Harzianic acid exerts antimicrobial activity against Gram-positive bacteria and targets the cell membrane.

The thermophilic fungus Oidiodendron flavum is a saprobe that is commonly isolated from soil. Here, we identified a Gram-positive bacteria-selective antimicrobial secondary metabolite from this fungal species, harzianic acid (HA). Using Bacillus subtilis strain 168 combined with dynamic bacterial morphology imaging, we found that HA targeted the cell membrane. To further study the antimicrobial activity of HA, we isolated an HA-resistant strain, Bacillus subtilis strain M9015, and discovered that the mutant had more translucent colonies than the wild type strain, showed cross resistance to rifampin, and harbored five mutations in the coding region of four distinct genes. Further analysis of these genes indicated that the mutation in atpE might be responsible for the translucency of the colonies, and mutation in mdtR for resistance to both HA and rifampin. We conclude that HA is an antimicrobial agent against Gram-positive bacteria that targets the cell membrane.

Protein Tyrosine Phosphatase Studies in Zebrafish.

The zebrafish is an ideal model for functional analysis of genes at the molecular, protein, cell, organ, and organism levels. We have used zebrafish to analyze the function of members of the protein tyrosine phosphatase (PTP) superfamily for more than two decades. The molecular genetic toolbox has significantly improved over the years. Currently, generating mutant lines that lack the function of a PTP gene is relatively straightforward by CRISPR/Cas9 technology-mediated generation of insertions or deletions in the target gene. In addition, generating point mutations using CRISPR/Cas9 technology and homology-directed repair (HDR) is feasible, albeit the success rate could be higher. Here, we describe the methods, including the tips and tricks, that we have used to generate knock-out and knock-in zebrafish lines in PTP genes successfully.

Paecilomycone Inhibits Quorum Sensing in Gram-Negative Bacteria.

Pseudomonas aeruginosa is an opportunistic pathogen that causes major health care concerns due to its virulence and high intrinsic resistance to antimicrobial agents. Therefore, new treatments are greatly needed. An interesting approach is to target quorum sensing (QS). QS regulates the production of a wide variety of virulence factors and biofilm formation in P. aeruginosa. This study describes the identification of paecilomycone as an inhibitor of QS in both Chromobacterium violaceum and P. aeruginosa. Paecilomycone strongly inhibited the production of virulence factors in P. aeruginosa, including various phenazines, and biofilm formation. In search of the working mechanism, we found that paecilomycone inhibited the production of 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS), but not 2'-aminoacetophenone (2-AA). Therefore, we suggest that paecilomycone affects parts of QS in P. aeruginosa by targeting the PqsBC complex and alternative targets or alters processes that influence the enzymatic activity of the PqsBC complex. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research. IMPORTANCE Antibiotics are becoming less effective against bacterial infections due to the evolution of resistance among bacteria. Pseudomonas aeruginosa is a Gram-negative pathogen that causes major health care concerns and is difficult to treat due to its high intrinsic resistance to antimicrobial agents. Therefore, new targets are needed, and an interesting approach is to target quorum sensing (QS). QS is the communication system in bacteria that regulates multiple pathways, including the production of virulence factors and biofilm formation, which leads to high toxicity in the host and low sensitivity to antibiotics, respectively. We found a compound, named paecilomycone, that inhibited biofilm formation and the production of various virulence factors in P. aeruginosa. The toxicity of paecilomycone toward eukaryotic cells and organisms was low, making it an interesting lead for further clinical research.

Loss of Shp1 impairs myeloid cell function and causes lethal inflammation in zebrafish larvae.

PTPN6 encodes SHP1, a protein tyrosine phosphatase with an essential role in immune cell function. SHP1 mutations are associated with neutrophilic dermatoses and emphysema in humans, which resembles the phenotype seen in motheaten mice that lack functional SHP1. To investigate the function of Shp1 in developing zebrafish embryos, we generated a ptpn6 knockout zebrafish line lacking functional Shp1. Shp1 knockout caused severe inflammation and lethality around 17 days post fertilization (dpf). During early development, the myeloid lineage was affected, resulting in a decrease in the number of neutrophils and a concomitant increase in the number of macrophages. The number of emerging hematopoietic stem and progenitor cells (HSPCs) was decreased, but due to hyperproliferation, the number of HSPCs was higher in ptpn6 mutants than in siblings at 5 dpf. Finally, the directional migration of neutrophils and macrophages was decreased in response to wounding, and fewer macrophages were recruited to the wound site. Yet, regeneration of the caudal fin fold was normal. We conclude that loss of Shp1 impaired neutrophil and macrophage function, and caused severe inflammation and lethality at the larval stage.

