1 Department of Biology, University of Konstanz, Fach M626, 78457 Konstanz, Germany
2 Program in Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center Box 290, 1275 York Avenue, New York, New York 10021, USA
*Author for correspondence (e-mail: martin.bastmeyer{at}uni-konstanz.de)
Accepted September 26, 2001
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SUMMARY |
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Key words: Carbohydrate, NCAM, Axon guidance, Commissural axon, Floorplate, Midline, Zebrafish, Danio rerio
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INTRODUCTION |
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PSA has the ability to weaken not only NCAM-mediated adhesion but also a variety of cell-cell interactions mediated by other molecules (Rutishauser and Landmesser, 1996). Its global effect on cell interactions includes an overall steric influence of PSA on membrane-membrane apposition (Yang et al., 1992). Additional mechanisms for PSA action have been proposed. For example, the ability of endo N to block induction of long-term potentiation and long-term depression in hippocampal slice cultures (Muller et al., 1996) has been attributed to an effect of PSA on the activity of brain-derived neurotrophic factor (Muller et al., 2000). There also is evidence from in vitro experiments that the ability of NCAM to bind to a matrix containing heparan sulphate proteoglycans is augmented by PSA (Storms and Rutishauser, 1998). In both of these cases, it is suggested that PSA has a specific binding affinity that can be competitively inhibited by PSA that is not attached to NCAM (that is, soluble and isolated PSA chains as found in colominic acid). This ability of soluble PSA to mimic the action of endo N, which does not occur with the steric effect of PSA on cell-cell interactions (Storms and Rutishauser, 1998; Muller et al., 2000), is therefore characteristic of a positive binding mode of PSA action.
The polysialylation of NCAM occurs in all vertebrates but appears to be absent in invertebrates (Rutishauser, 1998). However, nearly all studies of PSA function have been carried out in higher vertebrates, such as mammals and birds (Kiss and Rougon, 1997). We have chosen the zebrafish embryo for investigation of the function of PSA during development of the nervous system of lower vertebrates. In the past decade, embryonic zebrafish have become a popular model for studying the cellular and molecular bases of nervous system development (Eisen, 1996) and axonal pathfinding (Bernhardt, 1999). The anatomy of the embryonic nervous system has been described in detail (Chitnis and Kuwada, 1990; Wilson et al., 1990; Mendelson, 1986; Metcalfe et al., 1986) and the accessibility of the embryos allows for functional in vivo experiments (Weiland et al., 1997; Lauderdale et al., 1997; Bernhardt and Schachner, 2000; Ott et al., 2001).
In the present study, we have used PSA-specific antibodies and whole-mount immunocytochemistry to describe the spatiotemporal expression pattern of PSA during zebrafish CNS development. Injections of endo N into the developing CNS caused a disordered growth pattern of commissural axons in the hindbrain and the posterior commissure (pc). Whereas the disordered growth pattern of hindbrain commissures produced by endo N could be induced by injections of soluble PSA, the growth of axons in the pc was unaffected by such treatment. The results suggest that there are distinct mechanisms for PSA action during axon growth and pathfinding in the developing zebrafish CNS.
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MATERIALS AND METHODS |
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Purification of brain membranes
A membrane-enriched fraction of normal adult goldfish brain homogenate was obtained as described (Vielmetter et al., 1991). In brief, the brains were homogenised in homogenisation buffer (pH 7.4, 10 mM Tris-HCl, 1.5 mM CaCl2, 15 µg/ml 2,3-dehydro-2-desoxy-N-acetylneuraminic acid) containing the protease inhibitors spermidin (1 mM), aprotinin (25 µg/ml), leupeptin (25 µg/ml) and pepstatin (5 µg/ml). Cell surface membranes were enriched in the interband of a sucrose step gradient (upper phase 20%, lower phase 50% sucrose) by centrifugation (6x104 g, 10 minutes, 4°C). The membrane fragments were solubilised in buffer with the detergent octylglucoside (OG lysis buffer: 100 mM octylglucosid, 20 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml phenylmethylsulfonyl fluoride, pH 7.4) by sonication. The supernatant obtained after centrifugation (105 g, 1 hour, 4°C) was called OG-brain extract. OG-brain extract from zebrafish was obtained in the same way as described above.
