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.<br /

Stat5 as a diagnostic marker for leukemia

The Jak-Stat-Socs pathway is an important component of cytokine receptor signaling. Not surprisingly, perturbation of this pathway is implicated in diseases of hematopoietic and immune origin, including leukemia, lymphoma and immune deficiencies. This review examines the role of a key component of this pathway, Stat5. This has been shown to be activated in a variety of leukemias and myeloproliferative disorders, including downstream of a range of key oncogenes where it has been shown to play an important role in mediating their effects. Therefore, Stat5 represents a useful pan-leukemia/myeloproliferative disorder diagnostic marker and key therapeutic end point, as well as representing an attractive therapeutic target for these disorders.<br /

SOCS proteins in development and disease

Cytokine and growth factor signaling mediates essential roles in the differentiation, proliferation, survival and function of a number of cell lineages. This is achieved via specific receptors located on the surface of target cells, with ligand binding activating key intracellular signal transduction cascades to mediate the requisite cellular outcome. Effective resolution of receptor signaling is also essential, with excessive signaling having the potential for pathological consequences. The Suppressor of cytokine signaling (SOCS) family of proteins represent one important mechanism to extinguish cytokine and growth factor receptor signaling. There are 8 SOCS proteins in mammals; SOCS1-7 and the alternatively named Cytokine-inducible SH2-containing protein (CISH). SOCS1-3 and CISH are predominantly associated with the regulation of cytokine receptor signaling, while SOCS4-7 are more commonly involved in the control of Receptor tyrosine kinase (RTK) signaling. Individual SOCS proteins are typically induced by specific cytokines and growth factors, thereby generating a negative feedback loop. As a consequence of their regulatory properties, SOCS proteins have important functions in development and homeostasis, with increasing recognition of their role in disease, particularly their tumor suppressor and anti-inflammatory functions. This review provides a synthesis of our current understanding of the SOCS family, with an emphasis on their immune and hematopoietic roles

Metabolic profile analysis of zebrafish embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model

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. &copy; 2015 Ferrata Storti Foundation

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.<br /

Evolution of JAK-STAT pathway components : mechanisms and role in immune system development

BackgroundLying downstream of a myriad of cytokine receptors, the Janus kinase (JAK) &ndash; 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.ResultsOur 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.ConclusionDiversification 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.<br /

Exercise-induced activation of STAT3 signaling is increased with age

Activation of the transcription factor signal transducers and activators of transcription (STAT) 3 is common to many inflammatory cytokines and growth factors, with recent evidence of involvement in skeletal muscle regeneration. The purpose of this study was to determine whether STAT3 signaling activation is regulated differentially, at rest and following intense resistance exercise, in aged human skeletal muscle. Skeletal muscle biopsies were harvested from healthy younger (n = 11, 20.4 &plusmn; 0.8 years) and older men (n = 10, 67.4 &plusmn; 1.3 years) under resting conditions and 2 h after the completion of resistance exercise. No differences were evident at rest, whereas the phosphorylation of STAT3 was significantly increased in old (23-fold) compared to young (5-fold) subjects after exercise. This correlated with significantly higher induction of the STAT3 target genes including; interleukin-6 (IL-6), JUNB, c-MYC, and suppressor of cytokine signaling (SOCS) 3 mRNA in older subjects following exercise. Despite increased SOCS3 mRNA, cellular protein abundance was suppressed. SOCS3 protein is an important negative regulator of STAT3 activation and cytokine signaling. Thus, in aged human muscle, elevated responsiveness of the STAT3 signaling pathway and suppressed SOCS3 protein are evident following resistance exercise. These data suggest that enhanced STAT3 signaling responsiveness to proinflammatory factors may impact on mechanisms of muscle repair and regeneration.<br /

From transcriptome to biological function: environmental stress in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis

BackgroundOur understanding of the importance of transcriptional regulation for biological function is continuously improving. We still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. In this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. We exposed individuals of the coral reef fish Pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately 16,000 genes in liver. We also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures.ResultsWe identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. These common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. In response to heat exposure over five days, a total of 324 gene loci were differentially expressed. Many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. We were also able to identify groups of co-regulated genes, the genes within which shared similar functions.ConclusionThis is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. We have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. The functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. This study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future

Characterization of zebrafish polymerase iii promoters for the expression of short hairpin RNA interference molecules

RNA interference (RNAi) is a powerful, sequence specific, and long-lasting method of gene knockdown, and can be elicited by the expression of short-hairpin RNA (shRNA) molecules driven via polymerase III type 3 promoters from a DNA vector or transgene. To further develop RNAi as a tool in zebrafish, we have characterized the zebrafish U6 and H1 snRNA promoters and compared the efficiency of each of the promoters to express an shRNA and silence a reporter gene, relative to previously characterized U6 promoters from pufferfish, chicken, and mouse. Our results show that the zebrafish polymerase III promoters were capable of effective gene silencing in the zebrafish ZF4 cell line, but were ineffective in mammalian Vero cells. In contrast, mouse and chicken promoters were active in Vero but not ZF4 cells, highlighting the importance of homologous promoters to achieve effective silencing