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Role of acetylcholinesterase in the development of axon tracts within the embryonic vertebrate brain
Int. J. Devl Neuroscience, Vol. 17, No. 8, pp. 787±793, 1999 # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0736-5748/99 $20.00 + 0.00
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ROLE OF ACETYLCHOLINESTERASE IN THE DEVELOPMENT OF AXON TRACTS WITHIN THE EMBRYONIC VERTEBRATE BRAIN R.B. ANDERSON$ and B. KEY%* $Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Melbourne 3052, Australia; %Neurodevelopment Laboratory, Department of Anatomical Sciences, University of Queensland, Brisbane 4072, Australia (Received 26 April 1999; received in revised form 21 June 1999; accepted 23 June 1999) AbstractÐIn the developing vertebrate brain, acetylcholinesterase (AChE) expression coincides temporally with axon tract formation. Although AChE promotes neurite outgrowth in vitro, the role of this molecule in the development of axon tracts in vivo is unknown. To address this question, we examined the eects of the AChE inhibitor, BW284C51, on the formation of the early scaold of axon tracts in the embryonic Xenopus brain. In exposed Xenopus brain preparations, axons elongate and establish a normal topography of axon tracts. However, when brains were exposed to BW284C51, the thickness of the major longitudinal axon tract, the tract of the post-optic commissure decreased in a dose-dependent manner. When BW284C51 was removed from the culture media axon tract development returned to normal within 5 h. These ®ndings provide the ®rst evidence for a non-classical role of AChE in the initial formation of axon tracts within the developing vertebrate brain. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved. Key words: AChE; BW284C51; brain; axon tract; Xenopus.
INTRODUCTION The classical role of acetylcholinesterase (AChE) is to hydrolyse the neurotransmitter acetylcholine at cholinergic synapses. However, AChE is also expressed in regions that are not cholinergically innervated. Interestingly, the expression of AChE in the developing vertebrate brain corresponds temporally with the major period of axon elongation and path®nding.13,24,26,28 This has led to the suggestion that AChE may mediate important morphogenetic events that are independent of its enzymatic activity. In support of this, AChE inhibitors such as echothiopate and diisopropyl¯uorophosphate that block esterase activity have no eect on neurite outgrowth,18,23,29 whereas BW284C51 which blocks both the catalytic and the peripheral sites on AChE decreases neurite outgrowth.6,18,23,30 A similar reduction in neurite growth occurs when the peripheral site alone is blocked with Fasciculin II.9 Furthermore, neurite outgrowth is not aected when the catalytically active site of AChE is inactivated by insertional mutagenesis.31 AChE has been proposed to act as a cell or substrate adhesion molecule due to its homology with several known cell adhesion molecules (CAMs) which include Drosophila neurotactin,15 glutactin,25 gliotactin4 and rat neuroligin.16 Based on sequence homologies, AChE is believed to be a member of a new family of CAMs called the serine ester hydrolase family.21 Since AChE is expressed by neurons that give rise to the early scaold of axon tracts within the developing vertebrate brain10,28,32 we propose that AChE is involved in the initial development of axon tracts. In the present study we have examined the in vivo role of AChE during the early period of axonogenesis using an exposed Xenopus brain preparation.2 Our observations indicate that cell surface AChE is necessary for the normal development of axon tracts but is not involved in axon path®nding in the embryonic vertebrate brain. *To whom all correspondence should be addressed. Tel.: +61-3-9344-5796; fax: +61-3-9347-4190; E-mail: b.key@ anatomy.unimelb.edu.au 787
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EXPERIMENTAL PROCEDURES Immunohistochemistry The immunostaining of wholemount Xenopus brains was performed as previously described.1 Brie¯y, stage 32 Xenopus embryos were immersion ®xed in 4% paraformaldehyde and brains dissected and dehydrated in a graded ethanol series (70±100%). Embryos were washed in ethanol:propylene oxide, followed by two consecutive washes in 100% propylene oxide, and rehydrated through a graded ethanol series (70±100%) to Tris buered saline (TBS; 0.1 M, pH 7.4). Brains were incubated for 1 h in 2% Bovine serum albumin (BSA) and 0.3% Triton X100 to block non-speci®c binding, and then reacted with the anti-acetylated a-tubulin (Sigma Chemical Company, St Louis, MO) monoclonal antibody in 2% BSA in TBS. Embryos were then reacted with a biotinylated goat anti-mouse IgG (g-chain speci®c) secondary antibody (Sigma) and visualised using Extravidin-FITC (Sigma). Wholemounts of Xenopus brains were viewed using a Biorad 1024 scanning confocal