Function In Lower Vertebrates Research Articles

LEAP2 is a more conserved ligand than ghrelin for fish GHSRs

Recently, liver-expressed antimicrobial peptide 2 (LEAP2) was identified as an endogenous antagonist and an inverse agonist of the ghrelin receptor GHSR. However, its functions in lower vertebrates are not well understood. Our recent study demonstrated that both LEAP2 and ghrelin are functional towards a fish GHSR from Latimeria chalumnae, an extant coelacanth believed to be one of the closest ancestors of tetrapods. However, amino acid sequence alignment identified that the 6.58 position (Ballesteros-Weinstein numbering system) of most fish GHSRs are not occupied by an aromatic Phe residue, which is absolutely conserved in all known GHSRs from amphibians to mammals, and is responsible for human GHSR binding to its agonist, ghrelin. To test whether these unusual fish receptors are functional, we studied the ligand binding properties of three representative fish GHSRs, two from Danio rerio (zebrafish) and one from Larimichthys crocea (large yellow croaker). After overexpression in human embryonic kidney 293T cells, the three fish GHSRs retained normal binding to all tested LEAP2s, except for a second LEAP2 from L. crocea. However, they displayed almost no binding to all chemically synthesized n-octanoylated ghrelins, despite these ghrelins all retaining normal function towards human and coelacanth GHSRs. Thus, it seems that LEAP2 is a more conserved ligand than ghrelin towards fish GHSRs. Our results not only provided new insights into the interaction mechanism of GHSRs with LEAP2s and ghrelins, but also shed new light on the functions of LEAP2 and ghrelin in different fish species.

LEAP2 has antagonized the ghrelin receptor GHSR1a since its emergence in ancient fish.

Recent studies have demonstrated that liver-expressed antimicrobial peptide 2 (LEAP2) antagonizes the ghrelin receptor GHSR1a in mammals. However, its antagonistic function in lower vertebrates has not yet been tested. LEAP2 orthologs have been identified from a variety of fish species; however, previous studies all focused on their antimicrobial activity. To test whether LEAP2 functions as a GHSR1a antagonist in the lowest vertebrates, we studied the antagonism of a fish LEAP2 from Latimeria chalumnae, an extant coelacanth that is one of the closest living fish relatives of tetrapods. Using binding assays, we demonstrated that the coelacanth LEAP2 and ghrelin bound to the coelacanth GHSR1a with IC50 values in the nanomolar range. Using activation assays, we demonstrated that the coelacanth ghrelin activated the coelacanth GHSR1a with an EC50 value in the nanomolar range, and this activation effect was efficiently antagonized by a nanomolar range of the coelacanth LEAP2. In addition, we also showed that the human LEAP2 and ghrelin were as effective as their coelacanth orthologs towards the coelacanth GHSR1a; however, the coelacanth peptides had moderately lower activity towards the human GHSR1a. Thus, LEAP2 serves as an endogenous antagonist of the ghrelin receptor GHSR1a in coelacanth and the ghrelin-LEAP2-GHSR1a system has evolved slowly since its emergence in ancient fish.

Leukemia inhibitory factor signaling in Xenopus embryo: Insights from gain of function analysis and dominant negative mutant of the receptor

Leukemia inhibitory factor (LIF) is a cytokine member of the interleukin 6 family (IL6) of cytokines. It signals through a heterodimer receptor complex that consists of the LIF receptor (or LIFR formerly known as gp190) and the Interleukin 6 signal transducer (or IL6ST formerly known as gp130). LIF signaling is mediated mainly by signal transducer and activator of transcription 3 (STAT3) and has a wide variety of biological activities with pleiotropic effects on many cell types and organs among which are stem cell renewal and implantation process in mammalian embryo. Despite the wealth of data on LIF in mammalian cells, there is a paucity of information on its functions in lower vertebrates. Here, we provide information on the status and the function of LIF signaling in Xenopus amphibian. The IL6 cytokine family is highly conserved in Xenopus genome both at ligands and receptors levels. All cytokines and receptors of the family, except oncostatin M (OSM) and IL27, can be identified in the genome including the orthologs of LIF, cardiotrophin 1 (CTF1), ciliary neurotrophic factor (CNTF), cardiotrophin like cytokine factor 1 (CLCF1), LIFR, IL6ST, IL6R, IL11RA and CNTFR. Lif mRNA is zygotically expressed after midblastula transition while lifr and il6st are maternally expressed. We have investigated the functions of LIF in Xenopus early development with a gain-of-function analysis combined to the use of a dominant negative form of the receptor. The overexpression of Xenopus lif in embryo activates STAT3 phosphorylation and induces a dramatic phenotype where embryos are ventralised and show a reduction of anterior structures with microcephaly. This results mainly from BMP signal stimulation and antagonism towards IGF signals. In addition, most embryos develop tumor-like cell masses according to both autonomous and non-autonomous processes. Through the use of a dominant negative form of the receptor, we demonstrate for the first time that a functional LIF signaling is required for normal vertebrate kidney development. Owing to its experimental advantages, the Xenopus embryo constitutes a useful model to identify the molecular actors that may account for the pleiotropic functions of LIF and their role in vertebrate development.

