Tatsuya Ueki

Peripheral Development of Cranial Nerves in a Cyclostome, Lampetra japonica: Morphological Distribution of Nerve Branches and the Vertebrate Body Plan

The development of peripheral nerves was studied in a Japanese marine lamprey, Lampetra japonica, in whole‐mount and sectioned embryos from hatching until the earliest ammocoete. Nerve fibers were immunohistochemically stained with a monoclonal antibody against acetylated tubulin. Branchiomeric nerves first developed in a simple metamerical pattern, each associated with a single pharyngeal arch. Of those, the ophthalmicus profundus, maxillomandibular, and facial nerves later developed a highly modified branching pattern, whereas postotic nerves were less specialized and showed the stereotypical branching pattern of post‐trematic nerves. The early distribution of melanocytes in myotome‐free space largely overlapped with the morphology of the cranial nerve and ganglion anlage, and resembled the cephalic crest cell distribution pattern in the early chick embryo. It was suggested that the cephalic crest cell distribution, which is also inhibited by myotomes in the lamprey, would be the common basis for branchiomeric nerve patterning. In later development of the lamprey embryo, myotomes 1 through 3, which had originated in the postotic region, grew rostrally into the preotic region, laterally covering all of the branchiomeric nerves. This results in a deep position of the cranial nerves, which is not observed in gnathostomes. J. Comp. Neurol. 384:483–500, 1997.

Molecular biological approaches to the accumulation and reduction of vanadium by ascidians

Abstract About 90 years ago, Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples. His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists. Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained. To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes. To date, three types of vanadium-binding protein, designated as Vanabins, have been isolated, with molecular masses of 12.5, 15, and 16 kDa, along with the cDNAs encoding these proteins. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes. The pentose phosphate pathway participates in the reduction of V(V) to V(IV). The cDNA for each of the vacuolar-type H+ATPase (VATPase) A, B, C, and D subunits, which are located on the vacuolar membranes of vanadocytes, has been isolated and analyzed. VATPase generates a proton-motive force, and is thought to provide the energy for vanadium accumulation. To clarify the entire mechanism involved in the accumulation and reduction, much more genes and proteins expressed in the blood cells need to be systematically identified. Thus, we have performed an expressed sequence tag (EST) analysis of blood cells and have established the functional assay system to elucidate the functions of genes and proteins obtained from ascidian blood cells.

STEREOTYPED AXONAL BUNDLE FORMATION AND NEUROMERIC PATTERNS IN EMBRYOS OF A CYCLOSTOME, LAMPETRA JAPONICA

Early embryonic development of the nervous system of a lamprey, Lampetra japonica, was studied by using immunohistochemical techniques and by scanning electron microscopy. The earliest appearance of axons was detected at Taharas stage 21‐, when dorsolateral and ventral longitudinal fasciculi were present in the hindbrain and spinal cord regions. The branchiomeric nerve roots began to appear at stage 22; the fibers were joined to the dorsolateral fasciculus proximally and also extended distally into each pharyngeal arch. The anterior neural tube was divided into several neuromeres: the mid‐hindbrain sulcus became apparent first, then the portion rostral to this sulcus was subdivided into two portions by the syn‐parencephalic boundary. In the hindbrain around stage 23, rhombomeres developed transiently, of which, rhombomere 4 was the most distinctive. Putative crest cells forming the octavofacial nerve root anlage were selectively adhering to rhombomere 4, whereas no crest cells were found on rhombomere 3. The assignment of the crest‐derived nerve anlage to rhombomeres is conserved between gnathostomes and L. japonica. The neuromerical scheme of the neural tube of L. japonica is also mostly in accordance with that in gnathostomes, sharing the basic developmental patterning of axon bundles at early developmental stages. The most distinct difference between these two groups is the topographical relationships between the hindbrain neuraxis and pharyngeal arches, as well as the otic placode. J. Comp. Neurol. 391:99–114, 1998.

Vanadium-binding proteins (Vanabins) from a vanadium-rich ascidian Ascidia sydneiensis samea

Since the beginning of the last century, it has been known that ascidians accumulate high levels of a transition metal, vanadium, in their blood cells, although the mechanism for this curious biological function remains unknown. Recently, we identified three vanadium-binding proteins (vanabins), previously denoted as vanadium-associated proteins (VAPs) [Zool. Sci. 14 (1997) 37], from the cytoplasm fraction of vanadium-containing blood cells (vanadocytes) of the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we describe the cloning, expression, and analysis of the metal-binding ability of vanabins. Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1x10(-5) and 2.3x10(-5) M, respectively. The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(VI) ions. Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.

Otx cognates in a lamprey, Lampetra japonica.

Abstract Gnathostomes have two lineages of Otx genes, Otx1 and Otx2, as cognates of a Drosophila head gap gene, orthodenticle. Previous studies with mutant mice have demonstrated that they play essential roles in the development of rostral head. To shed lights on the evolution of the rostral head in vertebrates we isolated their cognates in the Japanese marine lamprey, Lampetra japonica. The lamprey genome appeared to have two Otx cognantes, LjOtxA and LjOtxB. Phylogenetic analyses suggest that LjOtxA clusters with gnathostome Otx2 genes, but LjOtxB does not belong to either the Otx1 or Otx2 lineage. LjOtxA was expressed in the forebrain and midbrain with the caudal limit possibly at the midbrain/hindbrain junction as gnathostome Otx cognates are, but LjOtxB was not expressed in the brain. No Otx1 or Otx2 cognates are known in gnathostomes that are not expressed in the brain. Both LjOtxA and LjOtxB were expressed in the olfactory placode, epiphysis, optic stalks, and lower and upper lips. LjOtxB was also expressed in the eyes, where no LjOtxA transcripts were detected. Thus, Otx1 and Otx2 functions for the development of forebrain and midbrain in gnathostomes appear to be shouldered by LjOtxA alone in the lamprey. LjOtxB may have diverged from the stem of the Otx1 and Otx2 lineages and evolved independently.