microscope. Images were assembled using both Photoshop 4.0 (Abode Systems Incorporated, CA, USA) and Coreldraw 7.0 (Corel Corporation Limited, Dublin, Ireland). Exposed Xenopus brain preparation In order to test the role of AChE in development of axon tracts we used an exposed Xenopus brain preparation. Stage 26 Xenopus embryos were anaesthetised with tricaine methanesulfate (0.4 mg/ml) and cultured in modi®ed Barth's solution (100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and 50 mg/ml gentamicin) containing 0.1% dimethyl sulfoxide as previously described.2 The ectoderm overlaying the left side of the embryo was removed to expose the underlying brain. These exposed brain preparations were then cultured in a de®ned media for 8 h. During this culture period axonogenesis begins in the forebrain and pioneer axons establish a scaold of axon tracts that provides a template for subsequent neural development. Within 8 h the scaold of axon tracts that forms in exposed brain preparations2 is identical to that produced during normal brain development.1 Thus, this preparation provides a unique means for assessing the role of speci®c molecules in early development of the rostral brain, particularly in the construction of axon tracts. Embryos were cultured in either unsupplemented media or in the presence of the highly speci®c membrane impermeable AChE inhibitor, 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51; Sigma) at concentrations ranging from 100 mM±1 mM for 8 h. Some animals were initially cultured in the presence of BW284C51 for 3 h and then transferred to unsupplemented media for the remaining 5 h. The reversible AChE inhibitor BW284C51 binds both the catalytic and peripheral anionic sites of AChE.18 Following the culture period, brains were ®xed and immunostained for acetylated a-tubulin, a marker for growing axons. Wholemounts of Xenopus brain were then viewed by confocal microscopy and para-sagittal optical sections were collected and compiled into a single image. These complied sections allow the trajectory of all axon tracts to be examined in a single image and provide a novel way of assessing the growth and development of axons in the early embryonic brain. Quanti®cation The width of the tract of the post-optic commissure (TPOC) was measured at its widest point within 100 mm caudal to the optic stalk. Brains were immunostained for acetylated a-tubulin and digital images were collected using the scanning confocal microscope. Lateral views of wholemounts of Xenopus brains were used to identify the dorsal- and ventral-most region of this tract. The width of the TPOC was determined directly from micrographs using a digitiser. A one-way analysis of variance (ANOVA) was performed comparing the means of control and treatment groups. A post hoc comparison was performed using a Schee-test to determine signi®cance between groups.
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RESULTS Control embryonic Xenopus brains cultured in unsupplemented media developed normally (Fig. 1A). The gross morphology of the brain was identical to that of an in vivo brain of similar age.1,2 At this age the telencephalic vesicle has not yet emerged and there are no clear morphological divisions between the prosencephalon and the mesencephalon. The brain is simply a hollow tube shaped like a pistol with a single ventral ¯exure. Immunostaining for acetylated a-tubulin revealed the trajectory of axon tracts which develop at the apical surface of the neuroepithelium during the culture period (Fig. 1A). The principal tract of the forebrain is the tract of the post-optic commissure (TPOC), which courses along the ventrolateral surface of the rostral brain where it merges in the midbrain with the ventral longitudinal tract (VLT). There are three smaller tracts arranged in the dorso-ventral plane that merge with the TPOC: the supraoptic tract (SOT), which arises from the nucleus of the presumptive telencephalon; the dorsoventral diencephalic tract (DVDT), which arises from the epiphysis; and the tract of the posterior commissure (TPC), which arises from a small nucleus in the dorsal midbrain. When exposed brain preparations were cultured in the presence of the AChE inhibitor, BW284C51, the topography of the early scaold of axon tracts appeared normal (Fig. 1B, C, E). In all cases the three main axon tracts, the TPOC, TPC, and DVDT, were present in treated brains. However, the TPOC, which normally increases considerably in width during the culture period due to an increase in axon number, seemed reduced in thickness in brains cultured in the presence of either 500 mM or 1 mM BW284C51. A one-way analysis of variance (ANOVA) and a post hoc Schee-test on the width of the TPOC, revealed a dose-dependent reduction in the size of this tract in response to the AChE inhibitor. Animals cultured in the presence of 100 mM BW284C51 (n=10) showed no statistical