Studies on the origin of the hypothalamus-pituitary endocrine axis and the evolution of glycoprotein hormones in amphioxus (ナメクジウオにおける視床下部―下垂体系の起源と 糖タンパク質ホルモンの進化に関する研究) Thesis submitted for the degree of Doctor of Philosophy (agriculture) of The University of Tokyo December 2009 Yukiko Tando Department of Aquatic Bioscience Graduate School of Agricultural and Life Sciences The University of Tokyo Contents Abbreviations and Notes ........................................................................................ i Chapter 1 General Introduction ………………………………………….…… 1 Chapter 2 Survey of Homologous Genes for Vertebrate Adenohypophysial …. 11 Hormones from Amphioxus Chapter 3 Structures of Glycoprotein Hormone Subunits in Amphioxus ……. 33 Chapter 4 The Structures and Phylogenetic Relation of Amphioxus GPH …... 64 Subunit Genes Chapter 5 Distribution of ampGPA2 and ampGPB5 in Amphioxus ………….. 77 Chapter 6 Expression of Genes Encoding Neurohypophysial Hormone and …. 93 Receptors for Hypothalamic Hormone and Sex Steroid in the Head of Amphioxus Chapter 7 General Discussion ……………………………….………………… 112 References …………………………………………………….…………………. 124 Acknowledgements …………………………………………….…………...…….. 136 Appendix …………………………………………….…………...……………….. I-XII i Abbreviations ACTH adrenocorticotropic hormone ampGPA2 amphioxus glycoprotein hormone α subunit 2 ampGPA2LP amphioxus glycoprotein hormone α subunit 2 like protein ampGPB5 amphioxus glycoprotein hormone β subunit 5 DDW deionized and distilled water DEPC dietyl pyrocarbonate ER estrogen receptor EST expressed sequence tag FSH follicle-stimulating hormone GH growth hormone GnRH gonadotropin-releasing hormone GPH glycoprotein hormone GTH gonadotropin hCG human chorionic gonadotropin HPA hypothalamus-pituitary axis IHC immunohistochemistry ISH in situ hybridization JGI Joint Genome Institute LH luteinizing hormone LMD laser microdissection NCBI National Center for Biotechnology Information, USA PCR polymerase chain reaction Pit-1 pituitary-specific transcription factor-1 ii POMC proopiomelanocortin PRL prolactin SL somatolactin SR steroid receptor TSH thyroid-stimulating hormone TRH thyrotropin-releasing hormone VT vasotocin Notes Animal Names: Usages of animal names in this thesis followed those used in the genome database of the NCBI. iii Chapter 1 General Introduction 1 1-1 The endocrine systems of vertebrates and invertebrates The endocrine system is one of the major systems that control physiological processes, such as growth, metabolism, reproduction, and osmoregulation by transmission of chemical signals, or hormonal signals through their specific receptors. This regulatory system is diversified among invertebrates and vertebrates. In all vertebrates, molecular structures and physiological functions of hormones and receptors differ quite often even in the same classes, and also the presence or absence of particular endocrine organs depends on species. This diversity was mostly derived from adaptation to environments where animals survived during the history of evolution. Nonetheless, fundamental regulatory mechanisms of the endocrine systems in vertebrates can be described as almost common when compared to those in invertebrates. In contrast to vertebrates, the endocrine systems of invertebrates are highly variable even in the same animal groups. The structure and function of their organs for endocrine regulations are much specialized, as cnidarians have scattered neurosecretory cells which secrete neuropeptides to control feeding, reproduction, and development (Mackie et al., 2003; Katsukura et al., 2004). Insects have the corpora allata and the prothoracic gland which regulate molt, metamorphosis and maturation of gonads (Moshtzky et al., 1996; Bollenbacher et al., 1975). Crustaceans have the X organ-sinus gland system and Y organ which regulate body color change and molt, and function of the androgenic gland (Keller, 1992), while octopuses have the optic gland which is involved in control of reproduction (Wells and Wells, 1969). However, most invertebrates do not have endocrine organs which are specialized for secretion of hormones. The neurosecretory systems are thus well developed in invertebrates. 