In Vivo Analysis of Ifn-γ1 and Ifn-γ2 Signaling in Zebrafish

Abstract

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-ϕ) and type II Ifns (Ifn-γ), together with Il-10 and its related cytokines (Il-20, Il-22, and Il-26)—only the Ifn-ϕ–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.

Koi Herpesvirus Encodes and Expresses a Functional Interleukin-10

“…Danio rerio IL-10R1 (also known as crfb7) has previously been shown to have no overt role in development or in the signal transduction for Danio rerio phi and gamma interferons (1,31). Our data confirm that IL-10R1 is functional during embryonic development in response to its cognate ligand, causing an increase in leukocytes.…”

miR-142-3p acts as an essential modulator of neutrophil development in zebrafish

“…For instance, crfb17, a necessary component of IFN-g receptor complexes in zebrafish, was upregulated by 6.35-fold. 43 We also identified obvious upregulation of 2 interferon regulatory factor (IRF) family members, irf1b (2.92-fold) and irf9 (2.15-fold) ( Figure 6D). …”

Expression regulation of zebrafish interferon regulatory factor 9 by promoter analysis

“…For overexpression, the coding regions of DrIFNu1, DrIFNc1, DrIFNc2 were inserted into the restriction sites Xho I/Xba I of pCS2+ vector (Promega) to make zebrafish IFN expression plasmid (Aggad et al, 2009(Aggad et al, , 2010. Similarly, other plasmids including DrIFNu2, DrIFNu3, DrIFNu4 were made by insertion of their coding regions into Xho I/Xba I sites of pCS2+ vector (Promega).…”

“…Two types of fish IFN genes have been identified (Zou and Secombes, 2011). For example, zebrafish Danio rerio genome has four IFNu genes (IFNu1/2/3/4, or named IFN1/2/ 3/4) (Aggad et al, 2009;Altmann et al, 2003) and two IFNc genes (IFNc1/2) (Aggad et al, 2010;Igawa et al, 2006). Although the former of zebrafish IFNs use distinct receptors from mammalian type I IFNs (Aggad et al, 2009), crystal structure analyses have revealed that they are evolutionarily closer to mammalian type I IFNs than to type III IFNs (Hamming et al, 2011).…”