferation, and osteoblast maturation via regulation of a multitude of secondary signals.

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Daniel Wehner, Christopher Jahn, Gilbert Weidinger

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta-catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

A Polyphenylene Dendrimer Drug Transporter with Precisely Positioned Amphiphilic Surface Patches

Rene Stangenberg, Yuzhou Wu, Jana Hedrich, Dennis Kurzbach, Daniel Wehner, Gilbert Weidinger, Seah Ling Kuan, Malin Insa Jansen, Fedor Jelezko, et al.

The design and synthesis of a polyphenylene dendrimer (PPD 3) with discrete binding sites for lipophilic guest molecules and characteristic surface patterns is presented. Its semi-rigidity in combination with a precise positioning of hydrophilic and hydrophobic groups at the periphery yields a refined architecture with lipophilic binding pockets that accommodate defined numbers of biologically relevant guest molecules such as fatty acids or the drug doxorubicin. The size, architecture, and surface textures allow to even penetrate brain endothelial cells that are a major component of the extremely tight blood-brain barrier. In addition, low to no toxicity is observed in in vivo studies using zebrafish embryos. The unique PPD scaffold allows the precise placement of functional groups in a given environment and offers a universal platform for designing drug transporters that closely mimic many features of proteins.

beta-Catenin-Dependent Control of Positional Information along the AP Body Axis in Planarians Involves a Teashirt Family Member

Hanna Reuter, Martin Maerz, Matthias C. Vogg, David Eccles, Laura Grifol-Boldu, Daniel Wehner, Suthira Owlarn, Teresa Adell, Gilbert Weidinger, et al.

Wnt/beta-catenin signaling regulates tissue homeostasis and regeneration in metazoans. In planarians-flatworms with high regenerative potential-Wnt ligands are thought to control tissue polarity by shaping a beta-catenin activity gradient along the anterior-posterior axis, yet the downstream mechanisms are poorly understood. We performed an RNA sequencing (RNA-seq)-based screen and identified hundreds of beta-catenin-dependent transcripts, of which several were expressed in muscle tissue and stem cells in a graded fashion. In particular, a teashirt (tsh) ortholog was induced in a beta-catenin-dependent manner during regeneration in planarians and zebrafish, and RNAi resulted in two-headed planarians. Strikingly, intact planarians depleted of tsh induced anterior markers and slowly transformed their tail into a head, reminiscent of beta-catenin RNAi phenotypes. Given that beta-catenin RNAi enhanced the formation of muscle cells expressing anterior determinants in tail regions, our study suggests that this pathway controls tissue polarity through regulating the identity of differentiating cells during homeostasis and regeneration.

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

Karina Geurtzen, Franziska Knopf, Daniel Wehner, Leonie F. A. Huitema, Stefan Schulte-Merker, Gilbert Weidinger

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.

Wnt/beta-Catenin Signaling Defines Organizing Centers that Orchestrate Growth and Differentiation of the Regenerating Zebrafish Caudal Fin

Daniel Wehner, Wiebke Cizelsky, Mohankrishna Dalvoy Vasudevaro, Guenes Oezhan, Christa Haase, Birgit Kagermeier-Schenk, Alexander Roeder, Richard I. Dorsky, Enrico Moro, Francesco Argenton, Michael Kuehl, Gilbert Weidinger

Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/beta-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that beta-catenin signaling is neither active in the epidermis nor the majority of the proliferative blastemal cells. Rather, tissue-specific pathway interference indicates that Wnt signaling in the nonproliferative distal blastema is required for cell proliferation in the proximal blastema, and signaling in cells lining the osteoblasts directs osteoblast differentiation. Thus, Wnt signaling regulates epidermal patterning, blastemal cell proliferation, and osteoblast maturation indirectly via secondary signals. Gene expression profiling, chromatin immunoprecipitation, and functional rescue experiments suggest that Wnt/beta-catenin signaling acts through Fgf and Bmp signaling to control epidermal patterning, whereas retinoic acid and Hedgehog signals mediate its effects on blastemal cell proliferation. We propose that Wnt signaling orchestrates fin regeneration by defining organizing centers that instruct cellular behaviors of adjacent tissues.

Lypd6 Enhances Wnt/beta-Catenin Signaling by Promoting Lrp6 Phosphorylation in Raft Plasma Membrane Domains

Guenes Oezhan, Erdinc Sezgin, Daniel Wehner, Astrid S. Pfister, Susanne J. Kuehl, Birgit Kagermeier-Schenk, Michael Kuehl, Petra Schwille, Gilbert Weidinger

Wnt/beta-catenin signaling plays critical roles during embryogenesis, tissue homeostasis, and regeneration. How Wnt-receptor complex activity is regulated is not yet fully understood. Here, we identify the Ly6 family protein LY6/PLAUR domain-containing 6 (Lypd6) as a positive feedback regulator of Wnt/beta-catenin signaling. lypd6 enhances Wnt signaling in zebrafish and Xenopus embryos and in mammalian cells, and it is required for wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Lypd6 is GPI anchored to the plasma membrane and physically interacts with the Wnt receptor Frizzled8 and the coreceptor Lrp6. Biophysical and biochemical evidence indicates that Lypd6 preferentially localizes to raft membrane domains, where Lrp6 is phosphorylated upon Wnt stimulation. lypd6 knockdown or mislocalization of the Lypd6 protein to nonraft membrane domains shifts Lrp6 phosphorylation to these domains and inhibits Wnt signaling. Thus, Lypd6 appears to control Lrp6 activation specifically in membrane rafts, which is essential for downstream signaling.

Notch signaling coordinates cellular proliferation with differentiation during zebrafish fin regeneration

Bartholomaeus Grotek, Daniel Wehner, Gilbert Weidinger

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.

Waif1/5T4 Inhibits Wnt/beta-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization

Birgit Kagermeier-Schenk, Daniel Wehner, Guenes Oezhan-Kizil, Hideki Yamamoto, Jian Li, Katharina Kirchner, Christian Hoffmann, Peter Stern, Akira Kikuchi, Alexandra Schambony, Gilbert Weidinger

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/beta-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits beta-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/beta-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances beta-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.

The D153del Mutation in GNB3 Gene Causes Tissue Specific Signalling Patterns and an Abnormal Renal Morphology in Rge Chickens

Hemanth Tummala, Stewart Fleming, Paul M. Hocking, Daniel Wehner, Zahid Naseem, Manir Ali, Christopher F. Inglehearn, Nikolai Zhelev, Douglas H. Lester

Background: The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the beta transducin subunit in the colour visual transduction process. A naturally occurring mutation 'D153del' in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye.<br> Principal Findings: Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the G beta 3 protein subunit that, together with different G gamma and G alpha subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected.<br> Significance: These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.