dierence in the thickness of the TPOC, compared to control cultures (Fig. 2). However, brains exposed to 500 mM (n=10) and 1 mM (n=10) BW284C51 exhibited a signi®cant dose-dependent reduction in the thickness of this tract (P < 0.000) (Fig. 2). Our observations could not exclude the possibility that the size of the TPOC was reduced as a result of neuronal death rather than from predicted changes in axon growth, even though previous reports indicate that BW284C51 selectively aects neurite outgrowth in vitro.6,18,23,30 In order to determine whether BW284C51 was causing the death of embryonic neurons that gives rise to the TPOC, we cultured exposed brain preparations in the presence of the AChE inhibitor for 3 h and then returned them to control media for a 5-h rescue period. The exposure of embryonic brains to 500 mM (n=8) or 1 mM (n=8) BW284C51 for 3 h decreased tract thickness by 38% and 75%, respectively. Thus, a very short exposure to BW284C51 was sucient to have a signi®cant eect on the development of axon tracts. When brain preparations exposed to 500 mM BW284C51 were transferred back into control media (n=10) the thickness of the TPOC returned to control levels within 5 h (Fig. 1D; Fig. 2). A similar eect was also observed for embryos exposed to 1 mM BW284C51 (n=10; Fig. 2). These results suggest that the reduced size of the TPOC is the result of either decreased AChE-mediated axonogenesis or axon elongation rather than neuronal death. DISCUSSION AChE begins to be expressed within the embryonic vertebrate brain just prior to the major period of axon outgrowth that leads to the formation of the initial scaold of axon tracts.10,24,26 We have shown here that cell surface AChE is required for the normal development of the major longitudinal tract of the forebrain, the tract of the post-optic commissure (TPOC) in Xenopus brain. When the membrane impermeable AChE inhibitor BW284C51 was added to exposed brain preparations of Xenopus the thickness of the TPOC decreased in a dosedependent manner. AChE does not play a role in axon guidance, since axons followed their normal stereotypical trajectory under all conditions. Instead AChE appears to be involved in the normal growth of axons within the embryonic vertebrate brain. How does AChE mediate this eect? One possibility is that AChE is essential for axonogenesis, the initial formation of the axon. Antisense suppression of AChE led to a
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Fig. 1. Immunostaining of axon tracts in exposed Xenopus brain preparations. (A) Confocal image of a wholemount Xenopus brain cultured in unsupplemented media and immunostained for acetylated atubulin to reveal all growing axon tracts. The development of the early scaold of axon tracts was normal. The lines represent the point at which the TPOC was measured. (B, C, E) Embryos cultured in the presence of 100 mM, 500 mM and 1 mM of BW284C51, respectively. These brains demonstrated a dosedependent reduction in the thickness of the TPOC. (D, F) Rescue experiments. Embryos initially cultured in the presence of BW284C51 and then allowed to develop in control media demonstrated substantial recovery in the thickness of the TPOC. DVDT, dorsoventral diencephalic tract; POC, postoptic commissure; SOT, supraoptic tract; TPC, tract of the posterior commissure; TPOC, tract of the postoptic commissure; VC, ventral commissure; VLT, ventral longitudinal tract. Scale=100 mm (A±F).
reduction in neurite production by PC12 cells.12 The decrease in AChE was accompanied by a loss of neurexin Ia, the receptor for neuroligin which possesses an AChE-like extracellular domain. Since neuritogenesis was rescued by overexpression of neuroligin in these AChE depleted PC12 cells it was argued that AChE was modulating lateral cis membrane interactions between neurexin and neuroligin.12 The involvement of neurexins in the molecular interactions underlying AChE mediated axon growth was further supported by evidence that levels of neurexin 1b were altered in transgenic mice expressing human AChE.3 The cell autologous eects of AChE were also demonstrated when AChE was transfected into glioma cells and produced extension of processes.19 Moreover, Xenopus spinal neurons misexpressing human AChE extend longer neurites than control neurons when grown in dispersed cultures.31 These
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Fig. 2. The eect of BW284C51 on axon tract formation. A one-way ANOVA and a post hoc Scheetest revealed a dose-dependent reduction in the thickness of the TPOC (P < 0.000). Rescue experiments, in which embryos were initially cultured in the presence of BW284C51 and then allowed to develop in control media, returned to either control levels (500 mM+Rescue) or near control levels (1 mM+Rescue). Values are mean 2 SEM; $ value is signi®cantly dierent from control cultures; % value is signi®cantly dierent from rescue cultures.
