The Pre-nervous Serotonergic System of Developing ... - Springer Link

Neurochemical Research, Vol. 30, Nos. 6/7, June/July 2005 (Ó 2005), pp. 825–837 DOI: 10.1007/s11064-005-6876-6

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence Gennady A. Buznikov,1,4 Robert E. Peterson,2,5 Lyudmila A. Nikitina,1 Vladimir V. Bezuglov,3 and Jean M. Lauder1,6 (Accepted March 16, 2005)

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT1 receptors, antagonists of 5-HT2, 5-HT3 or 5-HT4 receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter. KEY WORDS: 5-HT receptor; neurotransmitter; pre-nervous; SERT.

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3

4

5

6

INTRODUCTION Department of Cell and Dev. Biology, University of North Carolina School of Medicine, Chapel Hill, NC, 27599-7090, USA. Department of Biology, Coastal Carolina University, Conway, SC, USA. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117871, Moscow, Russia. N.K.Koltzov Institute of Developmental Biology, 117808, Moscow, Russia. Neuroscience Center, University of North Carolina School of Medicine, Chapel Hill, NC, 25799, USA. Address reprint requests to: Jean M. Lauder Department of Cell and Developmental Biology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7090, USA; Tel.: +1-919-966-5020; Fax: +1-919-966-1856; E-mail: [email protected]

Serotonin (5-HT), as well as other classical neurotransmitters, are present in oocytes, early (pre-nervous) embryos and larvae of all vertebrates and invertebrates studied, including mammals, birds, amphibians, teleosts, ascidians, echinoderms, insects, mollusks and nemertean worms (1–19). The most detailed results were obtained for pre-nervous 5-HT in sea urchins (20–26). In particular, it was shown that the concentration of 5-HT (or 5-HT-like substances) changes regularly in developing sea urchin embryos and larvae, starting from the first minutes after fertilization (1). It was also found that some 5-HT 825 0364-3190/05/0700–0825/0 Ó 2005 Springer Science+Business Media, Inc.

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Buznikov, Peterson, Nikitina, Bezuglov, and Lauder

neurochemicals inhibit or block the first cell divisions (cleavage divisions) and that exogenous 5-HT prevents these developmental malformations (7). It appears, therefore, that endogenous 5-HT is functionally active throughout pre-nervous embryogenesis in sea urchins, mediated by 5-HT receptors or transporter. However, little is known about the developmental dynamics of expression of these receptors, their pharmacologic properties, or signal transduction mechanisms utilized. The goal of the present study was to use a pharmacologic perturb-and-rescue strategy, together with immunocytochemistry using polyclonal antibodies, to obtain evidence for the existence of prenervous 5-HT receptors and the 5-HT transporter in

early sea urchin embryos and larvae. Results of these studies enhance our understanding of roles played by pre-nervous 5-HT acting as a multifunctional regulator of early embryogenesis.

EXPERIMENTAL PROCEDURES The main object of this study was the sea urchin Lytechinus variegatus. Adult animals were maintained in tanks with continuously filtered, circulating artificial seawater (ASW). Gametes were harvested, eggs fertilized and incubated, as described previously (27). Embryos and larvae were cultured in 12-well tissue culture plates (100–150 specimens per well). All experiments were conducted at approximately 70°F (21°C). Neurochemicals were

Table I. 5-HT Receptor Ligands Tested on L. variegatus Embryos and Larvae Pharmacological characteristics

Substance

Vehicle for stock solution

Source

Serotonin (5-HT.HCl) Tryptamine (T.HCl) 8-OH-DPAT 8-OH-PIPAT LY-165,163 R-(+)-UH-301 NAN-190 WAY 100135 WAY-100635 Pindobind 5-HT1A p-MPPF 5-(Nonyloxy)tryptamine CGS 12066B GR 55562 a-Methylserotonin ())-DOI BW 723C86 Clozapine

d.w. d.w. Ethanol or d.w. Ethanol or DMSO Ethanol or d.w. Ethanol DMSO d.w. d.w. Methanol d.w. d.w. d.w. d.w. d.w. d.w. DMSO DMSO or ethanol

Sigmaa Sigmaa Tocrisb Tocris Sigma Sigma Sigma Tocris Sigma Sigma Sigma Tocris Tocris Tocris Sigma Sigma Tocris Sigma

Cyproheptadine LY-53,857 Mianserin Cinanserin Pirenperone Ritanserin MDL-11,939 SB 206553 2-Methylserotonin 5-HTQ Quipazine 1-(m-chlorphenyl)-biguanide Tropanyl-3,5-dimethylbenzoate (TDB) Tropisetron Tropanyl-indole-3-carboxylate. CH3I 3-(4-allylpiperazin-1-yl)-2-quinoxalinecarbonitrile 5-methoxytryptamine GR 113808 SDZ-205,557 Fluoxetine Imipramine

Methanol Ethanol Ethanol Methanol Methanol Methanol Ethanol d.w. d.w. d.w. d.w. d.w. DMSO d.w. Methanol d.w. d.w. DMSO d.w. d.w. d.w.

