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Sea Urchin Embryos and Larvae as Biosensors for Neurotoxicants
Publication Name:
Current Protocols in Toxicology
Unit Number:
Unit 1.6
DOI:
10.1002/0471140856.tx0106s16
Online Posting Date:
August, 2003 Abstract
Sea urchin embryos and larvae provide an inexpensive high-throughput system for determining developmental actions of neuropharmacologic agents or environmental neurotoxins in both applied and basic biologic contexts. The use of this system for the testing of chlorpyrifos, 1-nicotine, lipophilic amides of choline, and ritanserin is described in detail.
Keywords: sea urchin embryos and larvae; chlorpyrifos; 1-nicotine; ritanserin
Table of Contents
- Unit Introduction
- Basic Protocol 1: Maintaining and Handling Adult Sea Urchins
- Basic Protocol 2: Harvesting Sea Urchin Gametes
- Basic Protocol 3: Fertilizing and Incubating Sea Urchin Eggs
- Basic Protocol 4: Embryonic and Larval Development of Sea Urchins
- Protocols for Testing Neurotoxicants
- Basic Protocol 5: Testing of Chlorpyrifos on Sea Urchin Embryos and Larvae
- Alternate Protocol 1: Testing of l-Nicotine on Sea Urchin Embryos and Larvae
- Alternate Protocol 2: Testing of Arachidonoylcholine and Docosahexaenoylcholine on Sea Urchin Embryos and Larvae
- Alternate Protocol 3: Testing of Ritanserin on Sea Urchin Embryos and Larvae
- Reagents and Solutions
- Commentary
- Literature Cited
- Figures
- Tables
Materials
Basic Protocol 5: Testing of Chlorpyrifos on Sea Urchin Embryos and Larvae
Materials
- Sea urchin embryos and larvae, stages 1a or 1b, 3 or 4, 10, 11, 13 (Tables
- ASW (see
- 20 mM chlorpyrifos (cpf; Chem Service) in 100% (v/v) methanol, store up to 1 to 2 weeks at –20°C
- Potential rescue compound, such as the polyenoic fatty acid derivatives (see
- Digital imaging system, including microscope (e.g., Leica), digital color videocamera (e.g., Spot RT; Diagnostic Instruments), and computer with appropriate software
- 12- or 24-well tissue culture plates
Alternate Protocol 1: Testing of l-Nicotine on Sea Urchin Embryos and Larvae
Additional Materials (also see Basic Protocol 5 )
- 20 mM l-nicotine bitartrate in 100% (v/v) methanol, store in dark up to 1 to 2 weeks at 4°C
- Potential rescue compound, such as:
- 20 mM d-tubocurarine chloride (Sigma), store up to 1 week at –20°C
- 20 mM imechine (Latoxan), store up to 1 to 2 weeks at 4°C
- 20 mM QX-222 (Astra Pharmaceuticals), store up to 1 to 2 weeks at 4°C
- 20 mM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7; Sigma), store for several days at –20°C
- CFASW (see
- 2 mM PMA (see
Alternate Protocol 2: Testing of Arachidonoylcholine and Docosahexaenoylcholine on Sea Urchin Embryos and Larvae
Additional Materials (also see Basic Protocol 5 )
- Polyenoic fatty acid derivatives (see
- Arachidonoylcholine (AA-choline)
- Docosahexaenoylcholine (DHA-choline)
Alternate Protocol 3: Testing of Ritanserin on Sea Urchin Embryos and Larvae
Additional Materials (also see Basic Protocol 5 )
- 20 mM ritanserin (Sigma) in methanol, store up to 2 days at 20°C
- 20 mM serotonin hydrochloride (5-HT), store in dark up to 24 hr at 4°C