Modeling (not so) rare developmental disorders associated with mutations in the protein-tyrosine phosphatase SHP2.

Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is a highly conserved protein tyrosine phosphatase (PTP), which is encoded by PTPN11 and is indispensable during embryonic development. Mutations in PTPN11 in human patients cause aberrant signaling of SHP2, resulting in multiple rare hereditary diseases, including Noonan Syndrome (NS), Noonan Syndrome with Multiple Lentigines (NSML), Juvenile Myelomonocytic Leukemia (JMML) and Metachondromatosis (MC). Somatic mutations in PTPN11 have been found to cause cancer. Here, we focus on the role of SHP2 variants in rare diseases and advances in the understanding of its pathogenesis using model systems.

Classification of antimicrobial mechanism of action using dynamic bacterial morphology imaging.

Antimicrobial resistance is a major threat to human health. Basic knowledge of antimicrobial mechanism of action (MoA) is imperative for patient care and for identification of novel antimicrobials. However, the process of antimicrobial MoA identification is relatively laborious. Here, we developed a simple, quantitative time-lapse fluorescence imaging method, Dynamic Bacterial Morphology Imaging (DBMI), to facilitate this process. It uses a membrane dye and a nucleoid dye to track the morphological changes of single Bacillus subtilis cells in response to antimicrobials for up to 60 min. DBMI of bacterial cells facilitated assignment of the MoAs of 14 distinct, known antimicrobial compounds to the five main classes. We conclude that DBMI is a simple method, which facilitates rapid classification of the MoA of antimicrobials in functionally distinct classes.

Gregatins, a Group of Related Fungal Secondary Metabolites, Inhibit Aspects of Quorum Sensing in Gram-Negative Bacteria.

Quorum sensing (QS) is a process that regulates gene expression based on cell density. In bacteria, QS facilitates collaboration and controls a large number of pathways, including biofilm formation and virulence factor production, which lead to lower sensitivity to antibiotics and higher toxicity in the host, respectively. Inhibition of QS is a promising strategy to combat bacterial infections. In this study, we tested the potential of secondary metabolites from fungi to inhibit bacterial QS using a library derived from more than ten thousand different fungal strains. We used the reporter bacterium, Chromobacterium violaceum, and identified 39 fungal strains that produced QS inhibitor activity. These strains expressed two QS inhibitors that had been described before and eight QS inhibitors that had not been described before. Further testing for QS inhibitor activity against the opportunistic pathogen Pseudomonas aeruginosa led to the identification of gregatins as an interesting family of compounds with QS inhibitor activity. Although various gregatins inhibited QS in P. aeruginosa, these gregatins did not inhibit virulence factor production and biofilm formation. We conclude that gregatins inhibit some, but not all aspects of QS.

Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects.

Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies.

Juvenile myelomonocytic leukaemia is a childhood blood cancer. It is more common in children with a genetic condition called Noonan Syndrome, which causes problems with development in many parts of the body. The most frequent cause is a mutation in a protein called Src homology region 2 domain-containing phosphatase-2, or SHP2 for short. Juvenile myelomonocytic leukaemia starts in the stem cells that normally become blood cells. In children with Noonan Syndrome, these cells show signs of problems before leukaemia begins. Recreating Noonan Syndrome in an animal could shed light on how this childhood cancer develops, but doing this is not straightforward. One option is to use zebrafish, a species of fish in which the embryos are transparent, allowing scientists to watch their blood cells developing under a microscope. They also share many genes with humans, including SHP2. Solman et al. genetically modified zebrafish so they would carry one of the most common mutations seen in children with Noonan Syndrome in the SHP2 protein. The fish had many of the typical features of the condition, including problems producing blood cells. Single cell analysis of the stem cells that become these blood cells showed that, in the mutated fish, these cells had abnormally high levels of activity in genes involved in inflammation. Treating the fish with an anti-inflammatory drug, dexamethasone, reversed the problem. When Solman et al. investigated stem cells from human patients with juvenile myelomonocytic leukaemia, they found the same high levels of activity in inflammatory genes. The current treatment for juvenile myelomonocytic leukaemia is a stem cell transplant, which is only successful in around half of cases. Finding a way to prevent the cancer from developing altogether could save lives. This new line of zebrafish allows researchers to study Noonan Syndrome in more detail, and to test new treatments. A next step could be to find out whether anti-inflammatory drugs have the same effects in mammals as they do in fish.

