Involvement of Huntingtin in Development and Ciliary Beating Regulation of Larvae of the Sea Urchin, Hemicentrotus pulcherrimus.

The multiple functions of the wild type Huntington's disease protein of the sea urchin Hemicentrotus pulcherrimus (Hp-Htt) have been examined using the anti-Hp-Htt antibody (Ab) raised against synthetic oligopeptides. According to immunoblotting, Hp-Htt was detected as a single band at around the 350 kDa region at the swimming blastula stage to the prism larva stage. From the 2-arm pluteus stage (2aPL), however, an additional smaller band at the 165 kDa region appeared. Immunohistochemically, Hp-Htt was detected in the nuclei and the nearby cytoplasm of the ectodermal cells from the swimming blastula stage, and the blastocoelar cells from the mid-gastrula stage. The Ab-positive signal was converged to the ciliary band-associated strand (CBAS). There, it was accompanied by several CBAS-marker proteins in the cytoplasm, such as glutamate decarboxylase. Application of Hp-Htt morpholino (Hp-Htt-MO) has resulted in shortened larval arms, accompanied by decreased 5-bromo-2-deoxyuridin (BrdU) incorporation by the ectodermal cells of the larval arms. Hp-Htt-MO also resulted in lowered ciliary beating activity, accompanied by a disordered swirling pattern formation around the body. These Hp-Htt-MO-induced deficiencies took place after the onset of CBAS system formation at the larval arms. Thus, Hp-Htt is involved in cell proliferation and the ciliary beating pattern regulation signaling system in pluteus larvae.

Involvement of Netrin/Unc-5 Interaction in Ciliary Beating and in Pattern Formation of the Ciliary Band-Associated Strand (CBAS) in the Sea Urchin, Hemicentrotus pulcherrimus.

The GABAergic neural circuit is involved in the motile activities of both larval and juvenile sea urchins. Therefore, its function is inherited beyond metamorphosis, despite large scale remodeling of larval organs during that period. However, the initial neural circuit formation mechanism is not well understood, including how glutamate decarboxylase-expressing blastocoelar cells (GADCs) construct the neural circuit along the circumoral ciliary band (a ciliary band-associated strand, CBAS) on the larval body surface. In this study, using whole-mount immunohistochemistry and 3D reconstructed imaging, the ontogenic process of CBAS patterning was studied by focusing on Netrin and the interaction with its receptor, Unc-5. During the early 2-arm pluteus stage, a small number of GADCs egress onto the apical surface of the larval ectoderm. Then, they line up on the circumoral side of the ciliary band, and by being inserted by a further number of GADCs, form longer multicellular strands along the Netrin stripe. Application of a synthetic peptide, CRFNMELYKLSGRKSGGVC of Hp-Netrin, that binds to the immunoglobulin domain of Unc-5 during the prism stage, causes stunted CBAS formation due to inhibition of GADC egression. This also results in reduced ciliary beating. Thus, the Netrin/Unc-5 interaction is involved in the construction and function of the CBAS.

Initial report of γ-aminobutyric acidergic locomotion regulatory system and its 3-mercaptopropionic acid-sensitivity in metamorphic juvenile of sea urchin, Hemicentrotus pulcherrimus.

The γ-aminobutyric acid (GABA) signal transmission system (GSTS) contributes to larval swimming through the regulation of ciliary beating. However, whether this system also contributes to the primary podia (PP)-generated motility of juveniles remained unclear. The present study aimed to elucidate the involvement of the GSTS in the motility of metamorphic juveniles (juveniles) (1) by immunohistochemically elucidating the location of molecular constituents of the PP, and (2) by inhibiting the activity of GΑΒΑ decarboxylase (GAD) with 3-mercaptopropionic acid (3-MPA). During metamorphosis, the echinus rudiment protrudes its PP out of the body surface in 8-arm plutei. The PP expresses immunopositive signal (-IS) of GAD, GABA, GABAA receptor and tropomyosin, and is constituted with the GABA-IS negative distal tip and the GABA/GAD-IS gaiter region. The latter radiates distal projections to the disc that contains a GAD-IS cellular network. The juvenile body cavity houses a GABA/ßIII-tubulin-IS Penta-radial ring (PrR) that extends branches into each PP and several bridges to the GAD/GABA-IS Penta-radial plate (PrP) on the oral side but does not reach to the gaiter region. 3-MPA reversibly inhibits the juvenile motility and GABA-IS expression in the PrR/PrP complex. This indicates that the complex is the major contributor to the GABAergic motility in juveniles.