Sigma Sigma Sigma Tocris Sigma Sigma Tocris Sigma Sigma Sigma Sigma Sigma Tocris Sigma Sigma Tocris Sigma Tocris Sigma EliLillyc Sigma

Agonist of all 5-HT receptors Agonists of 5-HT1A receptors

Antagonists of 5-HT1A receptors

Agonists of 5-HT1B receptors Antagonist of 5-HT1B receptors Agonist of 5-HT2 receptors Agonist of 5-HT2A receptors Agonist of 5-HT2B receptors Antagonist of some DA and 5-HT receptors, including 5-HT2A/2C Antagonists of 5-HT2 receptors

Antagonist of 5-HT2A receptors Antagonist of 5-HT2B/2C receptors Agonist of 5-HT3 receptors Antagonists of 5-HT3 receptors

Agonist of 5-HT4 receptors Antagonists of 5-HT4 receptors Inhibitors of serotonin transporter

Abbreviations: d.w. distilled-deionized H2O. a Sigma-Aldrich Corp. St.Louis, MO, USA. b Tocris Cookson Inc. Ellisville, MO, USA. c Eli Lilly and Co. Indianapolis, IN, USA.

Serotonin and Sea Urchin Embryogenesis introduced after the first cleavage division, at mid-blastula 2 stage, or later, at a final concentration of 100 lM or less. Each substance was tested on specimens obtained from at least 10–15 females (1 female per experiment). Control embryos/larvae were incubated in ASW or in ASW with the same volume of vehicle used to dissolve test chemicals (DMSO, ethanol, methanol or deionized/distilled water), depending on the substance used (Table I). Results (changes in cellular or embryonic phenotype) were documented using a Leitz Orthoplan microscope with brightfield optics and a Spot RT color digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). Imaging was performed when control larvae were at the stage of late gastrula or early prism. The effects of different doses of neurochemicals were determined by consistency for a particular concentration within each set of experiments (i.e., malformations were the same for 3000 or more animals), and were completely absent in vehicle-treated controls. Some neurochemicals were also tested on embryos and larvae of other sea urchin species (mainly Strongylocentrotus droebachiensis and sometimes S. purpuratus, S. pallidus, S. franciscanus, or Dendraster excentricus) using the same procedure, with similar results. Adult specimens of these species were maintained in tanks with running natural sea water at Friday Harbor Labs (FHL, Univ. Washington). Water temperature during these experiments was about 50°F (10°C). Substances used in these experiments are listed in Table I. In addition, arachidonoyl serotonin (AA-5-HT, a lipophilic 5-HT derivative, synthesized in the Laboratory of oxylipins, ShemyakinOvchinnikov Institute of Bioorganic Chemistry, Moscow, Russia) was used (Table III). The pharmacologic characteristics of these substances, given in Table I, are based on the accepted classification of vertebrate 5-HT receptors (28–30). Immunocytochemical localization of 5-HT, 5-HT receptors and the 5-HT transporter (SERT) was determined during early development of L. variegatus and S. droebachiensis, with special attention to late gastrula and early prism stages. The action of 5-HT receptor ligands (ritanserin, 8-OH-DPAT) or an inhibitor of 5-HT transport (fluoxetine) on localization of 5-HT, 5-HT receptors and SERT was determined. Control and neurochemical-treated larvae were fixed overnight in 4% paraformaldehyde in ASW (pH 7.4, 4–6°C) plus 0.1% glutaraldehyde (5-HT receptors, SERT) or 4% paraformaldehyde without glutaraldehyde (5-HT). Specimens were washed several times with ASW, rinsed with cold methanol (2 min) and transferred through an ascending ethanol (ETOH) series, then stored in 70% ethanol at )20°C until use. Prior to immunocytochemistry, specimens were washed three times (20 min each) in phosphate buffered saline plus 0.1% Triton-X-100 (PBS-TX, pH 7.4) and incubated overnight (4–10°C) in 10% normal goat or sheep serum containing PBS-TX. Following this, specimens were incubated in primary antibody or primary antibody preabsorbed with blocking peptide for 2 days at 4–10°C, followed by rinsing 3 15 min in PBS-TX, and incubation in Texas Red labeled secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) overnight in the dark, 4–10°C (dilution 1:500–1:1000; species matching that of primary antiserum). Specimens were rinsed 3 15 min in PBS and mounted on slides using Fluoromount. Specimens were viewed and imaged using a Leitz Orthoplan microscope with rhodamine fluorescence optics and a Spot RT color digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). Primary antibodies and blocking peptides were as follows: 1) 5-HT: rabbit polyclonal antibody (31), dilution 1:500 +/) BSA or