results indicate that AChE can stimulate neurite outgrowth by lateral membrane interactions. It does not, however, negate possible heterologous cell interactions in vivo. Another possibility is that AChE functions as a cell adhesion molecule and modulates axon elongation. AChE is a member of the serine ester hydrolase family, which comprises several known cholinesterase-like cell adhesion molecules. For example, neurotactin is a Drosophila transmembrane glycoprotein that contains a catalytically inactive cholinesterase-like extracellular domain5,9 which mediates cell adhesion.5 When the extracellular cholinesterase-like domain of neurotactin was replaced with the core domain of AChE, the resulting chimeric protein was capable of promoting cell adhesion.8 These results suggest that in addition to its enzymatic activity, AChE may also function as an adhesion molecule. The ®nding that substrate-bound AChE enhances neurite outgrowth of chick sympathetic neurons further indicates an adhesive non-catalytic mode of action for AChE.29 AChE may also mediate axon growth through its HNK-1 carbohydrate moiety.7 In vivo and in vitro studies have demonstrated that antibodies directed against the HNK-1 epitope can block axonal growth,11,14,20 while neurite outgrowth has been shown to be enhanced when cultured on a substrate containing HNK-1.27 The extracellular matrix glycoprotein laminin binds HNK-1 at a site localised to domain 2 within the E8 fragment.27 When this binding site is blocked, neural cells have been shown to lose their ability to attach to laminin.27 This raises the possibility that interactions between AChE and various extracellular matrix molecules could promote axon growth. However, HNK-1 could only mediate AChE dependent axon growth in the present study, if BW284C51 was non-speci®cally blocking receptor-carbohydrate binding interactions. Although it is possible that neuronal death contributes to the reduced size of the TPOC in the presence of BW284C51, our control experiments suggest otherwise. We showed that a 5-h rescue period was sucient to reverse the deleterious eects of BW284C51 on the size of the TPOC. If BW284C51 was causing neuronal death then elevated levels of cell proliferation
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followed by neuronal dierentiation and axon outgrowth must be restoring the TPOC to its normal size during the rescue period. However, 5 h is insucient for proliferating neuroepithelial cells to reconstitute the TPOC since it takes at least 10 h for cells to dierentiate and initiate axon outgrowth after they have exited the cell cycle.17,22 In conclusion, we have shown for the ®rst time that AChE is involved in the development of axon tracts within the embryonic vertebrate brain in vivo. AChE may be mediating its eect by reducing axonogenesis and/or attenuating axon elongation. The exposed brain preparation used here will provide a convenient experimental model to now determine the molecular mechanisms of AChE mediated axon growth in vivo. AcknowledgementsÐThis work was supported by an Australian N.H. & M.R.C. grant to B.K. and by a University of Melbourne Faculty of Medicine scholarship to R.B.A.
REFERENCES 1. Anderson, R. B. and Key, B., Expression of a novel N-CAM glycoform (NOC-1) on axon tracts in embryonic Xenopus brain. Dev. Dyn., 1996, 207, 263±269. 2. Anderson, R. B., Walz, A., Holt, C. E. and Key, B., Chondroitin sulfates modulate axon guidance in embryonic Xenopus brain. Dev. Biol., 1998, 202, 235±243. 3. Andres, C., Beeri, R., Friedman, A., Lev-Lehman, E., Henis, S., Timberg, R., Shani, M. and Soreq, H., Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin Ibeta gene expression followed by late-onset neuromotor deterioration. Proc. Natl. Acad. Sci. USA, 1997, 94, 8173±8178. 4. Auld, V. J., Fetter, R. D., Broadie, K. and Goodman, C. S., Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood±nerve barrier in Drosophila. Cell, 1995, 81, 757±767. 5. Barthalay, Y., Hipeau-Jacquotte, R., de la Escalera, S., Jimenez, F. and Piovant, M., Drosophila neurotactin mediates heterophilic cell adhesion. EMBO J., 1990, 9, 3603±3609. 6. Bataille, S., Portalier, P., Coulon, P. and Ternaux, J. P., In¯uence of acetylcholinesterase on embryonic spinal rat motoneurones growth in culture: a quantitative morphometric study. Eur. J. Neurosci., 1998, 10, 560±572. 7. Bon, S., Me¯ah, K., Musset, F., Grassi, J. and Massoulie, J., An immunoglobulin M monoclonal antibody, recognizing a subset of acetylcholinesterase molecules from electric organs of Electrophorus and Torpedo, belongs to the HNK-1 anti-carbohydrate family. J. Neurochem., 1987, 49, 1720±1731. 8. Darboux, I., Barthalay, Y., Piovant, M. and Hipeau-Jacquotte, R., The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties. EMBO J., 1996, 15, 4835± 4843. 9. de la Escalera, S., Bockamp, E. O., Moya, F., Piovant, M. and Jimenez, F., Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to cholinesterases. EMBO J., 1990, 9, 3593±3601. 10. Eagleson, G. W., Ubink, R., Jenks, B. G. and Roubos, E. W., Forebrain dierentiation and axonogenesis in amphibians: I. Dierentiation of the suprachiasmatic nucleus in relation to background adaptation behavior. Brain Behav. Evol., 1998, 52, 23±36. 11. Fischer, G., Kunemund, V. and Schachner, M., Neurite outgrowth patterns in cerebellar microexplant cultures are aected by antibodies to the cell surface glycoprotein L1. J. Neurosci., 1986, 6, 605±612. 12. Grifman, M. and Soreq, H., Dierentiation intensi®es the susceptibility of pheochromocytoma cells to antisense oligodeoxynucleotide-dependent suppression of acetylcholinesterase activity. Antisense Nucleic Acid Drug Dev., 1997, 7, 351±359. 13. Hanneman, E. and Wester®eld, M. J., Early expression of acetylcholinesterase activity in functionally distinct neurons of the Zebra®sh. Comp. Neurol., 1989, 284, 350±361. 14. Henke-Fahle, S. and Bonhoeer, F., Inhibition of axonal growth by a monoclonal antibody. Nature, 1983, 303, 65± 67. 15. Hortsch, M., Patel, N. H., Bieber, A. J., Traquina, Z. R. and Goodman, C. S., Drosophila neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis. Development, 1990, 110, 1327±1340. 16. Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C. and Sudhof, T. C., Neuroligin 1: a splice site-speci®c ligand for beta-neurexins. Cell, 1995, 81, 435±443. 17. Jacobson, M. and Huang, S., Neurite outgrowth traced by means of horseradish peroxidase inherited from neuronal ancestral cells in frog embryos. Dev. Biol., 1985, 110, 102±113. 18. Jones, S. A., Holmes, C., Budd, T. C. and Green®eld, S. A., The eect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain. Cell Tissue Res., 1995, 279, 323±330. 19. Karpel, R., Sternfeld, M., Ginzberg, D., Guhl, E., Graessmann, A. and Soreq, H., Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells. J. Neurochem., 1996, 66, 114±123. 20. Keilhauer, G., Faissner, A. and Schachner, M., Dierential inhibition of neurone±neurone, neurone±astrocyte and astrocyte±astrocyte adhesion by L1, L2 and N-CAM antibodies. Nature, 1985, 316, 728±730. 21. Krejci, E., Duval, N., Chatonnet, A., Vincens, P. and Massoulie, J., Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identi®cation of a catalytically essential aspartic acid. Proc. Natl. Acad. Sci. USA, 1991, 88, 6647±6651.