Purification of PSA glycoproteins
Immunoaffinity absorption of the glycoproteins was performed using a column of CNBr-Sepharose (Pharmacia) to which the monoclonal anti PSA antibody 735 had been covalently coupled (kindly provided by Rita Gerardy-Schahn). Goldfish OG-brain extract was passed over the affinity column (12 hours at 4°C under rotation). The affinity column containing the bound antigen was washed with OG lysis buffer followed by OG lysis buffer containing 0.5M NaCl, and then with pre-elution buffer (20 mM Tris-HCl, 150 mM NaCl, 100 mM octylglucosid, pH 8). The antigen was eluted with elution buffer (100 mM triethylamine, 100 mM octylglucosid, pH 11.5) into 2 M Tris-HCl, pH 6 (50 µl per 250 µl eluate) to neutralise the eluate.
The eluted antigen was subjected to SDS 8% PAGE under nonreducing conditions. For western blot analysis, the antigen was transferred to a nitrocellulose membrane (Hybond-C extra, Amersham), blocked with 3% fat-free milk powder in phosphate-buffered saline (PBS) and 0.05% Tween (1 hour, 37°C), incubated (overnight, 4°C) with monoclonal antibodies against PSA (mAb 735) or NCAM (mAb D3 (Schlosshauer, 1989)). After three washes (5 minutes each) with PBS containing 0.05% Tween, antibody binding was detected by horseradish peroxidase-coupled goat anti-mouse antibodies (Dianova, diluted 1:10000 in 3% milk powder, 0.05% Tween in PBS), and developed in staining solution (Super Signal, Pierce). For Silver stain analysis, the eluted PSA glycoproteins were digested with endo N (1 µg/ml eluate, 2 hours at 37°C).
Production of polyclonal antibodies against PSA glycoproteins
Polyclonal antibodies were produced by injecting rabbits subcutanously with approximately 3 µg of purified PSA glycoprotein three times at intervals of three weeks. The antigen was emulsified in adjuvant (MPL+TDM+CWS Adjuvant system, Sigma) according to the manufacturers protocol. Serum was collected on the eighth day after the third injection and IgG antibodies were purified with a protein A sepharose column (Pharmacia).
Antibody staining
Zebrafish embryos were processed for whole-mount immunohistochemistry as previously described (Weiland et al., 1997). Briefly, embryos were fixed for 4 hours at 4°C in 4% paraformaldehyde in fixation buffer (Westerfield, 1994) and permeabilised by exposure to acetone (20°C for 2-5 minutes). After incubation in blocking buffer (PBS with 1% bovine serum albumin (BSA), 1% DMSO, 2% goat serum, 2% donkey serum) for 1 hour at 37°C, they were exposed to primary antibodies overnight at 4°C.
In this study, three monoclonal antibodies against PSA were used: mAb 735 (an IgG) (Frosch et al, 1985), mAb 5A5 (an IgM) (Dodd et al, 1988) and mAb 12E3 (an IgM) (Seki and Arai, 1991). The specificity of the antibodies was tested on whole-mount staining with Endo N-injected embryos. In contrast to control embryos, which show a distinct staining pattern, there was no staining in embryos treated with endo N, indicating that the antibodies are specific for PSA and do not crossreact with other epitopes in fish material.
To visualise zebrafish axons, a monoclonal antibody against acetylated -tubulin (25 µg/ml, clone 6-11B-1, Sigma) and a polyclonal serum against zebrafish Tag-1 (Lang et al., 2001) were applied. Embryos were rinsed in wash buffer (PBS with 1% BSA, 1% DMSO) and incubated with either Alexa-488-coupled goat anti-rabbit (2 µg/ml; Molecular Probes), cyanin-3-coupled donkey anti-mouse IgG (H+L) (2 µg/ml; Dianova), or cyanin-3-coupled goat anti-mouse IgM, µ-chain specific (2 µg/ml, Dianova) for 1 hour at 37°C. Yolk sacs were removed and embryos were embedded between two coverslips in Mowiol containing n-propylgallate as an antifading agent.
DiI labeling
Embryos were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer overnight and embedded in 1% low melting point agarose (Sigma) in PBS. Excess agarose covering the hindbrain was removed. The tip of a pulled glass micropipette was dipped into a 1% DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, Molecular Probes) solution in ethanol and inserted into the dorsolateral aspect of the hindbrain, rostral to the otocyst. Embryos were stored in a wet chamber overnight at 4°C to allow diffusion of DiI along commissural axons. For analysis, embryos were re-embedded in 0.3% agarose (Gibco) in PBS and analysed with a confocal microscope.
