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First published online January 17, 2007
Journal of Experimental Biology 210, 403-412 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02666

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

Hideki Katow^*, Shunsuke Yaguchi and Keiichiro Kyozuka

Research Center for Marine Biology, Graduate School of Life Sciences, Tohoku University, Asamushi, Aomori, Aomori 039-3501, Japan

View larger version (69K): [in this window] [in a new window]   Fig. 1. Sequence of 5HThpr gene and protein and expected secondary structure. (A) 5HThpr gene consists of 2194 nucleotides comprising 543 bp of 5' untranslated region (UTR), 1395 bp of coding region and 254 bp of 3' UTR. The open reading frame encoded 465 amino acids that contained seven predicted transmembrane domains (shaded boxes) and an expected G-protein-coupled signature domain (underlined). Three potential N-glycosylation sites were shown (open boxes). (B) Schematic presentation of predicted secondary structure of 5HThpr based on analysis by SOSUI software program. First six transmembrane domains (1–6) from the N terminus (NH2) are primary helix, and seventh domain (7) nearest to the C terminus (COOH) is a secondary helix. A G-protein-coupled signature 1 locates between the third and fourth transmembrane domains. Cytoplasmic, inside the cell; extracellular, outside the cell.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Aligned sequences of transmembrane domains of serotonin receptors of five species. All transmembrane domains showed high homology, but the number of domains were different. Shaded boxes show location of transmembrane domains, based on SOSUI analysis. The transmembrane domains are numbered from the N terminus. Although serotonin receptor 5HT-1A of S. purpuratus lacked the 6th and 7th transmembrane domains (SOSUI analysis), two additional C-terminal transmembrane domains at equivalent regions to the other serotonin receptors were predicted both by SOSUI analysis using the C-terminal region alone (open rectangles) and TMpred (underlined), respectively. First domain of Scallop was at the N-terminal end from Met1 to Asp23, thus was not shown in this figure. 5Hthpr, H. pulcherrimus; Scallop, 5HT receptor of Mizuhopecten yessoensis; Aplysia, 5HT receptor of Aplysia californica; Fugu, 5HT receptor of Takifugu rubripes; S.p., S. purpuratus 5HT-1A.

View larger version (55K): [in this window] [in a new window]   Fig. 3. (A–G) Immunohistochemical localization of serotonin receptor cell processes (fibers) inserted into larval ectoderm. In plutei at 48 h post-fertilization (h.p.f.), fibers from serotonin receptor cell network are inserted into the ectoderm around the apical ganglion (Ab,B, arrows, H), extending from the oral lobe transverse tract (`oltrt' in B,H), at the left and right corners of oral lobe (Ac,C,H), at the tip of left and right arms (Ad,D,H), at the middle region of posterior body on both left and right sides (Ae,E,H), at ventral ciliary band (Af,F, arrows, H), and at the tip of posterior end of the body (Ag,G, arrow, H). Bars, 100 µm (A,h), 30 µm (B,E,F), 40 µm (C,D) and 20 µm(G). (H) Schematic ventral view of 48 h.p.f. pluteus larva summarizing major tracts of the serotonin receptor cell network and the sites where fibers are inserted into the ectoderm (green lines), based on present and previous observations (Katow et al., 2004). Letters b–g in gray rectangles correspond to the white rectangles in A and insets in C–G. This scheme shows tracts of serotonin receptor network shown by whole-mount immunohistochemistry (Ah), including left and right oral rod tracts (lort and rort), left and right anal rod tracts (lart and rart), dorsal stomach plexus (dsp) and dorsal central tract (dct). Digestive tracts are shown with light brown line.

View larger version (104K): [in this window] [in a new window]   Fig. 4. Elevation of cytoplasmic Ca2+ concentration ([Ca2+]i) by exogenous serotonin microinjected to the blastocoel. (A) Ventral view of a larva hooked at the tip of a glass needle (black arrow) near stomach (white arrow). (B) Propagation of [Ca2+]i wave in a same pluteus larva as in A. Microinjection of serotonin into the blastocoel near stomach (B, 0'', yellow arrow) triggered elevation of [Ca2+]i on the larval surface. The micropipette was kept inserted to the blastocoel without releasing serotonin for 15 s to monitor [Ca2+]i level before serotonin application. Initial elevation of [Ca2+]i occurred near serotonin injection site immediately after onset of serotonin release (B,16'', arrow 1), that transiently propagated posteriorly for about 350 µm in 2 s (C, double-headed arrow) on the left side (B, 16'', post), then traveled to the right side of larva by 17 s (B, 17'', arrow 2 and curved arrow), and the other one anteriorly (B, 16'', ant) for 50 µm before diminishing earlier than the posterior propagation at 20 s (B, 20''). The numbers shown on upper left corner in (B) show the time in second from when micropipette was inserted (time zero, 0') and thereafter. Transient [Ca2+]i elevation returned close to visual time zero level in 30 s (B, 30''), and to background level in 60 s as shown by a time-course of the wave intensity (C). [Ca2+]i elevation occurred soon after the completion of serotonin releasing (C, green arrow). A second fluorescence wave toward the anterior direction diminished in 2 s after the initial [Ca2+]i elevation (B, from 16'' to 18'', 18'' arrowhead). Rainbow-colored bar shows relative intensity of fluorescence, as described in Materials and methods. (C) Entire time-course of [Ca2+]i elevation from 15 s before onset of serotonin release (time zero, green arrow) to 60 s. (D) Double stained immunohistochemistry of serotonin (red) and serotonin receptor cell network (green). The serotonin receptor cell network is a major structure in the blastocoelar space. (E) Double stained immunohistochemistry of serotonin (negative immunoreaction) and serotonin receptor cell network (green) of p-chlorophenylalanine (pCPA)-treated larva. pCPA inhibited serotonin synthesis at the apical ganglion and perturbed the formation of serotonin receptor cell network. (F) Same pCPA-treated larvae as that used to examine [Ca2+]i elevation. Red arrow shows an oil droplet from the glass micropipette, and is used as a marker of injection. The stomach is smaller than that of normal larvae (black arrow). (G) pCPA-treated larva lacked intrinsic high level of [Ca2+]i at stomach (0', arrow). (H) 18 s after serotonin injection, no elevation of [Ca2+]i occurred. (I) Time course of [Ca2+]i elevation in pCPA-treated larva. No [Ca2+]i elevation occurred. Bars, 100 µm.

View larger version (100K): [in this window] [in a new window]   Fig. 5. Double-stained immunohistochemistry of serotonin (red) and synaptotagmin (green). (A) In intact larva, serotonin was synthesized at the apical ganglion (red) and synaptotagmin-possessed nerve cell network lined along the ciliary band (green). (B) In pCPA-treated larva, the apical ganglion did not produce serotonin, but formation of the synaptotagmin-possessing nerve cell network (green) was not affected (B). Bar, 100 µm.

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