A20 (tnfaip3) is a negative feedback regulator of RIG-I-Mediated IFN induction in teleost

Interferon production is tightly regulated in order to prevent excessive immune responses. The RIG-I signaling pathway, which is one of the major pathways inducing the production of interferon, is therefore finely regulated through the participation of different molecules such as A20 (TNFAIP3). A20 is a negative key regulatory factor of the immune response. Although A20 has been identified and actively studied in mammals, nothing is known about its putative function in lower vertebrates. In this study, we sought to define the involvement of fish A20 orthologs in the regulation of RIG-I signaling. We showed that A20 completely blocked the activation of IFN and ISG promoters mediated by RIG-I. Furthermore, A20 expression in fish cells was sufficient to reverse the antiviral state induced by the expression of a constitutively active form of RIG-I, thus allowing the efficient replication of a fish rhabdovirus, the viral hemorrhagic septicemia virus (VHSV). We brought evidence that A20 interrupted RIG-I signaling at the level of TBK1 kinase, a critical point of convergence for many different pathways that activates important transcription factors involved in the expression of many cytokines. Finally, we showed that A20 expression was directly induced by the RIG-I pathway demonstrating that fish A20 acts as a negative feedback regulator of this key pathway for the establishment of an antiviral state.

MicroRNA-146a promotes red spotted grouper nervous necrosis virus (RGNNV) replication by targeting TRAF6 in orange spotted grouper, Epinephelus coioides

MicroRNA-146a (miR-146a) has been demonstrated to function as a negative regulator of cellular immune responses against pathogens in mammals, however, little information focused on its functions in lower vertebrates. In this study, we investigated the regulatory roles of orange spotted grouper, Epinephelus coioides miR-146a during red spotted grouper nervous necrosis virus (RGNNV) infection. During RGNNV infection in grouper spleen (GS) cells, the endogenous expression level of miR-146a and tumor necrosis factor receptor-associated factor 6 (TRAF6) significantly increased along with the infection time. Overexpression of miR-146a significantly facilitated viral infection, evidenced by the increased transcription of viral CP and RdRp genes, while miR-146a knockdown by specific inhibitors decreased RGNNV replication. Using pMIR-REPORT Luciferase system, we found that the 3′ untranslated region (UTR) of grouper TRAF6 could be specifically targeted by miR-146a. Further studies showed that its downstream target gene pro-inflammatory cytokines, including TNF-α, IL-8 and IL-1β, were all significantly decreased in miR-146a mimic transfected cells, but increased in miR-146a inhibitors transfected cells during RGNNV infection. Thus, our results suggested and verified that holding the level of miR-146a exerted crucial roles in RGNNV infection through TRAF6-mediated inflammatory response.

Evidence for a role of heme oxygenase-1 in the control of cardiac function in zebrafish (Danio rerio) larvae exposed to hypoxia.

