Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

Developmental Biology 271 (2004) 291 – 305 www.elsevier.com/locate/ydbio

Role of cell adhesion molecule DM-GRASP in growth and orientation $ of retinal ganglion cell axons H.X. Avci, P. Zelina, K. Thelen, and G.E. Pollerberg * Department of Developmental Neurobiology, University of Heidelberg, D-69120 Heidelberg, Germany Received for publication 28 November 2003, revised 24 March 2004, accepted 25 March 2004 Available online 18 May 2004

Abstract The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP’s presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system. D 2004 Elsevier Inc. All rights reserved. Keywords: SC1; BEN; ALCAM; NgCAM; L1; Axonal CAMs; Growth cone; Axon navigation; Pathfinding; Embryonic retina; Central nervous system development

Introduction A variety of complex interacting mechanisms underlie axonal pathway- and target-finding during development of the vertebrate central nervous system. Among others, cell adhesion molecules (CAMs) of the immunoglobulin (Ig) superfamily have been demonstrated to play a role in axonal orientation: L1/NgCAM (Brittis et al., 1995; Castellani et al., 2000), NCAM (Pollerberg and Beck-Sickinger, 1993; Thanos et al., 1984), F11 (Falk et al., 2002), NrCAM (Stoeckli et al., 1997), and Axonin-1/TAG1 (Perrin et al., 2001). The CAM DM-GRASP (Burns et al., 1991; Pourquie et al., 1990; Tanaka and Obata, 1984) has been shown to play a role in higher vertebrates in processes such as cell $ Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ydbio.2004.03.035. * Corresponding author. Department of Developmental Neurobiology, University of Heidelberg, Im Neuenheimer Feld 232, D-69120 Heidelberg, Germany. Fax: +49-6221-546375. E-mail address: [email protected] (G.E. Pollerberg).

0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.03.035

adhesion (Burns et al., 1991; Tanaka et al., 1991), axon growth (DeBernardo and Chang, 1996; Pollerberg and Mack, 1994), migration (Heffron and Golden, 2000), differentiation (Stephan et al., 1999), and synapse formation (Chedotal et al., 1996; Yamagata et al., 1995). DM-GRASP is an integral membrane protein of 100 kDa which was first identified in chick; it has been also termed SC1, BEN, and JC7 (Burns et al., 1991; el-Deeb et al., 1992; Pourquie et al., 1990, 1992; Tanaka and Obata, 1984; Tanaka et al., 1991). DM-GRASP contains two unusual Ig domains of the V-type, in addition to three of the common C-type (Burns et al., 1991). A close homolog of DMGRASP, Gicerin, has been identified in chick (Taira et al., 1994). Mammalian orthologs of DM-GRASP have been found in human (ALCAM/CD166; Bowen et al., 1995), rodents (ALCAM/CD166; Kanki et al., 1994; SekineAizawa et al., 1998), and bovine (CD166; Konno et al., 2001). Orthologs were also found in fish (Neurolin; Laessing et al., 1994; Paschke et al., 1992), fly (IrreC-rst; Ramos et al., 1993), and worm (SYG-1; Shen and Bargmann, 2003). Neurolin, which shows 37% identity and 56%

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similarity to chick DM-GRASP and 20% and 47% to Gicerin, respectively, was demonstrated to play a role in axonal navigation of retinal and motor neurons (Leppert et al., 1999; Ott et al., 1998, 2001). For DM-GRASP (el-Deeb et al., 1992; Tanaka et al., 1991), as well as ALCAM (Bowen et al., 1995; van Kempen et al., 2001), homophilic interaction has been demonstrated using cell aggregation assays. Heterophilic interactions have also been described: DM-GRASP interacts with NgCAM during neurite extension (DeBernardo and Chang, 1996) and with CD6 in the hemopoietic system (Bowen et al., 1995, 1997). To study the role of DM-GRASP in navigating axons, we chose the chick embryo retina as a model system since it is a relative simple structure of the central nervous system with well-described developmental patterns and in addition provides a variety of experimental approaches (for review, see Thanos and Mey, 2001). Retinal ganglion cell (RGC) axons leave the eye and project to the optic tectum, the roof of the mesencephalon, following stereotype pathways (Halfter and Deiss, 1986; Stahl et al., 1990). Already within the developing retina, extending RGC axons have to make several directional decisions to find the optic fissure, the ‘‘exit’’ located in the central retina. Due to a strict developmental gradient from central to peripheral retina (Halfter et al., 1983; Snow and Robson, 1995), the first emerging RGC axons are located in the vicinity of the optic fissure. As axons protrude from the RGC somata, they first extend without axonal contact on the basal lamina (also termed inner limiting membrane or basal membrane) delimiting the vitreous. Growing towards the central retina, RGC axons meet, fasciculate, and gradually build up the optic fiber layer (OFL), extending selectively in the interphase between already formed RGC axons of the OFL and the basal lamina (Easter et al., 1984; Halfter and von Boxberg, 1992; Rager, 1980; Silver and Rutishauser, 1984). Having reached the central retina, RGC axons have to perform turns to grow into and along the optic fissure, towards optic nerve head, and finally into the optic nerve to leave the eye. Our previous studies indicated that DM-GRASP is most highly concentrated on RGC axons growing and fasciculating in the retina (Pollerberg and Mack, 1994). To study the role of DM-GRASP in RGC axon – axon interactions, we designed an in vitro assay where the extension of RGC axons on RGC axons can be selectively analyzed (Pollerberg and Mack, 1994): DM-GRASP inhibition resulted in a reduction of elongation. In contrast, growth of RGC axons on Laminin or retinal basal lamina preparations was unimpaired. These results suggested that DM-GRASP might ensure selective RGC axon extension in the OFL and thereby could be crucial for routing of RGC axons within the retina. Here, we report about the role of DM-GRASP for RGC axon growth and orientation, as could be observed by using several functional assays. We found that DM-GRASP is able to enhance RGC axon elongation and growth cone size on Laminin substrate. Given the choice between DM-GRASP/Laminin and Laminin lanes, RGC axons clearly prefer to grow on the DM-

GRASP-containing ones. Moreover, inhibition of DMGRASP in the developing retina results in RGC axon routing errors at the optic fissure and failure to leave the eye. Together, these findings demonstrate for the first time a role of DM-GRASP in axonal pathfinding during embryonic development of the higher vertebrate central nervous system.