Immunohistochemical and ultrastructural properties of the larval ciliary band-associated strand in the sea urchin Hemicentrotus pulcherrimus.

BACKGROUND: The swimming activity of sea urchin larvae is dependent on the ciliary band (CB) on the larval surface and is regulated by several neurotransmitters, including serotonin (5HT), dopamine, and γ-aminobutyric acid (GABA). However, the CB signal transmission mechanism remains unknown. The present study investigated the structural relationship between the CB and external signal receptors by immunohistochemical and transmission electron microscopic analyses of sea urchin, Hemicentrotus pulcherrimus, larvae. RESULTS: Glutamate decarboxylase (GAD; GABA synthetase) was detected in a strand of multiple cells along the circumoral CB in 6-arm plutei. The GAD-expressing strand was closely associated with the CB on the oral ectoderm side. The ciliary band-associated strand (CBAS) also expressed the 5HT receptor (5HThpr) and encephalopsin (ECPN) throughout the cytoplasm and comprised 1- to 2-µm diameter axon-like long stretched regions and sporadic 6- to 7-µm diameter bulbous nucleated regions (perikarya) that protruded into the oral ectoderm side. Besides the laterally polarized morphology of the CBAS cells, Epith-2, which is the epithelial lateral cell surface-specific protein of the sea urchin embryo and larva, was expressed exclusively by perikarya but not by the axon-like regions. The CBAS exposed its narrow apical surface on the larval epithelium between the CB and squamous cells and formed adherens junctions (AJs) on the apical side between them. Despite the presence of the CBAS axon-like regions, tubulins, such as α-, ß-, and acetylated α-tubulins, were not detected. However, the neuroendocrine cell marker protein synaptophysin was detected in the axon-like regions and in bouton-like protrusions that contained numerous small ultrastructural vesicles. CONCLUSIONS: The unique morphology of the CBAS in the sea urchin larva epithelium had not been reported. The CBAS expresses a remarkable number of receptors to environmental stimuli and proteins that are probably involved in signal transmission to the CB. The properties of the CBAS explain previous reports that larval swimming is triggered by environmental stimuli and suggest crosstalk among receptors and potential plural sensory functions of the CBAS.

Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos.

Sea urchin mesenchyme is composed of the large micromere-derived spiculogenetic primary mesenchyme cells (PMC), veg2-tier macromere-derived non-spiculogenetic mesenchyme cells, the small micromere-derived germ cells, and the macro- and mesomere-derived neuronal mesenchyme cells. They are formed through the epithelial-to-mesenchymal transition (EMT) and possess multipotency, except PMCs that solely differentiate larval spicules. The process of EMT is associated with modification of epithelial cell surface property that includes loss of affinity to the apical and basal extracellular matrices, inter-epithelial cell adherens junctions and epithelial cell surface-specific proteins. These cell surface structures and molecules are endocytosed during EMT and utilized as initiators of cytoplasmic signaling pathways that often initiate protein phosphorylation to activate the gene regulatory networks. Acquisition of cell motility after EMT in these mesenchyme cells is associated with the expression of proteins such as Lefty, Snail and Seawi. Structural simplicity and genomic database of this model will further promote detailed EMT research.

Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae.

The ontogenetic origin of blastocoelar glutamate decarboxylase (GAD)-expressing cells (GADCs) in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH) detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad) in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells.

Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos.

The epithelial cells of the sea urchin Hemicentrotus pulcherrimus embryo express an Epith-2, uncharacterized glycoprotein, on the lateral surface. Here, we describe internalization of Epith-2 during mesenchyme formation through the epithelial-to-mesenchymal transition (EMT). Epith-2 was first expressed on the entire egg surface soon after fertilization and on the blastomeres until the 4-cell stage, but was localized to the lateral surface of epithelial cells at and after the 16-cell stage throughout the later developmental period. However, primary mesenchyme cells (PMC) and secondary mesenchyme cells (SMC) that ingress by EMT lost Epith-2 from their cell surface by endocytosis during dissociation from the epithelium, which was associated with the appearance of cytoplasmic Epith-2 dots. The cytoplasmic Epith-2 retained a similar relative molecular mass to that of the cell surface immediately after ingression through the early period of the spreading to single cells. Then, Epith-2 was completely lost from the cytoplasm. Tyrosine residues of Epith-2 were phosphorylated. The endocytic retraction of Epith-2 was inhibited by herbimycin A (HA), a protein tyrosine kinase (PTK) inhibitor, and suramin, a growth factor receptor (GFR) inhibitor, suggesting the involvement of the GFR/PTK (GP) signaling pathway. These two GP inhibitors also inhibited PMC and SMC spreading to individual cells after ingression, but the dissociation of PMC and SMC from the epithelium was not inhibited. In suramin-treated embryos, dissociated mesenchyme cells migrated partially by retaining their epithelial morphology. In HA-treated embryos, no mesenchyme cells migrated. Thus, the EMT occurs in relation to internalization of Epith-2 from presumptive PMC and SMC.