Enzyme injections
Dechorionated embryos were oriented in a specialised sylgard matrix (Ott et al., 2001) on a heated stage at 28.5°C, allowing for the efficient injection of 6x30 animals. Pulled 5 µl glass micro pipettes were broken under optic control to short tips of 8 µm diameter and filled with solution using negative pressure. The pipette was manually inserted into the ventricle of the embryos and approximately 10 nl of solution was pressure injected using a microinjector (Eppendorf Transjector 5246). We injected either endo N (60 µg/ml, kindly provided by Rita Gerardy-Schahn), soluble PSA (colominic acid, 100 mg/ml in PBS, ICN Biomedicals), PSA-Trimer (N-Acetylneuraminic acid, Trimer, 100 mg/ml in PBS, Calbiochem) or control buffer (PBS). Single injections were performed at 20 hpf; embryos were fixed for immunocytochemistry at 32 hpf and for DiI labeling at 36 hpf.
Analysis of stained embryos
Stained embryos were analysed using a confocal microscope (LSM 510, Zeiss) equipped with a high aperture lens (C-Apochromat 40x/1.2W, Zeiss) and the appropriate lasers. Serial optical sections were flattened into projections. All images were further processed with Adobe Photoshop 5.02 software.
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RESULTS |
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Removal of PSA by endo N affects the growth pattern of the posterior commissure
To study the function of PSA during zebrafish CNS development, endo N was injected into the ventricle of 20 hpf embryos. Control embryos received an injection of buffer only. Immunocytochemistry revealed that a single injection of endo N was sufficient to remove PSA for at least 24 hours (Fig. 3A,B). After endo N injection at 20 hpf, the embryos appeared to develop normally and the overall appearance of the major axon tracts was not altered (Fig. 3C). The temporal development of the embryo is not slowed by PSA removal. The lateral line nerve reaches the same caudal spinal segments as in controls and the outgrowth pattern of secondary motoneurones, as determined by the most caudal somite with visible secondary motor axons (Ott et al., 2001), is also not delayed in the endo N-treated fish.
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Removal of PSA affects midline crossing by commissural axons in the hindbrain
The second axonal projections affected by endo N are PSA-negative hindbrain commissures. Hindbrain commissural axons originate from reticulospinal neurones (Mendelson, 1986; Metcalfe et al., 1986), follow a characteristically curved pathway and project into the contralateral medial longitudinal fascicle (mlf). In the zebrafish hindbrain, the first commissural axons cross the midline between 18-20 hpf (Weiland et al., 1997), before PSA is upregulated on floorplate cells. From 22 hpf onwards, at which time PSA is upregulated on floorplate cells, other commissural axons become visible, following a straight path across the midline (Weiland et al., 1997). These belong either to a group of axons that project rostrally via the mlf into the contralateral midbrain (Trevarrov et al., 1990) or are from commissural interneurones that project into the contralateral hindbrain segment. With further development, more commissural axons are added and form several bundles in each hindbrain segment. These bundles are negative for PSA (Fig. 5A,C) but can be visualised with anti-Tag-1 antibodies or with mAb 6-11B-1 against acetylated -tubulin (Fig. 5B,D). They cross the mlf and upon contact with the PSA-positive floorplate cells (Fig. 5C), defasciculate into single axons that cross the midline (Fig. 5D, arrowheads). This regular growth pattern was observed in all of the buffer-injected control embryos (n=56), but was markedly disturbed after enzymatic PSA removal in about 50% of endo N-injected embryos (n=60). That is, bundles of axons still grew ventrally and crossed the mlf, but fewer axons crossed the midline (Fig. 5E) and appeared to stop at the PSA-negative floorplate cells (Fig. 5F, arrowheads).
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DISCUSSION |
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The expression of PSA in the developing CNS of mouse or chick is dominated by the presence of high levels of heavily polysialylated NCAM on axonal processes in nearly every tract (Chuong and Edelman, 1984). As a result, immunohistochemistry for PSA in the brain of these species results in an almost uninterpretable mass of staining throughout much of the tissue. The selective expression of PSA on a small set of distinct tracts in the zebrafish was therefore a surprise, and indicates that the role of PSA in fish is probably different in scope and perhaps nature, when compared with avian and mammalian vertebrates. A more limited expression of PSA on zebrafish motoneurones, namely an absence on primary axons but heavy labelling of secondary axons (this study; U. R. and J. Eisen, unpublished), also supports such a phylogenetic difference in PSA use.