Materials and methods Animals Fertilized white leghorn chicken eggs and adult white leghorn chicken were obtained from a local provider. Chick embryos and post-hatching day (P)1 chicks were obtained by incubation of fertilized eggs at 38jC. Antibodies Monoclonal antibodies against DM-GRASP (mAb 4H5) and NCAM (252B5), as well as rabbit sera against DMGRASP were produced as described (Pollerberg and Mack, 1994). Nonspecific F(ab) fragments were generated from immunoglobulins (IgGs) purified from rabbit pre-immune serum. Serum against NgCAM was a kind gift of F.G. Rathjen and T. Bru¨mmendorf (Max-Delbru¨ck-Centrum, Berlin). Secondary antibodies were purchased from Jackson Laboratories (USA). Serum against Laminin (L9393) was purchased from Sigma (Germany). F(ab) fragments were generated according to Mage (1980). Affinity purification of DM-GRASP DM-GRASP was immunoaffinity-purified from brains of newly hatched chicks using a mAb 4H5 column as described before (Pollerberg and Mack, 1994). The purity of DM-GRASP was analyzed by SDS-PAGE and silver stainings, no traces of other proteins were detected. Quantitative Western blot analysis Retina homogenates of various developmental stages were prepared as described (Pollerberg and Beck-Sickinger, 1993). Equal amounts of protein were loaded for SDSPAGE and blotted on nitrocellulose filters. DM-GRASP was detected using DM-GRASP serum followed by peroxidaseconjugated goat anti-rabbit antibody and enhanced chemiluminescence (ECL, Amersham, Germany). Emitted light was detected by the Lumi-Imager system (Roche Diagnostics) and quantified using the Lumi-Analyst software (Roche Diagnostics). Three independent Western blots were analyzed. For each lane, the relative amount of DM-GRASP and the overall protein contents was determined by measuring the chemiluminescence values of the DM-GRASP bands and the overall Coomassie Brilliant Blue signals (SCION NIH Image software 4.0). Thereby, the variations in the

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overall protein contents of the single lanes (which were below 15%) could be determined; they were used to correct the DM-GRASP values. Retinal single cell cultures Single cell cultures of embryonic day (E) 6 retinal cells and E6 retina explant strips were prepared as described (Halfter et al., 1983; Rodriguez-Tebar et al., 1989) with minor modifications: Cultures were grown in DMEM/F12 (Invitrogen, Germany) containing 10% fetal bovine serum (FBS) or 10% basal medium supplement (BMS, Seromed, Germany) and 100 Ag/ml Gentamycin (Invitrogen). Cultures were fixed in 4% paraformaldehyde (PFA) for 30 min and stained by indirect immunofluorescence as described (Pollerberg et al., 1985). Immunohistochemical procedures Heads of E6 and E9 embryos were fixed in 4% PFA and 11% sucrose in PBS for 12 h, and in 25% sucrose in PBS for additional 12 h. The specimens were embedded (Tissue Tec O.C.T., Sakura, Netherlands) and sectioned with a cryostat (Reichert and Jung, Germany). Sections (12 Am) were stained by indirect immunofluorescence as described (Pollerberg et al., 1985). Axon growth assays Poly-L-lysine (PLL)-coated glass coverslips were incubated with either Laminin (5 Ag/ml; Invitrogen) alone or a

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mixture of DM-GRASP and Laminin (1.5 and 5 Ag/ml, respectively). For some experiments, substrate proteins were coated on PLL-free coverslips. Protein coating was monitored by indirect immunofluorescence labelings. For timelapse microscopy, medium was supplemented with 25 mM HEPES. After 18 – 24 h of pre-cultivation in the incubator, time-lapse studies were carried out for 60 min, using a selfmade incubation chamber with thermoregulation and CO2 regulation and an inverted microscope (Axiovert 200M, Zeiss, Germany) equipped with a digital camera (AxioCam, Zeiss). Only axons longer than 20 Am were included in the evaluation. Axon length, growth cone area, and perimeter were measured using SCION NIH Image software 4.0. Growth direction was defined by the distal-most 30 Am of the axon. Growth cone length was measured as the maximal extension from neck to tip in growth direction, and growth cone width was determined as the maximal extension perpendicular to the growth direction. Growth cones extending more than 60% of their width to one side of the growth direction were considered (left or right) asymmetrical; those with shifts below 60% as symmetrical. As a measure for the complexity of the growth cones, the ratio of perimeter and square root of area was calculated for each growth cone. Without any shape changes, an increase in growth cone area by a factor 1.8, for example, results in a proportional perimeter increase by a factor of 1.3 (square root of 1.8). For distribution analysis, complexity values were grouped into eight classes; a circle (the least complex possible figure) would score 3.7, for example. Significance of differences between mean values was determined by t tests and between variances by F tests.

Fig. 1. DM-GRASP expression during chick retina development. (a) Homogenates of embryonic (E4 – E18), post-hatching day 1 (P1), and adult (Ad) retina in Western blot analysis. DM-GRASP appears as a single band of 100 kDa. (b) Chemiluminescence analysis of DM-GRASP expression levels relative to the overall protein contents. Average values from three independent experiments are plotted in the histogram; therefore, it does not exactly represent the levels of the DM-GRASP band shown in (a). The highest value (P1) of each experiment (E4 – Ad) was set to 100%. Error bars represent standard deviations.

