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ABSTRACT: Serotonergic neurons differentiate in the neurogenic animal plate ectoderm of the sea urchin embryo. The regulatory mechanisms that control the specification or differentiation of these neurons in the sea urchin embryo are not yet understood, although, after the genome was sequenced, many genes encoding transcription factors expressed in this region were identified. Here, we report that zinc finger homeobox (zfhx1/z81) is expressed in serotonergic neural precursor cells, using double in situ hybridization screening with a serotonergic neural marker, tryptophan 5-hydroxylase (tph) encoding a serotonin synthase that is required for the differentiation of serotonergic neurons. zfhx1/z81 begins to be expressed at gastrula stage in individual cells in the anterior neuroectoderm, some of which also express delta. zfhx1/z81 expression gradually disappears as neural differentiation begins with tph expression. When the translation of Zfhx1/Z81 is blocked by morpholino injection, embryos express neither tph nor the neural marker synaptotagminB in cells of the animal plate, and serotonergic neurons do not differentiate. In contrast, Zfhx1/Z81 morphants do express fez, another neural precursor marker, which appears to function in the initial phase of specification/differentiation of serotonergic neurons. In addition, zfhx1/z81 is one of the targets suppressed in the animal plate by anti-neural signals such as Nodal as well as Delta-Notch. We conclude that Zfhx1/Z81 functions during the specification of individual anterior neural precursors and promotes the expression of tph and synaptotagminB, required for the differentiation of serotonergic neurons.
Developmental Biology 12/2011; 363(1):74-83. · 4.07 Impact Factor
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ABSTRACT: Recent studies of the sea urchin embryo have elucidated the mechanisms that localize and pattern its nervous system. These studies have revealed the presence of two overlapping regions of neurogenic potential at the beginning of embryogenesis, each of which becomes progressively restricted by separate, yet linked, signals, including Wnt and subsequently Nodal and BMP. These signals act to specify and localize the embryonic neural fields - the anterior neuroectoderm and the more posterior ciliary band neuroectoderm - during development. Here, we review these conserved nervous system patterning signals and consider how the relationships between them might have changed during deuterostome evolution.
Development 09/2011; 138(17):3613-23. · 6.60 Impact Factor
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ABSTRACT: Partitioning ectoderm precisely into neurogenic and non-neurogenic regions is an essential step for neurogenesis of almost all bilaterian embryos. Although it is widely accepted that antagonism between BMP and its inhibitors primarily sets up the border between these two types of ectoderm, it is unclear how such extracellular, diffusible molecules create a sharp and precise border at the single-cell level. Here, we show that Fez, a zinc finger protein, functions as an intracellular factor attenuating BMP signaling specifically within the neurogenic region at the anterior end of sea urchin embryos, termed the animal plate. When Fez function is blocked, the size of this neurogenic ectoderm becomes smaller than normal. However, this reduction is rescued in Fez morphants simply by blocking BMP2/4 translation, indicating that Fez maintains the size of the animal plate by attenuating BMP2/4 function. Consistent with this, the gradient of BMP activity along the aboral side of the animal plate, as measured by pSmad1/5/8 levels, drops significantly in cells expressing Fez and this steep decline requires Fez function. Our data reveal that this neurogenic ectoderm produces an intrinsic system that attenuates BMP signaling to ensure the establishment of a stable, well-defined neural territory, the animal plate.