827 Keyhole limpet hemocyanin-5-HT conjugate (50 lg/ml); 2) 5-HT1A receptor: rabbit polyclonal antibody, gift of John Raymond (32), dilution: 1:50 +/) blocking peptide 50 lg/ml); 3) 5-HT1B and 5-HT2A receptors: rabbit polyclonal antibodies (ImmunoStar, Inc., Hudson, WI, USA), dilution 1:300 +/) blocking peptide (50 lg/ml and 5 lg/ml, respectively; 4) 5-HT2B receptor: goat polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), dilution 1:100 +/) blocking peptide (50 lg/ml); 5) 5-HT3 receptor: rabbit polyclonal antibody (Oncogene Research Products, San Diego, CA, USA), dilution 1:500 +/) blocking peptide (10 lg/ml); 6) 5-HT4 receptor goat polyclonal antibody (Santa Cruz Biotechnology, Inc.), dilution 1:100 +/) blocking peptide (5 lg/ml); 7) SERT: rabbit polyclonal antibody (ImmunoStar, Inc), dilution 1:250 +/) blocking peptide (5 lg/ml). 8) Mesenchyme cells were identified with 1D5 (33), and the general mesenchyme marker, Meso1 (34,35), both monoclonal antibodies.

RESULTS Immunocytochemical Studies of Normal Sea Urchin Embyos/Larvae Serotonin and 5-HT receptors, as well as SERT were immunocytochemically localized at all pre-nervous stages of development in both L. variegatus and S. droebachiensis, beginning at the one-cell stage. Results shown here are from late gastrula and early prism stages, the last pre-nervous stages of sea urchin development. Specificity of immunocytochemical reactions was demonstrated by blocking with antigens (Fig. 1). All components of the serotonergic system were present in the primary gut (archenteron), from the very beginning of archenteron development, progressing from the vegetal to animal poles during gastrulation. At the developmental stages considered here, immunoreactivity (IR) for some components of the pre-nervous 5-HT system was expressed mainly in the animal part of the archenteron and in cells of the apical ectoderm (e.g., 5-HT itself, 5-HT1A and 5-HT2A receptors). Interestingly, 5-HT1A receptor IR appeared in the apical ectoderm earlier than other 5-HT-receptors (Figs. 1, 4 and 5). In the vegetal part of the archenteron and adjacent larval wall, IR for 5-HT2B , 5-HT3 and 5-HT4 receptors was observed (Figs. 1 and 6). SERT IR was present in both the apical and mid parts of archenteron and adjacent apical ectoderm (Fig. 1). Most of these serotonergic components also appeared to be expressed by mesenchyme-like cells, as judged by preliminary studies with sea urchin mesenchymal cell markers (33–35). In contrast, 5-HT IR was the only component seen in the paired apical organs of prism stage embryos, anlagen of the apical ganglia (Fig. 1).

828 Pharmacologic Studies of Teratogenicity of Serotonergic Neurochemicals Many 5-HT-receptor ligands, at concentrations of 5–20 lM, had embryotoxic/teratogenic activity, blocking gastrulation and eliciting hyper-production of mesenchyme-like cells that accumulated in the blastocoele (Figs. 2 and 3). These developmental malformations, abnormal changes in embryonic and cellular phenotypes, were similar, but not identical, for all 5-HT ligands tested. They were dose-dependent, incompatible with further development, and followed by death of embryos or larvae. The duration of embryonic or larval survival was shortened in


proportion to increasing concentration of ligand. The protective actions of corresponding antidotes (rescue compounds), described below, were also dosedependent. 5-HT1A Receptors Ligands for 5-HT1A receptors at concentrations, up to 100 lM, did not perturb cleavage divisions, and had no significant effects until the blastula stage (Table II), although they sometimes perturbed early cell–cell interactions, causing dissociation of the first blastomeres and formation of dwarf embryos. All agonists of these receptors (Table II) at concentra-

Fig. 1. Immunocytochemical expression of 5-HT (a), 5-HT-receptors 1A (b), 1B (c), 2A (d), 2B (e), 3 (f), 4 (g) and 5-HT transporter (SERT) (h) at the late gastrula or prism stage of sea urchin Lytechnus variegatus. 1 – Standard treatment; 2 – Treatment by primary antibody with blocking peptide. Scale bar 50 lm.

