Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. The CSF1R pathway is a promising therapeutic target in many human diseases, including neurological disorders and cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands (Csf1a, Csf1b, and Il34) in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors, Csf1ra and Csf1rb, however there is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Furthermore, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation, resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rbΔ4bp-deficient and il34Δ5bp-deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors, and the expression of csf1ra and csf1rb is nonoverlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.
- MeSH
- dánio pruhované * genetika MeSH
- hematopoéza MeSH
- ligandy MeSH
- receptor faktoru stimulujícího kolonie makrofágů * metabolismus MeSH
- signální transdukce MeSH
- transportní proteiny metabolismus MeSH
- tyrosinkinasové receptory metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The cellular pathology of schizophrenia and the potential of antipsychotics to target underlying neuronal dysfunctions are still largely unknown. We employed glutamatergic neurons derived from induced pluripotent stem cells (iPSC) obtained from schizophrenia patients with known histories of response to clozapine and healthy controls to decipher the mechanisms of action of clozapine, spanning from molecular (transcriptomic profiling) and cellular (electrophysiology) levels to observed clinical effects in living patients. Glutamatergic neurons derived from schizophrenia patients exhibited deficits in intrinsic electrophysiological properties, synaptic function and network activity. Deficits in K+ and Na+ currents, network behavior, and glutamatergic synaptic signaling were restored by clozapine treatment, but only in neurons from clozapine-responsive patients. Moreover, neurons from clozapine-responsive patients exhibited a reciprocal dysregulation of gene expression, particularly related to glutamatergic and downstream signaling, which was reversed by clozapine treatment. Only neurons from clozapine responders showed return to normal function and transcriptomic profile. Our results underscore the importance of K+ and Na+ channels and glutamatergic synaptic signaling in the pathogenesis of schizophrenia and demonstrate that clozapine might act by normalizing perturbances in this signaling pathway. To our knowledge this is the first study to demonstrate that schizophrenia iPSC-derived neurons exhibit a response phenotype correlated with clinical response to an antipsychotic. This opens a new avenue in the search for an effective treatment agent tailored to the needs of individual patients.
- Publikační typ
- časopisecké články MeSH
Kit ligand (Kitlg) is pleiotropic cytokine with a prominent role in vertebrate erythropoiesis. Although the role of Kitlg in this process has not been reported in Danio rerio (zebrafish), in the present study we show that its function is evolutionarily conserved. Zebrafish possess 2 copies of Kitlg genes (Kitlga and Kitlgb) as a result of whole-genome duplication. To determine the role of each ligand in zebrafish, we performed a series of ex vivo and in vivo gain- and loss-of-function experiments. First, we tested the biological activity of recombinant Kitlg proteins in suspension culture from zebrafish whole-kidney marrow, and we demonstrate that Kitlga is necessary for expansion of erythroid progenitors ex vivo. To further address the role of kitlga and kitlgb in hematopoietic development in vivo, we performed gain-of-function experiments in zebrafish embryos, showing that both ligands cooperate with erythropoietin (Epo) to promote erythroid cell expansion. Finally, using the kita mutant (kitab5/b5 or sparse), we show that the Kita receptor is crucial for Kitlga/b cooperation with Epo in erythroid cells. In summary, using optimized suspension culture conditions with recombinant cytokines (Epo, Kitlga), we report, for the first time, ex vivo suspension cultures of zebrafish hematopoietic progenitor cells that can serve as an indispensable tool to study normal and aberrant hematopoiesis in zebrafish. Furthermore, we conclude that, although partial functional diversification of Kit ligands has been described in other processes, in erythroid development, both paralogs play a similar role, and their function is evolutionarily conserved.
Small fish species, such as zebrafish and medaka, are increasingly gaining popularity in basic research and disease modeling as a useful alternative to rodent model organisms. However, the tracking options for fish within a facility are rather limited. In this study, we present an aquatic species tracking database, Zebrabase, developed in our zebrafish research and breeding facility that represents a practical and scalable solution and an intuitive platform for scientists, fish managers, and caretakers, in both small and large facilities. Zebrabase is a scalable, cross-platform fish tracking database developed especially for fish research facilities. Nevertheless, this platform can be easily adapted for a wide variety of aquatic model organisms housed in tanks. It provides sophisticated tracking, reporting, and management functions that help keep animal-related records well organized, including a QR code functionality for tank labeling. The implementation of various user roles ensures a functional hierarchy and customized access to specific functions and data. In addition, Zebrabase makes it easy to personalize rooms and racks, and its advanced statistics and reporting options make it an excellent tool for creating periodic reports of animal usage and productivity. Communication between the facility and the researchers can be streamlined by the database functions. Finally, Zebrabase also features an interactive breeding history and a smart interface with advanced visualizations and intuitive color coding that accelerate the processes.