Carbon monoxide (CO) is a gaseous neurotransmitter produced from the breakdown of heme via heme oxygenase-1 (HO-1; hypoxia-inducible isoform) and heme oxygenase-2 (HO-2; constitutively expressed isoform). In mammals, CO is involved in modulating cardiac function. The role of the HO-1/CO system in the control of heart function in fish, however, is unknown and investigating its physiological function in lower vertebrates will provide a better understanding of the evolution of this regulatory mechanism. We explored the role of the HO-1/CO system in larval zebrafish (Danio rerio) in vivo by investigating the impact of translational gene knockdown of HO-1 on cardiac function. Immunohistochemistry revealed the presence of HO-1 in the pacemaker cells of the heart at 4 days post-fertilization and thus the potential for CO production at these sites. Sham-treated zebrafish larvae (experiencing normal levels of HO-1) significantly increased heart rate (fH) when exposed to hypoxia (PwO2 =30 mmHg). Zebrafish larvae lacking HO-1 expression after morpholino knockdown (morphants) exhibited significantly higher fH under normoxic (but not hypoxic) conditions when compared with sham-treaded fish. The increased fH in HO-1 morphants was rescued (fH was restored to control levels) after treatment of larvae with a CO-releasing molecule (40 µmoll(-1) CORM). The HO-1-deficient larvae developed significantly larger ventricles and when exposed to hypoxia they displayed higher cardiac output ([Formula: see text]) and stroke volume (SV). These results suggest that under hypoxic conditions, HO-1 regulates [Formula: see text] and SV presumably via the production of CO. Overall, this study provides a better understanding of the role of the HO-1/CO system in controlling heart function in lower vertebrates. We demonstrate for the first time the ability for CO to be produced in presumptive pacemaker cells of the heart where it plays an inhibitory role in setting the resting cardiac frequency.

Characterization of the tilapia p53 gene and its role in chemical-induced apoptosis

Roles of p53 in mammals have been extensively studied but little is known about its functions in lower vertebrates. We have cloned and characterized the p53 gene from tilapia (Oreochromis niloticus), expressed it in Escherichia coli, generated a rabbit polyclonal antibody and examined the gene's expression patterns at both transcript and protein levels. The full-length p53 cDNA was 1288 bp with an ORF of 1,138 bp, encoding a polypeptide of 364 amino acids. The gene contains all four classical p53 family signature motifs but has low sequence similarity with other p53 genes. It was constitutively transcribed in all tissues examined, most abundantly in liver and blood cells. In addition, ATM, p53 and Bax proteins, associated with DNA damage responses and apoptosis, were all upregulated in tilapia liver after treatment with the stress inducers etoposide and CdCl(2). Tilapia p53 is thus both a constitutive and inducible acute-phase protein that is cooperatively activated via the ATM-p53 pathway in responses to stress and DNA-damaging signals.

MyD88 Interacts with Interferon Regulatory Factor (IRF) 3 and IRF7 in Atlantic Salmon (Salmo salar): TRANSGENIC SsMyD88 MODULATES THE IRF-INDUCED TYPE I INTERFERON RESPONSE AND ACCUMULATES IN AGGRESOMES

MyD88 is an intracellular adaptor protein that transmits signals downstream of immune receptors such as the IL-1 receptor and the majority of the known mammalian toll-like receptors. Homologs of MyD88 have been identified in many vertebrate species; however, the adaptor has been studied mostly in mammals, and little is known about its function in lower vertebrates. The results presented in the current paper demonstrate, for the first time, that the teleost MyD88, through its Toll/Interleukin-1 receptor domain, interacts with SsIRF3 and two SsIRF7 paralogs: transcription factors that are critically involved in the virus-induced IFN responses. The data further highlight the potential of transgenic SsMyD88 to modulate the IRF-induced type I IFN response as the adaptor synergized with SsIRF3 to activate IRF-E/IFN-stimulated response element-containing reporter gene constructs and endogenous myxovirus resistance homolog expression. Microscopy analyses demonstrated that, similar to mammalian MyD88, both endogenous and transgenic SsMyD88 accumulated in intracellular aggregates. However, unlike the endogenous SsMyD88 clusters, which co-localized with endocytosed CpGs and probably represented myddosomes, overexpressed SsMyD88 accumulated in aggresomes. Although these structures accumulated ubiquitinated proteins, they did not associate with the autophagosome markers p62 and light chain 3-like protein, indicating that they are most likely classical aggresomes rather than aggresome-like induced structures, aggregates of ubiquitinated proteins induced by toll-like receptor/MyD88 signaling in antigen-presenting cells. The significance of the accumulation of transgenic MyD88 in aggresomes is currently unknown; nevertheless it is tempting to speculate that it might represent a defense mechanism against the potentially harmful effects of excessive MyD88 signaling.

Functional analysis of carp interferon-γ: Evolutionary conservation of classical phagocyte activation

In teleost fish two IFN-γ gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-γ function is associated with the IFN-γ2 cluster. We investigated the evolutionary conservation of the IFN-γ function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-γ sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-γ2, in contrast to carp IFN-γ1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1β, TNFα and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-γ sensitive. These results corroborate an evolutionary conserved nature of IFN-γ function in lower vertebrates including classical activation of phagocytes.