Serotonin and Sea Urchin Embryogenesis tions of 20–40 lM or greater, when introduced at the mid-blastula stage, elicited identical developmental malformations, which we have termed the ‘‘5-HT1A syndrome’’. These malformations were characterized by inhibition of gastrulation, and hyperproduction of pigmented mesenchyme-like cells, that accumulated in the blastocoele near the primary mouth (blastopore) (Fig. 2a). These malformations were prevented to some extent by equimolar or higher concentrations of 5-HT or its hydrophilic analog, 5-HTQ, whereas the lipophilic analog, AA-5-HT, did not protect against 5-HT1A receptor agonists (Fig. 3a). Some protective action against agonists of 5-HT1A receptors were also found with equimolar concentrations of selective antagonists, such as NAN-190, WAY100135 and WAY-100635 (Table II). However, when used at high concentrations (50–100 lM), these antagonists also blocked gastrulation. Interestingly, the agonist 8-OH-PIPAT did not perturb the immunocytochemical expression of 5-HT1A receptors, but did strongly disrupt their patterning, such that 5-HT1A receptor IR in the region of apical ectoderm is no longer visible (Fig. 4b, c), causing these receptors to be expressed by the underdeveloped archenteron and mesenchyme-like cells. As shown in preliminary experiments, the sensitivity of S. droebachiensis and D. excentricus larvae to

829 8-OH-DPAT and 8-OH-PIPAT decreased during gastrulation and appeared to lose pharmacological specificity, since the protective actions of 5-HT and 5-HTQ were lost at the end of gastrulation. 5-HT1B Receptors Agonists of 5-HT1B receptors (at concentrations of 5–20 lM or greater) blocked cleavage divisions. When added at the mid-blastula stage, these agonists inhibited gastrulation and elicited accumulation of highly pigmented, mesenchyme-like cells in the blastocoele. These pigmented cells appeared to be different from the weakly pigmented mesenchyme-like cells of control larvae and larvae treated with 5-HT1A agonists. In addition, unlike the ‘‘5-HT1A syndrome’’, these pigmented cells did not immediately accumulate near the blastopore, but were displaced laterally (Fig. 2b). In some cases, the hyaline layer of treated larvae became exfoliated, such that the pigmented mesenchyme-like cells were extruded into the space between this layer and the larval surface. We have termed this type of malformation the ‘‘5-HT1B syndrome’’. One 5-HT1B receptor antagonist, GR 55562 (40–80 lM), elicited similar, but less severe malformations as 5-HT1B agonists, and at a lower concentration (20 lM). This antagonist partially protected

Fig. 2. Typical developmental malformations elicited by 5-HT ligands (5-HT1A agonists [8-OH-DPAT, 8-OH-PIPAT] – a; 5-HT1B agonists [CGS-12066B, 5-nonyloxytryptamine] – b; 5-HT2 antagonists ritanserin – c1; cinanserin et al – c2; 5-HT3 antagonist TDB - d; 5-HT4 antagonist GR-113808 – e; SERT inhibitors [fluoxetine, imipramine] – f. Experiments were performed on sea urchins L. variegatus (a, b) and Strongylocentrotus droebachiensis (c–i). Substances were introduced at the mid-blastula stage (Control 1; g). Development was usually blocked at the early gastrula 2 stage (Control 2; h). Abnormal neurochemical-treated larvae were digitized, when control larvae were at the late gastrula or prism stage (Control 3; i). Scale bar 50 lm.

830


Fig. 3. The action of hydrophilic (5-HTQ) and lipophilic (AA-5-HT) 5-HT derivatives on the sensitivity of sea urchin L.variegatus larvae to some 5-HT teratogens given at the mid-blastula 2 stage. imaging of images was made at prism stage in the control. (a) 1. Control; 2. 8-OHPIPAT 20 lM alone; 3. +5-HTQ 40 lM (good protection); 4. +AA-5-HT 40 lM (weak protection). (b) 1 Control; 2 Ritanserin 10 lM alone; 3. +5-HTQ (no protection); 4. +AA-5-HT (good protection); (c) 1. Control; 2. Fluoxetine 5 lM alone; 3. +5-HTQ (good protection); 4. +AA-5-HT (no protection). Scale bar 50 lm.