Development 08/2011; 138(19):4233-43. · 6.60 Impact Factor
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Takuji Yoshiyama-Yanagawa,
Sora Enya,
Yuko Shimada-Niwa, Shunsuke Yaguchi,
Yoshikazu Haramoto,
Takeshi Matsuya,
Kensuke Shiomi,
Yasunori Sasakura,
Shuji Takahashi,
Makoto Asashima,
Hiroshi Kataoka,
Ryusuke Niwa
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ABSTRACT: Steroid hormones play essential roles in a wide variety of biological processes in multicellular organisms. The principal steroid hormones in nematodes and arthropods are dafachronic acids and ecdysteroids, respectively, both of which are synthesized from cholesterol as an indispensable precursor. The first critical catalytic step in the biosynthesis of these ecdysozoan steroids is the conversion of cholesterol to 7-dehydrocholesterol. However, the enzymes responsible for cholesterol 7,8-dehydrogenation remain unclear at the molecular level. Here we report that the Rieske oxygenase DAF-36/Neverland (Nvd) is a cholesterol 7,8-dehydrogenase. The daf-36/nvd genes are evolutionarily conserved, not only in nematodes and insects but also in deuterostome species that do not produce dafachronic acids or ecdysteroids, including the sea urchin Hemicentrotus pulcherrimus, the sea squirt Ciona intestinalis, the fish Danio rerio, and the frog Xenopus laevis. An in vitro enzymatic assay system reveals that all DAF-36/Nvd proteins cloned so far have the ability to convert cholesterol to 7-dehydrocholesterol. Moreover, the lethality of loss of nvd function in the fruit fly Drosophila melanogaster is rescued by the expression of daf-36/nvd genes from the nematode Caenorhabditis elegans, the insect Bombyx mori, or the vertebrates D. rerio and X. laevis. These data suggest that daf-36/nvd genes are functionally orthologous across the bilaterian phylogeny. We propose that the daf-36/nvd family of proteins is a novel conserved player in cholesterol metabolism across the animal phyla.
Journal of Biological Chemistry 06/2011; 286(29):25756-62. · 4.77 Impact Factor
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ABSTRACT: The ciliary band is a distinct region of embryonic ectoderm that is specified between oral and aboral ectoderm. Flask-shaped ciliary cells and neurons differentiate in this region and they are patterned to form an integrated tissue that functions as the principal swimming and feeding organ of the larva. TGFβ signaling, which is known to mediate oral and aboral patterning of the ectoderm, has been implicated in ciliary band formation. We have used morpholino knockdown and ectopic expression of RNA to alter TGFβ signaling at the level of ligands, receptors, and signal transduction components and assessed the differentiation and patterning of the ciliary band cells and associated neurons. We propose that the primary effects of these signals are to position the ciliary cells, which in turn support neural differentiation. We show that Nodal signaling, which is known to be localized by Lefty, positions the oral margin of the ciliary band. Signaling from BMP through Alk3/6, affects the position of the oral and aboral margins of the ciliary band. Since both Nodal and BMP signaling produce ectoderm that does not support neurogenesis, we propose that formation of a ciliary band requires protection from these signals. Expression of BMP2/4 and Nodal suppress neural differentiation. However, the response to receptor knockdown or dominant-negative forms of signal transduction components indicate signaling is not acting directly on unspecified ectoderm cells to prevent their differentiation as neurons. Instead, it produces a restricted field of ciliary band cells that supports neurogenesis. We propose a model that incorporates spatially regulated control of Nodal and BMP signaling to determine the position and differentiation of the ciliary band, and subsequent neural patterning.
Developmental Biology 11/2010; 347(1):71-81. · 4.07 Impact Factor
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ABSTRACT: In sea urchin embryos, the apical tuft forms within the neurogenic animal plate. When FoxQ2, one of the earliest factors expressed specifically in the animal plate by early blastula stage, is knocked down, the structure of the apical tuft is altered. To determine the basis of this phenotype, we identified FoxQ2-dependent genes using microarray analysis. The most strongly down-regulated gene in FoxQ2 morphants encodes a protein with ankyrin repeats region in its N-terminal domain. We named this gene ankAT-1, Ankyrin-containing gene specific for Apical Tuft. Initially its expression in the animal pole region of very early blastula stage embryos is FoxQ2-independent but becomes FoxQ2-dependent beginning at mesenchyme blastula stage and continuing in the animal plate of 3-day larvae. Furthermore, like FoxQ2, this gene is expressed throughout the expanded apical tuft region that forms in embryos lacking nuclear β-catenin. When AnkAT-1 is knocked-down by injecting a morpholino, the cilia at the animal plate in the resulting embryos are much shorter and their motility is less than that of motile cilia in other ectoderm cells, and remains similar to that of long apical tuft cilia. We conclude that AnkAT-1 is involved in regulating the length of apical tuft cilia.
