A. Zapata

Positional cloning of zebrafish ferroportin1 identifies a conservedvertebrate iron exporter

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMT1 (refs 1,2,3). A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Early hematopoiesis and developing lymphoid organs in the zebrafish

In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5‐somite stage, about 11.5 hours post‐fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early blood‐forming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf. Dev Dyn 1999;214:323–336. 

Phylogeny of lower vertebrates and their immunological structures.

Vertebrate organisms have evolved sophisticated immunological mechanisms for the recognition of self versus nonself. The immune system is functionally characterized by specificity, diversity, and memory but is based at the molecular level on highly diverse antigen receptors (T-cell receptor, TcR; immunoglobulin, Ig), which together with major histocompatibility complex (MHC) molecules allow the activation of the so-called immunocompetent cells (T- and B-lymphocytes) during the immune response. Development and functional activation of immune cells occur in specialized tissue microenvironments provided by the primary and/or secondary lymphoid organs. These lymphoid organs then, represent a higher-order morphological structure culminating from complex molecular, cellular and tissue interactions, and are an invariable feature of the vertebrate immune system.

Lymphocyte development in fish and amphibians

Summary: Recently, molecular markers such as recombination activating genes (RAG), terminal deoxynucleotidyl transferase (TdT), stem cell leukemia hematopoletic transcription factor (SCL), Ikaros and gata‐binding protein (Gata) ‐family members have been isolated and characterized from key lower vertebrates, adding to our growing knowledge of lymphopolesis in ectotherms. In all gnathostomes there appear to be two main embryonic locations derived from the early mesoderm, both intra‐ and extraembryonic, which contribute to primitive and dermitive hematopolesis based upon their differential expression of SCL, Gaia‐1, Gata‐2 and tnyeloblasto‐sis oncogene (c‐myb). In teleosts. a unique intraembryonic location for hematopoletic stem cells termed the intermediate cell mass (ICM) of Oellacter appears to be responsible for primitive or definitive hematopolesis depending upon the species being investigated. In Xenopus, elegant grafting studies in combination with specific molecular markers has led to a better definition of the roles that ventral blood islands and dorsal lateral plate play in amphibian hematopolesis, that of primitive and deffinitive lymphopolesis. After the early embryonic contribution to hematopolesis. specialized tissues must assume the role of providing the proper microenvironment for T and B‐lymphocyte development from progenitor stem cells. In all gnathostomes, the thymus is the major site for T‐cell maturation as evidenced by strong expression of developmental markers such as Ikaros, Rag and TdT plus expression of T‐cell specific markers such as T‐cell receptor β and lck. In this respect, several zebrafish mutants have provided new insights on the development of the thymopoletic environment. On the other band, the sites for B‐cell lymphopolesis are less clear among the lower vertebrates. In elasmobranchs, the spleen, Leydigs organ and the spiral valve may all contribute to B‐cell development, although pre‐B cells have yet to be fully addressed in fish. In teleosts, the kidney is undeniably the major source of B‐cell development based upon functional, cellular and molecular indices. Amphibians appear to use several different sites (spleen, bone marrow and/or kidney) depending upon the species in question.

Shift from Goal-Directed to Habitual Cocaine Seeking after Prolonged Experience in Rats

The development of drug-seeking habits is implicated in the transition from recreational drug use to addiction. Using a drug seeking/taking chained schedule of intravenous cocaine self-administration and reward devaluation methods in rats, the present studies examined whether drug seeking that is initially goal-directed becomes habitual after prolonged drug seeking and taking. Devaluation of the outcome of the drug seeking link (i.e., the drug taking link of the chained schedule) by extinction significantly decreased drug seeking indicating that behavior is goal-directed rather than habitual. With, however, more prolonged drug experience, animals transitioned to habitual cocaine seeking. Thus, in these animals, cocaine seeking was insensitive to outcome devaluation. Moreover, when the dorsolateral striatum, an area implicated in habit learning, was transiently inactivated, outcome devaluation was effective in decreasing drug seeking indicating that responding was no longer habitual but had reverted to control by the goal-directed system. These studies provide direct evidence that cocaine seeking becomes habitual with prolonged drug experience and describe a rodent model with which to study the neural mechanisms underlying the transition from goal-directed to habitual drug seeking.

D2 Receptors Regulate Dopamine Transporter Function via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent and Phosphoinositide 3 Kinase-Independent Mechanism