Characterization of inhibin α subunit (inha) in the zebrafish: evidence for a potential feedback loop between the pituitary and ovary

Inhibin and activin are closely related disulphide-linked dimers that belong to the transforming growth factor beta superfamily. Although inhibin has been extensively studied in mammals, the information about its existence and function in lower vertebrates is very scarce. Using zebrafish as a model, the present study demonstrated that the inhibin-specific alpha subunit (inha) was predominantly expressed in the gonads and no transcript could be detected in other tissues including the pituitary and brain. In the ovary, the expression of inha was restricted to the somatic follicle cells surrounding the oocyte, together with the beta subunits (inhbaa and inhbb). This was further supported by the absence of its expression in the ovulated unfertilized eggs. During folliculogenesis, inha expression in the follicles slightly but steadily increased from primary growth to the mid-vitellogenic stage; however, its expression surged dramatically at the full-grown stage. Interestingly, the expression level of inha decreased significantly in the follicles whose oocytes were undergoing spontaneous maturation or germinal vesicle breakdown. When tested on cultured ovarian fragments, both goldfish pituitary extract and forskolin significantly stimulated inha expression. Further experiments showed that recombinant zebrafish FSH but not LH significantly increased inha expression in the same assay system. When tested in vitro, human inhibin A exhibited a slight but significant inhibitory effect on 17alpha, 20beta-dihydroxyprogesterone-induced oocyte maturation after 4 h incubation. The stimulation of inha expression by FSH and the potential inhibition of FSH by inhibin suggest a possible existence of a negative feedback loop between the pituitary and ovary in the zebrafish.

Pre-B cell colony-enhancing factor in lower vertebrates: First evidence of this cytokine being involved in antioxidant activity by reconstruction of a novel NAD salvage pathway in E. coli

The pre-B cell colony-enhancing factor identified in mammals is an important cytokine involved in multiple functions, such as immunoregulation, cellular proliferation and differentiation. However, little is known about its existence and function in lower vertebrates; therefore, we investigated the characterization, expression and especially the biofunction of this factor in Tetraodon nigroviridis, a model organism of lower vertebrates. We focus on the question of whether the pre-B cell colony-enhancing factor of lower vertebrates contributes to the NAD mediated antioxidant activity by its involvement in the biosynthesis of NAD through pyridine nucleotide cycles. Experimental data demonstrated that by transforming fish pre-B cell colony-enhancing factor into Escherichia coli cells, the amounts of NAD and NADP significantly increased, and cellular antioxidant activity greatly improved. This is the first report about vertebrate pre-B cell colony-enhancing factor acting as a nicotinamide phosphoribosyltransferase to reconstruct a pyridine nucleotide cycle III pathway in E. coli and playing an important role in antioxidant stress by up-regulation of NAD biosynthesis. We hope that our observations may enrich the study of pre-B cell colony-enhancing factor, and contribute to a better understanding of the molecular and functional evolution of the pre-B cell colony-enhancing factor family in both lower vertebrates and mammals as a whole.

Comparative Genome Analysis of the Neurexin Gene Family in Danio rerio: Insights into Their Functions and Evolution

Neurexins constitute a family of proteins originally identified as synaptic transmembrane receptors for a spider venom toxin. In mammals, the 3 known Neurexin genes present 2 alternative promoters that drive the synthesis of a long (alpha) and a short (beta) form and contain different sites of alternative splicing (AS) that can give rise to thousands of different transcripts. To date, very little is known about the significance of this variability, except for the modulation of binding to some of the Neurexin ligands. Although orthologs of Neurexins have been isolated in invertebrates, these genes have been studied mostly in mammals. With the aim of investigating their functions in lower vertebrates, we chose Danio rerio as a model because of its increasing importance in comparative biology. We have isolated 6 zebrafish homologous genes, which are highly conserved at the structural level and display a similar regulation of AS, despite about 450 Myr separating the human and zebrafish species. Our data indicate a strong selective pressure at the exonic level and on the intronic borders, in particular on the regulative intronic sequences that flank the exons subject to AS. Such a selective pressure could help conserve the regulation and consequently the function of these genes along the vertebrates evolutive tree. AS analysis during development shows that all genes are expressed and finely regulated since the earliest stages of development, but mark an increase after the 24-h stage that corresponds to the beginning of synaptogenesis. Moreover, we found that specific isoforms of a zebrafish Neurexin gene (nrxn1a) are expressed in the adult testis and in the earliest stages of development, before the beginning of zygotic transcription, indicating a potential delivery of paternal RNA to the embryo. Our analysis suggests the existence of possible new functions for Neurexins, serving as the basis for novel approaches to the functional studies of this complex neuronal protein family and more in general to the understanding of the AS mechanism in low vertebrates.