Developmental Biology 09/2010; 348(1):67-75. · 4.07 Impact Factor
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ABSTRACT: The mechanisms that regulate the organized swimming movements of sea urchin blastulae are largely unknown. Using immunohistochemistry, we found that dopamine (DA) and the Hemicentrotus pulcherrimus homolog of the dopamine receptor D1 (Hp-DRD1) were strongly co-localized in 1-2 microm diameter granules (DA/DRD1 granules). Furthermore, these granules were arranged across the entire surface of blastulae as they developed locomotory cilia before hatching, and remained evident until metamorphosis. DA/DRD1 granules were associated with the basal bodies of cilia, and were densely packed in the ciliary band by the eight-arm pluteus stage. The transcription of Hp-DRD1 was detected from the unfertilized egg stage throughout the period of larval development. Treatment with S-(-)-carbidopa, an inhibitor of aromatic-l-amino acid decarboxylase, for 20-24 h (i) from soon after insemination until the 20 h post-fertilization (20 hpf) early gastrula stage and (ii) from the 24 hpf prism larva stage until the 48 hpf pluteus stage, inhibited the formation of DA granules and decreased the swimming activity of blastulae and larvae in a dose-dependent manner. Exogenous DA rescued these deprivations. The formation of DRD1 granules was not affected. However, in 48 hpf plutei, the serotonergic nervous system (5HT-NS) developed normally. Morpholino antisense oligonucleotides directed against Hp-DRD1 inhibited the formation of DRD1 granules and the swimming of larvae, but did not disturb the formation of DA granules. Thus, the formation of DRD1 granules and DA granules occurs chronologically closely but mechanically independently and the swimming of blastulae is regulated by the dopaminergic system. In plutei, the 5HT-NS closely surrounded the ciliary bands, suggesting the functional collaboration with the dopaminergic system in larvae.
Journal of Experimental Biology 08/2010; 213(Pt 16):2808-19. · 3.00 Impact Factor
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ABSTRACT: Tc1/mariner superfamily transposons are used as transformation vectors in various model organisms. The utility of this transposon family is evidenced by the fact that Tc1/mariner transposons have loose host specificity. However, the activity of these transposons has been observed in only a few organisms, and a recent study in the ascidian Ciona intestinalis suggests that not all Tc1/ mariner transposons show loose host specificity. To understand host specificity, we used sea urchins, since they have a long history as materials of embryology and developmental biology. Transposon techniques have not been reported in this organism, despite the likelihood that these techniques would open up many experimental possibilities. Here we tested the activity of three Tc1/ mariner transposons (Minos, Sleeping Beauty, and Frog Prince) in the sea urchin Hemicentrotus pulcherrimus. Minos has both excision and transposition activity in H. pulcherrimus embryos, whereas no excision activity was detected for Sleeping Beauty or Frog Prince. This study suggests that Minos is active in a broad range of non-host organisms and can be used as a transformation tool in sea urchin embryos.
ZOOLOGICAL SCIENCE 03/2010; 27(3):256-62. · 0.95 Impact Factor
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ABSTRACT: We have cloned and studied Hp-ECPN, an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus. Hp-ecpn cDNA was produced and found to contain a 1461-bp open reading frame that encodes 486 amino acids. Accumulation of Hp-ecpn mRNA and protein expression occurred at the 14 h postfertilization (hpf) swimming blastula stage and thereafter. The Hp-ECPN protein was N-glycosylated, and the amino acid sequence was similar to that of vertebrate encephalopsins. Whole-mount immunohistochemistry revealed the presence of Hp-ECPN in cells (ECPN cells) that appeared initially around the tip of the archenteron in 20 hpf early gastrulae. By the 54 hpf pluteus stage, ECPN cells had spread through the aboral ectoderm, and, by the eight-arm pluteus stage, were restricted to the tips of the larval arms and the posterior end of the body. The number of ECPN cells increased under conditions of continuous light, but decreased under continuous dark. Knockdown of Hp-ecpn mRNA using morpholino antisense oligonucleotides decreased the number of ECPN cells considerably, and inhibited the vertical swimming of the larvae. This suggested that Hp-ECPN plays a role in photosensitive larval swimming vertical migration. In adult tissues, the ECPN cells were detected exclusively in tube feet.
