Stein, Cornelia
(2011).
Innate immune genes in the zebrafish, Danio rerio.
PhD thesis, Universität zu Köln.
Abstract
The zebrafish embryo offers the in vertebrates so far unparalled opportunity to study innate immune responses without the influence of an acquired response, which starts to operate much later in development. In the recent past the zebrafish has proven to be a versatile tool to study host-pathogen interactions and an excellent model for the study of human diseases. However, it has been unclear whether the defense mechanisms employed in the embryo are comparable to that known from mammals. In order to determine the extent of conservation between mammalian and fish innate immune pathways, a phylogenetic analysis was conducted to search the zebrafish genome and those of two pufferfish for putative orthologs of known mammalian innate immune genes. This revealed that those components of the intracellular signaling cascades, that transduce the signal downstream of the receptors, are conserved between fish and mammals. However, for the class II cytokines and their receptors the assembled data showed that orthologous relationships cannot easily be established. Within the class II cytokines it could be shown that fish have their own set of interferons (named interferon-φ), which are more closely related to each other than to the mammalian class II cytokine genes or to those shared by mammals and fish, suggesting that interferon genes expanded independently in zebrafish and mammals. The class II cytokine receptor genes are highly divergent. Mammals and fish have approximately the same number of receptor genes. Since syntenic relationships were found only between pufferfish and mammals and appeared to have been lost in the zebrafish, no reliable orthologies could be established. IFNγ is a key regulator of immune responses in mammals, but a possible role of the two zebrafish ifnγ genes during innate immunity is not well established. Infection studies in zebrafish embryos showed that both genes mediate responses to protect the embryo against pathogenic bacterial infection, but seem to function in a redundant manner. Analysis of putative interferon receptor genes revealed that zebrafish Ifnγ1 and Ifnγ2 use distinct receptor chains to transduce the signal to the conserved intracellular signaling pathway. By conducting knockdown experiments in combination with infection assays it was shown that ifnγ2 depends on crfb13 to protect the embryo against infection with Yersinia ruckeri. Among the NLR protein family, a large fish-specific expansion was found. The NLRs are intracellular pathogen sensors that have only recently been described in mammals. They react to various pathogens and are implicated in several human autoimmune disorders, such as Morbus Crohn. More than 200 novel zebrafish genes encoding NLRs were identified in this study. They were found to be closely related to each other but lack the identifiable effector domains that mediate the downstream events in mammals. A new conserved domain was identified, which is present in all fish NLRs and which was termed Fisna. Its functional role is however unknown. Taken together the results from the phylogentic analyses suggest that the signaling mediators are conserved in fish, whereas those proteins that possibly respond to or interact with pathogens appear to have diverged. In addition to the fish-specific NLRs, Nwd1/NACHT-P1 was identified as a novel NLR gene in fish and mammals. Analysis of the genomic organization and transcript structure of zebrafish nwd1 revealed that it encodes a predicted protein of 1620 amino acids that does not contain any known N-terminal effector domain. In situ hybridization in zebrafish showed that it is specifically expressed in neuromasts, which are mechanosensory organs found only in fish and amphibians. In RT-PCR studies, mouse Nwd1 was found expressed in tissues such as brain, thymus, or testis. These data suggest a functional role for this gene, which awaits further analysis.
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