Evolution-Informed Discovery of the Naphthalenone Biosynthetic Pathway in Fungi.

Fungi produce a wide diversity of secondary metabolites with interesting biological activities for the health, industrial, and agricultural sectors. While fungal genomes have revealed an unexpectedly high number of biosynthetic pathways that far exceeds the number of known molecules, accessing and characterizing this hidden diversity remain highly challenging. Here, we applied a combined phylogenetic dereplication and comparative genomics strategy to explore eight lichenizing fungi. The determination of the evolutionary relationships of aromatic polyketide pathways resulted in the identification of an uncharacterized biosynthetic pathway that is conserved in distant fungal lineages. The heterologous expression of the homologue from Aspergillus parvulus linked this pathway to naphthalenone compounds, which were detected in cultures when the pathway was expressed. Our unbiased and rational strategy generated evolutionary knowledge that ultimately linked biosynthetic genes to naphthalenone polyketides. Applied to many more genomes, this approach can unlock the full exploitation of the fungal kingdom for molecule discovery. IMPORTANCE Fungi have provided us with life-changing small bioactive molecules, with the best-known examples being the first broad-spectrum antibiotic penicillin, immunosuppressive cyclosporine, and cholesterol-lowering statins. Since the 1980s, exploration of chemical diversity in nature has been highly reduced. However, the genomic era has revealed that fungal genomes are concealing an unexpected and largely unexplored chemical diversity. So far, fungal genomes have been exploited to predict the production potential of bioactive compounds or to find genes that control the production of known molecules of interest. But accessing and characterizing the full fungal chemical diversity require rational and, thus, efficient strategies. Our approach is to first determine the evolutionary relationships of fungal biosynthetic pathways in order to identify those that are already characterized and those that show a different evolutionary origin. This knowledge allows prioritizing the choice of the pathway to functionally characterize in a second stage using synthetic-biology tools like heterologous expression. A particular strength of this strategy is that it is always successful: it generates knowledge about the evolution of bioactive-molecule biosynthesis in fungi, it either yields novel molecules or links the studied pathway to already known molecules, and it reveals the chemical diversity within a given pathway, all at once. The strategy is very powerful to avoid studying the same pathway again and can be used with any fungal genome. Functional characterization using heterologous expression is particularly suitable for fungi that are difficult to grow or not genetically tractable. Thanks to the decreasing cost of gene synthesis, ultimately, only the genome sequence is needed to identify novel pathways and characterize the molecules that they produce. Such an evolution-informed strategy allows the efficient exploitation of the chemical diversity hidden in fungal genomes and is very promising for molecule discovery.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Expanding the molecular spectrum of pathogenic SHOC2 variants underlying Mazzanti syndrome.

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

Berkchaetoazaphilone B has antimicrobial activity and affects energy metabolism.

Antimicrobial resistance has become one of the major threats to human health. Therefore, there is a strong need for novel antimicrobials with new mechanisms of action. The kingdom of fungi is an excellent source of antimicrobials for this purpose because it encompasses countless fungal species that harbor unusual metabolic pathways. Previously, we have established a library of secondary metabolites from 10,207 strains of fungi. Here, we screened for antimicrobial activity of the library against seven pathogenic bacterial strains and investigated the identity of the active compounds using ethyl acetate extraction, activity-directed purification using HPLC fractionation and chemical analyses. We initially found 280 antimicrobial strains and subsequently identified 17 structurally distinct compounds from 26 strains upon further analysis. All but one of these compounds, berkchaetoazaphilone B (BAB), were known to have antimicrobial activity. Here, we studied the antimicrobial properties of BAB, and found that BAB affected energy metabolism in both prokaryotic and eukaryotic cells. We conclude that fungi are a rich source of chemically diverse secondary metabolites with antimicrobial activity.