1-2 The hypothalamus-pituitary axis One of the inherent endocrine systems in vertebrates is the hypothalamus-pituitary axis 2 (HPA). This typical neuroendocrine axis is crucial for conversion of encephalic neural information to systemic chemical signals, which are conveyed to the target organs by bloodstream and regulate activities of target cells. This system is composed of two organs, the hypothalamus and the pituitary that are connected structurally and functionally. The hypothalamus is the basal part of the diencephalon lying below the thalamus (Swaab et al., 1993), whereas the two major subdivisions of the pituitary, adenohypophysis and neurohypophysis, locate ventrally to the brain and attached to the hypothalamus. Several hypothalamic neurosecretory centers project their axons to one of the subdivisions of the neurohypophysis, the median eminence, which is richly supplied with blood vessels that drain into the pituitary stalk, known as the hypophysial portal system (Popa and Fielding, 1930), although fishes have no median eminence and their hypothalamic neurosecretory axons project directly to the adenohypophysis. Basically, hypothalamic neurosecretory neurons secrete small neurohormones, such as neurohypophysisal hormones, releasing hormones and inhibiting hormones. They are released into blood capillaries, travel through the portal system, and reach the pituitary where they control secretion of pituitary hormones. Pituitary hormones target certain peripheral endocrine organs such as the gonads and the thyroid which in turn release their hormones into the blood. Peripheral hormones regulate functions of target organs, and further provide either positive or negative feedback effects on the hypothalamus and the pituitary. The two regions of the pituitary, the adenohypophysis and neurohypophysis, are derived from two different origins (Green, 1951). The adenohypophysis originates from an invagination of the oral ectoderm, or the Rathke’s pouch, beneath the diencephalon (Jacobson et al., 1979). This structure elongates to be constricted at its attachment to the oral epithelium. The adenohypophysis is thus primarily a glandular tissue, whereas the neurohypophysis originates from neuronal endings of hypothalamic neurons. The adjacent 3 region of the neural plate becomes a neuronal component like a funnel shaped process, which connects the Rathke’s pouch that guides the neurohypophysis. The adenohypophysis can be separated into three regions: the pars distalis, the pars tuberalis, and the pars intermedia; and two subregions can be identified in the neurohypophysis, that is, the median eminence and the pars nervosa. Recently, molecular mechanisms that determine the fates of adenohypophysial endocrine cells were investigated (Scully and Rosenfeld, 2002). This study elucidated many signals that induce differentiation of cell types in the adenohypophysis. The acquisition of the HPA is considered as a remarkable innovation in vertebrates. This endocrine system, which controls many complex endocrine functions, is consistently conserved throughout all classes of vertebrates, regardless of diverse patterns of life cycles and reproductive strategies. Such consistency in vertebrates has led an abundance of comparative researches on the mechanisms and roles of this system from the points of evolutionary views. However, why and how the HPA had emerged and acquired crucial functions only in the lineage of vertebrates are yet to be clarified. Here, it is noteworthy that the most primitive representatives of vertebrates, hagfish and lamprey which belong to agnathan, do not have the evident median eminence. Although histochemically comparable structures of the neurohypophysis were identified in agnathan fish, neither direct neuronal innervation seen in teleosts nor vascularization seen in tetrapods is apparent between the neurohypophysis and adenohypophysis (Gorbman et al., 1983; Kobayashi and Uemura, 1972). On the basis of these classical findings, many researchers believe that a manner of hypothalamic regulation of pituitary functions is diffusion of neurohormones from the neurohypophysis to the adenohypophysis across the connective tissues (Tsukahara et al., 1986; Nozaki et al., 1994). Although it is still uncertain whether the structural characteristics of the agnathan pituitary are the origin of the HPA or not, the abovementioned findings led researches to explore homologous structures of the 4 hypothalamus and the pituitary in more primitive and vertebrate-related species among invertebrates such as cephalochordates and urochordates. 1-3 Amphioxus as the model for an ancestor of vertebrates Amphioxus, which belongs to phylum Chordata, subphylum Cephalochordata, was first described by Pallas as a molluscan slug (Pallas, 1774). Cephalochordata consists of three genera (Branchiostoma, Epigonichtys, and Asymmetron) and widely distributed in tropical and temperate seas including Japanese coastal areas (Fig. 1-1). They are small worm-like marine animals that spend most of their lives in the sea floor, and filter-feed through the mouth (Fig. 1-2). In contrast to vertebrates, the notochord which runs along the antero-posterior axis is maintained for life. The nerve cord lies directly on the notochord. Its anterior tip is called the cerebral vesicle which shows distinctive designation (Wicht and Lacalli,