Clifford Liongue

Evolution of Class I cytokine receptors

BackgroundThe Class I cytokine receptors have a wide range of actions, including a major role in the development and function of immune and blood cells. However, the evolution of the genes encoding them remains poorly understood. To address this we have used bioinformatics to analyze the Class I receptor repertoire in sea squirt (Ciona intestinalis) and zebrafish (Danio rerio).ResultsOnly two Class I receptors were identified in sea squirt, one with homology to the archetypal GP130 receptor, and the other with high conservation with the divergent orphan receptor CLF-3. In contrast, 36 Class I cytokine receptors were present in zebrafish, including representative members for each of the five structural groups found in mammals. This allowed the identification of 27 core receptors belonging to the last common ancestor of teleosts and mammals.ConclusionThis study suggests that the majority of diversification of this receptor family occurred after the divergence of urochordates and vertebrates approximately 794 million years ago (MYA), but before the divergence of ray-finned from lobe-finned fishes around 476 MYA. Since then, only relatively limited lineage-specific diversification within the different Class I receptor structural groups has occurred.

Zebrafish granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration

Granulocyte colony-stimulating factor receptor (GCSFR) signaling participates in the production of neutrophilic granulocytes during normal hematopoietic development, with a particularly important role during emergency hematopoiesis. This study describes the characterization of the zebrafish gcsf and gcsfr genes, which showed broad conservation and similar regulation to their mammalian counterparts. Morpholino-mediated knockdown of gcsfr and overexpression of gcsf revealed the presence of an anterior population of myeloid cells during primitive hematopoiesis that was dependent on GCSF/GCSFR for development and migration. This contrasted with a posterior domain that was largely independent of this pathway. Definitive myelopoiesis was also partially dependent on a functional GCSF/GCSFR pathway. Injection of bacterial lipopolysaccharide elicited significant induction of gcsf expression and emergency production of myeloid cells, which was abrogated by gcsfr knockdown. Collectively, these data demonstrate GCSF/GCSFR to be a conserved signaling system for facilitating the production of multiple myeloid cell lineages in both homeostatic and emergency conditions, as well as for early myeloid cell migration, establishing a useful experimental platform for further dissection of this pathway.

Granulocyte colony-stimulating factor receptor: Stimulating granulopoiesis and much more

The granulocyte colony-stimulating factor receptor (G-CSFR) plays an important role in the production, survival and activation of neutrophilic granulocytes during both normal and emergency hematopoiesis. The G-CSFR also participates in the development of other myeloid lineages, the mobilization of hematopoietic stem cells and myeloid cell migration. This has lead to several important clinical applications for its ligand, G-CSF. More recently, additional important roles for G-CSFR have emerged outside the hematopoietic system, such as in the protection and repair of a diverse range of tissues, including muscle, liver and neural tissue, providing further scope for developing G-CSF as a therapeutic agent. The G-CSFR has also been implicated in the etiology of disease, with mutations/variants of G-CSFR implicated in neutropenia, myelodysplasia and leukemia. Additionally, autocrine/paracrine stimulation of G-CSFR may be important in the biology of solid tumors, including metastasis.

Evolution of JAK-STAT Pathway Components: Mechanisms and Role in Immune System Development

Background Lying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) – Signal transducer and activator of transcription (STAT) pathway is pivotal for the development and function of the immune system, with additional important roles in other biological systems. To gain further insight into immune system evolution, we have performed a comprehensive bioinformatic analysis of the JAK-STAT pathway components, including the key negative regulators of this pathway, the SH2-domain containing tyrosine phosphatase (SHP), Protein inhibitors against Stats (PIAS), and Suppressor of cytokine signaling (SOCS) proteins across a diverse range of organisms. Results Our analysis has demonstrated significant expansion of JAK-STAT pathway components co-incident with the emergence of adaptive immunity, with whole genome duplication being the principal mechanism for generating this additional diversity. In contrast, expansion of upstream cytokine receptors appears to be a pivotal driver for the differential diversification of specific pathway components. Conclusion Diversification of JAK-STAT pathway components during early vertebrate development occurred concurrently with a major expansion of upstream cytokine receptors and two rounds of whole genome duplications. This produced an intricate cell-cell communication system that has made a significant contribution to the evolution of the immune system, particularly the emergence of adaptive immunity.

Evolution of the JAK-STAT pathway

The JAK-STAT pathway represents a finely tuned orchestra capable of rapidly facilitating an exquisite symphony of responses from a complex array of extracellular signals. This review explores the evolution of the JAK-STAT pathway: the origins of the individual domains from which it is constructed, the formation of individual components from these basic building blocks, the assembly of the components into a functional pathway, and the subsequent reiteration of this basic template to fulfill a variety of roles downstream of cytokine receptors.

Koi herpesvirus encodes and expresses a functional interleukin-10

ABSTRACT Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish.

Background Chromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated. Design and Methods TEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro. Results Injection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis. Conclusions This study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.

A novel zebrafish jak2aV581F model shared features of human JAK2V617F polycythemia vera

OBJECTIVE The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV. MATERIALS AND METHODS Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites. RESULTS Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019). CONCLUSION The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.

Suppressor of Cytokine Signaling 1 Regulates Embryonic Myelopoiesis Independently of Its Effects on T Cell Development

Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2–Stat5 pathway.

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages.