Chondroitin sulfate disrupts axon pathfinding in ... - Wiley Online Library

Chondroitin Sulfate Disrupts Axon Pathfinding in the Optic Tract and Alters Growth Cone Dynamics Andreas Walz,1,2 Richard B. Anderson,3 Atsushi Irie,3,* Chi-Bin Chien,1,4 Christine E. Holt1,3 1

University of California San Diego, Department of Biology, La Jolla, California 92093

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The Rockefeller University, 1230 York Avenue, New York, New York 10021

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University of Cambridge, Department of Anatomy Downing Street, Cambridge CB2 3DY

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University of Utah Medical Center, Department of Neurobiology and Anatomy, 50 North Medical Drive, Salt Lake City, Utah 84132

Received 21 February 2002; accepted 2 July 2002

ABSTRACT:

Little is known about the cues that guide retinal axons across the diencephalon en route to their midbrain target, the optic tectum. Here we show that chondroitin sulfate proteoglycans are differentially expressed within the diencephalon at a time when retinal axons are growing within the optic tract. Using exposed brain preparations, we show that the addition of exogenous chondroitin sulfate results in retinal pathfinding errors. Retinal axons disperse widely from their normal trajectory within the optic tract and extend aberrantly

INTRODUCTION To reach the optic tectum, retinal ganglion cell (RGC) axons must correctly exit the eye, cross the dience*Present address: The Tokyo Metropolitan Institute of Medical Science, Department of Biochemical Cell Research, Tokyo 1138613, Japan Correspondence to: C. E. Holt ([email protected]) Contract grant sponsor: Pew Scholars Award Contract grant sponsor: NIH; contract grant number: NS23780 Contract grant sponsor: MRC Programme Grant (to C.E.H) Contract grant sponsor: American Cancer Society Fellowship (to C-B.C.) Contract grant sponsor: NH&MRC C.J. Martin/R.G.Menzies Fellowship (to R.B.A) Contract grant sponsor: the Tokyo Metropolitan Institute of Medical Science (to A.I) This article includes Supplementary Material available via the Internet at http://journals.wiley.com/neu © 2002 Wiley Periodicals, Inc. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/neu.10113

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into inappropriate regions of the forebrain. Time-lapse analysis of retinal growth cone dynamics in vivo shows that addition of exogenous chondroitin sulfate causes intermittent stalling and increases growth cone complexity. These results suggest that chondroitin sulfate may modulate the guidance of retinal axons as they grow through the diencephalon towards the optic tectum. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 330 –342, 2002

Keywords: glycosaminoglycans; proteoglycans; axon guidance; neurite extension; retinotectal pathway

phalic midline, and grow through the contralateral diencephalon. Numerous molecules have been shown to guide retinal axons at these various choice points throughout their trajectory. For example, chondroitin sulfate proteoglycans (CSPGs) have been implicated in the intraretinal growth of axons towards the central retina (Snow et al., 1991; Brittis et al., 1992; Snow and Letourneau, 1992; Brittis and Silver, 1995); netrin-1 and the EphBs are involved in retinal axons exiting the eye (de la Torre et al., 1997; Deiner et al., 1997; Hopker et al., 1999; Birgbauer et al., 2000); ephrin-As, ephrin-Bs, netrin-1, Slits and CSPGs are involved in guiding retinal axons at the optic chiasm (Deiner and Sretavan, 1999; Dutting et al., 1999; Chung et al., 2000a, 2000b; Erskine et al., 2000; Marcus et al., 2000; Nakagawa et al., 2000; Niclou et al., 2000; Hutson and Chien, 2002; Plump et al., 2002); and fibroblast growth factor 2 (FGF2) and heparan sulfate (HS) proteoglycan sidechains are in-

CS and Retinal Pathfinding

volved in target recognition and innervation of the optic tectum (McFarlane et al., 1995, 1996; Walz et al., 1997; Irie et al., 2002). However, little is known about the cues that guide retinal axons within the optic tract as they grow through the diencephalon. One class of candidate molecules that has been implicated in this process are the chondroitin sulfate proteoglycans (CSPGs). CSPGs are typically thought of as inhibitory molecules that form barriers to axon growth. In vitro studies have demonstrated that CSPGs can inhibit neurite growth, even on otherwise permissive substrates (Snow et al., 1990a; Freidlander et al., 1994; Yamada et al., 1997; Hynds and Snow, 1999; Niederost et al., 1999; Wilson and Snow, 2000; Ichijo and Kawabata, 2001; Becker and Becker, 2002). In vivo, CSPGs are often localized to sites avoided by growing axons. For instance, CSPGs are localized within the posterior sclerotome, which is repulsive to sensory axons (Keynes and Stern, 1984; Snow et al., 1990b; Oakley and Tosney, 1991; Perris et al., 1991) as well as in the cortical plate at a time when thalamic afferents accumulate in the underlying subplate region (Emerling and Lander, 1996). Thalamic afferents only enter the cortical plate as levels of CSPGs fall in the deeper cortical layers (Emerling and Lander, 1994). However, not all axons are inhibited by CSPGs. Low levels of a brain-derived chondroitin sulfate (CS) have been shown to promote rather than inhibit neurite outgrowth from neocortical neurons (Iijima et al., 1991). During development of thalamocortical projections, thalamic axons initially grow through the subplate, a region rich in CSPGs. In vitro assays revealed that the CSs present in the subplate are stimulatory for both neuronal adhesion and neurite outgrowth (Emerling and Lander, 1996). This dichotomy in the action of CSs has been shown to depend on the mode by which they are presented (Snow and Letourneau, 1992; Challacombe and Elam, 1997; Hynds and Snow, 1999); the composition of their side chains (Faissner et al., 1994; Braunewell et al., 1995; Clement et al., 1998; Nadanaka et al., 1998) and on the neuronal cell type (Snow and Letourneau, 1992; Feraud-Espinosa et al., 1994; Dou and Levine, 1995). In the diencephalon, CSPGs have been shown to be expressed within the optic tract (McAdams and McLoon, 1995; Chung et al., 2000a), raising the possibility that they may be involved in guiding retinal axons towards the optic tectum. Enzymatic removal of endogenous CSs resulted in retinal pathfinding errors within the forebrain (Ichijo and Kawabata, 2001), further supporting the idea that CSPGs may play a role in guiding retinal axons within the diencephalon. In the present study, we examined the role of CSs

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in the guidance of retinal axons as they grow through the diencephalon towards their target, the optic tectum. We show that CSPGs are differentially expressed within the diencephalon and that retinal axons extend through a CS-rich environment. Using exposed brain preparations, we have found that exogenously applied CS causes gross retinal pathfinding errors within the optic tract. In vivo time-lapse analysis revealed that retinal pathfinding errors are accompanied by a more complex growth cone morphology and saltatory growth dynamics. Thus, these results suggest that CSs may modulate the guidance of retinal axons as they grow through the diencephalon.

METHODS Embryos Xenopus laevis embryos were obtained from hormone-induced mating of adult frogs. Embryos were raised in 10% Holtfreter’s solution; the temperature was varied between 14 and 27°C to control their speed of development. Staging was done according to the criteria of Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Embryos were anesthetized with tricaine (ethyl-3-aminobenzoate methanesulfonic acid, Aldrich) before experimental manipulations.

Histochemistry Fixed embryos were embedded in O.C.T. (Miles) and cryostat sectioned at 12 ␮M. Slides were prepared for labeling with 3-B-3, 2-B-6, and 1-B-5 primary antibodies by digestion with 0.01 units/mL of chondroitinase ABC (Sigma) for 45 min. For single labeling, sections were blocked in PBT (phosphatebuffered saline and 0.5% Triton X-100) containing 5% normal goat serum. Primary antibodies were incubated overnight at 4°C and then visualized using a fluorescein-coupled secondary antibody (Jackson ImmunoResearch Laboratories). To visualize RGC axons in double-labeling experiments, sections were incubated with the anti-HRP antibody for 1 h at room temperature followed by staining with a rhodamine-coupled secondary antibody (Jackson ImmunoResearch Laboratories) prior to incubation with the CS primary antibody. Slides were coverslipped in glycerol with an antibleaching agent (0.1% paraphenylenediamine) and viewed under either a Nikon Optiphot-2 microscope attached to a cooled CCD camera (Spectrasource) or a Noran Odyssey confocal scanning laser microscope. Color confocal images were assembled using Photoshop (Adobe Inc.). Primary antibodies used in this study include: CS-56, which recognizes both chondroitin-4-sulfate and chondroitin-6-sulfate at a 1:100 dilution (Sigma); 3-B-3, 2-B-6, and 1-B-5, which recognize chondroitin-6-sulfate, chondroitin-4-sulfate, and chondroitin, respectively (ICN), at 1:50; an anti-HRP polyclonal antibody (Sigma) at 1:500; R5, which labels radial glia cells (generously provided by

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Dr. U. Drager; Drager et al.,1984) at 1:1; 6F11, which recognizes the extracellular domain of NCAM at 1:10 (Sakaguchi et al.,1989); 6-11B-1, which recognizes acetylated tubulin at 1:200; and an anti-BrdU monoclonal antibody (Sigma) at 1:1000. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories. For BrdU labeling, 5 mg/mL BrdU (Sigma) was injected into the abdomen of stage 40 embryos that had previously been exposed (between stages 33/34 and 40) to CSC. Embryos were left for 3 h at 25°C and then fixed in 4% paraformaldehyde; 0.5% Hoechst nuclear stain (Molecular Probes) was applied to cryostat sections of exposed embryos for 10 min at 1:1000 dilution. For viability studies, 1% solution of Trypan blue (Sigma) was added to CStreated embryos for 1 h before fixation in 4% paraformaldehyde. Dead cells were identified by their positive staining with Trypan blue.

Exposed Brain Preparations and HRP Labeling of Optic Fibers Exposed brain experiments were performed as previously described (Chien et al., 1993; Anderson et al., 1998). All surgeries were carried out in 100% modified Ringers (MMR): 100 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 0.4 mg/mL tricaine, 1% Fungibact, 50 ␮g/mL gentamicin sulfate (Gemini Products), 10 ␮g/mL phenol red (pH 7.4). Briefly, St 33/34 embryos were immobilized by pinning in a Sylgard petri dish and the eye and epidermis overlying the diencephalon and tectum were removed from the left side of the head to expose the underlying brain. However, in some cases, the epidermis covering the ventral one-third of the diencephalon remained intact or grew back rapidly after surgery. Embryos were transferred to either experimental or control solutions and allowed to develop until stage 40. To make experimental solutions, CSA from bovine trachea, CSB from bovine mucosa, and CSC from shark cartilage were used at 1–15 mg/mL (Sigma); KS from bovine cornea at 10 –15 mg/mL and Heparin from bovine lung at 0.1–1 mg/mL (Sigma). All experiments were performed using CSC unless otherwise stated. Chondroitinase ABC (Seikagaku) was added to control solutions at 5 U/mL. Heat-inactivated CSC was prepared by boiling for 30 min, and fragmentation of CSC was performed using 1 U/mL of Chondroitinase ABC in 0.1 M Tris-HCl (pH 8.0) and 0.03 M sodium acetate at 37°C for 2 h. The reaction was stopped by boiling the sample for 5 min. Retinal axons were visualized by anterograde transport of horseradish peroxidase (HRP; Sigma) as described previously (Cornel and Holt, 1992). Briefly, the lens of the right eye was surgically removed and a plug of HRP in 1% L-␣-lysolecithin solution was placed in the eye cavity. After 25 min the embryos were then fixed in 4% paraformaldehyde. Embryos were either prepared for cryostat sectioning, or their brains were dissected and reacted with diaminobenzidine (DAB) for wholemount preparations. DAB-reacted

brains were dehydrated, cleared with 2:1 benzyl-benzoate/ benzyl alcohol, and mounted in Coverbond (BSP).

Quantitative Analysis of Optic Tract To quantify the effect of CS on tract formation, width measurements were taken along the entire length of the optic tract. Camera lucida drawings were made of HRPfilled axons in whole-mount brain preparations. Samples were included only if they had densely filled tracts and were mounted in a lateral orientation. Drawings were digitized with a flatbed scanner (ScanJet IIc, Hewlett-Packard) and macros written in NIH Image were used to normalize the brains by rotating and scaling them according to a line drawn from the optic chiasm to the posterior pole of the tectum (two easily recognizable landmarks). This line was then aligned to a standard reference line. The reference line was used to define an artificial unit length, with one CTU (chiasm–tectum unit) corresponding to ⬃620 ␮m in an unfixed brain (Chien et al., 1993). Concentric reference circles were placed at 0.1 CTU intervals centered on the optic chiasm [0.1, 0.2. 0.3 . . .CTU; see insert in Fig. 4(A)]. Tract widths were measured as the distance between the two outermost axons intersecting each circle.

In Vivo Time-Lapse and Growth-Cone Analysis In vivo time-lapse experiments were conducted as previously described (Chien et al., 1993). Briefly, right eyes were labeled at stage 32 with a small piece of DiI (Molecular Probes) and left to recover for 12 h at 17°C. At stage 35/36, the left side of the head was exposed and embryos displaying only a few brightly labeled retinal axons were decapitated and mounted in a perfusion chamber (Chien et al., 1993). Labeled growth cones were observed with a Nikon 40⫻ objective, using neutral density filters to reduce the illumination. Images were captured at 2.5-min intervals with a silicon-intensified target camera (DAGE-MTI) and digitized with a Scion LG-3 frame grabber and NIH Image software. To visualize retinal growth cones, DiI crystals were placed between the lens and the retina of stage 35/36 embryos, which were then exposed to either CSC containing bathing solution or control solution for 2 h at 27°C and then fixed in 4% paraformaldehyde. Brains were dissected and mounted in phosphate buffer for viewing under a Nikon Diaphot 200 inverted microscope attached to a Noran Odyssey confocal scanning laser microscope. Growth cones were captured using a 60⫻ objective. Growth cones were only counted if they were clearly separate from other axons within the optic tract. Mean filopodial numbers and lengths were compared with those of controls using a Student’s t test.


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Figure 1 CS expression in the Xenopus embryonic optic pathway. Confocal micrographs of transverse sections of stage 37/38 embryos immunostained for endogenous CSs. (A) CS immunostaining, as visualized by the CS-56 antibody, is localized in the basal lamina surrounding the diencephalon and to a lesser extent in the neuropil (Np) and the neuroepithelium (Ne). There is also abundant CS expression in the mesenchyme outside the nervous system. Arrowheads represent the area enlarged in panel E. (B–D) Specific expression of the CS subtypes. (B) Chondroitin is expressed at higher levels in the neuropil than in the neuroepithelium. (C) Chondroitin-4-sulfate is expressed in both the neuropil and neuroepithelium. (D) Chondroitin-6-sulfate is expressed primarily in the neuroepithelium and at a lower degree in the neuropil. (E) A double-labeled section showing HRP-filled RGC axons (red) growing in the CS-rich region (green) of the diencephalic neuropil. Dorsal is up. Scale bar is 50 ␮m in (A)–(D) and 25 ␮m in (E).

RESULTS CSs Are Differentially Expressed in the Developing Optic Pathway In the developing visual system of Xenopus laevis, RGC axons enter the base of the contralateral diencephalon at stage 33/34, grow dorsally to form the optic tract through stages 33/34 and 35/36 and begin to enter the optic tectum at stage 37/38 (Holt, 1984,

1989). To correlate the expression of CSs with the advancing RGC axons in the developing optic tract, sections of Xenopus embryonic brains were immunostained with the CS-56 monoclonal antibody (Avnur and Geiger, 1985). In the diencephalon, strong CS-56 immunostaining was localized to the pial surface and to a lesser degree on neuroepithelial cells and in the neuropil [Fig. 1(A)]. Because CS sugar chains consist of tandem repeats of glucuronic acid (GlcA) and

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Figure 2 The addition of exogenous CS disrupts retinal axon pathfinding. Lateral (A, B, D, E, and F) and dorsal (C) views of stage 40 whole-mount Xenopus brains showing the trajectories of HRP-filled RGC axons. Brains were exposed to different glycosaminoglycans during stage 33/34 to 40. (A) Control projection forms a defined optic tract (ot) in the diencephalon (Di) and correctly enters the tectum (Tec). Black dotted curve shows the approximate border of the tectum. (B) Projections exposed to CS (10 mg/mL) disperse widely from their normal trajectory and extend aberrantly in the telencephalon (Tel), diencephalon, and tectum. (C) CS treated retinal axons grew across the dorsal midline and into the contralateral tectum. White dotted line shows the dorsal midline. (D) Disruption in retinal axon pathfinding caused by CS exposure. RGC axons show aberrant growth in both the dorsal and ventral forebrain. (E) RGC axons exposed to heparin (100 ␮g/mL) extend correctly through the diencephalon but veer dorsally at the tectal border and bypass the tectum. (F) Brains exposed to keratan sulfate (10 mg/mL) have normal optic projections that enter the tectum. Pi, pineal; dorsal is up, anterior to the left. Scale bar is 100 ␮m in (A)–(F).

N-acetyl-galactosamine (GalNAc), and are heterogeneously modified by sulfation at C4 or C6 of GalNAc residues (Prydz and Dalen, 2000), we then examined the expression patterns of chondroitin (nonsulfated CS), 4-sulfated and 6-sulfated CSs using subtypespecific monoclonal antibodies. Strong expression of chondroitin-6-sulfate (C-6-S) was detected in the neuroepithelium and to a lesser extent in the neuropil [Fig. 1(D)], mirroring the expression of chondroitin, which was greater in the neuropil than in the neuroepithelium [Fig. 1(B)]. Chondroitin-4-sulfate (C-4-S) was present in both the neuropil and neuroepithelium [Fig. 1(C)]. All CSs are expressed as early as stage 24, peak at stage 35/36, and remain strong up to stage 42 (data not shown). To visualize CS expression in relation to the RGC projection, sections were doublelabeled with antibodies against CS-56 and horseradish peroxidase (HRP) following anterograde transport of HRP from the retina. This analysis revealed an overlapping expression pattern of CS and retinal axons

within the optic tract [Fig. 1(E)]. Thus, RGC axons extend through a region expressing chondroitin, chondroitin-4-sulfate, and chondroitin-6-sulfate.

Addition of Exogenous CS Disrupts Pathfinding in the Diencephalon To determine whether CSs play a role in guiding RGC axons to the tectum, CS sugar chains were applied to exposed Xenopus brains during the period when they first navigate to and innervate the tectum. CSC (10 mg/mL) was added to the bathing medium at stage 33/34 and the optic tract was visualized with HRP histochemistry at stage 40. Instead of forming the tightly defined optic tract characteristic of normal projections [Fig. 2(A)], retinal axons in CS treated brains were widely dispersed and invaded inappropriate regions of the diencephalon and telencephalon [Fig. 2(B) and (D)]. Some retinal axons that entered the tectum were observed crossing the dorsal midline


Figure 3 CS-induced pathfinding errors result in a wider optic tract. The widths of control and CS-treated optic projections were quantified on normalized scans of camera lucida drawings made from wholemount brains. (A) Widths measured at 0.1 CTU intervals (for definition of CTU refer to Materials and Methods) show that CS treatment resulted in a significantly wider optic tract along its entire length (p ⬍ .001 at each point). (B) Dose–response curve of CStreated optic tracts. Tract widths were measured at mid-tract (0.4 CTU) and show that concentrations greater than 5 mg/mL resulted in the formation of a wider optic tract.

into the contralateral tectum [Fig. 2(C)]. All three types of CS tested (CSA, CSB, and CSC) produced similar disruptions in axonal pathfinding (data not shown). CS-induced pathfinding errors were reflected by significantly wider optic tracts compared to controls, when measured at different points along the length of the projection [Fig. 3(A)]. CS (5 mg/mL) did not disorganize the tract significantly, while 10 –15 mg/mL caused severe disruptions in retinal axon pathfinding [Fig. 3(B)]. The need for high concentrations of exogenous CS to induce pathfinding errors may arise from the inability of large and highly negatively charged CS molecules to penetrate the neuroepithelium effectively. When fluorescently labeled CS was applied to exposed brains, only a small fraction of the labeled CS was seen to penetrate the exposed part of

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the brain (data not shown), indicating that the actual concentration of exogenous CS reaching the retinal growth cones was significantly lower. To determine whether the misrouting phenotype was specific to CS, other glycosaminoglycans were tested for their ability to induce comparable pathfinding errors. We have previously shown that exogenous application of heparin causes retinal axons to bypass their final target, the optic tectum (Walz et al., 1997; Irie et al., 2002). The addition of exogenous heparin resulted in retinal axons extending dorsally through the diencephalon and failing to enter the tectum [Fig. 2(E)]. In contrast, CS-treated retinal axons were more widely dispersed throughout the optic tract, with some retinal axons entering the tectum [Fig. 2(B)–(D)]. Thus, the addition of exogenous heparin generated a phenotype markedly different from that of CS. Another glycosaminoglycan, keratan sulfate, was also tested and found to have no effect on retinal axon guidance or target recognition [Fig. 2(F)] when compared to control embryos [Fig. 2(A)]. Heat-treated CS was just as effective as untreated CS in generating pathfinding errors, thus ruling out the possibility that heat sensitive contaminants were responsible for the phenotype (data not shown). Furthermore, CS treated with Chondroitinase ABC did not cause pathfinding errors (data not shown). Taken together, these results show that the addition of exogenous CS causes severe retinal pathfinding errors within the optic tract, generating a phenotype that is specific to CS sugar chains and not a nonspecific response of retinal axons to the presence of exogenous glycosaminoglycans.

Organization of Neuroepithelium Is Unaltered by CS Treatment The application of exogenous CS to the exposed diencephalon could indirectly cause pathfinding errors by perturbing the neuroepithelial substrate through which retinal axons grow. To examine whether CS treatment affects the neuroepithelium, the number of dividing cells within the telencephalon and diencephalon were examined using BrdU. No difference was detected between control and CS-treated embryos either in the location of dividing cells [Fig. 4(A) and (B)] or in the number of proliferating cells (24.5 ⫾ 2.2 per 10 ␮m cross-section in CS-treated embryos compared to 26.1 ⫾ 2.4 in controls; p ⬎ .1, Student’s t test, n ⫽ 26 sections each). Furthermore, cell viability (as analyzed by trypan blue uptake and Hoechst nuclear stain) was not impaired by CS treatment. No changes were observed in the expression of markers of neuronal or glial differentiation (anti-NCAM and anti-R5; data not shown); however, it is possibility

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Figure 4 CS treatment does not alter neuroepithelial organization. Embryos were exposed to either control or CS solutions (10 mg/mL) at stage 33/34 and examined at stage 40. (A)–(B) Confocal images of control (A) and CS (B)treated brain sections stained with anti-BrdU following BrdU injections at stage 40. BrdU incorporation was restricted to the ventricular surface (vs) of the neuroepithelium and was unaffected by CS treatment. Exposed side is to the left. (C)–(D) Major axon tracts were not affected by CS treatment as seen with antiacetylated tubulin staining in wholemount control (C) and CS-treated (D) brains. Dorsal is up, anterior to the left. Scale bar is 100 ␮m in (C) and (D) and 50 ␮m in (A) and (B).