Lack of p53 induction in fish cells by model chemotherapeutics

Although p53 has been extensively studied in mammalian models, relatively little is known about its specific function in lower vertebrates. It has long been assumed that p53 pathways characterized in mammals apply to other vertebrates as well. Fish provide a useful model for the study of environmental carcinogenesis, and populations of fish inhabiting highly polluted environments provide information on the etiology of pollutant-mediated cancer. In this study, we investigated p53 protein and apoptosis induction in PLHC-1 (desert topminnow hepatocellular carcinoma), RTL-W1 (rainbow trout normal liver), and primary rainbow trout hepatocytes exposed to model chemotherapeutics. All of the chemicals used in these studies have been demonstrated to increase p53 protein levels and induce apoptosis in mammalian cell lines. In contrast, PLHC-1 p53 protein was not induced in response to any model mammalian p53 inducers following 24 h exposures. Additionally, both trout cell types demonstrated this same lack of p53 induction upon exposure to model chemotherapeutic drugs. PLHC-1 cells demonstrated an induction of apoptosis as measured by caspase-3 activation following exposure to all of the chemotherapeutics tested. Our results suggest that fish p53 may be activated differently from that of their mammalian counterparts, and that important differences may exist between phyla in the function and regulation of p53 as well as other mechanisms involved in environmental carcinogenesis.

The siphon controversy counterpoint: the brain need not be “baffling”

The application of siphon principles to the cerebral circulation has engendered a surprising amount of controversy (1–3, 11, 19, 22, 23). The reluctance to apply siphon principles to the cerebral circulation probably stems more from its inescapable, but counterintuitive, corollary: if the

Neuroglobin and Cytoglobin: Genes, Proteins and Evolution

Hemoglobin and myoglobin are oxygen transport and storage proteins of most vertebrates. Neuroglobin (Ngb) and cytoglobin (Cygb)--two recent additions to the vertebrate globin superfamily--have still disputed functions. Combining the data from all available resources, we investigate the evolution of these novel globins. Both Ngb and Cygb show little sequence variation in vertebrate evolution, suggesting conserved structures and functions, and an important role in the animal's metabolism. Exon-intron patterns remained unchanged in Ngb and Cygb, with the exception of the addition of a 3' exon to Cygb early in mammalian evolution. In phylogenetic analyses, Ngb forms a common branch with globin X, another recently identified globin with undefined function in lower vertebrates, and with some invertebrate nerve globins. This shows an early divergence of this branch in animal evolution. Cygb is related to myoglobin, and associated with an eye-specific globin from birds. The pattern of globin evolution shows that proteins with clear respiratory roles evolved independently from intracellular globins with uncertain functions. This result suggests either multiple independent functional changes or a yet undefined respiratory role of tissue globins like Ngb and Cygb.

Cell and molecular cell biology of melanin-concentrating hormone

Recent advances in the study of melanin-concentrating hormone (MCH) have depended largely on molecular biological techniques. In mammals, which have attracted the most attention, novel findings concern (i) the MCH gene, which can yield several peptides by either posttranslational cleavage or alternative splicing, as well as bidirectional transcription; (ii) the identification of two G protein-coupled MCH receptors in the brain and peripheral tissues; and (iii) the evidence for subpopulations of MCH neurons in the central nervous system, characterized by their chemical phenotypes, connections, and individual physiological responses to different physiological paradigms. The involvement of central MCH in various functions, including feeding, reproduction, stress, and behavior patterns, is reviewed. The stage during evolution at which MCH may have acquired hypophysiotrophic and hormonal functions in lower vertebrates is considered in light of morphological data. Evidence that MCH also has peripheral paracrine/autocrine effects in mammals is provided.