- Polyenoic fatty acid derivatives (see
- Serotonamide of arachidonic acid (arachidonoyl serotonin; AA-5-HT)
- Serotonamide of docosahexaenoic acid (DHA-5-HT)
- Serotonamide of eicosapentaenoic acid (EPA-5-HT)
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Figures
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Figure 1.6.1Developmental stages of the sea urchin L. variegatus: (A) stage 0, (B) stage 1a, (C) stage 1b, (D) stage 2a, (E) stage 3, (F) stage 4, (G) stage 5, (H) stage 6, and (I) stage 7. Abbreviations: cf, cleavage furrow; fe, fertilization envelope; hl, hyaline layer; ma, macromeres; me, mesomeres; mi, micromeres; nc, nucleus; pn, pronucleus. Scale bar, 50 µm. -
Figure 1.6.2Developmental stages of the sea urchin L. variegatus: (A) stage 8, (B) stage 9, (C) stage 10, (D) stage 11, (E) stage 12, (F) stage 13, (G) stage 14, (H) stage 15, and (I) stage 16. Abbreviations: ah, after hatching; ar, archenteron; bh, before hatching; bp, blastopore; pm, primary mesenchyme; sm, secondary mesenchyme. Scale bar, 50 µm. -
Figure 1.6.3Developmental stages of the sea urchin L. variegatus: (A) stage 17, (B) stage 18, (C) stage 19, (D) stage 20, (E) stage 21, (F) stage 22, (G) stage 23, (H) stage 24, and (I) stage 25. Abbreviations: a1, first pair of arms; a2, second pair of arms; ar, archenteron; bp, blastopore; pm, primary mesenchyme; rd2, rudiment of second pair of arms; sm, secondary mesenchyme. Scale bars, 50 µm (A-F) and 100 µm (G-I). -
Figure 1.6.4Effects of chlorpyrifos (cpf) introduced at stage 11 (25 hr 30 min after fertilization, 10°C or 30 hr 00 min, 8°C) on development of the sea urchin, S. droebachiensis. (A) Mushroom-like (defect III) larvae after exposure to 20 µM cpf. (B) Practically normal (defect 0) larvae (stage 20) after exposure to 0.5 µM cpf. They are similar to controls, but with a small retardation in development. Imaging was done 62 hr after fertilization (10°C). Scale bar, 200 µm. Modified from Buznikov et al., -
Figure 1.6.5Structure of the choline and dimethylaminoethyl esters of polyenoic fatty acid. Abbreviations: AA-choline, arachidonoylcholine; AA-DMAE, dimethylaminoethyl arachidonate; DHA-choline, docosahexaenoylcholine; DHA-DMAE, dimethylaminoethyl docosahexaenoate. -
Figure 1.6.6Effects of chlorpyrifos (cpf) introduced at stage 11 (25 hr 30 min after fertilization, 10°C) on the development of the sea urchin, S. droebachiensis. (A) Extruded, transformed (pigmented) cells cover the entire surface of larvae (defect IV) after exposure to 40 µM cpf. (B) Transformed cells (>50% of all larval cells) form an extralarval cluster (defect III, first phase) after exposure to 20 µM cpf. (C) Exogastrulation (defect II, second phase) after exposure to 40 µM cpf. (D) Transformed cells accumulate in the blastocoel without extruding (defect I) after exposure to 5 µM cpf. (E) Exogastrulation (defect I, second phase) after exposure to 5 µM cpf. (F) At stage 19, larvae appear normal after exposure to 40 µM cpf with 40 µM dimethylaminoethyl arachidonate (AA-DMAE). Imaging was done 62 hr after fertilization (A,B,D,F) or 10 hr later (C,E). Abbreviations: dl, dwarf larva; ec, extralarval cell cluster; tr, transformed (pigmented) cells. Scale bar, 50 µm. -
