We identified orthologues of all mammalian Janus kinase (JAK) and signal transducer and activator of transcription (STAT) genes in teleostean fishes, indicating that these protein families were already largely complete before the teleost tetrapod split, 450 million years ago. In mammals, the STAT repertoire consists of seven genes (STAT1, -2, -3, -4, -5a, -5b, and -6). Our phylogenetic analyses show that STAT proteins that are recruited downstream of endocrine hormones (STAT3 and STAT5a and -5b) show a markedly higher primary sequence conservation compared with STATs that convey immune signals (STAT1-2, STAT4, and STAT6). A similar dichotomy in evolutionary conservation is observed for the JAK family of protein kinases, which activate STATs. The ligands to activate the JAK/STAT-signalling pathway include hormones and cytokines such as GH, prolactin, interleukin 6 (IL6) and IL12. In this paper, we examine the evolutionary forces that have acted on JAK/STAT signalling in the endocrine and immune systems and discuss the reasons why the JAK/STAT cascade that conveys classical immune signals has diverged much faster compared with endocrine JAK/STAT paralogues.

The class-I helical cytokines constitute a large group of signalling molecules that play key roles in a plethora of physiological processes including host defence, immune regulation, somatic growth, reproduction, food intake and energy metabolism, regulation of neural growth and many more. Despite little primary amino acid sequence similarity, the view that all contemporary class-I helical cytokines have expanded from a single ancestor is widely accepted, as all class-I helical cytokines share a similar three-dimensional fold, signal via related class-I helical cytokine receptors and activate similar intracellular signalling cascades. Virtually all of our knowledge on class-I helical cytokine signalling derives from research on primate and rodent species. Information on the presence, structure and function of class-I helical cytokines in non-mammalian vertebrates and non-vertebrates is fragmentary. Consequently, our ideas about the evolution of this versatile multigene family are often based on a limited comparison of human and murine orthologs. In the last 5 years, whole genome sequencing projects have yielded draft genomes of the early vertebrates, pufferfish (Takifugu rubripes), spotted green pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio). Fuelled by this development, fish orthologs of a number of mammalian class-I helical cytokines have recently been discovered. In this review, we have characterised the mammalian class-I helical cytokine family and compared it with the emerging class-I helical cytokine repertoire of teleost fish. This approach offers important insights into cytokine evolution as it identifies the helical cytokines shared by fish and mammals that, consequently, existed before the divergence of teleosts and tetrapods. A ‘fish–mammalian’ comparison will identify the class-I helical cytokines that still await discovery in fish or, alternatively, may have been evolutionarily recent additions to the mammalian cytokine repertoire.

We describe duplicate leptin genes in zebrafish (Danio rerio) that share merely 24% amino acid identity with each other and only 18% with human leptin. We were also able to retrieve a second leptin gene in medaka (Oryzias latipes). The presence of duplicate leptin genes in these two distantly related teleosts suggests that duplicate leptin genes are a common feature of teleostean fishes. Despite low primary sequence conservation, we are confident in assigning orthology between mammalian and zebrafish leptins for several reasons. First, both zebrafish leptins share their characteristic gene structure and display key features of conserved synteny with mammalian leptin genes. Secondly, the cysteine residues that make up leptin's single disulphide bridge are equally spaced in mammalian and zebrafish leptins and are unique among all members of the class-I helical cytokine family. Thirdly, the zebrafish leptins cluster with other fish leptins and mammalian leptins in phylogenetic analysis, supported by high bootstrap values. Within the leptin cluster, leptin-b forms a separate clade with the leptin-b orthologue from medaka. Finally, our prediction of the tertiary structures shows that both leptins conform to the typical four α-helix bundle structure of the class-I α-helical cytokines. The zebrafish leptins are differentially expressed; the liver shows high leptin-a expression (in concordance with what we observed for carp leptins), while leptin-b is expressed at much lower levels, which are downregulated further upon fasting. The finding of duplicate leptin genes in teleosts adds to our understanding of the evolution of leptin physiology in the early vertebrate lineage.

There is great interest in generating functionally mature beta cells from stem cells, as loss of functional beta cell mass contributes to the pathophysiology of diabetes. Identifying markers of beta cell maturity is therefore very helpful for distinguishing stem cells that have been successfully differentiated into fully mature beta cells from stem cells that did not. Urocortin 3 (UCN3) is a peptide hormone whose expression is associated with the acquisition of functional maturity in beta cells. The onset of its expression occurs after other beta cell maturity markers are already expressed and its loss marks the beginning of beta cell dedifferentiation. Its expression pattern is therefore tightly correlated with beta cell maturity. While this makes UCN3 an excellent marker of beta cell maturity, it is not established whether UCN3 is required for beta cell maturation. Here, we compared gene expression and function of beta cells from Ucn3-null mice relative to WT mice to determine whether beta cells are functionally mature in the absence of UCN3. Our results show that genetic deletion of Ucn3 does not cause a loss of beta cell maturity or an increase in beta cell dedifferentiation. Furthermore, virgin beta cells, first identified as insulin-expressing, UCN3-negative beta cells, can still be detected at the islet periphery in Ucn3-null mice. Beta cells from Ucn3-null mice also exhibit normal calcium response when exposed to high glucose. Collectively, these observations indicate that UCN3 is an excellent mature beta cell marker that is nevertheless not necessary for beta cell maturation.