Unc-5/netrin-mediated axonal projection during larval serotonergic nervous system formation in the sea urchin, Hemicentrotus pulcherrimus.

The molecular structure and role of two splice-isoforms of Unc-5 (Hp-Unc-5v1 and v2) in Unc-5/netrin interaction during serotonergic axonal projection were elucidated in this study. Hp-Unc-5v1 was found to be comprised of two immunoglobulin-like domains, two thrombospondin domains in the extracellular region, and ZU-5, DB, and Death domains in the cytoplasmic region, whereas Hp-Unc-5v2 lacked one thrombospondin domain, the transmembrane domain, and all cytoplasmic domains. Hp-Unc-5v1 was transcribed in unfertilized eggs, which continued until the 3-day post-fertilization (-dpf) 2-arm pluteus stage, but was suspended at the mesenchyme blastula stage (mB1), whereas Hp-Unc-5v2 was not transcribed in unfertilized eggs, but was from after fertilization to the same developmental stage of mB1 as Hp-Unc-5v1. Relative accumulation of transcripts of both splice-isoforms peaked at the prism stage and declined thereafter, and they were localized at the vegetal pole region of early gastrulae, around the blastopore in mid- to late gastrulae, at fore- and mid-gut regions and on the basal side of dorsal ectoderm in 28-hour post-fertilization prism larvae, and within axons at and after the 2-dpf pluteus stage. Hp-Unc-5v2:GFP was detected in the entire serotonergic cell body and extracellularly on the basal surface of oral ectoderm in 2-dpf plutei and exclusively within axons in 4-dpf plutei. Overexpression of Hp-Unc-5v2 resulted in decreased axonal projection in plutei. Knockdown of Hp-Unc-5v1 by morpholino antisense oligonucleotide resulted in severe deficiency of axonal projection. Interference of Unc-5/netrin interaction using an exogenous synthetic SQDFGKTW peptide from the VI domain in Hp-netrin inhibited axonal projection and larval swimming.

Development of the GABA-ergic signaling system and its role in larval swimming in sea urchin.

The present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning glutamate decarboxylase (Hp-gad), GABAA receptor (Hp-gabrA) and GABAA receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2 days post-fertilization (d.p.f.) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by RT-PCR during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2 d.p.f. pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4 d.p.f. plutei but strongly from fertilized eggs to 2 d.p.f. plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the two-arm pluteus stage. Hp-gabarap transcription and protein expression were detected from the swimming blastula stage. Inhibition of the GABAA receptor by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17 and 34 d.p.f. plutei) than in younger ones (1 d.p.f. prism larvae).

Spatiotemporal expression pattern of gonad-stimulating substance-like peptide of the sea cucumber, Apostichopus japonicus.

The spatiotemporal expression pattern of gonad-stimulating substance-like peptide-containing polypeptide (GSSLP) in the sea cucumber Apostichopus japonicus was examined using immunochemistry. The GSSLP was detected in the gonads from shortly before the empirical breeding season (May and June) to July. On the basis of immunoblotting analysis, GSSLP showed considerable polymorphism among the organs examined in this study, particularly in the gonads, in which the polymorphism was associated with N-glycosylation and the formation of intra-molecular disulfide bonds. In the ovary, GSSLP was expressed from March to June and corresponded to two bands at 113 and 100 kDa under reducing conditions. In July, only the larger band weakly remained. In testis, GSSLP was detected first in April as two bands of 245 and 190 kDa under reducing conditions. The number of bands increased to five in June but decreased to three smeared bands in July. In the radial nerve and circumoral nerve ring, GSSLP corresponded to a single peptide of 170 kDa with little N-glycosylation and its expression level hardly changed throughout a year with no correlation with the breeding season. GSSLP was detected mainly in the morula cells in all the organs examined. In addition, GSSLP was detected in the follicle cells of the ovary and, for a brief period, in the jelly space, but never in the ooplasm. In testis, the morula cells were localized close to the invaginated inner epithelium, but never in the male gametes. In July animals, gonadal morula cells were rarely observed.