Figure 1.6.7Effects of chlorpyrifos (cpf) introduced at stage 1b (30 min after fertilization, 10°C) on the development of the sea urchin, S. droebachiensis. (A) Exposure to 160 µM cpf. (B) Control stage 8 (8 hr 45 min after fertilization) specimen. (C) Exposure to 40 µM cpf. (D) Control stage 10 (22 hr 25 min after fertilization) specimen. (E) Mushroom-like larvae after exposure to 40 µM cpf. (F) Control stage 20 (64 hr 30 min after fertilization) specimen. Abbreviations: dl, dwarf larva; ec, extralarval cell cluster; tr, transformed (pigmented) cells. Scale bar, 50 µm. Modified from Buznikov et al., -
Figure 1.6.8Effect of l-nicotine introduced at stage 12 (35 hr after fertilization, 8°C) on development of the sea urchin, S. droebachiensis. (A-C) Development of extralarval cell cluster near the vegetal pole of larvae after exposure to 200 µM l-nicotine. Imaging was done (A) 47, (B) 51, and (C) 60 hr after fertilization. (D) Normal larva at stage 15 (51 hr after fertilization) after exposure to 200 µM l-nicotine and 50 µM imechine. (E) Nearly normal larva at stage 15 after exposure to 200 µM l-nicotine and 40 µM dimethylaminoethyl docosahexaenoate (DHA-DMAE). (F) Extralarval cell cluster near the animal pole of a larva 60 hr after fertilization following exposure to 50 µM l-nicotine and 0.2 µM phorbol 12-myristate 13-acetate (PMA). Abbreviations: bp, blastopore; ec, extralarval cell cluster; hl, hyaline layer (enfoliated). Scale bar, 50 µm. -
Figure 1.6.9Effects of 40 µM arachidonoylcholine (AA-choline) on development of the sea urchin, S. droebachiensis and protective action of 40 µM dimethylaminoethyl arachidonate (AA-DMAE). (A) AA-choline produces one-cell multinuclear embryos, which are beginning to undergo cell lysis. (B) AA-choline with AA-DMAE produces normal hatched larvae (stage 11). (C) AA-choline produces typical mushroom-like larvae at later stages. (D) AA-choline with AA-DMAE produces normal larvae (stages 12 to 13). Substances were introduced at stage 1b (15 min after fertilization; A, B) or at stage 10 (28 hr after fertilization; C,D). Specimens were imaged 3 hr (A) or 27 hr (B) later or 40 hr after fertilization (C,D). Abbreviations: ap, animal pole; ec, extralarval cell cluster; vp, vegetal pole. Scale bars, 50 µm. -
Figure 1.6.10Effect of ritanserin introduced at stage 11 (9 hr 15 min after fertilization, 21°C) on development of the sea urchin, L. variegatus. (A) Exposure to 10 µM ritanserin produces blastulae with multilayered cell walls. (B) Exposure to 10 µM ritanserin with 40 µM serotonamide of arachidonic acid (AA-5-HT) produces almost normal larvae at stage 15. (C) Exposure to 10 µM ritanserin with 40 µM N,N,N-trimethylserotonin iodide (5-HTQ) provides no protection. (D) Control larvae, stage 15. Imaging was done 15 hr after fertilization. Scale bar, 50 µm. -
Figure 1.6.11Structure of serotonamides of polyenoic fatty acid. Abbreviations: AA-5-HT, serotonamide of arachidonic acid; DHA-5-HT, serotonamide of docosahexanoic acid; EPA-5-HT, serotonamide of eicosapentanoic acid.