that other molecules within the neuroepithelium may have been affected by the addition of exogenous CS. Finally, CS treatment did not alter the gross organization or number of newly formed axons within the Xenopus brain, as visualized by antiacetylated tubulin [Fig. 4(C) and (D)]. These results suggest that CS induced pathfinding errors are not due to nonspecific toxicity or changes in the diencephalic neuroepithelial substrate, but rather are likely to arise from specific interactions between exogenous CS and molecules involved in RGC axon pathfinding.

CS Causes Enlarged Growth Cones and Saltatory Growth of RGC Axons Previous studies in Xenopus have shown that retinal axon pathfinding errors similar to those observed with exogenous CS can be induced by cytochalasin treat-

ment, which disrupts microfilaments and reduces the filopodial complexity of retinal growth cones (Chien et al., 1993). To determine whether a similar mechanism is responsible for the pathfinding errors seen in CS treated embryos, time-lapse microscopy was used to visualize the dynamic growth and behavior of individual retinal axons labeled with the lipophilic dye DiI during CS application (see movie in supplementary material, http://journals.wiley.com/neu). In these experiments, individual axons were seen to display complex growth patterns with active growth cones [Fig. 5(A)–(D)]. Instead of the regular and steady advance seen in control axons, CS-treated retinal axons showed a saltatory growth pattern. Retinal axons underwent periods where their growth rates were comparable to control axons, interspersed by long stalling periods [Fig. 5(E)]. During these periods of halted growth, growth cones were often highly active, extending and retracting numerous filopodia [Fig. 5(C)–(D)]. This behavior was reflected in an overall increase in number and length of filopodial processes, suggesting that exogenous CS augmented rather than decreased growth cone complexity [Fig. 5(F) and (G)]. The frequent stalling of CS treated axons resulted in a significantly slower growth rate (41.6 ⫾ 3.4 ␮m/h, n ⫽ 10 growth cones vs. 69.9 ⫾ 3.4 ␮m/h in controls, n ⫽ 6 growth cones; p ⬍ .01). Nevertheless, the final axonal length of CS treated axons [Fig. 2(B)] were found to be comparable to control axons [Fig. 2(A)] by stage 40. Saltatory growth patterns were observed within 30 min of adding exogenous CS (average speed before CS application 79.2 ⫾ 6.4 ␮m/h and 34.6 ⫾ 3.6 ␮m/h after CS application; n ⫽ 4 growth cones each, p ⬍ .01). Conversely, once retinal axons were exposed to CS, they continued to display CS-typical growth behavior for up to 4 h after CS application was discontinued. In addition, brains exposed to fluorescently labeled CS retained strong labeling within the neuroepithelium even after prolonged periods of washing (data not shown). These observations may reflect the ability of exogenous CS to bind to numerous extracellular matrix molecules with high affinity. This could result in CS becoming immobilized within the neuroepithelium, thus causing RGC axons to continue to display CS typical misrouting behavior.

Elimination of Endogenous CS Does Not Cause Pathfinding Errors To test for an endogenous role of CSPGs in guiding retinal axons through the diencephalon, native CSs


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Figure 5 Exogenous CS induces saltatory growth rates and altered growth cone morphology. (A–D) A sequence of individual DiI labeled RGC axons growing through the diencephalon. Dorsal is up, anterior to the left, tectum is located in the upper right corner. One axon [asterisks in (A)] makes clear pathfinding errors [arrows in (C)] as it turns anteriorly away from the tectum. Exogenous CS did not cause growth cone morphology to simplify. In fact, the growth cone becomes more complex during times of stalled elongation [arrow in (D)]. (E) Axon length versus time for the RGC axon (black squares) displayed in panels (A)–(D), compared to a control axon (open squares; images not shown). Unlike control axons that advance at a relatively steady rate, those exposed to CS display a saltatory growth pattern with frequent long stalling periods. (F)–(G). High magnification confocal images of DiI-labeled growth cones in fixed preparations. (F) A control growth cone showing an average number and length of filopodial extensions (3.5 ⫾ 0.3 filopodia/growth cone and 5.8 ⫾ 0.4 ␮m average filopodial length, n ⫽ 67). (G) A growth cone treated with exogenous CS shows an enlarged and more complex growth cone morphology. The number of filopodial extensions was also increased (4.9 ⫾ 0.4 filopodia/growth cone and 7.2 ⫾ 0.5 ␮m average filopodial length, n ⫽ 62; p ⬍ .05, Student’s t test). Scale bar is 20 ␮m in (A)–(D) and10 ␮m in (F)–(G).

were removed from the optic tract by treatment of exposed brains with Chondroitinase ABC (5 U/mL). The elimination of CS from the exposed neuroepithe-

lium was confirmed by CS-56 antibody staining (data not shown). Retinal axons extending through a CSfree environment showed no significant difference in

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their overall trajectory, when compared to control embryos (data not shown).