In fish, the hypothalamus–pituitary–interrenal axis (HPI-axis), the equivalent of the hypothalamus–pituitary–adrenal axis (HPA-axis) in mammals, is activated during stress and leads to production and release of cortisol by the interregnal cells in the head kidney. In mammals, the cytokine interleukin-1β (IL-1β) takes a key position in the innate immune and inflammatory responses and influences the HPA-axis. In fish, studies that address the effects of cytokines on HPI-axis activation are limited. We quantitatively assessed expression of IL-1β and its receptor, IL-1RI (the latter was cloned and sequenced), in an acute restraint stress paradigm in common carp, Cyprinus carpio. We also considered expression of the pituitary hormones prolactin (PRL) and GH that have been shown to be structurally related to cytokines and have immunomodulatory actions. Pituitary PRL expression increased fourfold during stress; GH mRNA levels were unaffected. Following restraint, hypothalamic IL-1β expression was upregulated; in head kidney and pituitary pars intermedia, IL-1RI expression significantly increased. We suggest that during acute stress IL-1β signalling in the HPI-axis becomes more sensitive, since both ligand and receptor expressions are enhanced. In vitro, recombinant carp IL-1β stimulates release of α-MSH and N-Ac β-endorphin from the pituitary gland. This observation concurs with increased in vivo plasma levels of α-MSH and N-Ac β-endorphin following restraint. Our findings combined lead us to conclude that IL-1β affects the activity of the HPI-axis and, in turn, expression profiles of genes encoding IL-1β and its receptor are modified during acute stress. Our study provides convincing evidence for bi-directional communication of the HPI-axis and the immune system in fish.

Pancreatic alpha cell activity and glucagon secretion lower as glucose levels increase. While part of the decrease is regulated by glucose itself, paracrine signaling by their neighboring beta and delta cells also plays an important role. Somatostatin from delta cells is an important local inhibitor of alpha cells at high glucose. Additionally, urocortin 3 (UCN3) is a hormone that is co-released from beta cells with insulin and acts locally to potentiate somatostatin secretion from delta cells. UCN3 thus inhibits insulin secretion via a negative feedback loop with delta cells, but its role with respect to alpha cells and glucagon secretion is not understood. We hypothesize that the somatostatin-driven glucagon inhibition at high glucose is regulated in part by UCN3 from beta cells. Here, we use a combination of live functional Ca²⁺ and cAMP imaging as well as direct glucagon secretion measurement, all from alpha cells in intact mouse islets, to determine the contributions of UCN3 to alpha cell behavior. Exogenous UCN3 treatment decreased alpha cell Ca²⁺ and cAMP levels and inhibited glucagon release. Blocking endogenous UCN3 signaling increased alpha cell Ca²⁺ by 26.8 ± 7.6%, but this did not result in increased glucagon release at high glucose. Furthermore, constitutive deletion of Ucn3 did not increase Ca²⁺ activity or glucagon secretion relative to controls. UCN3 is thus capable of inhibiting mouse alpha cells, but, given the subtle effects of endogenous UCN3 signaling on alpha cells, we propose that UCN3-driven somatostatin may serve to regulate local paracrine glucagon levels in the islet instead of inhibiting gross systemic glucagon release.

Corticotropin-releasing factor (CRF) plays a central role in the regulation of the stress axis. In mammals, CRF as well as its receptors and its CRF-binding protein (CRF-BP) are expressed in a variety of organs and tissues outside the central nervous system. One of these extrahypothalamic sites is the adrenal gland, where the paracrine actions of adrenal CRF influence cortical steroidogenesis and adrenal blood flow. Although the central role of CRF signaling in the initiation and regulation of the stress response has now been established throughout vertebrates, information about the possible peripheral presence of CRF in earlier vertebrate lineages is scant. We established the expression of CRF, CRF-BP, and the CRF receptor 1 in a panel of peripheral organs of common carp (Cyprinus carpio). Out of all the peripheral organs tested, CRF and CRF-BP are most abundantly expressed in the carp head kidney, the fish equivalent of the mammalian adrenal gland. This expression localizes to chromaffin cells. Furthermore, detectable quantities of CRF are released from the intact head kidney following in vitro stimulation with 8-bromo-cAMP in a superfusion setup. The presence of CRF and CRF-BP within the chromaffin compartment of the head kidney suggests that a pathway homologous to the mammalian intra-adrenal CRF system is present in the head kidney of fish. It follows that such a system to locally fine-tune the outcome of the centrally initiated stress response has been an integral part of the vertebrate endocrine system since the common ancestor of teleostean fishes and mammals.