embryos against 5-HT1B agonists, in most experiments. The protective action of 5-HTQ, 5-HT and AA-5-HT against agonists of 5-HT1B receptors was incomplete, but was observed in most experiments (Table II). 5-HT2A/2B/2C Receptors Ritanserin, an antagonist of 5-HT2A/2B/2C receptors (36,37) at concentrations of 2.5 lM and higher, inhibited both cleavage divisions and gastrulation, depending on the stage at which it was intro-

duced (Table III). These malformations, termed the ‘‘ritanserin syndrome’’, were unusual, since they consisted not only of a blockade of gastrulation, but also involved formation of a multilayered blastula wall (Figs. 2c-1 and 3b) compared to the wall of normal larvae which is a single-cell layer. Within a short period of time (1–2 h for L. variegatus, 6–12 h for S. droebachiensis), cells of the multilayered wall began to be extruded into the blastocoele, eventually filling it completely. Such larvae were incapable of further development. These mesenchyme-like cells had the immunocytochemical appearance of PMC

Serotonin and Sea Urchin Embryogenesis and/or SMC, as shown in preliminary experiments with markers to these cell types (33–35). Ritanserin did not inhibit the immunocytochemical expression of 5-HT2A and 5-HT2B receptors, but did perturb

831 their normal patterning, since, similar to 5-HT1A receptors after 8-OH-PIPAT treatment, these receptors were localized to the underdeveloped archenteron and associated mesenchyme-like cells, as judged

Fig. 4. Immunocytochemical expression of 5-HT1A receptors in larvae of L. variegates treated by 8-OH-PIPAT 20 lM (b) and 50 lM (c). 1st control (a) corresponds to the stage when development is blocked by 8-OH-PIPAT; 2nd control (d ) corresponds to the stage of normal development when 8-OH-PIPAT-treated larvae were digitized. Scale bar 50 lm.

Fig. 5. Immunocytochemical expression of 5-HT2A receptors in normal L. variegatus larvae (a) and larvae treated by ritanserin 10 lM (b) and 20 lM (c) given at the mid-blastula 2 stage. Imaging of images was made at late gastrula 2 stage Scale bar 50 lm.

Fig. 6. Immunocytochemical expression of 5-HT2B receptors in normal L. variegatus larvae (a) and larvae treated by ritanserin, 10 lM (b) and 20 lM (c) given at the mid-blastula 2 stage. Imaging of images was made at late gastrula 2 stage. Scale bar 50 lm.

832


by preliminary studies with sea urchin mesenchymal cell markers (33–35) and were absent from the apical ectoderm. (Compare Figs. 5c, 6b,c with Fig. 4). Other antagonists of 5-HT2 receptors (e.g., cyproheptadine, mianserin, cinanserin) caused other types of defects, where the formation of a multilayered larval wall was followed immediately by extrusion of these cells into the blastocoele (Fig. 2c-2, Table III). Some 5-HT2 antagonists blocked cleavage divisions, whereas others did not (Table III). MDL 11,939, acting on the adult mammalian nervous system mainly as a 5-HT2A receptor antagonist (38), and CB206,553, a 5-HT2B/2C receptor antagonist (39), only perturbed development after the late blastula stage. These developmental malformations have not yet been studied in detail, but they appear dissimilar to malformations caused by ritanserin, cyproheptadine, mianserin or cinanserin. AA-5-HT was most effective in protecting against these malformations, whereas 5-HTQ was ineffective (Fig. 3b), except against MDL 11,939 (Table III). A pan-5-HT2 receptor agonist, a-methylserotonin, protected against all antagonists of 5-HT2 receptors, whereas DOI, a 5-HT2A,2C agonist protected only against the effects of MDL 11,939, whereas B723C86, a 5-HT2B agonist, only protected against CB-206,553 (Table III). The sensitivity of embryos/larvae to ritanserin did not change significantly from cleavage to the early pluteus stage, as shown in preliminary experiments with S. droebachiensis and D. excentricus). Developmental malformations were not the same if ritanserin was given at different stages of development, although formation of a multilayered larval wall was always observed, and many cells in the wall had unusually strong pigmentation if ritanserin was