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22. Lamborghini, J. E., Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation. J. Comp. Neurol., 1980, 189, 323±333. 23. Layer, P. G., Weikert, T. and Alber, R., Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism. Cell. Tissue Res., 1993, 273, 219±226. 24. Layer, P. G., Cholinesterases during development of the avian nervous system. Cell Mol. Neurobiol., 1991, 11, 7±33. 25. Olson, P. F., Fessler, L. I., Nelson, R. E., Sterne, R. E., Campbell, A. G. and Fessler, J. H., Glutactin, a novel Drosophila basement membrane-related glycoprotein with sequence similarity to serine esterases. EMBO J., 1990, 9, 1219±1227. 26. Ross, L. S., Parrett, T. and Easter, S. S., Axonogenesis and morphogenesis in the embryonic zebra®sh brain. J. Neurosci., 1992, 12, 467±482. 27. Schachner, M. and Martini, R., Glycans and the modulation of neural-recognition molecule function. Trends Neurosci., 1995, 18, 183±191. 28. Schlaggar, B. L., De Carlos, J. A. and O'Leary, D. D., Acetylcholinesterase as an early marker of the dierentiation of dorsal thalamus in embryonic rats. Dev. Brain Res., 1993, 75, 19±30. 29. Small, D. H., Reed, G., White®eld, B. and Nurcombe, V., Cholinergic regulation of neurite outgrowth from isolated chick sympathetic neurons in culture. J. Neurosci., 1995, 15, 144±151. 30. Srivatsan, M. and Peretz, B., Acetylcholinesterase promotes regeneration of neurites in cultured adult neurons of Aplysia. Neuroscience, 1997, 77, 921±931. 31. Sternfeld, M., Ming, G., Song, H., Sela, K., Timberg, R., Poo, M. and Soreq, H., Acetylcholinesterase enhances neurite growth and synapse development through alternative contributions of its hydrolytic capacity, core protein, and variable C termini. J. Neurosci., 1998, 18, 1240±1249. 32. Wilson, S. W., Ross, L. S., Parrett, T. and Easter, S. S., The development of a simple scaold of axon tracts in the brain of the embryonic zebra®sh, Brachydanio rerio. Development, 1990, 108, 121±145.
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ROLE OF ACETYLCHOLINESTERASE IN THE DEVELOPMENT OF AXON TRACTS WITHIN THE EMBRYONIC VERTEBRATE BRAIN R.B. ANDERSON$ and B. KEY%* $Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Melbourne 3052, Australia; %Neurodevelopment Laboratory, Department of Anatomical Sciences, University of Queensland, Brisbane 4072, Australia (Received 26 April 1999; received in revised form 21 June 1999; accepted 23 June 1999) AbstractÐIn the developing vertebrate brain, acetylcholinesterase (AChE) expression coincides temporally with axon tract formation. Although AChE promotes neurite outgrowth in vitro, the role of this molecule in the development of axon tracts in vivo is unknown. To address this question, we examined the eects of the AChE inhibitor, BW284C51, on the formation of the early scaold of axon tracts in the embryonic Xenopus brain. In exposed Xenopus brain preparations, axons elongate and establish a normal topography of axon tracts. However, when brains were exposed to BW284C51, the thickness of the major longitudinal axon tract, the tract of the post-optic commissure decreased in a dose-dependent manner. When BW284C51 was removed from the culture media axon tract development returned to normal within 5 h. These ®ndings provide the ®rst evidence for a non-classical role of AChE in the initial formation of axon tracts within the developing vertebrate brain. # 1999 ISDN. Published by Elsevier Science Ltd. All rights reserved. Key words: AChE; BW284C51; brain; axon tract; Xenopus.