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Axon preference assays Substrate lanes were generated as described (Vielmetter et al., 1990) with minor modifications: Silicone matrices (provided by J. Jung and F. Bonhoeffer, Max-Planck-Institute for Developmental Biology, Tu¨bingen) were placed on PLLcoated coverslips, and the substrate protein solution (3 Ag/ ml in PBS) was injected into the channels of the matrices, followed by an incubation of 1 h at 37jC. Injection and incubation was repeated once, unbound protein was removed by rinsing with PBS. Matrices were removed and the coverslips were incubated with a Laminin solution (5 Ag/ml in PBS) for 2 h at 37jC. This Laminin concentration ensures substantial growth of RGC axons. For controls, IgGs were coated instead of DM-GRASP. In additional experiments, bovine serum albumin was employed with identical results. E6 retinal explants were placed onto the coverslips and incubated for 2 d at 37jC and 5% CO2. To test for DMGRASP-specific preference, the coated coverslips were in-

cubated for 1 h with DM-GRASP-specific F(ab) fragments (0.5 mg/ml) or as a control with nonspecific F(ab) fragments (0.5 mg/ml) before cultivation of retinal explants. NgCAMspecific F(ab) fragments (0.5 mg/ml) or as a control nonspecific F(ab) fragments (0.5 mg/ml) were added to medium to test for a role of this CAM. Explants were fixed with 4% PFA in PBS and stained by indirect immunofluorescence. A substrate lane was counted as exerting axonal preference when containing axons or axon bundles and at the same time being neighbored by a lane containing no axons, that is, if its axons respected the substrate borders with the exception of a very few. A retinal explant strip was considered showing preference if RGC axons respected the borders of at least 50% of the lanes of a given substrate. Eye organ culture and retinal flat-mounts Eyes of E4.5 embryos were isolated (connective tissue and pigment epithelium were removed) and cultured in 200

Fig. 2. Localization of DM-GRASP in the developing chick retina. (a, d, e, f) E6 and (b) E9 retina cryostat sections stained with DM-GRASP (a, b, d) and Laminin (e) antibodies; (c) growth cone in vitro stained for DM-GRASP. (a) In E6 retina, DM-GRASP is present on RGC axons forming the optic fiber layer (OFL) and optic nerve (ON). The ganglion cell layer (GCL) and neuroepithelial cells (NEC) are negative. (b) In E9 retina, DM-GRASP is present in OFL, ON, and forming inner plexiform layer (IPL); GCL and forming inner nuclear layer (INL) are negative. (c) DM-GRASP is present on axon and growth cone, including lamellipodia and filopodia, as detected on RGCs in single cell culture. (d) A high magnification of a retina section shows the restriction of DMGRASP to the OFL; somata of RGCs and NECs are negative. (e) Laminin is highest concentrated in the basal lamina (BL), but also present in decreasing amounts in OFL, GCL, and NEC. (f) The merged picture of the double staining (d, e) depicts the interphase of the Laminin- and DM-GRASP-richest zones, providing an optimal growth substrate for RGC axons. Magnification bars: (a and b) 100 Am; (c – f) 5 Am.

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Al organ culture medium (DMEM supplemented with 10% FBS and 2% chicken serum) for 24 h at 37jC as described (Halfter and Deiss, 1986; Pollerberg and Beck-Sickinger, 1993). For inhibition experiments, eyes were incubated with DM-GRASP F(ab) fragments (1 mg/ml). As controls, nonspecific F(ab) fragments (1 mg/ml) or no F(ab) fragments were added. Normal growth and development of organcultured eyes per se was not affected by presence of DM-

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GRASP-F(ab) fragments or nonspecific F(ab) fragments. After the organ culture period, lens and vitreous were removed and the retina was spread out flat on a nitrocellulose filter (Schleicher and Schu¨ll, Germany) and fixed for 1 h in 4% PFA. DiO (Sigma, Germany) crystals were placed on one half of the flat-mounted retina, in a distance of 400– 500 Am to the optic fissure to visualize axons growing to the optic fissure and further. Retinae were evaluated using an

Fig. 3. Occurrence of DM-GRASP in the embryonic retina. (a) Scanning micrographs of a flat-mounted retina (E4.5) stained for DM-GRASP. Due to the developmental gradient from central to peripheral retina, only single axons and thin axon bundles are visualized in the periphery, converging to increasingly thicker fascicles towards the optic fissure, an elongated structure (marked by two arrows) in the center. The upper-left square delineates the peripheral region of the retina shown enlarged in (b); the lower-right square the central region shown in (c). (b) In the peripheral retina, DM-GRASP is present on somata (one marked by arrow) and axons of young RGCs having reached a more central RGC (top panel). The side view (1-Am optical section along the dashed line in the upper panel) allows for the visualization of DM-GRASP delineating the entire RGC soma (bottom panel). (c) In the central retina, DM-GRASP is present in the dense network of axon bundles (top panel). The side view reveals that in the mature RGCs of the central retina, DM-GRASP is confined to the axons and not present on the somata (bottom panel). D, dorsal; V, ventral; T, temporal; N, nasal; OFL, optic fiber layer; GCL, ganglion cell layer; NEC, neuroepithelial cells. Magnification bars: (a) 200 Am; (b and c) 100 Am.

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inverted microscope (Axiovert 200M, Zeiss) equipped with a digital camera (AxioCam, Zeiss). To analyze the distribution of DM-GRASP, flat-mounted retinae of E4.5 embryos were stained with DM-GRASP serum followed by incubation with biotin-conjugated goat anti-rabbit IgG and Cy3conjugated streptavidin. Images were captured using a laser scanning confocal microscope (TCS SP2, Leica, Germany) and annotated with Adobe Photoshop 7.0.

OFL, GCL, and NEC (Fig. 2e). The zone where RGC growth cones come into contact with both basal lamina molecules such as Laminin and axonal proteins such as DM-GRASP (Fig. 2f) presumably represents the optimal growth environment for extending RGC axons. In flat-mounted retinae, a developmental gradient from central to peripheral retina becomes clearly visible (Fig. 3). At E 4.5, the central retina is already covered by RGC axon bundles which are DM-GRASP-positive, all converging towards the optic fissure, where they grow into the optic

Results DM-GRASP expression correlates with RGC axon growth To investigate the expression levels of DM-GRASP in the developing retina, Western blot analyses of chick retina homogenates of embryonic day (E) 4 to adult were performed (Fig. 1a). Quantification of DM-GRASP protein relative to the overall protein contents revealed three maxima in DM-GRASP expression. The first comprises a period from E4 to E10, a second from E12 to E18, and a third at the time of hatching (Fig. 1b). During the first maximum, expression of DM-GRASP is almost doubled within 2 days (E4 – E6). Between E6 and E10, DM-GRASP presence in the retina is declining by more than 60%. At E12, expression of DM-GRASP rises again and reaches a maximum at E16, with an almost 5-fold increase compared to the minimum at E10. This maximum declines by about one fifth towards E18. The third increase is detected immediately after hatching, and a considerable level is maintained throughout adulthood. To correlate DM-GRASP expression levels with retinal histogenesis, immunolabelings of retina sections were performed to visualize its distribution patterns (Fig. 2). At E6, DM-GRASP is exclusively present in the OFL and optic nerve, that is, selectively on RGC axons (Fig. 2a). RGC somata, forming the ganglion cell layer (GCL), as well as undifferentiated neuroepithelial cells (NECs) are devoid of DM-GRASP. Therefore, the first peak of DM-GRASP expression (see Fig. 1b) can be clearly attributed to its presence selectively on RGC axons, correlating with the maximum number of RGC growth cones and distal axons extending in the retina. From E8 on, DM-GRASP’s presence is decreasing in the OFL, concomitantly to the distal RGC axon parts leaving the retina. As differentiation of the retina proceeds, other fiber-containing layers form and begin to exhibit DMGRASP; the inner plexiform layer (IPL) starts to show DMGRASP (Fig. 2b), followed by the forming outer plexiform layer (OPL, not shown), together causing the second expression peak observed around E16 (see Fig. 1b). Moreover, immunostainings of RGCs sending out axons in sparse single cell cultures on Laminin reveal that DM-GRASP is not only present on RGC axons but also on their growth cones (Fig. 2c). While DM-GRASP is only present in the OFL (Fig. 2d), Laminin is found in highest concentrations in the basal lamina and also—at considerably lower levels—in

Fig. 4. RGC axon growth on Laminin and DM-GRASP. (a) Length of RGC axons grown on the indicated substrates, plotted as percentages of axons falling into four length classes. (b) The percentage of RGC axons (ordinate) longer than a given length (abscissa). For axons growing on DM-GRASP/ Laminin, the length distribution curve is shifted towards larger values. (c) Axon elongation determined in time-lapse analysis (n = 10 each) depicted in a cumulative plot of axon length increase (t = 0 start of observation period). Axons extending on DM-GRASP/Laminin show a stronger length increase than those grown on Laminin. Error bars represent standard deviations.

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nerve. In the periphery, thin axon bundles positive for DMGRASP can be detected (Fig. 3a). Also, single RGC axons extending in the very periphery and having reached another, more centrally located RGC (Fig. 3b), display DM-GRASP. Young RGCs in the periphery of the retina are positive for DM-GRASP on both soma and axon (Fig. 3b), whereas in more centrally located, more mature RGCs, DM-GRASP is restricted to the axonal compartment (Fig. 3c). Together, the expression studies show that DM-GRASP is present at the right time and place in the developing retina and on the decisive structures to play a role for growing and navigating RGC axons.

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DM-GRASP promotes axonal growth and modulates growth cones Since in vivo RGC axons extend in contact with both DM-GRASP-containing OFL and Laminin-rich basal lamina, we investigated the impact of DM-GRASP and Laminin—offered as substrates—on RGC axons in single cell culture (Fig. 4a). On glass coverslips coated only with polyL-lysine (PLL), RGCs form very short axons (axon length 37 F 20 Am, n = 58). Coating, in addition, DM-GRASP does not have any significant effect on axon lengths (37 F 18 Am, n = 62), indicating that DM-GRASP alone is not

Fig. 5. Effects of DM-GRASP on RGC growth cones. (a) On DM-GRASP substrate, areas covered by growth cones are increased by a factor of 1.8 compared to those on Laminin (174 F 82 Am2 and 98 F 50 Am2; n = 35; t test: P < 0.00001). Perimeters increase 1.3-fold, that is, proportionally (149 F 56 and 113 F 81 Am; t test: P < 0.03). (b) Perimeters divided by the square root of the corresponding areas, as a measure of growth cone complexity are similar on both substrates (11.5 F 2.5 and 10.7 F 5.5 Am; t test: P < 0.46). (c) Grouping of values according to their complexity levels reveals a significant decrease in variation range of growth cone complexity on DM-GRASP (four and seven complexity levels, F test, P < 0.000004). (d) Both forward (12 F 4 to 17 F 4 Am; n = 35; t test, P < 0.00001) and lateral (15 F 8 Am to 22 F 6 Am; t test P < 0.0003) dimensions—together a measure of growth cone shape—increase. The standard deviation of growth cone width is significantly decreased ( F test, P < 0.05); in contrast, this is not the case for growth cone length. (e) The length/ width ratio, however, is not significantly changed due to a proportional increase in both dimensions by a factor of 1.4. (f) The relative number of symmetrical growth cones and of those forming an unilateral extension to either left or right side (i.e. asymmetrically shaped growth cones) was not influenced by presence of DM-GRASP (40% and 37% asymmetric growth cones on Laminin and DM-GRASP, respectively, n = 35; P < 0.1). Error bars represent standard deviations. For details of evaluation, see Materials and methods. *P < 0.05; ***P < 0.001.

