Kaoru Kubokawa

The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

Cephalochordate Melanopsin: Evolutionary Linkage between Invertebrate Visual Cells and Vertebrate Photosensitive Retinal Ganglion Cells

Animal photoreceptor cells can be classified into two distinct types, depending on whether the photopigment is borne on the membrane of a modified cilium (ciliary type) or apical microvilli (rhabdomeric type) [1]. Ciliary photoreceptors are well known as vertebrate rods and cones and are also found in several invertebrates. The rhabdomeric photoreceptor, in contrast, is a predominant type of invertebrate visual cell, but morphologically identifiable rhabdomeric photoreceptors have never been found in vertebrates. It is hypothesized that the rhabdomeric photoreceptor cell had evolved to be the photosensitive retinal ganglion cell for the vertebrate circadian photoentrainment [2, 3 and 4] owing to the fact that some molecules involved in cell differentiation are common among them [5]. We focused on the cephalochordate amphioxus because it is the closest living invertebrate to the vertebrates, and interestingly, it has rhabdomeric photoreceptor cells for putative nonvisual functions [6]. Here, we show that the amphioxus homolog of melanopsin [7, 8 and 9], the circadian photopigment in the photosensitive retinal ganglion cells of vertebrates, is expressed in the rhabdomeric photoreceptor cells of the amphioxus and that its biochemical and photochemical properties, not just its primary structure, are considerably similar to those of the visual rhodopsins in the rhabdomeric photoreceptor cells of higher invertebrates. The cephalochordate rhabdomeric photoreceptor represents an evolutionary link between the invertebrate visual photoreceptor and the vertebrate circadian photoreceptor.

Amphioxus homologs of Go-coupled rhodopsin and peropsin having 11-cis- and all-trans-retinals as their chromophores

Because of low contents in the native organs and failure of the expression in cultured cells, the chromophore configurations of the pigments in Go‐coupled opsin and peropsin groups in the opsin family are unknown. Here we have succeeded in expression of the amphioxus homologs of these groups in HEK293s cells and found that they can be regenerated with 11‐cis‐ and all‐trans‐retinals, respectively. Light isomerized the chromophores of these opsins into the all‐trans and 11‐cis forms, respectively. The results strongly suggest that the physiological function of peropsin would be a retinal photoisomerase, while 11‐cis configuration is necessary for the Go‐coupled opsin groups.

Endogenous green fluorescent protein (GFP) in amphioxus.

remains to be learned about the taxonomic distribution and biological function of these proteins in nature. To date, GFPs have been found in only two major groups in the metazoan tree: specifically, in a number of cnidarians, rel atively near the base of the tree, and in a few copepod crustaceans, relatively derived within the protostome branch (2, 3). The cnidarian GFPs are often associated with biolu minescence, but those found so far in copepods are not. We now report that the limited taxonomic distribution of ani mals with endogenous GFPs may be partially due to inad equate sampling efforts, because we have found such mol ecules in the cephalochordate amphioxus. About 10 years

PROTEIN TYROSINE KINASE CDNAS FROM AMPHIOXUS, HAGFISH, AND LAMPREY : ISOFORM DUPLICATIONS AROUND THE DIVERGENCE OF CYCLOSTOMES AND GNATHOSTOMES

Abstract. Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications.

Estrogen-Dependent Transactivation of Amphioxus Steroid Hormone Receptor via Both Estrogen and Androgen Response Elements

Estrogens are necessary for ovarian differentiation during critical developmental windows in most vertebrates and promote the growth and differentiation of the adult female reproductive system. Estrogen actions are largely mediated through the estrogen receptors (ERs), which are ligand-activated transcription factors. To understand the molecular evolution of sex steroid hormone receptors, we isolated cDNAs encoding two steroid receptors from Japanese amphioxus, Branchiostoma belcheri: an ER ortholog and a ketosteroid receptor (SR) ortholog. Reporter gene assays revealed that the SR ortholog has molecular functions similar to those of the vertebrate ER. Surprisingly, the ER ortholog is an estrogen-insensitive repressor of SR-mediated transcription. Furthermore, we found that the SR ortholog can bind to both estrogen-responsive elements (EREs) and androgen-responsive elements (AREs) and mediates transcriptional activation by estrogens through both types of elements. Our findings suggest that the ancestral SR, but not ER, could bind estrone and induce the ERE- and ARE-dependent transactivation and that it gained the ability to be regulated by ketosteroid and recognize ARE specifically before jawless vertebrates split. These results highlight the importance of comparative experimental approaches for the evolutionary study of endocrine systems.

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus

SummaryImmunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LHβ or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.

Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan

Novel bacterial species were isolated from sediments adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of 226-246 m. The isolated strains, JAMM 0745T, JAMM 1380, JAMM 1475 and JAMM 1610, were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or subterminal flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of the novel isolates indicated a relationship to a symbiotic bacterial clone of the polychaete Osedax japonicus (99.6-99.9 % sequence similarity) and these bacteria were closely related to members of the genus Neptunomonas (95.6-96.0 % similarity) within the class Gammaproteobacteria. The novel strains were able to produce isoprenoid quinone Q-8 as the major quinone component. The predominant fatty acids were C16 : 0, C16 : 1 and C18 : 1, with C18 : 2 and C20 : 2 present in smaller amounts. The DNA G+C contents of the four novel strains were about 43.6-43.8 mol%. Based on the taxonomic differences observed, the four isolated strains appear to represent a novel species of the genus Neptunomonas. The name Neptunomonas japonica sp. nov. (type strain JAMM 0745T=JCM 14595T=DSM 18939T) is proposed for the novel strains.

Expression of the gene for ancestral glycoprotein hormone β subunit in the nerve cord of amphioxus

Amphioxus belongs to the subphylum cephalochordata, a clade of chordates phylogenetically placed at the most basal position. Despite many studies on the endocrine system of amphioxus, there were no confident lines of evidence on the presence of pituitary hormones, whereas recent amphioxus genome analysis reported that amphioxus has no pituitary hormone except for thyrostimulin, which is a glycoprotein hormone in the pituitary, brain, and other organs of vertebrates. In the present study, we cloned cDNA for one glycoprotein hormone beta subunit (GPB) from amphioxus, AmpGPB5, and phylogenetically indicated that AmpGPB5 is the ancestral molecule of glycoprotein hormone beta subunits of vertebrates including pituitary glycoprotein hormones. Synteny analyses showed conservation of chromosomal location of genes near GPB genes from amphioxus through human. The AmpGPB5 gene was expressed in a restricted region of the dorsal part of the nerve cord, glandular atrial cells of gills, and pre-vitellogenic oocytes in amphioxus. However, expression was not detected in the Hatscheks pit which is considered to be a primitive pituitary gland. On the basis of present results, we hypothesize that a portion of vertebrate pituitary hormones might be derived from an ancestral glycoprotein hormone of amphioxus that functions as a neuroendocrine hormone.

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene‐pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle‐stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α‐dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.