Embryologia 02/2010; 52(2):195-207. · 2.21 Impact Factor
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ABSTRACT: Two major signaling centers have been shown to control patterning of sea urchin embryos. Canonical Wnt signaling in vegetal blastomeres and Nodal signaling in presumptive oral ectoderm are necessary and sufficient to initiate patterning along the primary and secondary axes, respectively. Here we define and characterize a third patterning center, the animal pole domain (APD), which contains neurogenic ectoderm, and can oppose Wnt and Nodal signaling. The regulatory influence of the APD is normally restricted to the animal pole region, but can operate in most cells of the embryo because, in the absence of Wnt and Nodal, the APD expands throughout the embryo. We have identified many constituent APD regulatory genes expressed in the early blastula and have shown that expression of most of them requires Six3 function. Furthermore, Six3 is necessary for the differentiation of diverse cell types in the APD, including the neurogenic animal plate and immediately flanking ectoderm, indicating that it functions at or near the top of several APD gene regulatory networks. Remarkably, it is also sufficient to respecify the fates of cells in the rest of the embryo, generating an embryo consisting of a greatly expanded, but correctly patterned, APD. A fraction of the large group of Six3-dependent regulatory proteins are orthologous to those expressed in the vertebrate forebrain, suggesting that they controlled formation of the early neurogenic domain in the common deuterostome ancestor of echinoderms and vertebrates.
Development 05/2009; 136(7):1179-89. · 6.60 Impact Factor
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ABSTRACT: The primary (animal-vegetal) (AV) and secondary (oral-aboral) (OA) axes of sea urchin embryos are established by distinct regulatory pathways. However, because experimental perturbations of AV patterning also invariably disrupt OA patterning and radialize the embryo, these two axes must be mechanistically linked. Here we show that FoxQ2, which is progressively restricted to the animal plate during cleavage stages, provides this linkage. When AV patterning is prevented by blocking the nuclear function of beta-catenin, the animal plate where FoxQ2 is expressed expands throughout the future ectoderm, and expression of nodal, which initiates OA polarity, is blocked. Surprisingly, nodal transcription and OA differentiation are rescued simply by inhibiting FoxQ2 translation. Therefore, restriction of FoxQ2 to the animal plate is a crucial element of canonical Wnt signaling that coordinates patterning along the AV axis with the initiation of OA specification.
Developmental Cell 02/2008; 14(1):97-107. · 14.03 Impact Factor
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ABSTRACT: Nodal functions in axis and tissue specification during embryogenesis. In sea urchin embryos, Nodal is crucial for specification of oral ectoderm and is thought to pattern neurogenesis in the animal plate. To determine if Nodal functions directly in suppressing neuron differentiation we have prepared mutant forms of Sp-Smad2/3. Expressing an activated form produces embryos similar to embryos overexpressing Nodal, but with fewer neurons. In chimeras in which Nodal is suppressed, cells expressing activated Sp-Smad2/3 form oral ectoderm, but not neurons. In embryos with vegetal signaling blocked, neurons do not form if activated Smad2/3 is co-expressed. Expression of dominant negative mutants produces embryos identical to those resulting from blocking Nodal expression. In chimeras overexpressing Nodal, cells expressing dominant negative Sp-Smad2/3 form aboral ectoderm and give rise to neurons. In permanent blastula chimeras dominant negative Sp-Smad2/3 is able to suppress the effects of Nodal permitting neuron differentiation. In these chimeras Nodal expression in one half suppresses neural differentiation across the interface. Anti-phospho-Smad3 reveals that the cells adjacent to cells expressing Nodal have nuclear immunoreactivity. We conclude Sp-Smad2/3 is a component of the Nodal signaling pathway in sea urchins and that Nodal diffuses short distances to suppress neural differentiation.