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sufficient to enhance axonal elongation. On Laminin-coated coverslips, much longer axons are formed (79 F 73 Am; n = 215). If DM-GRASP is coated in addition to Laminin, average axon length is significantly increasing by about 25% (99 F 103 Am; n = 266, P < 0.05) in comparison to Laminin substrate alone (Fig. 4b). The proportion of RGC axons on DM-GRASP/Laminin longer than 100 Am is clearly increased by 76% compared to axons on Laminin (37% and 21%, respectively). Time-lapse analysis revealed that DM-GRASP causes an accelerated elongation (Fig. 4c). On DM-GRASP/Laminin, RGC axons extend 35% faster than on Laminin (elongation rates 1.32 F 0.07 and 0.97 F 0.07 Am/min, respectively). This enhancement might also take place in the retina as soon as an extending RGC axon has reached a DM-GRASP-positive RGC. To study whether DM-GRASP not only enhances axonal elongation but also possesses the capacity to induce initial axon outgrowth, we employed PLL-free coverslips since PLL alone induces outgrowth of short axons. On glass, retinal cells adhered but only generated growth cone-like protrusions at the soma. Only rarely very short axons were formed; none of them longer than 20 Am were observed (not shown). The same was found when the coverslips were coated with DMGRASP, indicating that DM-GRASP by itself has no capacity to induce axon formation. This suggests that, in vivo, the basal lamina provides substrate molecules—such as Laminin—for initial RGC axon outgrowth. We also tested whether DM-GRASP not only affects elongation rates of RGC axons, but also the morphology and dynamics of their growth cones (Fig. 5). For this, single extending axons were followed by time-lapse microscopy.

The average growth cone size (area covered) was increased 1.8-fold for axons extending on DM-GRASP/Laminin compared to Laminin (Fig. 5a). To quantify growth cone complexity, that is, the degree of indentations and protrusions formed, the perimeters of the growth cones were determined. DM-GRASP causes an increase in perimeter proportional to the size increase (Fig. 5a), indicating that average growth cone complexity is not affected (Fig. 5b). The variation range of growth cone complexity, however, is decreased on DMGRASP, indicating that the growth cones become more uniform (Fig. 5c). The overall growth cone shape is maintained, as both forward and lateral dimensions of growth cones increase on DM-GRASP (Fig. 5d) with a constant length/width ratio (Fig. 5e). Formation of asymmetrical growth cone extensions to one side of the growth direction—as a measure for turning activity—is not influenced by DM-GRASP (Fig. 5f). Our studies show that DM-GRASP is capable of increasing elongation rate and growth cone size of RGC axons extending on Laminin, at the same time reducing the variation range in growth cone complexity. In vivo, this might give rise to a more swift and straightforward elongation once the RGC axon has reached the OFL, the only structure providing both DM-GRASP and high levels of Laminin. DM-GRASP is capable of guiding RGC axons in vitro To address the question whether DM-GRASP is not only capable of influencing axon growth but also mediates axonal guidance, we employed a modified in vitro preference assay. It reflects the in vivo situation of RGC axons in the retina,

Fig. 6. Preference of RGC axons for DM-GRASP. RGC axons extending from retinal explant strips on alternating substrate lanes of Laminin (dark lanes) vs. Laminin plus the indicated protein, stained by the respective antibody (green lanes). (a) In 63% of the DM-GRASP lanes, axons show preference for this substrate (2439 borders evaluated; n = 56 independent experiments). (b) In contrast, in only 7% of IgG-containing lanes axons respect the substrate border (1341 borders; n = 20). (c) Substrate pre-incubation with DM-GRASP F(ab) fragments decreases axonal preference for DM-GRASP lanes to 6% (3007 borders; n = 45). (d) In contrast, nonspecific F(ab) fragments do not decrease axonal preference for DM-GRASP; 77% of the lanes showed preferential axon elongation (3009 borders, n = 46). DM, DM-GRASP; IgG, immunoglobulin; LN, Laminin. Magnification bar: 200 Am.

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which has the choice between a DM-GRASP/Laminin combination (OFL) and Laminin only (GCL, NEC). For this, RGC axons extending from retinal explant strips were challenged with alternating substrate lanes containing either Laminin or DM-GRASP/Laminin. The majority of explants (73%, n = 56) shows a clear preference for DM-GRASP/ Laminin, that is, their axons respect the borders of at least 50% of the DM-GRASP lanes (Fig. 6a). When immunoglobulins class G (IgGs) are coated instead of DM-GRASP (Fig. 6b), RGC axons display random growth patterns (0% of explants exhibiting preference, n = 20). To further substantiate that the axonal preference for the DM-GRASP containing lanes is indeed specifically caused by DM-GRASP,

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coverslips were pre-incubated with DM-GRASP F(ab) fragments to make the substrate-DM-GRASP inaccessible for axonal binding partners (Fig. 6c). In this case preference is completely abolished (0% preference, n = 45). In contrast, incubation of substrates with nonspecific F(ab) fragments (Fig. 6d) does not decrease axonal preference for DMGRASP (85% preference, n = 46), indicating that preincubation with F(ab) fragments per se does not result in unspecific abolishment of the DM-GRASP preference. Together, these results indicate that DM-GRASP is capable of causing a preference of RGC axons to grow where this CAM is present. Axons stay on DM-GRASP-containing lanes once they have reached them. In vivo DM-GRASP

Fig. 7. Involvement of NgCAM in DM-GRASP preference. Retinal explants grown on alternating substrate lanes (Laminin vs. DM-GRASP/Laminin) in presence or absence of NgCAM F(ab) fragments. (a) In presence of NgCAM F(ab) fragments, number of explants displaying preference for DM-GRASP in at least 50% of the lanes declines to 20% (2248 borders; n = 40), whereas in presence of nonspecific F(ab) fragments (1588 borders; n = 36) or without F(ab) addition (2242 borders; n = 66), about 60% of the explants display preference. (b) Preference distribution plots for these three experimental conditions show percentage of explants (ordinate) with at least the indicated percentage of lanes showing axonal preference (abscissa). Axon preference for DM-GRASP is significantly reduced in the presence of NgCAM F(ab) fragments (t test; P < 0.00003).