In contrast to axon tracts, CNS cell bodies in the zebrafish display PSA quite broadly. PSA expression on neural precursors has been widely documented (Miragall, 1988; Murakami et al., 1991) and is, in some cases, related to the ability of these cells to migrate from their site of origin to distinct parts of the brain (Ono et al., 1994; Wang et al., 1994; Hu et al., 1996). Although we did not investigate cell migration in this study, it is possible that PSA function on neural precursors is more similar in vertebrates than its effect on the subsequent elaboration of axonal processes.
In all previous studies on axonal tracts of other vertebrates, PSA was found to influence either the fasciculation pattern of axon bundles (Tang et al., 1992; Yin et al., 1995; Honig and Rutishauser, 1996) or the formation of collateral branches (Daston et al., 1996), but had little or no effect on axonal elongation. Although the contexts differed widely, in each case PSA could be viewed as a steric negative regulator of membrane-membrane apposition, which serves to attenuate a variety of cell-cell interactions (Fig. 8A) (Rutishauser, 1998). This mechanism requires that the PSA be membrane bound and thus is eliminated by exposure of the cells to endo N but is unaffected by injection of soluble PSA. In the case of axons, the reduced interaction produced by expression of cell-surface PSA behaves as a permissive factor that promotes either the dispersal of a fascicle by loosening axon-axon adhesions (Tang et al., 1994) (Fig. 8B), or conversely the growth of axons along other axons by shielding the axon from even stronger adhesive influences such as those that lead to synapse formation (Seki and Rutishauser, 1998) (Fig. 8B). Which of these two behaviours occurs depends primarily on two variables: the presence of PSA on the axons and/or the environment, and the nature and susceptibility to PSA of cell-cell interactions offered to the axons by that environment.
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By contrast, the inability of specific hindbrain commissural axons to cross the midline is a new phenomenon for endo N-treated embryos and is clearly distinct from the behaviour induced in the posterior commissure. Axon guidance at the midline choice point is a complex process that requires a variety of molecules (Stoeckli, 1998; Kaprielian et al., 2001). Midline floorplate cells do express both attractive and repulsive components (Stoeckli et al., 1997) that interact with axonal receptors to regulate the growth pattern of ipsi- and contratateral projections. In the zebrafish hindbrain, commissural axons of interneurones are affected upon PSA removal, whereas reticulospinal axons, which cross the midline before PSA is upregulated, are not affected. Whether commissural axons in the zebrafish spinal cord were also affected after PSA removal could not be analysed in this study because of technical difficulties. Hindbrain commissural axons, like most tracts in the developing zebrafish, do not express PSA. Floorplate cells, however, do express PSA and thus are the likely source of the perturbation. In the mouse spinal cord, both commissural axons and floorplate cells express PSA (Boisseau et al., 1991). Whether the removal of PSA also affects commissural axon growth in chick and mice has not been reported.
Remarkably, injection of endo N induced an abrupt cessation of axon elongation at the midline that was anatomically indistinguishable from that produced by soluble PSA. As indicated in the Introduction, such an effect is indicative of a very different role for PSA, namely that of promoting rather than inhibiting interaction between cells. Fig. 8 illustrates two non-steric modes by which PSA could in principle act during midline crossing of commissural axons in the hindbrain. In the first mode (Fig. 8C), PSA binds a secreted component necessary for midline crossing, thus keeping it at a high concentration at the surface of floorplate cells. Removal of PSA would therefore reduce the local concentration of this compound and hinder crossing. In the second mode (Fig. 8C), PSA combines with a receptor on floorplate cells in order to bind to and trigger an second axon-associated receptor that permits midline crossing by that axon.
There is some precedent for each of these binding-based mechanisms. For example, binding of brain-derived neurotrophic factor (BDNF) has been proposed to be a mechanism by which PSA can influence long-term potentiation in the hippocampus (Muller et al., 2000). However, PSA appears to be a second binding component in the interaction of NCAM-expressing cells with heparan sulphate proteoglycans (Storms and Rutishauser, 1998). Although the available data are not sufficient to choose between these two possibilities, manipulation of these new potential binding partners should be useful in future studies.
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ACKNOWLEDGMENTS |
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