ABSTRACT
Zebrafish is a powerful model for forward genetics. Reverse genetic approaches are limited by the time required to generate stable mutant lines. We describe a system for gene knockout that consistently produces null phenotypes in G0 zebrafish. Yolk injection of sets of four CRISPR/Cas9 ribonucleoprotein complexes redundantly targeting a single gene recapitulated germline-transmitted knockout phenotypes in >90% of G0 embryos for each of 8 test genes. Early embryonic (6 hpf) and stable adult phenotypes were produced. Simultaneous multi-gene knockout was feasible but associated with toxicity in some cases. To facilitate use, we generated a lookup table of four-guide sets for 21,386 zebrafish genes and validated several. Using this resource, we targeted 50 cardiomyocyte transcriptional regulators and uncovered a role of zbtb16a in cardiac development. This system provides a platform for rapid screening of genes of interest in development, physiology, and disease models in zebrafish.
Subject(s)
Gene Knockout Techniques/methods , Heart/embryology , Promyelocytic Leukemia Zinc Finger Protein/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Base Sequence , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , GTP-Binding Protein alpha Subunits, G12-G13/genetics , Genetic Engineering/methods , Morpholinos/genetics , Myocytes, Cardiac/cytology , Transcription, Genetic/genetics , Zebrafish/embryologyABSTRACT
The transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) family of cytokines critically regulates vascular morphogenesis and homeostasis. Impairment of TGF-ß or BMP signaling leads to heritable vascular disorders, including hereditary hemorrhagic telangiectasia (HHT). Drosha, a key enzyme for microRNA (miRNA) biogenesis, also regulates the TGF-ß and BMP pathway through interaction with Smads and their joint control of gene expression through miRNAs. We report that mice lacking Drosha in the vascular endothelium developed a vascular phenotype resembling HHT that included dilated and disorganized vasculature, arteriovenous fistulae, and hemorrhages. Exome sequencing of HHT patients who lacked known pathogenic mutations revealed an overrepresentation of rare nonsynonymous variants of DROSHA Two of these DROSHA variants (P100L and R279L) did not interact with Smads and were partially catalytically active. In zebrafish, expression of these mutants or morpholino-directed knockdown of Drosha resulted in angiogenesis defects and abnormal vascular permeability. Together, our studies point to an essential role of Drosha in vascular development and the maintenance of vascular integrity, and reveal a previously unappreciated link between Drosha dysfunction and HHT.
Subject(s)
Gene Expression Regulation , Mutation , Neovascularization, Pathologic , Ribonuclease III/genetics , Ribonuclease III/physiology , Telangiectasia, Hereditary Hemorrhagic/genetics , Animals , Case-Control Studies , Cells, Cultured , Child , Cohort Studies , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Female , Humans , Male , Mice , Mice, Knockout , Morphogenesis , Pedigree , Phenotype , Ribonuclease III/metabolism , Signal Transduction , Telangiectasia, Hereditary Hemorrhagic/metabolism , Telangiectasia, Hereditary Hemorrhagic/pathology , Zebrafish/embryology , Zebrafish/physiologyABSTRACT
Mechanisms that sense and regulate epithelial morphogenesis, integrity, and homeostasis are incompletely understood. Protease-activated receptor 2 (Par2), the Par2-activating membrane-tethered protease matriptase, and its inhibitor, hepatocyte activator inhibitor 1 (Hai1), are coexpressed in most epithelia and may make up a local signaling system that regulates epithelial behavior. We explored the role of Par2b in matriptase-dependent skin abnormalities in Hai1a-deficient zebrafish embryos. We show an unexpected role for Par2b in regulation of epithelial apical cell extrusion, roles in regulating proliferation that were opposite in distinct but adjacent epithelial monolayers, and roles in regulating cell-cell junctions, mobility, survival, and expression of genes involved in tissue remodeling and inflammation. The epidermal growth factor receptor Erbb2 and matrix metalloproteinases, the latter induced by Par2b, may contribute to some matriptase- and Par2b-dependent phenotypes and be permissive for others. Our results suggest that local protease-activated receptor signaling can coordinate cell behaviors known to contribute to epithelial morphogenesis and homeostasis.
Subject(s)
Cell Proliferation/physiology , Epithelial Cells/metabolism , Serine Endopeptidases/metabolism , Signal Transduction/physiology , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Epithelial Cells/cytology , Homeostasis/physiology , Morphogenesis/physiology , Receptor, PAR-2/genetics , Receptor, PAR-2/metabolism , Serine Endopeptidases/genetics , Zebrafish Proteins/geneticsABSTRACT
Sphingosine 1-phosphate (S1P) is a bioactive lipid that acts via G protein-coupled receptors. The S1P receptor S1P1, encoded by S1pr1, is expressed in developing heart but its roles there remain largely unexplored. Analysis of S1pr1 LacZ knockin embryos revealed ß-galactosidase staining in cardiomyocytes in the septum and in the trabecular layer of hearts collected at 12.5 days post coitus (dpc) and weak staining in the inner aspect of the compact layer at 15.5 dpc and later. Nkx2-5-Cre- and Mlc2a-Cre-mediated conditional knockout of S1pr1 led to ventricular noncompaction and ventricular septal defects at 18.5 dpc and to perinatal lethality in the majority of mutants. Further analysis of Mlc2a-Cre conditional mutants revealed no gross phenotype at 12.5 dpc but absence of the normal increase in the number of cardiomyocytes and the thickness of the compact layer at 13.5 dpc and after. Consistent with relative lack of a compact layer, in situ hybridization at 13.5 dpc revealed expression of trabecular markers extending almost to the epicardium in mutants. Mutant hearts also showed decreased myofibril organization in the compact but not trabecular myocardium at 12.5 dpc. These results suggest that S1P signaling via S1P1 in cardiomyocytes plays a previously unknown and necessary role in heart development in mice.