Phosphatidylinositol-3 kinase signaling controls survival and stemness of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos lacking functional pten, an important tumor suppressor with a central role in cell signaling. Through in vivo live imaging, we discovered that in pten mutant embryos a proportion of the HSPCs died upon emergence from the VDA, an effect rescued by inhibition of phosphatidylinositol-3 kinase (PI3K). Surprisingly, inhibition of PI3K in wild-type embryos also induced HSPC death. Surviving HSPCs colonized the caudal hematopoietic tissue (CHT) normally and committed to all blood lineages. Single-cell RNA sequencing indicated that inhibition of PI3K enhanced survival of multipotent progenitors, whereas the number of HSPCs with more stem-like properties was reduced. At the end of the definitive wave, loss of Pten caused a shift to more restricted progenitors at the expense of HSPCs. We conclude that PI3K signaling tightly controls HSPCs survival and both up- and downregulation of PI3K signaling reduces stemness of HSPCs.

Thermal Proteome Profiling in Zebrafish Reveals Effects of Napabucasin on Retinoic Acid Metabolism.

Thermal proteome profiling (TPP) allows for the unbiased detection of drug-target protein engagements in vivo. Traditionally, 1 cell type is used for TPP studies, with the risk of missing important differentially expressed target proteins. The use of whole organisms would circumvent this problem. Zebrafish embryos are amenable to such an approach. Here, we used TPP on whole zebrafish embryo lysate to identify protein targets of napabucasin, a compound that may affect signal transducer and activator of transcription 3 (Stat3) signaling through an ill-understood mechanism. In zebrafish embryos, napabucasin induced developmental defects consistent with inhibition of Stat3 signaling. TPP profiling showed no distinct shift in Stat3 upon napabucasin treatment, but effects were detected on the oxidoreductase, Pora, which might explain effects on Stat3 signaling. Interestingly, thermal stability of several aldehyde dehydrogenases was affected. Moreover, napabucasin activated aldehyde dehydrogenase enzymatic activity in vitro. Aldehyde dehydrogenases have crucial roles in retinoic acid metabolism, and functionally, we validated napabucasin-mediated activation of the retinoic acid pathway in zebrafish in vivo. We conclude that TPP profiling in whole zebrafish embryo lysate is feasible and facilitates direct correlation of in vivo effects of small molecule drugs with their protein targets.

Cercosporamide inhibits bone morphogenetic protein receptor type I kinase activity in zebrafish.

Zebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein receptor (BMPR) type I kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin, induced by cercosporamide were strikingly similar to the phenotypes caused by renowned small-molecule BMPR type I kinase inhibitors and inactivating mutations in zebrafish BMPRs. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of type I BMPRs [also called activin receptor-like kinases (ALKs)]. In mammalian cells, cercosporamide selectively inhibited constitutively active BMPR type I-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. We believe that cercosporamide could be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMPR signaling, including fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.This article has an associated First Person interview with the first author of the paper.

A new perspective on fungal metabolites: identification of bioactive compounds from fungi using zebrafish embryogenesis as read-out.

There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development.

Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo.

Understanding how body weight is regulated at the molecular level is essential for treating obesity. We show that female mice genetically lacking protein tyrosine phosphatase (PTP) receptor type α (PTPRA) exhibit reduced weight and adiposity and increased energy expenditure, and are more resistant to diet-induced obesity than matched wild-type control mice. These mice also exhibit reduced levels of circulating leptin and are leptin hypersensitive, suggesting that PTPRA inhibits leptin signaling in the hypothalamus. Male and female PTPRA-deficient mice fed a high-fat diet were leaner and displayed increased metabolic rates and lower circulating leptin levels, indicating that the effects of loss of PTPRA persist in the obese state. Molecularly, PTPRA down-regulates leptin receptor signaling by dephosphorylating the receptor-associated kinase JAK2, with which the phosphatase associates constitutively. In contrast to the closely related tyrosine phosphatase ε, leptin induces only weak phosphorylation of PTPRA at its C-terminal regulatory site Y789, and this does not affect the activity of PTPRA toward JAK2. PTPRA is therefore an inhibitor of hypothalamic leptin signaling in vivo and may prevent premature activation of leptin signaling, as well as return signaling to baseline after exposure to leptin.-Cohen-Sharir, Y., Kuperman, Y., Apelblat, D., den Hertog, J., Spiegel, I., Knobler, H., Elson, A. Protein tyrosine phosphatase alpha inhibits hypothalamic leptin receptor signaling and regulates body weight in vivo.

Shp2-Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration.

Regeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a-/- ptpn11b-/- zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a-/- ptpn11b-/- zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a-mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration.

Differential oxidation of protein-tyrosine phosphatases during zebrafish caudal fin regeneration.

Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.