Early development of the peripheral nervous system in a lancelet species

The developmental pattern of the lancelet (amphioxus) peripheral nervous system from embryos to larvae has been studied by using wholemount immunostaining and transmission electron microscopy. The peripheral nerves first appeared on the anterior dorsal surface of the medulla at the middle neurula stage, when the anterior nerve cord was just closing. A single axon with a large growth cone was the progenitor of each nerve. The nerve roots adopted an asymmetric arrangement soon after. The first nerve, likely a pair of pure sensory nerves, sprouted from the anterior tip of the nerve cord. This nerve may be comparable topographically to the preoptic nerve (the posterior branch of the terminal nerve) in lungfishes. However, the neuron that first extends its axon was located in the medulla, as in the other posterior nerves. One of the extramedullary primary sensory neurons, the corpuscles of de Quatrefages, appeared in larvae with the mouth and two anterior gill pores. Their axons were seemingly fasciculated with the efferent axon of the first nerve. The second nerve, the most complex one to appear during embryonic and early larval development, innervated the preoral pit and the buccal region. The third and fourth nerves on the left side also innervated the buccal region. The larval innervation patterns in the anterior region differed from the adult organization, suggesting a segmental rearrangement of the nerve supply during development. There was no evidence to dichotomize the peripheral nerves into cranial and spinal nerves, as exist in vertebrates. These characteristics of the peripheral nervous system in the lancelet indicate that this animal has a rather derived or primitive developmental system of peripheral nerves, making the analysis of homology with vertebrates difficult. J. Comp. Neurol. 393:415–425, 1998.

Rostral truncation of a cyclostome, Lampetra japonica, induced by all-trans retinoic acid defines the head/trunk interface of the vertebrate body

The effect of all‐trans retinoic acid on embryogenesis was studied in a cyclostome, Lampetra japonica. Treatment with 0.05–0.5 μM retinoic acid on early gastrula and early neurula resulted in loss of the pharynx and in the rostral truncation of the neural tube. The mouth, pharynx, esophagus, heart, endostyle, and rostral brain were missing with graded severity. In the severest case, the embryo consisted only of trunk segments, especially myotomes that extended to the rostral end of the axis. The effect appeared to be dose‐ and stage‐dependent: Rostral pharyngeal arches were more vulnerable to a lower amount of retinoic acid, and earlier treatment resulted in severer defects. The initial protrusion of the anterior axis started equally in control and retinoic acid‐treated embryos, implying that the head morphogenesis is omitted in treated embryos. By identifying the number of myotomes based on the differentiation of hypobranchial muscles, there seemed to be no myotomes lost by retinoic acid‐induced truncation. The rostral truncation, therefore, was not simply a limitation of the anterior axis but was restricted to the ventral portion; only the branchial arches disappeared with normally developing myotomes dorsally. The absent region can be defined as the vertebrate head in a morphological sense, including the branchiomeric and preotic paraxial regions as well as the heart. The results suggest the presence of distinct programs between somitomeric and branchiomeric portions of the body, providing a developmental basis for the dual‐metamerical body plan of vertebrates. Dev. Dyn. 1998;211:35‐51.

Spatio-Temporal Expression Patterns of Eight Epidermis-Specific Genes in the Ascidian Embryo

Abstract During embryogenesis of the ascidian Halocynthia roretzi, exactly eight-hundred epidermal cells are formed in the larva, and the lineage of the cells has been almost completely described. In the present study, we examined the spatio-temporal expression patterns of eight epidermis-specific genes which we already isolated. In situ hybridization of whole-mount specimens unambiguously demonstrated that the expression patterns of the eight genes were not identical, and that they were categorizable into several types. Transcripts of seven genes were restricted to presumptive epidermal cells, although transcripts of one gene were evident in the presumptive neural cells in addition to the presumptive epidermal cells. Therefore, most of the epidermis-specific genes in ascidian embryos are expressed in lineage-associated manner. We discuss these results in relation to the question of whether (a) epidermis-specific genes are expressed exclusively in presumptive epidermal cells prior to neural induction, or (b) the genes are first expressed in both epidermal precursors and precursors of the central nervous system, then the gene expression is downregulated in the latter after the completion of neural induction. Interestingly, cells of the anterior-most region as well as the dorsal midline of the tailbud embryo did not express most of the epidermis-specific genes, suggesting regional differences in the embryonic epidermis. Some other genes might be expressed in a complementary pattern in these regions.

Scanning X-ray Microscopy of Living and Freeze-Dried Blood Cells in Two Vanadium-Rich Ascidian Species, Phallusia mammillata and Ascidia sydneiensis samea

Abstract Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 107 times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchrotron Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

6-Phosphogluconate Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Form a Supramolecular Complex in Human Neutrophils That Undergoes Retrograde Trafficking during Pregnancy

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme’s intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.