A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish.

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.

Protease signaling regulates apical cell extrusion, cell contacts, and proliferation in epithelia.

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.

Mechanical vessel injury in zebrafish embryos.

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.

Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death.

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.

Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.

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.

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant.

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.

Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.

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.