Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

Zebrafish Galectin 3 binding protein is the target antigen of the microglial 4C4 monoclonal antibody.

BACKGROUND: Two decades ago, the fish-specific monoclonal antibody 4C4 was found to be highly reactive to zebrafish microglia, the macrophages of the central nervous system. This has resulted in 4C4 being widely used, in combination with available fluorescent transgenic reporters to identify and isolate microglia. However, the target protein of 4C4 remains unidentified, which represents a major caveat. In addition, whether the 4C4 expression pattern is strictly restricted to microglial cells in zebrafish has never been investigated. RESULTS: Having demonstrated that 4C4 is able to capture its native antigen from adult brain lysates, we used immunoprecipitation/mass-spectrometry, coupled to recombinant expression analyses, to identify its target. The cognate antigen was found to be a paralog of Galectin 3 binding protein (Lgals3bpb), known as MAC2-binding protein in mammals. Notably, 4C4 did not recognize other paralogs, demonstrating specificity. Moreover, our data show that Lgals3bpb expression, while ubiquitous in microglia, also identifies leukocytes in the periphery, including populations of gut and liver macrophages. CONCLUSIONS: The 4C4 monoclonal antibody recognizes Lgals3bpb, a predicted highly glycosylated protein whose function in the microglial lineage is currently unknown. Identification of Lgals3bpb as a new pan-microglia marker will be fundamental in forthcoming studies using the zebrafish model.

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes.

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, csf1r deficiency disrupts embryonic to adult macrophage development. Zebrafish deficient for csf1r are viable and permit analyzing the consequences of macrophage loss throughout life.

Immune cells called macrophages are found in all organs in the body. These cells are highly effective at eating and digesting large particles including dead cells and debris, and microorganisms such as bacteria. Macrophages are also instrumental in shaping developing organs and repairing tissues during life. Macrophages were, until recently, thought to be constantly replenished from cells circulating in the bloodstream. However, it turns out that separate populations of macrophages become established in most tissues during embryonic development and are maintained throughout life without further input. Previous studies of zebrafish, rodents and humans have shown that, when a gene called CSF1R is non-functional, macrophages are absent from many organs including the brain. However, some tissue-specific macrophages still persist, and it was not clear why these cells do not rely on the CSF1R gene while others do. Kuil et al. set out to decipher the precise requirement for the CSF1R gene in macrophage development in living zebrafish. The experiments used zebrafish that make a green fluorescent protein in their macrophages. As these fish are transparent, this meant that Kuil et al. could observe the cells within the living fish and isolate them to determine which genes are switched on and off. This approach revealed that zebrafish with a mutated version of the CSF1R gene make macrophages as embryos but that these cells then fail to multiply and migrate into the developing organs. This results in fewer macrophages in the zebrafish's tissues, and an absence of these cells in the brain. Kuil et al. went on to show that new macrophages did emerge in zebrafish that were about two to three weeks old. However, unexpectedly, these new cells were not regular macrophages. Instead, they were a new recently identified cell-type called metaphocytes, which share similarities with macrophages but have a completely different origin, move faster and do not eat particles. Zebrafish lacking the CSF1R gene thus lose nearly all their macrophages but retain metaphocytes. These macrophage-free mutant zebrafish constitute an unprecedented tool for further studies looking to discriminate the different roles of macrophages and metaphocytes.

The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish.

The mononuclear phagocytic system consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate mononuclear phagocytic system subsets from other hematopoietic lineages. Macrophage-expressed gene (Mpeg)1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since then more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation, and function. Whereas the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8null myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception is skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the mononuclear phagocytic system in adult zebrafish.

Physiological Responses to Swimming-Induced Exercise in the Adult Zebrafish Regenerating Heart.

Exercise promotes a set of physiological responses known to provide long-term health benefits and it can play an important role in cardioprotection. In the present study, we examined cardiac responses to exercise training in the adult zebrafish and in the context of cardiac regeneration. We found that swimming-induced exercise increased cardiomyocyte proliferation and that this response was also found under regenerating conditions, when exercise was performed either prior to and after ventricular cryoinjury (CI). Exercise prior to CI resulted in a mild improvement in cardiac function and lesion recovery over the non-exercise condition. Transcriptomic profiling of regenerating ventricles in cryoinjured fish subjected to exercise identified genes possibly involved in the cardioprotective effects of exercise and that could represent potential targets for heart regeneration strategies. Taken together, our results suggest that exercise constitutes a physiological stimulus that may help promote cardiomyogenic mechanisms of the vertebrate heart through the induction of cardiomyocyte proliferation. The zebrafish exercise model may be useful for investigating the potential cardioprotective effects of exercise in teleost fish and to contribute to further identify and develop novel avenues in basic research to promote heart regeneration.

