Daniel Wehner

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

Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/β-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that β-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/β-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.

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/β-catenin signaling is already emerging as a central player. Surprisingly, Wnt/β-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.

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

Wnt/β-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 β-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 β-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 β-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 β-catenin RNAi phenotypes. Given that β-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.

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

ABSTRACT 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. Summary: Regeneration of spinal motor neurons following mechanical lesion or genetic ablation occurs at the expense of oligodendrogenesis and is promoted by the innate immune system.

A Polyphenylene Dendrimer Drug Transporter with Precisely Positioned Amphiphilic Surface Patches

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.

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

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/β-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/β-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/β-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/β-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/β-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.Following spinal injury in zebrafish, non-neural cells establish an extracellular matrix to promote axon re-growth but how this is regulated is unclear. Here, the authors show that Wnt/β-catenin signaling in fibroblast-like cells at a lesion activates axon re-growth via deposition of Collagen XII.

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

Background The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β 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. 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 Gβ3 protein subunit that, together with different Gγ and Gα 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. 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.

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

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.