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could thereby make RGC axons grow along and keep to preexisting axons—due to its restricted presence on these— and thus contribute to the navigation of axons towards the optic fissure and into the optic nerve. NgCAM is involved in axonal DM-GRASP preference Since DM-GRASP is known to interact with NgCAM, which is also present on RGC axons (DeBernardo and Chang, 1996; Morales et al., 1996; Pollerberg and Mack, 1994), we tested whether NgCAM is involved in preference of axons for DM-GRASP. For this, we performed preference assays on DM-GRASP/Laminin vs. Laminin-containing lanes, with RGC axons extending in presence of NgCAM F(ab) fragments (Fig. 7). Presence of these F(ab) fragments results in a highly significant reduction (by about 2/3; P < 0.00003) in the number of explant strips performing preference (20%, n = 40) as compared to controls incubated with nonspecific F(ab) fragments (61%, n = 36) or untreated controls (62%, n = 66) (Figs. 7a and b). These data show that NgCAM in the axonal membrane plays a role in preference of RGC axons for DMGRASP, potentially acting as a heterophilic binding partner. The remaining preference for DM-GRASP indicates that in addition to NgCAM, other axonal membrane proteins play a role in mediating DM-GRASP preference, most likely DMGRASP itself.

DM-GRASP is required for axonal orientation at the optic fissure To analyze the role of DM-GRASP for axons growing in their in vivo context, we inhibited this CAM in eyes developing in organ culture. For this, isolated eyes of E4.5 chick embryos were kept in vitro in presence or absence of DM-GRASP F(ab) fragments. Retinae were then spread out as flat-mounts, and the axons of small groups of RGCs were labeled by DiO crystal application (Fig. 8), allowing for the detection of deviations from normal RGC pathfinding. In eyes organ-cultured under standard conditions (no addition of F(ab) fragments) as well as in presence of nonspecific F(ab) fragments, RGC axons extend directly towards the optic fissure, turn, and dive into the nerve head to grow into the optic nerve (Figs. 8a and b). In only a minor fraction of the retinae (9%, n = 46), axons were observed to extend onto the opposite side of the retina. When organcultured eyes were incubated in presence of DM-GRASP F(ab) fragments, axons grow aberrantly across the optic fissure in a considerable number of retinae (43%, n = 14). Navigation of RGC axons towards the optic fissure per se, however, was not affected by DM-GRASP inhibition. Most of the misrouting axons extend a substantial distance onto the opposite side of the retina, following the trajectories of the RGC axons in an antiparallel manner, and heading

Fig. 8. Axonal misrouting by DM-GRASP inhibition. E4.5 eyes organ-cultured in presence of nonspecific F(ab) fragments (a, b) or DM-GRASP F(ab) fragments (c, d). Inlet in (b) shows three DiO crystals on a flat-mount; dashed line, position of the optic fissure; arrow, optic nerve head; D, dorsal region of the retina. Dye crystals label groups of RGC axons growing towards the optic fissure. The majority of axons has already reached the optic fissure and dived into the optic nerve head before the inhibition period. (a, b) Under control conditions, RGC axons enter the optic fissure (dashed line) and leave the retina at the optic nerve head (arrow). (c, d) In presence of DM-GRASP F(ab) fragments, a subpopulation of axons fails to enter the optic nerve, strays away from the preexisting, correctly orientating axons and extend a considerable distance on the opposite side of the retina. Initial navigation of the RGC axons towards the optic fissure, however, appears to be unaffected by DM-GRASP inhibition. Magnification bars: 50 Am in (a – d); 500 Am in inlet.

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towards the periphery (Fig. 8c). Some axons show a more erratic growth pattern in the vicinity of the optic fissure, however, also failing to leave the retina (Fig. 8d). Taken together, these results indicate that DM-GRASP plays an important role in axonal pathfinding at the optic fissure. Inhibition of DM-GRASP results in a loss of the RGC axon’s capability to turn into the optic nerve head and leave the retina; earlier processes—before reaching the optic fissure—remain unimpaired.

Discussion Our results provide insight into the role of DM-GRASP in processes crucial for the formation of the central nervous system. Employing the developing chick retina as a model system, we performed spatiotemporal expression analyses, in vitro studies on axon dynamics and substrate preference, as well as studies on axonal routing in the developing eye. Together, these data show for the first time that DMGRASP plays a crucial role for axonal pathfinding in higher vertebrates. DM-GRASP’s spatiotemporal distribution patterns In the early retina, DM-GRASP is selectively present on axons of RGCs, the only neuron type projecting from eye to mesencephalon. A few other CAMs are also restricted to RGC axons in this phase: NgCAM/L1 (Pollerberg and Mack, 1994; Rathjen et al., 1987b), NrCAM (de la Rosa et al., 1990; Drenhaus et al., 2003), Neurofascin (Rathjen et al., 1987a), TAG1/Axonin-1 (Ruegg et al., 1989), Thy-1 (Sheppard et al., 1988), and F11/Contactin (Rathjen et al., 1987b). Presence of most CAMs on RGC axons is persistent throughout development and in adulthood, only DM-GRASP, NrCAM, and Axonin-1 disappear from the OFL at the time when the growing axon tips leave the retina, pointing to a more specific role of these CAMs in early axonal tasks. With the formation of IPL and OPL, which contain the forming RGC dendrites and also processes of other retinal neurons, DM-GRASP expression increases again to reach a second maximum in the retina. DM-GRASP appears in these layers 2 days ahead of the onset of synaptogenesis, as also observed in other brain regions (Pollerberg and Mack, 1994), pointing to a second role of DM-GRASP in synapse formation and/or in steps preceding synapse formation. A third increase in expression of DM-GRASP is around hatching; it might be connected to refinement of synaptic connections induced by the onset of visual input. The maintenance of expression throughout adulthood is commonly found for IgCAMs, also for those with crucial axonal functions during early development, for example, L1/NgCAM, F11, NrCAM, and Neurofascin; it has been attributed to long-term processes maintaining the histotypic structure of the retina (Drenhaus et al., 2003; Pollerberg and Mack, 1994; Rathjen et al., 1987a).