Literature Cited
| Literature Cited | |
| Bezuglov, V.V., Zinchenko, G.N., Nikitina, L.A., and Buznikov, G.A. 2001. Arachidonoylcholine and N,N-dimethylaminoethyl arachidonate are new cholinergic compounds. Bioorg. Khim. 27:227-230. | |
| Bottger, S.A. and McClintock, J.B. 2001. The effects of organic and inorganic phosphates on fertilization and early development in the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 129:307-315. | |
| Buznikov, G.A. 1990. Neurotransmitters in Embryogenesis. Harwood Academic Publishers, Chur, Switzerland. | |
| Buznikov, G.A. and Podmarev, V.I. 1990. "The sea urchins Strongylocentrotus droebachiensis, S. nudus, and S. intermedius". In Animal Species for Developmental Studies, Vol. 1. Invertebrates (T.A. Dettlaff and S.G. Vassetzky, eds.) pp. 253-285. Plenum, New York and London. | |
| Buznikov, G.A., Shmukler, Y.B., and Lauder, J.M. 1996. From oocyte to neuron: Do neurotransmitters function in the same way throughout development Cell. Mol. Neurobiol. 16:533-559. | |
| Buznikov, G.A., Jokanovic, M., Kovacevic, N., and Rakic, L.J. 1997a. Sea urchin embryos and larvae as biosensors for screening and detailed study of pharmacologically active substances. Arch. Toxicol. Kinet. Xenobiot. Metab. 5:393-400. | |
| Buznikov, G.A., Koikov, L.N., Shmukler, Y.B., and Whitaker, M.J. 1997b. Nicotine antagonists (piperidines and quinuclidines) reduce the susceptibility of early sea urchin embryos to agents evoking calcium shock. Gen. Pharmacol. 29:49-53. | |
| Buznikov, G.A., Marshak, T.L., Malchenko, L.A., Nikitina, L.A., Shmukler, Y.B., Buznikov, A.G., Rakic, L.J., and Whitaker, M.J. 1998. Serotonin and acetylcholine modulate the sensitivity of early sea urchin embryos to protein kinase C activators. Comp. Biochem. Physiol. A 120:457-462. | |
| Buznikov, G.A., Lambert, H.W., and Lauder, J.M. 2001a. Serotonin and serotonin-like substances as regulators of early embryogenesis and morphogenesis. Cell Tissue Res. 305:177-186. | |
| Buznikov, G.A., Nikitina, L.A., Bezuglov, V.V., Lauder, J.M., Padilla, S., and Slotkin, T.A. 2001b. An invertebrate model of the developmental neurotoxicity of insecticides: Effects of chlorpyrifos and dieldrin in sea urchin embryos and larvae. Environ. Health Persp. 109:651-661. | |
| Buznikov, G.A., Bezuglov, V.V., Nikitina, L.A., Slotkin, T.A., and Lauder, J.M. 2001c. Cholinergic regulation of sea urchin embryonic and larval development. Ross. Fiziol. Zh. Im. I. M. Sechenova 87:1548-1556. | |
| Cameron, R.A., Mahairas, G., Rast, J.P., Martinez, P., Biondi, T.R., Swartzell, S., Wallace, J.C., Poutska, A.J., Livingston, B.T., Wray, G.A., Ettensohn, C.A., Lehrach, H., Britten, R.J., Davidson, E.H., and Hood, L. 2000. A sea urchin genome project: Sequence scan, virtual map, and additional resources. Proc. Natl. Acad. Sci. U.S.A. 97:9514-9518. | |
| Czihak, G. (ed.) 1975. The Sea Urchin Embryo: Biochemistry and Morphogenesis. Springer, New York. | |
| Dettlaff, T.A. and Dettlaff, A.A. 1961. On relative dimensionless characteristics of development duration in embryology. Arch. Biol. 72:1-16. | |
| Dinnel, P.A., Link, J.M., Stober, Q.J., Letourneau, M.W., and Roberts, W.F. 1989. Comparative sensitivity of sea urchin sperm bioassay to metals and pesticides. Arch. Environ. Contam. Toxicol. 18:748-755. | |
| Falugi, C. 1993. Localization and possible role of molecules associated with the cholinergic system during “non-nervous” developmental events. Eur. J. Histochem. 37:287-294. | |
| Harvey, E.B. 1956. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, N.J. | |
| Ivonnet, P.I. and Chambers, P.L. 1997. Nicotinic acetylcholine receptors of neuronal type occur in plasma membrane of sea urchin eggs. Zygote 5:277-287. | |
| Slotkin, T.A. 1998. Fetal nicotine or cocaine exposure: Which one is worse J. Pharmacol. Exp. Ther. 285:931-945. | |
| Slotkin, T.A. 1999. Developmental cholinotoxicants: Nicotine and chlorpyrifos. Environ. Health Persp. 10(Suppl. 1):71-80. | |
| Stephens, R.E. 1972. Studies on the development of the sea urchin Strongylocentrotus droebachiensis. I. Ecology and normal development. Biol. Bull. 142:132-144. | |
| Internet Resources | |
| http://www.stanford.edu/group/Urchin | |
| Describes all procedures related to maintaining adult sea urchins and obtaining and handling sea urchin embryos and larvae. | |
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