Developmental Biology 03/2007; 302(2):494-503. · 4.07 Impact Factor
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ABSTRACT: A full-length serotonin receptor mRNA from the 5Hthpr gene was sequenced from larvae of the sea urchin, Hemicentrotus pulcherrimus. The DNA sequence was most similar to 5HT-1A of the sea urchin Strongylocentrotus purpuratus found by The Sea Urchin Genome Project, and the protein sequence predicted the presence of seven transmembrane domains. Immunohistochemistry with anti-5HThpr antibodies indicated that the protein was expressed on blastocoelar cells that comprised the major blastocoelar network (serotonin receptor cell network). These network cells inserted their processes into the ectoderm in various regions, including the ciliary band region. Serotonin injected into the blastocoel stimulated a transient elevation of cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in the ectoderm, as detected by Oregon-Green dextran, injected earlier in development. The calcium transient propagated as a wave at about 175 microm s(-1), but was not detectable in the serotonin receptor-positive cell network. In larvae treated with p-chlorophenylalanine, a potent and irreversible serotonin synthesis inhibitor, serotonin application did not stimulate [Ca(2+)](i), the serotonin receptor cell network did not develop properly, and the swimming behavior of the larvae was abnormal. However, formation of a different nervous system comprising synaptotagmin-possessed neurites was not affected by p-chlorophenylalanine treatment. These results imply that serotonin secreted from the apical ganglion into the blastocoel stimulates the elevation of [Ca(2+)](i) in the larval ectodermal cells through the serotonin receptor cell network.
Journal of Experimental Biology 03/2007; 210(Pt 3):403-12. · 3.00 Impact Factor
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ABSTRACT: The genome of the sea urchin Strongylocentrotus purpuratus has recently been sequenced because it is a major model system for the study of gene regulatory networks. Embryonic expression patterns for most genes are unknown, however.
Using large-scale screens on arrays carrying 50% to 70% of all genes, we identified novel territory-specific markers. Our strategy was based on computational selection of genes that are differentially expressed in lithium-treated embryos, which form excess endomesoderm, and in zinc-treated embryos, in which endomesoderm specification is blocked. Whole-mount in situ hybridization (WISH) analysis of 700 genes indicates that the apical organ region is eliminated in lithium-treated embryos. Conversely, apical and specifically neural markers are expressed more broadly in zinc-treated embryos, whereas endomesoderm signaling is severely reduced. Strikingly, the number of serotonergic neurons is amplified by at least tenfold in zinc-treated embryos. WISH analysis further indicates that there is crosstalk between the Wnt (wingless int), Notch, and fibroblast growth factor signaling pathways in secondary mesoderm cell specification and differentiation, similar to signaling cascades that function during development of presomitic mesoderm in mouse embryogenesis. We provide differential expression data for more than 4,000 genes and WISH patterns of more than 250 genes, and more than 2,400 annotated WISH images.
Our work provides tissue-specific expression patterns for a large fraction of the sea urchin genes that have not yet been included in existing regulatory networks and await functional integration. Furthermore, we noted neuron-inducing activity of zinc on embryonic development; this is the first observation of such activity in any organism.
Genome biology 02/2007; 8(5):R85. · 6.63 Impact Factor
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Erica Sodergren,
George M Weinstock,
Eric H Davidson,
R Andrew Cameron,
Richard A Gibbs,
Robert C Angerer,
Lynne M Angerer,
Maria Ina Arnone,
David R Burgess,
Robert D Burke, [......],
Sandra Lee,
Lora Lewis,
George Miner,
Margaret Morgan,
Lynne V Nazareth,
Geoffrey Okwuonu,
David Parker,
Ling-Ling Pu,
Rachel Thorn,
Rita Wright
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ABSTRACT: We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus purpuratus, a model for developmental and systems biology. The sequencing strategy combined whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones, aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome. The genome encodes about 23,300 genes, including many previously thought to be vertebrate innovations or known only outside the deuterostomes. This echinoderm genome provides an evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes.