given from the mid-blastula to the prism stage. AA5-HT prevented ritanserin-induced malformations up to the early pluteus stage, unlike 5-HTQ. 5-HT3 Receptors All agonists and antagonists of 5-HT3 receptors, except tropanyl 3,5-dimethylbenzoate (TDB), tested at concentrations up to 100 lM, did not act on either embryonic or larval development. However, there were some signs of their pharmacologic activity. For example, 5-HTQ, generally considered to be a 5-HT3 agonist, partially or completely prevented malformations caused by 5-HT1 receptor agonists, the 5-HT2A antagonist, MDL 11,939, as well as by the 5-HT uptake inhibitor, fluoxetine (see below). Moreover, as shown in earlier experiments on L. pictus, the 5-HT3 receptor antagonist, tropanylindole-3-carboxylate methiodide, elicited transient regression of the first cleavage furrow (40). In experiments on S. droebachiens, another 5-HT3 receptor antagonist, TDB (10–20 lM), caused formation of elongated larvae with very short archenterons (Fig. 2d). These malformations were partially prevented by 5-HTQ or AA-5-HT. 5-HT4 Receptors Only two antagonists of 5-HT4 receptors, GR 113,808 and SDZ-205,667 were tested at (20–40 lM). When introduced after the mid-blastula stage, abnormal larvae were observed with reduced archenterons and a cluster of transformed mesenchymelike cells accumulating near the animal pole, and later filling the blastocoele (Fig. 2e). GR 113,808 (40– 80 lM), but not SDZ-203,667, also inhibited cleavage

Table II. Action of 5-HT1 Ligands on the Sea Urchin Development Protective effects of 5-HT-ergics and 5-HT1 antagonists

Sensitive periods Agonists 5-HTR1A 8-OH-DPAT 8-OH-PIPAT LY-165,163 R(+)UH-301 5-HTR1B 5-nonyloxy-T CGS 12066B

Cleavage

After mid-blastula

WAY WAY GR 5-HTQ 5-HT AA-5-HT 100135 100635 NAN-190 Pindobind p-MPPF 55562

No

Yes (5-HT1A syndrome)

++

++

0

Yes

Yes (5-HT1B syndrome)

+

+

+

++ strong but incomplete protective action. + moderate but obvious protective action. 0 no protection.

++

++

++

+

0

0

0

+

0

+ or 0

Serotonin and Sea Urchin Embryogenesis

833

Table III. Action of 5-HT2 Receptor Ligands on Sea Urchin Development Protective effects of 5-HT-ergics and 5-HT2 agonists

Sensitive periods Antagonists 5-HTR2 Ritanserin Cyproheptadine LY-53,857 Mianserin Cinanserin Clozapine Pirenperone 5-HTR2A MDL 11,939 5-HTR2B/2C CB 206553

Cleavage

After mid-blastula

5-HTQ

+(>2.5 lM) +(>10 lM) 0 (80 lM) 0 (80 lM) +(>10 lM) 0 (50 lM) 0 (80 lM) 0 (80 lM) 0 (50 lM)

+(RSa) +CSb +(CSb) +(CSb) +(CSb) +(CSb) +c +c +c

0 0 0 0 0 0 0 ++ 0

5-HT + + + + 0 or + ++ 0 or + + 0 or +

AA-5-HT

a-CH3–5-HT

())-DOI

BW723C86

+++ +++ +++ ++ ++ ++ ++ + ++

+ + +

0 0 0

0 0 0

+ 0

0 +

+ +

+++ full normalization of development. ++, + and 0 as in Table II. a ritanserin syndrome – see Figs. 2 and 3b. b cyproheptadine syndrome – see Fig. 2. c developmental malformations are not identical to RS or CS.

divisions. An agonist of 5-HT4 receptors, 5-methoxytryptamine (100 lM) did not cause malformations in any sea urchin species studied, but did prevent malformations caused by above mentioned 5-HT4 antagonists. The ability of other 5-HT receptor ligands to prevent effects of GR 113,808 and SDZ-203,667 has not yet been studied.

5-HT Transporter (SERT) Fluoxetine (5–20 lM) or imipramine (10– 40 lM), when introduced at the mid-blastula stage, caused a different type of malformation (‘‘fluoxetine syndrome’’) consisting of motile, spherical larvae without any signs of gastrulation, where the blastocoele was filled with uniformly distributed, pigmented mesenchyme-like cells (Figs. 2f and 3c). These abnormal larvae later became elongated (lemon-like) and appear to extrude these cells through the layer of apical ectoderm. When added before the mid-blastula stage, the same 5-HT uptake inhibitors blocked cleavage divisions. The effects of both fluoxetine and imipramine were prevented by 5-HTQ, whereas the protective action of 5-HT or AA-5-HT was minimal (Fig. 3c).