A CuI/CuIII prototypical organometallic mechanism for the deactivation of an active pincer-like CuI catalyst in Ullmann-type couplings.

Unraveling the mechanistic details of copper-catalyzed arylation of nucleophiles (Ullmann-type couplings) is a very challenging task. It is a matter of intense debate whether it is a radical-based process or an organometallic redox-based process. The ancillary ligand choice in Ullmann-type couplings plays a key role in such transformations and can strongly influence the catalytic efficiency as well as the mechanism. Here, we show how a predesigned tridentate pincer-like catalyst undergoes a deactivation pathway through a CuI/CuIII prototypical mechanism as demonstrated by helium-tagging infrared photodissociation (IRPD) spectroscopy and DFT studies, lending a strong support to the existence of an aryl-CuIII species in the Ullmann couplings using this tridentate ligand.

Trifluoromethylation of a Well-Defined Square-Planar Aryl-NiII Complex involving NiIII /CF3. and NiIV -CF3 Intermediate Species.

Ni-mediated trifluoromethylation of an aryl-Br bond in model macrocyclic ligands (Ln -Br) has been thoroughly studied, starting with an oxidative addition at Ni0 to obtain well-defined aryl-NiII -Br complexes ([Ln -NiII ]Br). Abstraction of the halide with AgX (X=OTf- or ClO4- ) thereafter provides [Ln -NiII ](OTf). The nitrate analogue has been obtained through a direct C-H activation of an aryl-H bond using NiII salts, and this route has been studied by X-ray absorption spectroscopy (XAS). Crystallographic XRD and XAS characterization has shown a tight macrocyclic coordination in the aryl-NiII complex, which may hamper direct reaction with nucleophiles. On the contrary, enhanced reactivity is observed with oxidants, and the reaction of [Ln -NiII ](OTf) with CF3+ sources afforded Ln -CF3 products in quantitative yield. A combined experimental and theoretical mechanistic study provides new insights into the operative mechanism for this transformation. Computational analysis indicates the occurrence of an initial single electron transfer (SET) to 5-(trifluoromethyl)dibenzothiophenium triflate (TDTT), producing a transient L1 -NiIII /CF3. adduct, which rapidly recombines to form a [L1 -NiIV -CF3 ](X)2 intermediate species. A final facile reductive elimination affords L1 -CF3 . The well-defined square-planar model system studied here permits to gain fundamental knowledge on the rich redox chemistry of nickel, which is sought to facilitate the development of new Ni-based trifluoromethylation methodologies.

Exercise-Induced Hypertrophic and Oxidative Signaling Pathways and Myokine Expression in Fast Muscle of Adult Zebrafish.

Skeletal muscle is a plastic tissue that undergoes cellular and metabolic adaptations under conditions of increased contractile activity such as exercise. Using adult zebrafish as an exercise model, we previously demonstrated that swimming training stimulates hypertrophy and vascularization of fast muscle fibers, consistent with the known muscle growth-promoting effects of exercise and with the resulting increased aerobic capacity of this tissue. Here we investigated the potential involvement of factors and signaling mechanisms that could be responsible for exercise-induced fast muscle remodeling in adult zebrafish. By subjecting zebrafish to swimming-induced exercise, we observed an increase in the activity of mammalian target of rapamycin (mTOR) and Mef2 protein levels in fast muscle. We also observed an increase in the protein levels of the mitotic marker phosphorylated histone H3 that correlated with an increase in the protein expression levels of Pax7, a satellite-like cell marker. Furthermore, the activity of AMP-activated protein kinase (AMPK) was also increased by exercise, in parallel with an increase in the mRNA expression levels of pgc1α and also of pparda, a ß-oxidation marker. Changes in the mRNA expression levels of slow and fast myosin markers further supported the notion of an exercise-induced aerobic phenotype in zebrafish fast muscle. The mRNA expression levels of il6, il6r, apln, aplnra and aplnrb, sparc, decorin and igf1, myokines known in mammals to be produced in response to exercise and to signal through mTOR/AMPK pathways, among others, were increased in fast muscle of exercised zebrafish. These results support the notion that exercise increases skeletal muscle growth and myogenesis in adult zebrafish through the coordinated activation of the mTOR-MEF2 and AMPK-PGC1α signaling pathways. These results, coupled with altered expression of markers for oxidative metabolism and fast-to-slow fiber-type switch, also suggest improved aerobic capacity as a result of swimming-induced exercise. Finally, the induction of myokine expression by swimming-induced exercise support the hypothesis that these myokines may have been produced and secreted by the exercised zebrafish muscle and acted on fast muscle cells to promote metabolic remodeling. These results support the use of zebrafish as a suitable model for studies on muscle remodeling in vertebrates, including humans.