Development of a dopaminergic system in sea urchin embryos and larvae.

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.

Spatiotemporal expression pattern of an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus during early development, and its potential role in larval vertical migration.

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.

The neurotoxic effects of monocrotophos on the formation of the serotonergic nervous system and swimming activity in the larvae of the sea urchin Hemicentrotus pulcherrimus.

The neurotoxicity of monocrotophos (MCP) in the development of the serotonergic nervous system and swimming activity of larvae of the sea urchin, Hemicentrotus pulcherrimus, was examined. Lethal dose 50% of MCP was 43µg/ml. Overall morphology was not affected in larvae that received up to 30µg/ml of MCP soon after fertilization until the 53h post-fertilization pluteus stage. However, while 70±0.6% of larvae in 5µg/ml MCP swam actively, the proportion decreased to 30±1.7% in 30µg/ml MCP. Accordingly, immunoblotting indicated that MCP decreased the relative intensity of immunoreaction of serotonin receptor protein. Whole-mount immunohistochemistry indicated that MCP inhibited serotonergic axon growth, reduced the number of serotonergic cells at the apical ganglion, and perturbed formation of the serotonin receptor cell network. The present study demonstrated that sea urchin larva is a useful model for evaluating the working mechanism of environmental toxicants in neurogenesis and behavior.

Gonad-stimulating substance-like molecule from the radial nerve of the sea cucumber.

Gonad-stimulating substance-like molecule (GSSL) was isolated from the radial nerve of the sea cucumber, Apostichopus japonicus (Aj-GSSL), and its partial DNA and protein sequences were characterized. The smaller part of the molecule that also retains GSSL activity was estimated. Radial nerve extract (RNE) induced germinal vesicle breakdown (GVBD) at 3 mg/ml in 85% of immature ovarian oocytes. Similar intensity of GSSL activity to RNE was seen in a fraction that contained peptides between 3 kDa and 10 kDa (3-10 kDa-fraction) separated by ultrafiltlation membrane. MALDI-TOF MS analysis and silver-stained 18% SDS-PAGE slab gels identified a major peptide at around 4.6 kDa in a 3-10 kDa-fraction, and that was subjected to internal protein sequencing. The resulting 12-amino acid sequence was not found in the BLAST database to date. Immunohistochemistry using antiserum raised against the 12-amino acid peptide located the peptide to granular cells in the hyponeural part of the radial nerve and in the epineural sinus beneath the radial nerve. Sequence data was obtained using degenerate primers designed from the 12-amino acid sequence and 5 and 3 RACEs. These resulted in a 148 bp cDNA that coded a 43-amino acid sequence of H2N-VLSKQAHHHHHEGWSLPGVPAEIDDLAGNIDYNIFKEQREKIK-COOH. The synthetic 43-amino acid Aj-GSSL generated from this sequence induced GVBD in 50% of immature ovarian oocytes at 6 microM. An N-terminal 21-amino acid peptide of the synthetic partial Aj-GSSL (Aj-GSSL-P1) induced GVBD to 80% of immature ovarian oocytes at 12 microM. This indicated that Aj-GSSL-P1 is of sufficient length for GSSL activity.

Development of nervous systems to metamorphosis in feeding and non-feeding echinoid larvae, the transition from bilateral to radial symmetry.

The development of nervous system (NS) in the non-feeding vestibula larva of the sea urchin, Holopneustes purpurescens, and the feeding echinopluteus larva of Hemicentrotus pulcherrimus was examined by focusing on fate during metamorphosis. In H. purpurescens, the serotonergic NS (SerNS) appeared simultaneously and independently in larval tissue and adult rudiment, respectively, from 3-day post-fertilization. In 4-day vestibulae, an expansive aboral ganglion (450 x 100 mum) was present in the larval mid region that extended axons toward the oral ectoderm. These axons diverged near the base of the primary podia. An axonal bundle connected with the primary podia and the rim of vestopore on the oral side. Thus, the SerNS of the larva innervated the rudiment at early stage of development of the primary podia. This innervation was short-lived, and immediately before metamorphosis, it disappeared from the larval and adult tissue domains, whereas non-SerNS marked by synaptotagmin remained. The NS of 1-month post-fertilization plutei of H. pulcherrimus comprised an apical ganglion (50 x 17 mum) and axons that extended to the ciliary bands and the adult rudiment (AR). A major basal nerve of serotonergic and non-serotonergic axons and a minor non-serotonergic nerve comprised the ciliary band nerve. In 3-month plutei, axonal connection among the primary podia in the neural folds completed. The SerNS never developed in the AR. Thus, there was distinctive difference between feeding- and non-feeding larvae of the above sea urchins with respect to SerNS and the AR.