INTRODUCTION The classical role of acetylcholinesterase (AChE) is to hydrolyse the neurotransmitter acetylcholine at cholinergic synapses. However, AChE is also expressed in regions that are not cholinergically innervated. Interestingly, the expression of AChE in the developing vertebrate brain corresponds temporally with the major period of axon elongation and path®nding.13,24,26,28 This has led to the suggestion that AChE may mediate important morphogenetic events that are independent of its enzymatic activity. In support of this, AChE inhibitors such as echothiopate and diisopropyl¯uorophosphate that block esterase activity have no eect on neurite outgrowth,18,23,29 whereas BW284C51 which blocks both the catalytic and the peripheral sites on AChE decreases neurite outgrowth.6,18,23,30 A similar reduction in neurite growth occurs when the peripheral site alone is blocked with Fasciculin II.9 Furthermore, neurite outgrowth is not aected when the catalytically active site of AChE is inactivated by insertional mutagenesis.31 AChE has been proposed to act as a cell or substrate adhesion molecule due to its homology with several known cell adhesion molecules (CAMs) which include Drosophila neurotactin,15 glutactin,25 gliotactin4 and rat neuroligin.16 Based on sequence homologies, AChE is believed to be a member of a new family of CAMs called the serine ester hydrolase family.21 Since AChE is expressed by neurons that give rise to the early scaold of axon tracts within the developing vertebrate brain10,28,32 we propose that AChE is involved in the initial development of axon tracts. In the present study we have examined the in vivo role of AChE during the early period of axonogenesis using an exposed Xenopus brain preparation.2 Our observations indicate that cell surface AChE is necessary for the normal development of axon tracts but is not involved in axon path®nding in the embryonic vertebrate brain. *To whom all correspondence should be addressed. Tel.: +61-3-9344-5796; fax: +61-3-9347-4190; E-mail: b.key@ anatomy.unimelb.edu.au 787
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EXPERIMENTAL PROCEDURES Immunohistochemistry The immunostaining of wholemount Xenopus brains was performed as previously described.1 Brie¯y, stage 32 Xenopus embryos were immersion ®xed in 4% paraformaldehyde and brains dissected and dehydrated in a graded ethanol series (70±100%). Embryos were washed in ethanol:propylene oxide, followed by two consecutive washes in 100% propylene oxide, and rehydrated through a graded ethanol series (70±100%) to Tris buered saline (TBS; 0.1 M, pH 7.4). Brains were incubated for 1 h in 2% Bovine serum albumin (BSA) and 0.3% Triton X100 to block non-speci®c binding, and then reacted with the anti-acetylated a-tubulin (Sigma Chemical Company, St Louis, MO) monoclonal antibody in 2% BSA in TBS. Embryos were then reacted with a biotinylated goat anti-mouse IgG (g-chain speci®c) secondary antibody (Sigma) and visualised using Extravidin-FITC (Sigma). Wholemounts of Xenopus brains were viewed using a Biorad 1024 scanning confocal microscope. Images were assembled using both Photoshop 4.0 (Abode Systems Incorporated, CA, USA) and Coreldraw 7.0 (Corel Corporation Limited, Dublin, Ireland). Exposed Xenopus brain preparation In order to test the role of AChE in development of axon tracts we used an exposed Xenopus brain preparation. Stage 26 Xenopus embryos were anaesthetised with tricaine methanesulfate (0.4 mg/ml) and cultured in modi®ed Barth's solution (100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, and 50 mg/ml gentamicin) containing 0.1% dimethyl sulfoxide as previously described.2 The ectoderm overlaying the left side of the embryo was removed to expose the underlying brain. These exposed brain preparations were then cultured in a de®ned media for 8 h. During this culture period axonogenesis begins in the forebrain and pioneer axons establish a scaold of axon tracts that provides a template for subsequent neural development. Within 8 h the scaold of axon tracts that forms in exposed brain preparations2 is identical to that produced during normal brain development.1 Thus, this preparation provides a unique means for assessing the role of speci®c molecules in early development of the rostral brain, particularly in the construction of axon tracts. Embryos were cultured in either unsupplemented media or in the presence of the highly speci®c membrane impermeable AChE inhibitor, 1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284C51; Sigma) at concentrations ranging from 100 mM±1 mM for 8 h. Some animals were initially cultured in the presence of BW284C51 for 3 h and then transferred to unsupplemented media for the remaining 5 h. The reversible AChE inhibitor BW284C51 binds both the catalytic and peripheral anionic sites of AChE.18 Following the culture period, brains were ®xed and immunostained for acetylated a-tubulin, a marker for growing axons. Wholemounts of Xenopus brain were then viewed by confocal microscopy and para-sagittal optical sections were collected and compiled into a single image. These complied sections allow the trajectory of all axon tracts to be examined in a single image and provide a novel way of assessing the growth and development of axons in the early embryonic brain. Quanti®cation The width of the tract of the post-optic commissure (TPOC) was measured at its widest point within 100 mm caudal to the optic stalk. Brains were immunostained for acetylated a-tubulin and digital images were collected using the scanning confocal microscope. Lateral views of wholemounts of Xenopus brains were used to identify the dorsal- and ventral-most region of this tract. The width of the TPOC was determined directly from micrographs using a digitiser. A one-way analysis of variance (ANOVA) was performed comparing the means of control and treatment groups. A post hoc comparison was performed using a Schee-test to determine signi®cance between groups.