Characterization and Reactivity Studies of a Terminal Copper-Nitrene Species.

High-valent terminal copper-nitrene species have been postulated as key intermediates in copper-catalyzed aziridination and amination reactions. The high reactivity of these intermediates has prevented their characterization for decades, thereby making the mechanisms ambiguous. Very recently, the Lewis acid adduct of a copper-nitrene intermediate was trapped at -90 °C and shown to be active in various oxidation reactions. Herein, we describe for the first time the synthesis and spectroscopic characterization of a terminal copper(II)-nitrene radical species that is stable at room temperature in the absence of any Lewis acid. The azide derivative of a triazamacrocyclic ligand that had previously been utilized in the stabilization of aryl-CuIII intermediates was employed as an ancillary ligand in the study. The terminal copper(II)-nitrene radical species is able to transfer a nitrene moiety to phosphines and abstract a hydrogen atom from weak C-H bonds, leading to the formation of oxidized products in modest yields.

Orthogonal Discrimination among Functional Groups in Ullmann-Type C-O and C-N Couplings.

The copper-catalyzed arylation of nucleophiles has been established as an efficient methodology for the formation of C-C and C-heteroatom bonds. Considering the advances during the last two decades, the ligand choice plays a key role in such transformations and can strongly influence the catalytic efficiency. The applicability of these Ullmann-type coupling reactions regarding the orthogonal selectivity of different functional groups constitutes a challenging subject for current synthetic strategies. Herein, we report a useful toolkit of Cu-based catalysts for the chemoselective arylation of a wide-range of nucleophiles in competitive reactions using aryl iodides and bromides. We show in this work that the arylation of all kinds of amides can be orthogonal to that of amines (aliphatic or aromatic) and phenol derivatives. This high chemoselectivity can be governed by the use of different ligands, yielding the desired coupling products under mild conditions. The selectivity trends are maintained for electronically biased iodobenzene and bromobenzene electrophiles. Radical clock experiments discard the occurrence of radical-based mechanisms.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Swimming-induced exercise promotes hypertrophy and vascularization of fast skeletal muscle fibres and activation of myogenic and angiogenic transcriptional programs in adult zebrafish.

BACKGROUND: The adult skeletal muscle is a plastic tissue with a remarkable ability to adapt to different levels of activity by altering its excitability, its contractile and metabolic phenotype and its mass. We previously reported on the potential of adult zebrafish as a tractable experimental model for exercise physiology, established its optimal swimming speed and showed that swimming-induced contractile activity potentiated somatic growth. Given that the underlying exercise-induced transcriptional mechanisms regulating muscle mass in vertebrates are not fully understood, here we investigated the cellular and molecular adaptive mechanisms taking place in fast skeletal muscle of adult zebrafish in response to swimming. RESULTS: Fish were trained at low swimming speed (0.1 m/s; non-exercised) or at their optimal swimming speed (0.4 m/s; exercised). A significant increase in fibre cross-sectional area (1.290±88 vs. 1.665±106 µm2) and vascularization (298±23 vs. 458±38 capillaries/mm2) was found in exercised over non-exercised fish. Gene expression profiling by microarray analysis evidenced the activation of a series of complex transcriptional networks of extracellular and intracellular signaling molecules and pathways involved in the regulation of muscle mass (e.g. IGF-1/PI3K/mTOR, BMP, MSTN), myogenesis and satellite cell activation (e.g. PAX3, FGF, Notch, Wnt, MEF2, Hh, EphrinB2) and angiogenesis (e.g. VEGF, HIF, Notch, EphrinB2, KLF2), some of which had not been previously associated with exercise-induced contractile activity. CONCLUSIONS: The results from the present study show that exercise-induced contractile activity in adult zebrafish promotes a coordinated adaptive response in fast muscle that leads to increased muscle mass by hypertrophy and increased vascularization by angiogenesis. We propose that these phenotypic adaptations are the result of extensive transcriptional changes induced by exercise. Analysis of the transcriptional networks that are activated in response to exercise in the adult zebrafish fast muscle resulted in the identification of key signaling pathways and factors for the regulation of skeletal muscle mass, myogenesis and angiogenesis that have been remarkably conserved during evolution from fish to mammals. These results further support the validity of the adult zebrafish as an exercise model to decipher the complex molecular and cellular mechanisms governing skeletal muscle mass and function in vertebrates.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray.