Science 12/2006; 314(5801):941-52. · 31.20 Impact Factor
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ABSTRACT: The animal plate of the sea urchin embryo becomes the apical organ, a sensory structure of the larva. In the absence of vegetal signaling, an expanded and unpatterned apical organ forms. To investigate the signaling that restricts the size of the animal plate and patterns neurogenesis, we have expressed molecules that regulate specification of ectoderm in embryos and chimeras. Enhancing oral ectoderm suppresses serotonergic neuron differentiation, whereas enhancing aboral or ciliary band ectoderm increases differentiation of serotonergic neurons. In embryos in which vegetal signaling is blocked, Nodal expression does not reduce the size of the thickened animal plate; however, almost no neurons form. Expression of BMP in the absence of vegetal signaling also does not restrict the size of the animal plate, but abundant serotonergic neurons form. In chimeras in which vegetal signaling is blocked in the entire embryo, and one half of the embryo expresses Nodal, serotonergic neuron formation is suppressed in both halves. In similar chimeras in which vegetal signaling is blocked and one half of the embryo expresses Goosecoid (Gsc), serotonergic neurons form only in the half of the embryo not expressing Gsc. We propose that neurogenesis is specified by a maternal program that is restricted to the animal pole by signaling that is dependent on nuclearization of beta-catenin and specifies ciliary band ectoderm. Subsequently, neurogenesis in the animal plate is patterned by suppression of serotonergic neuron formation by Nodal. Like other metazoans, echinoderms appear to have a phase of neural development during which the specification of ectoderm restricts and patterns neurogenesis.
Development 07/2006; 133(12):2337-46. · 6.60 Impact Factor
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ABSTRACT: Neural patterning genes that are expressed along the anterior-posterior axis of deuterostomes are expressed late in larval development in echinoderms and are thought to function in establishing the highly-derived, adult body plan. We have used genomic resources to clone an engrailed gene (SpEn) from Strongylocentrotus purpuratus, and with this we have developed an antibody specific for SpEn. SpEn is expressed late in embryogenesis in the developing larval nervous system. At the prism stage, a small number of neuroblasts in the oral ectoderm on the edge of the larval mouth begin expressing SpEn. The cells are in bilaterally symmetric positions. The expression of SpEn precedes the expression of the neural markers, synaptotagmin and serotonin in the SpEn immunoreactive cells. The SpEn cells are located on the margin of the domain of cells expressing SpNK2.1, but they do not have nuclear SpNK2.1. Expression of engrailed in a pair of bilateral neural structures in early development appears to be a shared feature of bilaterians.
Gene Expression Patterns 07/2006; 6(5):566-71. · 2.02 Impact Factor
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ABSTRACT: Interest in chordate evolution has emphasized a need for a better understanding of the comparative neuroanatomy of invertebrate deuterostomes. However, molecular and genetic approaches to neurobiological studies in these groups are hampered by a lack of neuron-specific molecular markers. A monoclonal antibody, 1E11, is neuron specific and is useful in identification of neural structures in larvae and adults of echinoderms, hemichordates, and urochordates. To identify a neuron-specific gene product, we have characterized the antigen recognized by 1E11. In immunoblots and immunoprecipitations of neural tissue from adult Strongylocentrotus purpuratus, 1E11 recognizes a 57-kDa band. Tandem mass spectrometry of trypsin digests of the 57-kDa band permitted peptide mass mapping and sequencing of five peptides. All of the sequenced peptides, and 12 additional mass-mapped peptides, are found within the open reading frame of a cDNA encoding synaptotagmin B (Sp-SynB). In situ RNA hybridizations with synaptotagmin B probes with S. purpuratus larvae reveal a pattern of expression that is similar to that revealed by the antibody 1E11. Antibodies produced against a bacterially expressed Sp-SynB protein recognize a 57-kDa protein and colocalize with 1E11. When a full-length Sp-SynB cDNA is expressed in chicken embryonic cells, the cells become immunoreactive to 1E11. We conclude that synaptotagmin B is a gene expressed in neurons that has conserved epitopes in other invertebrate deuterostomes.