DISCUSSION The results of this study suggest that CSs may play a role in guiding retinal axons through the diencephalon to their midbrain target, the optic tectum. First, CSs are expressed within the visual pathway during the period when retinal axons extend through the diencephalon. Second, exogenous application of CS interferes with retinal pathfinding. RGC axons disperse widely from their normal trajectory within the optic tract and extend aberrantly into inappropriate regions of the forebrain. Third, addition of exogenous CS causes a rapid decrease in RGC axon extension rate and a concurrent increase in the complexity of growth cone morphology in vivo. CSs are known to promote as well as inhibit axonal outgrowth (Maeda and Noda, 1996; Garwood et al., 1999). This dualism of function has been attributed to the heterogeneity of CSPGs and differences in sensitivity between different neuronal cell types. In the case of retinal axons, CSs have been shown to elicit inhibitory effects on retinal axons within the retina (Snow et al., 1991; Brittis et al., 1992), across the optic chiasm (Chung et al., 2000b), in the diencephalon (Ichijo and Kawabata, 2001), and at the dorsal midline of the optic tectum (Jhaveri, 1993; Jhaveri and Hoffman-Kim, 1996; Hoffman-Kim et al 1998). However, not all retinal axons are inhibited by CSPGs. Chick retinal axons have been shown to grow through CS-rich environments in vivo (McAdams and McLoon, 1995; Ring et al., 1995). In the present study, we have shown that the developing Xenopus visual system expresses CSPGs and that retinal axons project through a CS-rich environment within the diencephalon. In addition, CS is expressed within the central Xenopus retina (data not shown), which is in contrast to the mammalian retina where CS expression regresses as RGCs begin to differentiate and send out their axons (Brittis et al., 1992; Brittis and Silver, 1995). These data, together with the observation that exogenously applied CS, does not inhibit RGC neurite extension in vitro (Walz and Holt, unpublished observations), suggest that CSs are not inhibitory to Xenopus retinal axon growth in vivo. Retinal axons exposed to exogenously applied CS sugar chains displayed dramatic pathfinding errors. Instead of forming a tight optic tract following a stereotypical pathway towards the tectum, retinal axons exposed to exogenous CS invaded inappropriate regions of the telencephalon and diencephalon. Con-

sistent with these results are recent findings in the chick visual system where removal of endogenous CSs resulted in the anterior enlargement of the optic tract, with retinal axons projecting aberrantly within the telencephalon (Ichijo and Kawabata, 2001). In addition to the aberrant growth observed within the forebrain, retinal axons that entered the tectum were often observed crossing the tectal midline. CSs have been shown to be highly expressed at the hamster tectal midline, and are thought to act as a barrier, either directly or via CS-binding molecules, to maintain laterality of retinal axons (Hoffman-Kim et al., 1998). Although the expression of CSs within the Xenopus tectum was not examined, this raises the possibility that the addition of exogenous CS may have disrupted the formation of such an inhibitory barrier at the dorsal tectum, possibly by competing off CS-binding molecules from endogenous CSPGs. It is becoming increasingly clear that the specific pattern of sulfate modification determines the proteinbinding specificity of HS (Lindahl, 2000; Rong et al., 2001). We have recently shown that specific sequences rather than gross structural composition of HS glycosaminoglycan side chains are important for retinal axon targeting in vivo (Irie et al., 2002). Therefore, it will be interesting to determine the precise saccharide structures of CS that are required for retinal axon guidance. We found that the addition of different types of CSs resulted in similar retinal pathfinding errors, suggesting a possible conservation of key CS sequence motifs. Because these results were obtained from non-nervous system derived CS, we also tried a cocktail of brain-derived CSPGs (neurocan, phosphacan, versican, and aggrecan; Chemicon). However, no retinal pathfinding errors were observed in the presence of these brain-derived CSPGs (data not shown). The lack of pathfinding errors is perhaps not surprising, because it was not possible to use brain-derived CSPGs at the same concentrations as that required by CS glycosaminoglycan side chains to cause retinal navigational errors. Further work is required to determine which specific sequences in the CS glycosaminoglycan side chains are involved in retinal axon guidance. In contrast to the effects observed in the optic tract by the addition of exogenous CS, removal of endogenous CS did not result in retinal pathfinding errors. One possible explanation is that endogenous CSs are not directly involved in guiding retinal axons through the diencephalon. Pathfinding errors observed by the addition of exogenous CS may have resulted from disruptions to CS-binding molecules present in the optic pathway. CSs have been reported to bind numerous cell adhesion molecules and growth factors