BACKGROUND: Senegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution. RESULTS: A comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB. CONCLUSIONS: Transcriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

Aryl-copper(III)-acetylides as key intermediates in Csp2-Csp model couplings under mild conditions.

The mechanism of copper-mediated Sonogashira couplings (so-called Stephens-Castro and Miura couplings) is not well understood and lacks clear comprehension. In this work, the reactivity of a well-defined aryl-Cu(III) species (1ClO4) with p-R-phenylacetylenes (R = NO2, CF3, H) is reported and it is found that facile reductive elimination from a putative aryl-Cu(III)-acetylide species occurs at room temperature to afford the Caryl-Csp coupling species (IR), which in turn undergo an intramolecular reorganisation to afford final heterocyclic products containing 2H-isoindole (P NO2, P CF3, PHa) or 1,2-dihydroisoquinoline (PHb) substructures. Density Functional Theory (DFT) studies support the postulated reductive elimination pathway that leads to the formation of C sp2-Csp bonds and provide the clue to understand the divergent intramolecular reorganisation when p-H-phenylacetylene is used. Mechanistic insights and the very mild experimental conditions to effect Caryl-Csp coupling in these model systems provide important insights for developing milder copper-catalysed Caryl-Csp coupling reactions with standard substrates in the future.

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression.

BACKGROUND: Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: Individual zebrafish (n = 10) were able to swim at a critical swimming speed (U(crit)) of 0.548±0.007 m s(-1) or 18.0 standard body lengths (BL) s(-1). The optimal swimming speed (U(opt)) at which energetic efficiency is highest was 0.396±0.019 m s(-1) (13.0 BL s(-1)) corresponding to 72.26±0.29% of U(crit). The cost of transport at optimal swimming speed (COT(opt)) was 25.23±4.03 µmol g(-1) m(-1). A group-wise experiment was conducted with zebrafish (n = 83) swimming at U(opt) for 6 h day(-1) for 5 days week(-1) for 4 weeks vs. zebrafish (n = 84) that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb), insulin-like growth factor 1 receptor a (igf1ra), troponin C (stnnc), slow myosin heavy chain 1 (smyhc1), troponin I2 (tnni2), myosin heavy polypeptide 2 (myhz2) and myostatin (mstnb). CONCLUSIONS/SIGNIFICANCE: From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.

Aplicación del modelo de auditoría operativa a los Servicios de Anatomía Patológica / Operational audit methodology applied to Pathology Departments

Una organización está formada por un conjunto de individuos que se desarrollan en unas coordenadas espacio/tiempo y que interactúan entre sí orientando su actividad hacia un fin determinado. El tiempo es siempre su vector; sobre él lleva a cabo sus procesos productivos y de servicios, desgrana sus objetivos y crea valor, conocimiento y tecnología. Una organización se puede considerar como un conjunto de relaciones cliente/proveedor siendo ésta la forma natural de ordenación del trabajo en los servicios sanitarios. La organización sanitaria, y la hospitalaria en particular, puede ser estudiada en sus parámetros operativos usando metodología procedente de otros sectores productivos una vez ajustada a su realidad particular. En este artículo se presenta el resultado obtenido de aplicar al entorno hospitalario la metodología auditora operativa; se discute su interés y propósito, se desarrolla el método seguido, se detallan los resultados obtenidos y los instrumentos de gestión que comporta y se plantea su proyección y extensión. El elevado grado de incorporación de las sugerencias de la Auditoría a la dinámica de los Servicios de Anatomía Patológica nos induce a valorar la experiencia de manera positiva. Este es su indicador de interés: el hecho de que los de los profesionales médicos y de enfermería hagan suyas las recomendaciones aportadas en el proceso auditor (AU)