In vivo analysis of Ifn-γ1 and Ifn-γ2 signaling in zebrafish.

The zebrafish genome contains a large number of genes encoding potential cytokine receptor genes as judged by homology to mammalian receptors. The sequences are too divergent to allow unambiguous assignments of all receptors to specific cytokines, and only a few have been assigned functions by functional studies. Among receptors for class II helical cytokines-i.e., IFNs that include virus-induced Ifns (Ifn-) and type II Ifns (Ifn-γ), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)-only the Ifn--specific complexes have been functionally identified, whereas the receptors for the two Ifn-γ (Ifn-γ1 and Ifn-γ2) are unknown. In this work, we identify conditions in which Ifn-γ1 and Ifn-γ2 (also called IFNG or IFN-γ and IFN-gammarel) are induced in fish larvae and adults. We use morpholino-mediated loss-of-function analysis to screen candidate receptors and identify the components of their receptor complexes. We find that Ifn-γ1 and Ifn-γ2 bind to different receptor complexes. The receptor complex for Ifn-γ2 includes cytokine receptor family B (Crfb)6 together with Crfb13 and Crfb17, whereas the receptor complex for Ifn-γ1 does not include Crfb6 or Crfb13 but includes Crfb17. We also show that of the two Jak2 paralogues present in the zebrafish Jak2a but not Jak2b is involved in the intracellular transmission of the Ifn-γ signal. These results shed new light on the evolution of the Ifn-γ signaling in fish and tetrapods and contribute toward an integrated view of the innate immune regulation in vertebrates.

Hemicentin-1 is an essential extracellular matrix component of the dermal-epidermal and myotendinous junctions.

The extracellular matrix architecture is composed of supramolecular fibrillar networks that define tissue specific cellular microenvironments. Hemicentins (Hmcn1 and Hmcn2) are ancient and very large members (> 600 kDa) of the fibulin family, whose short members are known to guide proper morphology and functional behavior of specialized cell types predominantly in elastic tissues. However, the tissue distribution and function of Hemicentins within the cellular microenvironment of connective tissues has remained largely unknown. Performing in situ hybridization and immunofluorescence analyses, we found that mouse Hmcn1 and Hmcn2 show a complementary distribution throughout different tissues and developmental stages. In postnatal dermal-epidermal junctions (DEJ) and myotendinous junctions (MTJ), Hmcn1 is primarily produced by mesenchymal cells (fibroblasts, tenocytes), Hmcn2 by cells of epithelial origin (keratinocytes, myocytes). Hmcn1-/- mice are viable and show no overt phenotypes in tissue tensile strength and locomotion tests. However, transmission electron microscopy revealed ultrastructural basement membrane (BM) alterations at the DEJ and MTJ of Hmcn1-/- mice, pointing to a thus far unknown role of Hmcn1 for BM and connective tissue boundary integrity.

Long-chain Acyl-CoA synthetase 4A regulates Smad activity and dorsoventral patterning in the zebrafish embryo.

Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl-CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated bone morphogenetic protein (Bmp) signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 mitogen-activated protein kinase and the Akt-mediated inhibition of glycogen synthase kinase 3, critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a-deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes.

The role of gamma interferon in innate immunity in the zebrafish embryo.

The zebrafish genome contains ten genes that encode class II cytokine-like peptides, of which the two that are related most closely to mammalian interferon gamma (IFN-gamma) were named IFN-gamma1 and IFN-gamma2. Although the zebrafish has become a popular model system to study immune mechanisms, and although interferons are central regulators of immunity, which zebrafish cytokines correspond functionally to mammalian IFN-gamma has not been established. We used zebrafish embryos to assay the functions of IFN-gamma1 and IFN-gamma2, and have identified a subset of zebrafish homologs of the mammalian IFN-responsive genes as IFN-gamma targets in the zebrafish embryo: these genes are upregulated in response to raised levels of either IFN-gamma1 or IFN-gamma2. Infection studies using two different pathogens show that IFN-gamma signalling is required for resistance against bacterial infections in the young embryo and that the levels of IFN-gamma need to be regulated tightly: raising IFN-gamma levels sensitizes fish embryos against bacterial infection. Embryos injected with high doses of Escherichia coli are able to clear the bacteria within a day, and the gamma-interferons are necessary for this defence reaction. The protective response to Yersinia ruckeri, a natural fish pathogen that is lethal at low doses, also requires IFN-gamma. As in the induction of target genes, the two interferons act at least partly redundantly. Together with the previously demonstrated type III interferon response, these results show that the counterparts of the mammalian viral and bacterial interferon-dependent defence functions are in place in zebrafish embryos, and suggest that zebrafish IFN-gamma1 and IFN-gamma2 are functionally equivalent to mammalian IFN-gamma.

Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish.

BACKGROUND: The zebrafish has become a widely used model to study disease resistance and immunity. Although the genes encoding many components of immune signaling pathways have been found in teleost fish, it is not clear whether all components are present or whether the complexity of the signaling mechanisms employed by mammals is similar in fish. RESULTS: We searched the genomes of the zebrafish Danio rerio and two pufferfish for genes encoding components of the Toll-like receptor and interferon signaling pathways, the NLR (NACHT-domain and leucine rich repeat containing) protein family, and related proteins. We find that most of the components known in mammals are also present in fish, with clearly recognizable orthologous relationships. The class II cytokines and their receptors have diverged extensively, obscuring orthologies, but the number of receptors is similar in all species analyzed. In the family of the NLR proteins, the canonical members are conserved. We also found a conserved NACHT-domain protein with WD40 repeats that had previously not been described in mammals. Additionally, we have identified in each of the three fish a large species-specific subgroup of NLR proteins that contain a novel amino-terminal domain that is not found in mammalian genomes. CONCLUSION: The main innate immune signaling pathways are conserved in mammals and teleost fish. Whereas the components that act downstream of the receptors are highly conserved, with orthologous sets of genes in mammals and teleosts, components that are known or assumed to interact with pathogens are more divergent and have undergone lineage-specific expansions.