DISCUSSION Using immunocytochemistry with polyclonal antibodies, we found evidence for the presence of 5-HT (or a 5-HT-like substance) and several different types of 5-HT-receptors (5-HT1A, 5-HT1B, 5-HT2A, 5-HT2B, 5-HT3, 5-HT4 or corresponding receptor-like

proteins) and SERT in pre-nervous sea urchin embryos and larvae. Evidence that these components of the pre-nervous serotonergic system are functionally active was provided by a developmental pharmacologic perturb-and-rescue strategy, where malformations during gastrulation were observed for all groups of drugs tested, including agonists and antagonists of vertebrate 5-HT receptors and inhibitors of SERT. In a sense, these malformations were drug-specific, since they could be prevented by drugs with opposite actions (e.g., agonist vs. antagonist), as well as by 5-HT itself or some hydrophilic or lipophilic 5-HT analogs. Not unexpectedly, the pharmacology of these drugs was not identical to that known for the vertebrate nervous system, as illustrated in Tables II–IV, raising the possibility that pre-nervous 5-HT receptors and transporters may represent phylogenetically old forms of their vertebrate counterparts, with ‘‘hybrid’’ characteristics, such that they exhibit mixed pharmacologic properties. This possibility is supported by data from the Sea Urchin Genome Project (http:// sugp.caltech.edu). Molecular-biologic information available on this website regarding the presence of 5-HT receptors and the 5-HT transporter in the sea urchin S. purpuratus can be retrieved using the search word ‘‘serotonin’’. Most of this information is derived from BAC sequences pulled from a cDNA library, which were blasted against the NCBI GenBank database. These sequences are grouped on the basis of homology to 5-HT receptor or transporter sequences in this NCBI database. Interestingly, the first six groups include sequences identified as 5-HT6, 5-HT transporter, 5-HT1A or 5-HT2C receptors, followed by groups that consist of a mix of various

834

No Yes Near animal pole Uniformly Yes Yes

Yes Yes

Yes Yes

Cell surfaceCytoplasm

Not studied Cell surface

No

Not studied Yes No Yes Yes Yes Cell surface Cytoplasm

MDL- 11,939 Other 5-HTR2 antagonists TDB, 5-HTR3 antagonist 5-HTR4 antagonists Fluoxetine

No No

No

Not studied Yes (delayed)

No No No

Near the blastopore Near the lateral wall Along inner surface of blastocoele Not studied Along inner surface of blastocoele Near the blastopore Yes Yes Yes (delayed) 5-HTR1A agonists 5-HTR1B agonists Ritanserin

Yes Yes Yes Cell surface Cell surfaceCytoplasm Cytoplasm

Substances

No No Yes

Hyper-production of mesenchyme-like cells Formation of multilayer larval wall Block or damage of gastrulation Putative localization of receptors

Post-blastulation phenotype

Table IV. Abnormal Phenotypes Elicited by Different 5-HT Ligands

Distribution of mesenchyme-like cells in the blastocoele

Extrusion of cells in the ASW


5-HT receptors, including 5-HT5B and 5-HT2B, together with muscarinic ACh, tyramine, octopamine, dopamine D2, D1, somatostatin, opsin, tachykinin, neurokinin, neuromedim, melanocortin, gonadtropin, or adrenergic receptor sequences. This lends support to our hypothesis that the pre-nervous serotonergic system of sea urchins consists of receptors and transporters that are structural hybrids of their mammalian counterparts and other neurotransmitter receptors. This scenario raises the possibility that sea urchin pre-nervous neurotransmitter machinery may represent an evolutionary precursor to the neurotransmitter systems of the vertebrate nervous system. Taken together, the results of the present study suggest that many components of the serotonergic system are functionally active during pre-nervous development of sea urchins, where they regulate key morphogenic events through post-gastrulation stages. Clearly, the precise classification of these pre-nervous 5-HT receptor subtypes and transporters will require cloning to structurally characterize them. Immunocytochemistry with polyclonal antibodies demonstrated similar distributions of different components of the pre-nervous serotonergic system (5-HT, 5-HT receptors, SERT) up to the mid-blastula stage, whereas after the onset of gastrulation, the distribution of specific components differed, as illustrated by the unequal distribution of different 5-HT receptor subtypes along the archenteron and the apical ectoderm (Fig. 1). Consistent with this, developmental malformations elicited by ligands for different 5-HT receptors or SERT, when added at the mid-blastula stage, were generally similar, but not exactly (Figs. 2 and 3). Interestingly, many of these ligands blocked gastrulation and caused the accumulation of pigmented mesenchyme-like cells in the blastocoele, suggesting that the drugs had deleterious effects on the developing endomesoderm system. In some cases (5-HT1A agonists and the pan5-HT2antagonist, ritanserin) the drugs elicited the accumulation of pigmented cells in the blastocoele, which exhibited immunocytochemical characteristics of primary (PMC) and/or secondary (SMC) cells. Interestingly none of the drugs decreased the amount of immunoreactivity (IR) for the corresponding 5-HT receptors, but rather perturbed their patterning, such that IR was diminished or absent in the apical ectoderm (Figs. 4b,c; 5b,c; 6b,c). There were, however, differences in the types of teratogenic effects caused by different drugs, prompting us to discuss them as comprising different ‘‘syndromes’’ (Figs. 2 and 3, Tables II–IV).