AChE in axon tract formation
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RESULTS Control embryonic Xenopus brains cultured in unsupplemented media developed normally (Fig. 1A). The gross morphology of the brain was identical to that of an in vivo brain of similar age.1,2 At this age the telencephalic vesicle has not yet emerged and there are no clear morphological divisions between the prosencephalon and the mesencephalon. The brain is simply a hollow tube shaped like a pistol with a single ventral ¯exure. Immunostaining for acetylated a-tubulin revealed the trajectory of axon tracts which develop at the apical surface of the neuroepithelium during the culture period (Fig. 1A). The principal tract of the forebrain is the tract of the post-optic commissure (TPOC), which courses along the ventrolateral surface of the rostral brain where it merges in the midbrain with the ventral longitudinal tract (VLT). There are three smaller tracts arranged in the dorso-ventral plane that merge with the TPOC: the supraoptic tract (SOT), which arises from the nucleus of the presumptive telencephalon; the dorsoventral diencephalic tract (DVDT), which arises from the epiphysis; and the tract of the posterior commissure (TPC), which arises from a small nucleus in the dorsal midbrain. When exposed brain preparations were cultured in the presence of the AChE inhibitor, BW284C51, the topography of the early scaold of axon tracts appeared normal (Fig. 1B, C, E). In all cases the three main axon tracts, the TPOC, TPC, and DVDT, were present in treated brains. However, the TPOC, which normally increases considerably in width during the culture period due to an increase in axon number, seemed reduced in thickness in brains cultured in the presence of either 500 mM or 1 mM BW284C51. A one-way analysis of variance (ANOVA) and a post hoc Schee-test on the width of the TPOC, revealed a dose-dependent reduction in the size of this tract in response to the AChE inhibitor. Animals cultured in the presence of 100 mM BW284C51 (n=10) showed no statistical dierence in the thickness of the TPOC, compared to control cultures (Fig. 2). However, brains exposed to 500 mM (n=10) and 1 mM (n=10) BW284C51 exhibited a signi®cant dose-dependent reduction in the thickness of this tract (P < 0.000) (Fig. 2). Our observations could not exclude the possibility that the size of the TPOC was reduced as a result of neuronal death rather than from predicted changes in axon growth, even though previous reports indicate that BW284C51 selectively aects neurite outgrowth in vitro.6,18,23,30 In order to determine whether BW284C51 was causing the death of embryonic neurons that gives rise to the TPOC, we cultured exposed brain preparations in the presence of the AChE inhibitor for 3 h and then returned them to control media for a 5-h rescue period. The exposure of embryonic brains to 500 mM (n=8) or 1 mM (n=8) BW284C51 for 3 h decreased tract thickness by 38% and 75%, respectively. Thus, a very short exposure to BW284C51 was sucient to have a signi®cant eect on the development of axon tracts. When brain preparations exposed to 500 mM BW284C51 were transferred back into control media (n=10) the thickness of the TPOC returned to control levels within 5 h (Fig. 1D; Fig. 2). A similar eect was also observed for embryos exposed to 1 mM BW284C51 (n=10; Fig. 2). These results suggest that the reduced size of the TPOC is the result of either decreased AChE-mediated axonogenesis or axon elongation rather than neuronal death. DISCUSSION AChE begins to be expressed within the embryonic vertebrate brain just prior to the major period of axon outgrowth that leads to the formation of the initial scaold of axon tracts.10,24,26 We have shown here that cell surface AChE is required for the normal development of the major longitudinal tract of the forebrain, the tract of the post-optic commissure (TPOC) in Xenopus brain. When the membrane impermeable AChE inhibitor BW284C51 was added to exposed brain preparations of Xenopus the thickness of the TPOC decreased in a dosedependent manner. AChE does not play a role in axon guidance, since axons followed their normal stereotypical trajectory under all conditions. Instead AChE appears to be involved in the normal growth of axons within the embryonic vertebrate brain. How does AChE mediate this eect? One possibility is that AChE is essential for axonogenesis, the initial formation of the axon. Antisense suppression of AChE led to a
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Fig. 1. Immunostaining of axon tracts in exposed Xenopus brain preparations. (A) Confocal image of a wholemount Xenopus brain cultured in unsupplemented media and immunostained for acetylated atubulin to reveal all growing axon tracts. The development of the early scaold of axon tracts was normal. The lines represent the point at which the TPOC was measured. (B, C, E) Embryos cultured in the presence of 100 mM, 500 mM and 1 mM of BW284C51, respectively. These brains demonstrated a dosedependent reduction in the thickness of the TPOC. (D, F) Rescue experiments. Embryos initially cultured in the presence of BW284C51 and then allowed to develop in control media demonstrated substantial recovery in the thickness of the TPOC. DVDT, dorsoventral diencephalic tract; POC, postoptic commissure; SOT, supraoptic tract; TPC, tract of the posterior commissure; TPOC, tract of the postoptic commissure; VC, ventral commissure; VLT, ventral longitudinal tract. Scale=100 mm (A±F).