Induction of nitric oxide and respiratory burst response in activated goldfish macrophages requires potassium channel activity

Potassium channel activity is important for modulating mammalian macrophage antimicrobial functions. The involvement of potassium channels in mediation of immune cell function in lower vertebrates, such as teleost, has not been explored. Since relatively little is known about the types of potassium channels present in fish macrophages, pharmacological blockers with broad ranges of activity were tested: 4-aminopyridine (4-AP), quinine, and tetraethylammonium chloride (TEA). The potassium channel blockers inhibited reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) production by goldfish macrophages activated with bacterial lipopolysaccharide (LPS) and/or macrophage activating factor (MAF)-containing supernatants. Quinine was the most potent inhibitor with an IC 50 of 50 μM, while the other blockers, 4-AP and TEA, had IC 50 of 1.2 and 0.6 mM, respectively. A reversible depolarization of the goldfish macrophage plasma membrane potential (Vm) was observed following treatments with potassium channel blockers, and was related to transcriptional changes in the inducible nitric oxide synthase gene (iNOS). Down-regulation of antimicrobial activities and depolarization of the goldfish macrophage plasma membrane were not a consequence of reduced cell number or viability, suggesting that potassium channels are required for generation of appropriate goldfish macrophage antimicrobial functions.

Morphological and electrophysiological properties of dissociated primate retinal cells

Although isolated retinal cell preparations have been used widely to study retinal function in lower vertebrates, dissociated cells from primate retina have not been developed for routine physiological experiments. In this study, we demonstrated the feasibility of obtaining viable and identifiable dissociated cells from the primate retina. In addition, we characterized voltage-dependent membrane currents in each type of primate retinal cell with the whole-cell patch-clamp technique. Multiple types of ionic conductance with distinctive current profiles were recorded in various types of primate retinal neurons. Photoreceptors exhibited an inward I H activated by membrane hyperpolarization and an outward current activated at depolarized potentials. Two types of potassium currents (transient potassium current, I K(A), and delayed rectifier potassium current, I K(V)) were recorded from bipolar cells. I K(A) dominated the current response in putative midget bipolar cells, and I K(V) was mainly associated with putative rod bipolar cells. L-type calcium currents ( I Ca) were observed in primate bipolar cells with axon terminals, but not in axotomized bipolar cells. Large voltage-dependent sodium currents ( I Na) were only recorded from ganglion cells. Müller cells exhibited I K(V) and large potassium inward rectifier current ( I K(IR)), and occasionally a small I Na. Neurons with electrophysiological signatures of amacrine cells and horizontal cells were also studied even though their morphological features were lost during cell dissociation. By using both morphological and physiological criteria outlined in this report, it is possible to use the dissociated retinal cell preparation as an in vitro system for physiological, biochemical and pharmacological studies of the primate visual system.

Evolution of antigen binding receptors.

This review addresses issues related to the evolution of the complex multigene families of antigen binding receptors that function in adaptive immunity. Advances in molecular genetic technology now permit the study of immunoglobulin (Ig) and T cell receptor (TCR) genes in many species that are not commonly studied yet represent critical branch points in vertebrate phylogeny. Both Ig and TCR genes have been defined in most of the major lineages of jawed vertebrates, including the cartilaginous fishes, which represent the most phylogenetically divergent jawed vertebrate group relative to the mammals. Ig genes in cartilaginous fish are encoded by multiple individual loci that each contain rearranging segmental elements and constant regions. In some loci, segmental elements are joined in the germline, i.e. they do not undergo genetic rearrangement. Other major differences in Ig gene organization and the mechanisms of somatic diversification have occurred throughout vertebrate evolution. However, relating these changes to adaptive immune function in lower vertebrates is challenging. TCR genes exhibit greater sequence diversity in individual segmental elements than is found in Ig genes but have undergone fewer changes in gene organization, isotype diversity, and mechanisms of diversification. As of yet, homologous forms of antigen binding receptors have not been identified in jawless vertebrates; however, acquisition of large amounts of structural data for the antigen binding receptors that are found in a variety of jawed vertebrates has defined shared characteristics that provide unique insight into the distant origins of the rearranging gene systems and their relationships to both adaptive and innate recognition processes.

Neuroscience of the mechanisms of mind and behavior

Reflexes, compound movements, and innate behavior are basic neural functions in lower vertebrates, and are executed by the spinal cord and brain stem. These functions are made goal-oriented by the limbic system, stable by the basal ganglia, adaptive by the cerebellum, and restorable by the wakefulness-sleep centers in the brain stem. Elaborate sensorimotor functions of the cerebral neocortex have evolved after birds, and mental functions have developed in the association cortex of primates, particularly in humans. To understand the neuronal mechanisms operating in each of these executive and regulatory systems of brains, a number of obstacles must be overcome, as reviewed here from the perspective of recent studies on the cerebellum.