- MeSH
- automatizované zpracování dat MeSH
- chov zvířat metody organizace a řízení MeSH
- dánio pruhované * MeSH
- databáze faktografické MeSH
- laboratorní zvířata * MeSH
- monitorování životního prostředí MeSH
- software * MeSH
- vodní hospodářství metody organizace a řízení MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.
- MeSH
- buněčné kultury MeSH
- cytokiny genetika MeSH
- dánio pruhované krev MeSH
- hematopoetické kmenové buňky cytologie MeSH
- hematopoéza * MeSH
- kapři krev MeSH
- kultivační média chemie MeSH
- molekulární biologie metody MeSH
- proteiny dánia pruhovaného genetika MeSH
- rekombinantní proteiny genetika MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Vertebrate erythrocytes and thrombocytes arise from the common bipotent thrombocytic-erythroid progenitors (TEPs). Even though nonmammalian erythrocytes and thrombocytes are phenotypically very similar to each other, mammalian species have developed some key evolutionary improvements in the process of erythroid and thrombocytic differentiation, such as erythroid enucleation, megakaryocyte endoreduplication, and platelet formation. This brings up a few questions that we try to address in this review. Specifically, we describe the ontology of erythro-thrombopoiesis during adult hematopoiesis with focus on the phylogenetic origin of mammalian erythrocytes and thrombocytes (also termed platelets). Although the evolutionary relationship between mammalian and nonmammalian erythroid cells is clear, the appearance of mammalian megakaryocytes is less so. Here, we discuss recent data indicating that nonmammalian thrombocytes and megakaryocytes are homologs. Finally, we hypothesize that erythroid and thrombocytic differentiation evolved from a single ancestral lineage, which would explain the striking similarities between these cells.
- MeSH
- buněčná diferenciace fyziologie MeSH
- erytrocyty cytologie MeSH
- lidé MeSH
- megakaryocyty cytologie MeSH
- obratlovci fyziologie MeSH
- trombocyty cytologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.
- MeSH
- buněčná diferenciace MeSH
- dánio pruhované embryologie fyziologie MeSH
- embryo nesavčí MeSH
- geneticky modifikovaná zvířata MeSH
- hematopoetické kmenové buňky fyziologie MeSH
- hematopoéza genetika MeSH
- kultivované buňky MeSH
- proliferace buněk MeSH
- thrombopoetin genetika MeSH
- trombocyty fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
OBJECTIVES: Faecal Escherichia coli strains were isolated from great cormorants (Phalacrocorax carbo) and mallards (Anas platyrhynchos), which are commonly occurring waterbirds in Europe, and studied for resistance to cephalosporins and fluoroquinolones. METHODS: Cloacal swabs or faeces from great cormorants and mallards in Central Europe were cultivated to isolate Escherichia coli strains with extended-spectrum β-lactamase (ESBL) and plasmid-mediated quinolone resistance (PMQR) genes. RESULTS: Ten ESBL-producing E. coli with the bla(CTX-M-15) or bla(CTX-M-27) gene were isolated from eight great cormorants (1.6%, n = 499). The bla(CTX-M) genes were harboured by plasmids of F and I1 incompatibility groups. CTX-M-27-producing isolates were identified as the epidemiologically important B2-O25b-ST131 clone. No ESBL-producing E. coli was isolated from 305 mallards. Eight E. coli isolates with PMQR genes [six aac(6')-Ib-cr and two qnrS1] were detected in six great cormorants (1.2%). Seventeen strains with qnrS1 were detected in 17 mallards (6%). The PMQR genes were located on plasmids of incompatibility groups F, N or X2. ESBL and PMQR genes were found on conjugative plasmids, enabling the horizontal spread of resistance. CONCLUSIONS: Both great cormorants and mallards can spread epidemiologically important antimicrobial-resistant E. coli isolates to water bodies throughout Europe.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- beta-laktamasy sekrece MeSH
- cefalosporiny farmakologie MeSH
- chinolony farmakologie MeSH
- Escherichia coli enzymologie genetika izolace a purifikace MeSH
- feces mikrobiologie MeSH
- kloaka mikrobiologie MeSH
- plazmidy analýza MeSH
- ptáci mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
The ultrafast dynamic processes initiated by ioni¬zing radiation in water are discussed. At low photon energies, the primary processes are photoexcitation and photoionization. Both reaction channels ultimately lead to the formation of reactive species such as OH•radical or solvated electron. Ejection of low-lying electrons initiate further electron processes such as the Auger decay or the recently identified intermolecular Coulomb decay. All these processes contribute to radiation damage of biomolecules. Novel experimental and theoretical methods used for the study of primary events in radiation chemistry of water are described.
- Klíčová slova
- radiační chemie, Augerův rozpad, intermolekulární coulombovský rozpad, solvatovaný elektron, neadiabatická dynamika,
- MeSH
- voda MeSH