reduction in neurite production by PC12 cells.12 The decrease in AChE was accompanied by a loss of neurexin Ia, the receptor for neuroligin which possesses an AChE-like extracellular domain. Since neuritogenesis was rescued by overexpression of neuroligin in these AChE depleted PC12 cells it was argued that AChE was modulating lateral cis membrane interactions between neurexin and neuroligin.12 The involvement of neurexins in the molecular interactions underlying AChE mediated axon growth was further supported by evidence that levels of neurexin 1b were altered in transgenic mice expressing human AChE.3 The cell autologous eects of AChE were also demonstrated when AChE was transfected into glioma cells and produced extension of processes.19 Moreover, Xenopus spinal neurons misexpressing human AChE extend longer neurites than control neurons when grown in dispersed cultures.31 These
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Fig. 2. The eect of BW284C51 on axon tract formation. A one-way ANOVA and a post hoc Scheetest revealed a dose-dependent reduction in the thickness of the TPOC (P < 0.000). Rescue experiments, in which embryos were initially cultured in the presence of BW284C51 and then allowed to develop in control media, returned to either control levels (500 mM+Rescue) or near control levels (1 mM+Rescue). Values are mean 2 SEM; $ value is signi®cantly dierent from control cultures; % value is signi®cantly dierent from rescue cultures.
results indicate that AChE can stimulate neurite outgrowth by lateral membrane interactions. It does not, however, negate possible heterologous cell interactions in vivo. Another possibility is that AChE functions as a cell adhesion molecule and modulates axon elongation. AChE is a member of the serine ester hydrolase family, which comprises several known cholinesterase-like cell adhesion molecules. For example, neurotactin is a Drosophila transmembrane glycoprotein that contains a catalytically inactive cholinesterase-like extracellular domain5,9 which mediates cell adhesion.5 When the extracellular cholinesterase-like domain of neurotactin was replaced with the core domain of AChE, the resulting chimeric protein was capable of promoting cell adhesion.8 These results suggest that in addition to its enzymatic activity, AChE may also function as an adhesion molecule. The ®nding that substrate-bound AChE enhances neurite outgrowth of chick sympathetic neurons further indicates an adhesive non-catalytic mode of action for AChE.29 AChE may also mediate axon growth through its HNK-1 carbohydrate moiety.7 In vivo and in vitro studies have demonstrated that antibodies directed against the HNK-1 epitope can block axonal growth,11,14,20 while neurite outgrowth has been shown to be enhanced when cultured on a substrate containing HNK-1.27 The extracellular matrix glycoprotein laminin binds HNK-1 at a site localised to domain 2 within the E8 fragment.27 When this binding site is blocked, neural cells have been shown to lose their ability to attach to laminin.27 This raises the possibility that interactions between AChE and various extracellular matrix molecules could promote axon growth. However, HNK-1 could only mediate AChE dependent axon growth in the present study, if BW284C51 was non-speci®cally blocking receptor-carbohydrate binding interactions. Although it is possible that neuronal death contributes to the reduced size of the TPOC in the presence of BW284C51, our control experiments suggest otherwise. We showed that a 5-h rescue period was sucient to reverse the deleterious eects of BW284C51 on the size of the TPOC. If BW284C51 was causing neuronal death then elevated levels of cell proliferation
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followed by neuronal dierentiation and axon outgrowth must be restoring the TPOC to its normal size during the rescue period. However, 5 h is insucient for proliferating neuroepithelial cells to reconstitute the TPOC since it takes at least 10 h for cells to dierentiate and initiate axon outgrowth after they have exited the cell cycle.17,22 In conclusion, we have shown for the ®rst time that AChE is involved in the development of axon tracts within the embryonic vertebrate brain in vivo. AChE may be mediating its eect by reducing axonogenesis and/or attenuating axon elongation. The exposed brain preparation used here will provide a convenient experimental model to now determine the molecular mechanisms of AChE mediated axon growth in vivo. AcknowledgementsÐThis work was supported by an Australian N.H. & M.R.C. grant to B.K. and by a University of Melbourne Faculty of Medicine scholarship to R.B.A.
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