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The restricted presence of DM-GRASP on RGC axons and absence from their somata might contribute to RGC axon guidance, with the DM-GRASP-negative compact GCL functioning as a ‘‘no entry zone’’ preventing axons from leaving the OFL. Very young RGCs in the peripheral retina, however, exhibit DM-GRASP-positive somata. This might be due to some further maturation steps needed for the compartmentalized distribution of DM-GRASP. The mechanisms underlying the axonal restriction of DMGRASP are yet unknown; it lacks sorting motifs present in other axonal CAMs, for example, Thy-1 and L1 (Winckler and Mellman, 1999). DM-GRASP-positive somata in the peripheral retina might be advantageous for just emerging, more peripheral axons to find these RGCs, since the somata are bigger targets, providing a more substantial amount of DM-GRASP than just the axons. No DM-GRASP is found in the basal lamina forming the interphase between OFL and vitreous. This structure is strongly positive for Laminin and other outgrowth-promoting molecules (Halfter et al., 2000), suggesting that emerging RGC axons that have not yet reached a more centrally located RGC use the basal lamina as their first growth substrate. Interestingly, evaluating more than 1000 axons, we never observed DM-GRASP-positive axons that had not yet reached a more central and more mature, DM-GRASPexpressing RGC, indicating that the very first outgrowth of the axon from the RGC soma is independent of DMGRASP. This is in concordance with previous studies which showed in vitro that RGC axonal growth on basal lamina preparations or on Laminin is independent of DM-GRASP (Pollerberg and Mack, 1994). DM-GRASP’s impact on axons and growth cones Since RGC axons extend in contact with both the basal lamina, containing Laminin, and with older RGC axons, carrying DM-GRASP, we tested what impact Laminin and DM-GRASP have on RGC axons. DM-GRASP by itself cannot induce initial outgrowth of axons from RGC somata, as also observed by others (DeBernardo and Chang, 1996), nor enhance the very limited axonal elongation on PLL. The substantial axonal growth observed on Laminin, however, is significantly enhanced by DM-GRASP and growth cones become almost double their size. Increased extension of axons on an extracellular matrix protein (Fibronectin) has been also reported for the axonal CAM F11/Contactin (Treubert and Brummendorf, 1998). Enlarged RGC growth cones have been observed on substrate CAMs L1 and N-Cadherin (Burden-Gulley et al., 1995) too. The significant decrease in the variation range of growth cone complexity and of lateral extensions by DM-GRASP points to an instructive signal on growth cones supporting a more focused straightforward growth. This would be an extremely useful signal for RGC axons having reached a DM-GRASP-positive structure (a more centrally located RGC) since from then on they only have to track on older RGC axons to reach the tectum.

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The mechanisms underlying increased speed and growth cones size on DM-GRASP could be due to homophilic interactions of substrate and membrane DM-GRASP acting in concert with the Laminin-induced effects. A direct interaction of DM-GRASP with the Laminin receptor in the RGC axon membrane, Integrin a6h1 (de Curtis and Reichardt, 1993; Ivins et al., 2000), seems unlikely, since DM-GRASP does not contain any known binding motifs allowing for direct physical interactions of CAMs with Integrins, such as, for example, a Collagen-like site or a RGD sequence (D’Souza et al., 1991; Staatz et al., 1991). It is conceivable, however, that substrate DM-GRASP binds (homo- or heterophilically) to axonal membrane proteins, activating them to enhance the intracellular signaling elicited by Integrins. NgCAM/L1, the binding partner of DMGRASP (DeBernardo and Chang, 1996), has been shown to potentiate Integrin-mediated cell migration via inside-out signaling (Thelen et al., 2002), conceivably a similar mechanism could result in accelerated growth cone advance. Our findings that inhibition of NgCAM reduces preference of RGC axons for DM-GRASP substrate lanes (see below) support this notion. DM-GRASP-induced axonal preference To test whether the selective presence of DM-GRASP in the OFL might provide a guidance function for RGC axons, they were given the choice between lanes containing both DM-GRASP/Laminin and lanes providing Laminin only. This in vitro assay mimics the in vivo environment of the RGC axon: the OFL, containing DM-GRASP and Laminin, and the other layers of the early retina (GCL, NEC, basal lamina), containing no DM-GRASP but Laminin. The DMGRASP concentration we offered in vitro was considerably lower than the one present in vivo (in the OFL) or on single RGC axons in vitro as detected by immunofluorescence labelings. RGC axons clearly prefer to grow on DMGRASP-containing substrate lanes, suggesting that also, RGC axons in vivo are kept in the OFL and are prevented from straying into other retinal layers due to their preference for DM-GRASP. RGC growth cones extend in contact with the basal lamina, where Laminin is most concentrated, and the OFL, which provides axonal CAMs. This interphase presumably represents the optimal substrate combination present in the retina, in addition ‘‘rewarding’’ RGC axons by an enhanced elongation rate (see above). Since the binding partner of DM-GRASP, NgCAM, is also restricted to the OFL in the early retina (Morales et al., 1996; Pollerberg and Mack, 1994), we tested whether this CAM might be involved in the preference of RGC axons for DM-GRASP. Inhibition of NgCAM in the axonal membrane results in a clear decrease in axonal preference for DMGRASP lanes, indicating that NgCAM contributes to the DM-GRASP preference of RGCs. NgCAM might contribute to preference for DM-GRASP by acting as a direct transinteraction partner for substrate-DM-GRASP. Alternatively,