The Journal of Comparative Neurology 06/2006; 496(2):244-51. · 3.81 Impact Factor
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ABSTRACT: A gene encoding the serotonin (5-hydroxytryptamine, 5-HT) receptor (5-HT-hpr) was identified from the sea urchin, Hemicentrotus pulcherrimus. Partial amino acid sequence deduced from the cDNA showed strong similarity to Aplysia californica 5-HT2 receptor. Immunoblotting analysis of this 5-HT-hpr protein (5-HT-hpr) with an antibody raised against a deduced peptide showed two bands. Their relative molecular masses were 69 and 53 kDa, respectively. The larger band alone disappeared after N-glycopeptidase F digestion, indicating the protein was N-glycosylated. Immunolocalization analysis showed that cells expressing the 5-HT-hpr (SRC) first appeared near the tip of the archenteron in 33-h post-fertilization (33 hpf) prism larvae. Their cell number doubled in 2 h, and 5-HT-hpr protein expression increased further without cell proliferation. SRC spread ventrally on the basal surface of the oral ectoderm in 36 hpf prism larvae, and then clockwise on the ventral ectoderm to the posterior region to complete formation of the SRC network in 48 hpf early plutei. The SRC network was comprised of 7 main tracts: 4 spicule system-associated tracts and 3 spicule system-independent tracts. The network extended short fibers to the larval body surface through the ectoderm, implicating a signal transmission system that receives exogenous signal. Double-stain immunohistochemistry with antibodies to primary mesenchyme cells showed that SRC were not stained by the antibody. In embryos deprived of secondary mesenchyme cell (SMC) by microsurgery, the number of SRC decreased considerably. These two data indicate that SRC are SMC descendants, adding a new member to the SMC lineage.
Mechanisms of Development 05/2004; 121(4):325-37. · 2.83 Impact Factor
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ABSTRACT: Tryptophan 5-hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of serotonin. cDNA cloning of TPH was carried out, and the occurrence of spatiotemporal transcription of TPH message was examined in larvae of the sea urchin, Hemicentrotus pulcherrimus (HpTPH), with in situ hybridization by using the tyramide signal amplification (TSA) technique and Northern hybridization. Based on deduced amino acids sequence of HpTPH, phylogenetically sea urchin locates at the closest position to vertebrates among invertebrates, and HpTPH had common conserved sequences in a catalytic domain. Initiation of HpTPH transcription occurred at the late gastrula stage exclusively in serotonin cells of apical ganglion (SAG) that was composed of a cluster of HpTPH-positive cells and the negative cells in between. In situ hybridization showed that the mRNA expression pattern was similar to the immunohistochemical localization of serotonin cells reported before (Bisgrove and Burke [1986] Dev. Growth Differ. 28:557-569; Yaguchi et al. [2000] Dev. Growth Differ. 42:479-488). p-Chlorophenylalanine (CPA), an irreversible inhibitor of TPH activity, considerably decreased serotonin content in the serotonin cells, whereas the HpTPH expression pattern and timing, and the extension of neurofibers from SAG cells were apparently unaffected, suggesting CPA exclusively perturbed synthesis of serotonin but not nervous system organization. CPA-treated larvae did not swim, despite the occurrence of ciliary beating in culture chamber, suggesting that proper serotonin synthesis is necessary for normal swimming of the larvae.
The Journal of Comparative Neurology 12/2003; 466(2):219-29. · 3.81 Impact Factor