The dopamine transporter (DAT) terminates dopamine (DA) neurotransmission by reuptake of DA into presynaptic neurons. Regulation of DA uptake by D2 dopamine receptors (D2R) has been reported. The high affinity of DA and other DAT substrates for the D2R, however, has complicated investigation of the intracellular mechanisms mediating this effect. The present studies used the fluorescent DAT substrate, 4-[4-(diethylamino)-styryl]-N-methylpyridinium iodide (ASP+) with live cell imaging techniques to identify the role of two D2R-linked signaling pathways, extracellular signal-regulated kinases 1 and 2 (ERK1/2), and phosphoinositide 3 kinase (PI3K) in mediating D2R regulation of DAT. Addition of the D2/D3 receptor agonist quinpirole (0.1–10 μM) to human embryonic kidney cells coexpressing human DAT and D2 receptor (short splice variant, D2SR) induced a rapid, concentration-dependent and pertussis toxin-sensitive increase in ASP+ accumulation. The D2/D3 agonist (S)-(+)-(4aR, 10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride (PD128907) also increased ASP+ accumulation. D2SR activation increased phosphorylation of ERK1/2 and Akt, a major target of PI3K. The mitogen-activated protein kinase kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) prevented the quinpirole-evoked increase in ASP+ accumulation, whereas inhibition of PI3K was without effect. Fluorescence flow cytometry and biotinylation studies revealed a rapid increase in DAT cell-surface expression in response to D2R stimulation. These experiments demonstrate that D2SR stimulation increases DAT cell surface expression and therefore enhances substrate clearance. Furthermore, they show that the increase in DAT function is ERK1/2-dependent but PI3K-independent. Our data also suggest the possibility of a direct physical interaction between DAT and D2R. Together, these results suggest a novel mechanism by which D2SRautoreceptors may regulate DAT in the central nervous system.

Deletion of the M5 muscarinic acetylcholine receptor attenuates morphine reinforcement and withdrawal but not morphine analgesia

Little is known about the physiological roles of the M5 muscarinic receptor, the last member of the muscarinic receptor family (M1–M5) to be cloned. In the brain, the M5 receptor subtype is preferentially expressed by dopaminergic neurons of the substantia nigra and the ventral tegmental area. Dopaminergic neurons located in the ventral tegmental area are known to play important roles in mediating both the rewarding effects of opiates and other drugs of abuse and the manifestations of opiate/drug withdrawal symptoms. We therefore speculated that acetylcholine-dependent activation of M5 receptors might modulate the manifestations of opiate reward and withdrawal. This hypothesis was tested in a series of behavioral, biochemical, and neurochemical studies using M5 receptor-deficient mice (M5−/− mice) as novel experimental tools. We found that the rewarding effects of morphine, as measured in the conditioned place preference paradigm, were substantially reduced in M5−/− mice. Furthermore, both the somatic and affective components of naloxone-induced morphine withdrawal symptoms were significantly attenuated in M5−/− mice. In contrast, the analgesic efficacy of morphine and the development of tolerance to the analgesic effects of morphine remained unaltered by the lack of M5 receptors. The finding that M5 receptor activity modulates both morphine reward and withdrawal processes suggests that M5 receptors may represent a novel target for the treatment of opiate addiction.

1 - Cells and Tissues of the Immune System of Fish

This chapter focuses on the cell and tissues of the immune system of fish. In teleosts, three types of granulocytes—heterophils, acidophils, and basophils—have been reported. There is enormous variation within the teleosts in both relative abundance and staining reaction of the granulocytes. In the carp, Cyprinus carpio, all three types of granulocytes are found in the blood. Among them, the heterophils and basophils are the least numerous and constitute 1% of the total eukocyte count. The acidophilic granulocytes are rather abundant and constitute 8% of the total leukocyte count. The acidophilic granules are peroxidase positive and contain round or irregular granules with heterogeneous contents. Migrating leukocytes in the basal mucosa and submucosa, as well as in the epithelium but not lymphoid aggregates, have been detected in the hagfish intestine. In the stingray, Dasyatis akajei , meningeal lymphohemopoietic masses appear predominantly in the telencephalon, diencephalon, and mesencephalon. Antigen-binding cells and antibody-producing cells have been found in the lymphohemopoietic tissue of the teleost kidney Recently, a higher number of Ig-positive cells have been found in the head kidney than in the spleen. They occur as scattered cells or forming small pyroninophilic cell clusters, especially after antigenic stimulation. It has been observed that that the cells of the pyroninophilic clusters corresponded to the plasma cell lineage with Ig-producing capacity and to the large lymphoid type plasma cells that gradually differentiate in these cell clusters.

Ultrastructural study of the teleost fish kidney.

Abstract The ultrastructure of the stroma and vascularization of hematopoietic tissue in the kidney of two fresh water teleost fish are described. The presence of reticular and phagocytic reticular cells, similar to those in the bone marrow of higher vertebrates, and sinusoids and thin-walled arteries, support the idea of a phylogenetic relationship between this organ and the bone marrow. The kidney does not appear to be homologous with mammalian lymph nodes.

Mesenchymal stem cells: biological properties and clinical applications

Importance of the field: In the last decade, knowledge of mesenchymal stem cells (MSCs) has evolved rapidly; their immunomodulatory properties and paracrine interactions with specific cell types in damaged tissues and promising results in some clinical applications have made these cells an attractive option for the treatment of certain diseases. Areas covered in this review: We present some relevant methodological issues and biological properties of MSCs, as well as clinical applications of MSC therapies with particular emphasis in the treatment of graft versus host disease (GVHD), complex perianal fistula and refractory metastatic neuroblastoma. Other topical aspects relevant to the application of cellular therapies such as biosafety studies and cellular production of MSCs are also discussed in this review. What the reader will gain: The growing optimism regarding MSCs research is based on the promising results obtained in in vitro and in vivo studies. The rapid translational research with MSCs necessitated standardization of methodology and terminology and greater focus on other aspects such as biosafety and cellular production, especially for clinical use of MSCs. Take home message: Much has been learned about the biology and applications of MSCs and much remains to be learned.