NgCAM might cis-interact with DM-GRASP in the axonal membrane (directly or indirectly) resulting in an assisted homophilic DM-GRASP trans-interaction. The preference for DM-GRASP, which remains under NgCAM inhibition, could be due to homophilic DM-GRASP interactions or (also) to binding of—yet unknown—DM-GRASP interaction partners in the axonal membrane. DM-GRASP’s role in axonal orientation Up to now, two orthologs of DM-GRASP have been studied with respect to axonal navigation in fish and fly. Antibody inhibition of Neurolin in adult goldfish retinae resulted in some axons growing halfway towards the optic disk, but then looping or turning and growing towards the periphery (Leppert et al., 1999; Ott et al., 1998). In DMGRASP-inhibited embryonic chick retinae, RGC axons grow correctly the entire way to the optic fissure and only then misnavigate, crossing onto the opposite side of the retina. Reasons for this apparent discrepancy could be technical differences, as the fish retinae were labeled by Neurolin stainings or dye crystals placed all around the retina (not unilaterally as in our experiments), which makes it very difficult to detect potential misrouting axons at the optic disk. The main reasons for the discrepancy in observations in the middle/peripheral retina are probably species differences. The fish retina is growing throughout life. In adult fish, Neurolin is only present on peripheral RGCs sending out axons, older axons are Neurolin negative and also negative for other orientation-related proteins (Bastmeyer et al., 1990; Vielmetter et al., 1991). Therefore, the growing axons in fish form only a minor, young subpopulation in a ‘‘mature’’ environment. In the chick retina like in other higher vertebrates, all RGC axons grow in a relatively short embryonic phase and all axons are positive for DMGRASP and a variety of other proteins involved in orientation. It is therefore conceivable that alternative orientation proteins present on both the growing and the older chick axons (which have already taken the correct way before inhibition) partially counterbalance the DM-GRASP inhibition, whereas the older fish axons cannot provide such proteins since they do not display them any more. Having evaluated more than 300 optic fields in 3D scans, we are sure that axonal looping and misrouting towards the peripheral retina would not have escaped our attention. Interestingly, also the presumed ortholog in fly, IrreC, was shown to play a role for axonal projections in the insect visual system, in addition to one in programmed cell death (Ramos et al., 1993). Our studies on RGC axons in the embryonic chick retina demonstrate a role of DM-GRASP in axonal navigation in higher vertebrates. The data obtained from RGC axons growing in their in vivo environment are in concordance with our in vitro findings. Substrate preference assays show that RGC axons keep to DM-GRASP-containing lanes; in vivo, RGC axons stay in the only DM-GRASP-positive

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structure, the OFL. Under DM-GRASP inhibition, RGC axons in vitro leave the DM-GRASP lanes, in vivo, however, they remain restricted to the OFL. This is most likely due to the presence of other, orientation-providing molecules in the OFL and other layers of the retina (see below), which can counterbalance in vivo. At the optic fissure, however, RGC axons react to DM-GRASP inhibition similar as they do in vitro: they stray away from the DMGRASP-positive substrate provided by older axons, which have already left the retina before the inhibition period, correctly diving into the optic nerve. Here, where turns are necessary to follow the preexisting axons, the remaining navigational cues are probably not able to functionally counterbalance the loss of DM-GRASP function. An explanation could be that the interference with homo- or heterophilic DM-GRASP interactions weakens the adhesion between growing and preexisting RGC axons. This would not affect the rather straightforward axonal elongation towards the optic fissure considerably, with the converging axon bundles providing additional haptic/mechanical guidance. At the optic fissure, however, reduced adhesion is likely to result in RGC axons drifting away from the already established pathway formed by older axons since unimpeded adhesion is probably necessary to follow their approximately rectangular turns. Alternatively, DM-GRASP, which is not an abundant CAM, might not mediate substantial adhesion but rather have an instructive effect on projecting RGCs, for example, by acting on targets such as membrane receptors and/or cytoskeletal components of axon and growth cone, as also indicated by our in vitro data. Inhibition of DM-GRASP is probably counterbalanced by other molecules involved in axonal orientation. The ubiquitous CAMs NCAM and Cadherin have been demonstrated to play a role in axonal routing (Matsunaga et al., 1988; Monnier et al., 2001; Pollerberg and Beck-Sickinger, 1993). Inhibition of L1/NgCAM in rat retinae resulted in some RGC axons heading towards the peripheral retina in flat-mounts (Brittis et al., 1995). In L1-deficient mice, RGC axons bypass the correct targets in the anterior superior colliculus; effects on axon navigation in the retina have not yet been reported (Demyanenko and Maness, 2003). A role in axonal navigation for the axonal CAMs NrCAM, Axonin-1, and F11 has been demonstrated in other regions of the nervous system (Falk et al., 2002; Perrin et al., 2001). It is therefore conceivable that these CAMs, which are present on RGC axons, might contribute to axonal orientation in the developing retina. Also, molecules others than CAMs have been shown to be involved in axonal navigation of RGCs, such as Slit-1, EphB2/3, DCC, and Netrin-1 (Birgbauer et al., 2000; Deiner et al., 1997; Jin et al., 2003). Besides membrane proteins, intracellular signaling molecules, such as Rac and MAP kinase (Schaefer et al., 1999; Schmid et al., 2000), for example, and cytoskeletal components, which are known to be modulated by or linked to CAMs (Davis and Bennett, 1994; Pollerberg et al., 1986), are involved in axonal navigation. It remains to be elucidated, however,

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how they are interacting with DM-GRASP and how these complex interactions govern the growth cone’s behavior, thereby contributing to axonal navigation.

Acknowledgments We thank Thomas Bru¨mmendorf and Fritz G. Rathjen (Max-Delbru¨ck-Centrum, Berlin) for NgCAM antibodies; Friedrich Bonhoeffer and Ju¨rgen Jung (Max-Planck-Institute for Develpomental Biology, Tuebingen) for silicone matrices; Susanne Bergmann, Claudia Brandel, Heide Schiffbauer, and Monika Zieher-Lorenz for excellent technical assistance; Ursula Klingmu¨ller and Marcel Schilling (DKFZ, Heidelberg) for help with Western Blot Quantification; Lutz Edler and Werner Rittgen for help with statistics; and Kerry Tucker (IZN Heidelberg) for critical reading of the manuscript. The work was supported by the German Research Foundation DFG (SFB 488 and Graduate Program 484).

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