(Yamaguchi et al., 1990; Grumet et al., 1993; Friedlander et al., 1994; Fager et al., 1995; Emerling and Lander, 1996; Milev et al., 1996; Reichsman et al., 1996; Sakurai et al., 1996; Anderson et al., 1998; Soussi-Yanicostas et al., 1998), which could indirectly influence retinal axon guidance. Alternatively, endogenous CSs may directly guide retinal axons in conjunction with other guidance cues present in the pathway. The elimination of a single molecule from a complete set of guidance cues may not result in detectable pathfinding errors, as other molecules may compensate for its absence by performing a similar function. One class of molecules that may compensate for the absence of endogenous CS is the heparan sulfate proteoglycans (HSPGs). HSPGs are expressed within the optic tract during the period in which retinal axons grow through the diencephalon (Walz et al., 1997) and bind some of the same guidance cues as CSPGs, such as netrin-1 (Serafini et al., 1994; Litwack et al., 1995). Moreover, HS has been shown to impart directional information to growing retinal axons within the optic tract (Walz et al., 1997). In vivo time-lapse analysis revealed that individual retinal axons displayed saltatory growth patterns in the presence of exogenous CS. In contrast to the smooth, uninterrupted advance of control axons, CStreated retinal axons often showed long stalling periods followed by periods of normal growth speeds. Interestingly, inappropriate pathfinding decisions were often observed after a stalling period, suggesting that exogenous CS may prevent retinal growth cones from identifying their correct navigational cues. In addition, application of exogenous CS also resulted in alterations in growth cone morphology. Changes from a simple growth cone to an enlarged, more complex morphology were observed during these periods of stalling. This was similar to the phenotype observed in the developing mammalian retina when retinal axons were deflected from a CS-free central path into the CS-rich periphery (Brittis and Silver, 1995). Numerous studies of growth cone morphology and axon pathfinding have shown that increases in growth cone complexity and the slowing of growth often occur at important choice points (Caudy and Bentley, 1986; Bovolenta and Mason, 1987; Holt, 1989; Godement et al., 1994). Thus, the complex morphology in CStreated projections together with the frequent stalling of growth cones suggest that exogenous CSs may “blind” retinal growth cones to guidance cues present within the diencephalon. This would cause them to abandon their simple configuration in an attempt to explore the environment for directional information. The precise mechanism by which CS affects retinal pathfinding is not known. CSs are known to modulate

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adhesive interactions mediated by several neural cell adhesion molecules. For example, the neural specific CSPG neurocan inhibits neuronal adhesion and neurite outgrowth (Rauch et al., 1992; Friedlander et al., 1994). The binding of neurocan to the neural cell adhesion molecules N-CAM and Ng-CAM can be inhibited by the addition of free CS (Friedlander et al. 1994). Thus, CS may modulate the activity of specific adhesion molecules along the optic tract. Alternatively, CS may bind soluble molecules that in turn provide localized guidance cues for retinal axons within the diencephalon. The CSPG decorin has been shown to bind netrin-1 with high affinity and potentiate its activity in vitro through its CS side chains (Litwack et al., 1995). Xenopus netrin-1 localizes to the optic nerve head and has been shown to be involved in guiding retinal axons out of the eye (Hopker et al., 1999). Furthermore, netrin-1 is expressed in the diencephalon, demarcating the presumptive optic tract (de la Torre et al., 1997). Thus, the addition of exogenous CS might affect retinal axon guidance by competing off bound netrin-1 from endogenous CSPGs within the diencephalon. Another candidate molecule is Slit-2, which has been shown to bind the glycosaminoglycan side chains of the heparan sulfate proteoglycan glypican-1 (Liang et al., 1999; Ronca et al., 2001). However, the function of Slit-2 in Xenopus RGC axon guidance is not yet known. A third candidate molecule is Semaphorin 3A (Sema3A), which has been shown to act as a directional guidance cue in the growth cone turning assay for retinal growth cones (Campbell et al., 2001). Sema3A is strongly expressed along the dorsal border of the optic tract (Campbell et al., 2001), suggesting a possible role in retinal axon guidance. In conclusion, we propose that CSs may modulate the guidance of retinal axons, either directly or indirectly via CS-binding molecules, as they grow through the diencephalon en route to their target, the optic tectum. In the future, it will be important to identify the individual CSPGs involved and to determine their specific actions in the development of the Xenopus visual system. The authors would like to thank Asha Dwivedy for her excellent technical assistance.

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