Subject(s)
Heart/embryology , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Receptors, Lysosphingolipid/genetics , Receptors, Lysosphingolipid/metabolism , Animals , Cell Proliferation/genetics , Gene Knockout Techniques , Heart Septal Defects, Ventricular/genetics , Mice , Mice, Transgenic , Myocytes, Cardiac/cytology , Myofibrils/genetics , Myofibrils/metabolism , Myosin Light Chains/genetics , Signal Transduction , Sphingosine-1-Phosphate ReceptorsABSTRACT
Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.
Subject(s)
Blood Vessels/injuries , Disease Models, Animal , Zebrafish/embryology , Animals , Hemostasis , Microscopy, Video/methods , Time-Lapse Imaging/methods , Wound HealingABSTRACT
Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.
Subject(s)
Granuloma/immunology , Macrophages/immunology , Macrophages/microbiology , Mycobacterium Infections, Nontuberculous/immunology , Mycobacterium marinum/immunology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Tumor Necrosis Factors/metabolism , Animals , Apoptosis , Cell Death , Cell Movement , Cytokines/immunology , Cytokines/metabolism , Embryo, Nonmammalian , Granuloma/metabolism , Granuloma/microbiology , Immunity, Innate , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium marinum/growth & development , Mycobacterium marinum/physiology , Receptors, Tumor Necrosis Factor, Type I/immunology , Tumor Necrosis Factors/immunology , Zebrafish/immunology , Zebrafish/microbiologyABSTRACT
In tuberculosis, infecting mycobacteria are phagocytosed by macrophages, which then migrate into deeper tissue and recruit additional cells to form the granulomas that eventually contain infection. Mycobacteria are exquisitely adapted macrophage pathogens, and observations in the mouse model of tuberculosis have suggested that mycobacterial growth is not inhibited in macrophages until adaptive immunity is induced. Using the optically transparent and genetically tractable zebrafish embryo-Mycobacterium marinum model of tuberculosis, we have directly examined early infection in the presence and absence of macrophages. The absence of macrophages led rapidly to higher bacterial burdens, suggesting that macrophages control infection early and are not an optimal growth niche. However, we show that macrophages play a critical role in tissue dissemination of mycobacteria. We propose that residence within macrophages represents an evolutionary trade-off for pathogenic mycobacteria that slows their early growth but provides a mechanism for tissue dissemination.
Subject(s)
Fish Diseases/microbiology , Macrophages/microbiology , Mycobacterium Infections, Nontuberculous/microbiology , Mycobacterium marinum , Zebrafish/microbiology , Animals , Embryo, Nonmammalian/microbiology , Zebrafish/embryologyABSTRACT
One of the strengths of the zebrafish is the ease with which in situ hybridization can be performed to determine spatial and temporal patterns of gene expression in whole embryos. Thus far, colorimetric detection methods are mainly used for these analyses. Here we describe a fluorescent in situ hybridization (FISH) protocol for whole-mount zebrafish embryos using tyramide signal amplification (TSA). An optimal set of reagents was identified that allows for simultaneous localization of gene expression patterns of two genes within the same embryo, permitting identification of colocalized expression within single cells. This protocol can be extended to perform multiplex studies by repetition of the TSA-based detection for each target sequentially with a different fluorescent dye label. To this effect, we demonstrate that this approach can be combined with standard horseradish peroxidase (HRP)-mediated immunocytochemistry procedures in addition to FISH.
ABSTRACT
Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.
Subject(s)
Mycobacterium Infections/microbiology , Mycobacterium tuberculosis/pathogenicity , Ranidae/microbiology , Tuberculoma/microbiology , Tuberculosis/microbiology , Animals , Cell Death , Cell Line , Cells, Cultured , Chemotaxis , Granuloma/microbiology , In Situ Nick-End Labeling , Macrophage Activation , Macrophages/metabolism , Macrophages/microbiology , Mice , Microscopy, Video , Mutation , Mycobacterium Infections/pathology , Time Factors , Virulence , ZebrafishABSTRACT
Infection of vertebrate hosts with pathogenic Mycobacteria, the agents of tuberculosis, produces granulomas, highly organized structures containing differentiated macrophages and lymphocytes, that sequester the pathogen. Adult zebrafish are naturally susceptible to tuberculosis caused by Mycobacterium marinum. Here, we exploit the optical transparency of zebrafish embryos to image the events of M. marinum infection in vivo. Despite the fact that the embryos do not yet have lymphocytes, infection leads to the formation of macrophage aggregates with pathological hallmarks of granulomas and activation of previously identified granuloma-specific Mycobacterium genes. Thus, Mycobacterium-macrophage interactions can initiate granuloma formation solely in the context of innate immunity. Strikingly, infection can redirect normal embryonic macrophage migration, even recruiting macrophages seemingly committed to their developmentally dictated tissue sites.