Serotonin and Sea Urchin Embryogenesis The most informative differences in pharmacologic profiles gleaned from our perturb-and-rescue strategy were related to the differential protective actions of 5-HT compared to lipophilic or hydrophilic 5-HT analogs (Fig. 3, Tables II and III). For example, the 5-HT derivative, AA-5-HT, which is highly lipophilic, readily permeates into the cytoplasm of embryonic/larval cells, whereas 5-HTQ, which is hydrophilic, only acts at the cell surface. Serotonin itself, on the other hand, can act either at the cell surface or be transported intracellularly, placing it intermediate between 5-HTQ and AA-5HT in terms of its potential sites of action (41). Therefore, our pharmacologic perturb-and-rescue strategy provides useful clues to the possible localization of corresponding receptors (or binding sites) for 5-HT ligands. For example, if AA-5-HT protects against a certain ligand, whereas 5-HTQ is ineffective, as in the case of the ‘‘Ritanserin Syndrome’’, this suggests that the corresponding binding sites (5-HT2like receptors) are likely to located intracellularly. On the other hand, if 5-HTQ and 5-HT act as effective antidotes against a teratogenic ligand, and AA-5-HT is ineffective, this suggests that the binding sites (5-HT1A-receptors, SERT) are located at the cell surface. On the contrary, functionally active 5-HT1Band 5-HT3-like receptors may be located both intracellularly and at the cell surface, judging by the approximately equal protective actions of AA-5-HT, 5-HT and 5-HTQ against drugs that target these receptors (Tables II–IV). These findings are consistent with co-existence of intracellular and cell surface 5-HT receptors in pre-nervous embryos/larvae we proposed earlier based (1,19,22) on other pharmacological data. Theoretically, it is possible that intracellular 5-HT2 receptors represent cell surface-receptors that have been internalized during receptor turnover (recycling). However, this seems unlikely, since lipophilic AA-5-HT was an effective antidote against ritanserin at all stages studied, whereas 5-HTQ was ineffective, suggesting that 5-HT2-like receptors are located intracellularly from the earliest stages of development in sea urchin embryos/larvae. Moreover, we found no evidence for down-regulation of these receptors as a result of chronic ritanserin treatment, as is usual for receptor recycling in vertebrate neurons (42–48). As to the developmental dynamics of sensitivity to different 5-HT ligands, some ligands (agonists of 5-HT1B receptors, antagonists of 5-HT2 receptors, including ritanserin and inhibitors of SERT), not

835 only perturbed gastrulation, but also blocked cleavage divisions, whereas other ligands were only active beginning at the mid-blastula to mid-gastrula stages (Tables II and III). Although the phenotypes seen for the ‘‘ritanserin syndrome’’ depended on the developmental period of ritanserin exposure, the main characteristic was formation of a multi-layered larval wall, which was observed through the prism stage. At the same time, 5-HT1A receptor agonists were only able to elicit the typical ‘‘5-HT1A receptor syndrome’’ during the first half of gastrulation, consistent with the finding that 5-HT1A receptors appear to be present in cleaving sea urchin embryos, but do not act until later in development. This suggests that the functional characteristics of these receptors may change immediately prior to gastrulation. When using sea urchin embryos and larvae as biosensors for 5-HT neurochemicals (49), we must take into account these developmental dynamics and select appropriate stages for exposure to different test substances. Taken together, the results of the present study suggest that pre-nervous 5-HT may act as a multifunctional regulator of early embryonic development, including cleavage divisions, blastulation and gastrulation, mediated by different groups of 5-HT receptors, as well as by a 5-HT transporter (SERT). It is possible that these receptors are coupled to different signaling cascades and that they may act together at certain developmental stages, and at other times act independently. The abnormal phenotypes produced by different 5-HT ligands provide further evidence for the diversity of cellular targets during pre-nervous development, and for their functional coupling to different signaling cascades.

ACKNOWLEDGMENTS This study was supported by a grant from the Johns Hopkins center for Alternatives to Animal Testing to JML.

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