Spatio-temporal expression of a Netrin homolog in the sea urchin Hemicentrotus pulcherrimus (HpNetrin) during serotonergic axon extension.

A netrin homolog of the sea urchin, Hemicentrotus pulcherrimus (HpNetrin) was characterized in terms of its expression behavior. The predicted amino acid sequence was an ortholog of hemichordate netrin-1. Reverse transcriptase-PCR, immunoblotting, and whole mount immunohistochemistry showed that gene transcription and protein expression occurred from 15-hour post-fertilization (hpf) swimming blastula stage to, at least 53-hpf pluteus stage. HpNetrin was detected on the entire basal surface of the ectoderm in swimming and 16-hpf mesenchyme blastulae. However, by 24-hpf prism stage, immunoreaction on the oral ectoderm decreased, whereas it increased in the aboral ectoderm including near the animal plate ectoderm (area-I). By 48-hpf pluteus stage, the HpNetrin-rich area-I comprised a 40mm wide dorsal midline belt (DMB) that stretched from the dorsal posterior edge of the animal plate to the posterior end of the larval body. Serotonergic cells were first detected in the HpNetrin-moderate area between the anterior DMB and the HpNetrin-poor oral ectoderm near the animal plate in 24-hpf prism larvae. By 48-hpf pluteus stage, these cells extended axons toward the middle-ridge to form a neural plexus of the apical ganglion. At 53-hpf pluteus stage, these axons extended further away from the apical ganglion directly or through the DMB toward the HpNetrin-poor contralateral ectoderm. The protein expression and axon extension pattern were reproduced in embryonic cell-aggregates formed from artificially dissociated 20-hpf gastrulae and resembled small blastula. In Hpnetrin morpholino anti-sense oligonucleotide-injected plutei, serotonergic axon extension was severely inhibited. Thus, HpNetrin functions as a serotonergic axon guidance cue in this basal deuterostome.

Serotonin stimulates [Ca2+]i elevation in ciliary ectodermal cells of echinoplutei through a serotonin receptor cell network in the blastocoel.

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.

Exclusive expression of hedgehog in small micromere descendants during early embryogenesis in the sea urchin, Hemicentrotus pulcherrimus.

Hedgehog (hh) is a multifunctional extracellular protein, and known as an essential signal molecule in morphogenetic movement in animal embryos. We have cloned, sequenced, and studied dynamic localization of Hphh, a hedgehog homologue of the sea urchin, Hemicentrotus pulcherrimus. The origin of Hphh transcribing cells was also verified during early embryogenesis. The amino acid sequence of Hphh shows high homology to Lvhh, an hh homologue cloned in the sea urchin, Lytechinus variegatus. Reverse transcriptase polymerase chain reaction showed that the transcription of Hphh occurred at and after 19 h post-fertilization (19 hpf) mesenchyme blastula stage until, at least, 69 hpf 4-arm pluteus stage. Whole mount in situ hybridization showed Hphh transcription sites in a few cells at the tip of archenteron in 30 hpf gastrulae. At around 45 hpf 2-arm pluteus stage, the number of Hphh transcribed cells was 8, and unequally split to two groups, 5 cells in left coelomic sac and 3 cells in right coelomic sac. A cell lineage tracing by staining the small micromeres with 5-Bromo-2-deoxyuridine showed that Hphh was transcribed exclusively in all the small micromere descendants and comprised the coelomic sacs in 69 hpf plutei.

The 5-HT receptor cell is a new member of secondary mesenchyme cell descendants and forms a major blastocoelar network in sea urchin larvae.

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.

Expression of tryptophan 5-hydroxylase gene during sea urchin neurogenesis and role of serotonergic nervous system in larval behavior.

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.