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Shared and distinct functions of RAGS and ELF-1 in guiding retinal axons

1997; Springer Nature; Volume: 16; Issue: 6 Linguagem: Inglês

10.1093/emboj/16.6.1258

1460-2075

Bruno Monschau, Claus Kremoser, Kunimasa Ohta, Hideaki Tanaka, Tomomi Kaneko, Tomoko Yamada, Claudia Handwerker, Martin R. Hornberger, Jürgen Löschinger, Elena B. Pasquale, Doyle A. Siever, Michael F. Verderame, Bernhard Müller, Friedrich Bonhoeffer, Uwe Drescher,

Retinal Development and Disorders

Article15 March 1997free access Shared and distinct functions of RAGS and ELF-1 in guiding retinal axons Bruno Monschau Bruno Monschau Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, GermanyB.Monschau and C.Kremoser contributed equally to this work Search for more papers by this author Claus Kremoser Claus Kremoser Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, GermanyB.Monschau and C.Kremoser contributed equally to this work Search for more papers by this author Kunimasa Ohta Kunimasa Ohta Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Hideaki Tanaka Hideaki Tanaka Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Tomomi Kaneko Tomomi Kaneko Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Tomoko Yamada Tomoko Yamada Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Claudia Handwerker Claudia Handwerker Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Martin R. Hornberger Martin R. Hornberger Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Jürgen Löschinger Jürgen Löschinger Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Elena B. Pasquale Elena B. Pasquale The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Doyle A. Siever Doyle A. Siever Department of Microbiology and Immunology, The Pennsylvania State University, College of Medicine, Hershey, PA, 17033 USA Search for more papers by this author Michael F. Verderame Michael F. Verderame Department of Microbiology and Immunology, The Pennsylvania State University, College of Medicine, Hershey, PA, 17033 USA Search for more papers by this author Bernhard K. Müller Bernhard K. Müller Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Friedrich Bonhoeffer Friedrich Bonhoeffer Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Uwe Drescher Corresponding Author Uwe Drescher Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Bruno Monschau Bruno Monschau Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, GermanyB.Monschau and C.Kremoser contributed equally to this work Search for more papers by this author Claus Kremoser Claus Kremoser Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, GermanyB.Monschau and C.Kremoser contributed equally to this work Search for more papers by this author Kunimasa Ohta Kunimasa Ohta Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Hideaki Tanaka Hideaki Tanaka Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Tomomi Kaneko Tomomi Kaneko Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Tomoko Yamada Tomoko Yamada Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan Search for more papers by this author Claudia Handwerker Claudia Handwerker Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Martin R. Hornberger Martin R. Hornberger Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Jürgen Löschinger Jürgen Löschinger Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Elena B. Pasquale Elena B. Pasquale The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Doyle A. Siever Doyle A. Siever Department of Microbiology and Immunology, The Pennsylvania State University, College of Medicine, Hershey, PA, 17033 USA Search for more papers by this author Michael F. Verderame Michael F. Verderame Department of Microbiology and Immunology, The Pennsylvania State University, College of Medicine, Hershey, PA, 17033 USA Search for more papers by this author Bernhard K. Müller Bernhard K. Müller Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Friedrich Bonhoeffer Friedrich Bonhoeffer Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Uwe Drescher Corresponding Author Uwe Drescher Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany Search for more papers by this author Author Information Bruno Monschau1, Claus Kremoser1, Kunimasa Ohta2, Hideaki Tanaka2, Tomomi Kaneko2, Tomoko Yamada2, Claudia Handwerker1, Martin R. Hornberger1, Jürgen Löschinger1, Elena B. Pasquale3, Doyle A. Siever4, Michael F. Verderame4, Bernhard K. Müller1, Friedrich Bonhoeffer1 and Uwe Drescher 1 1Department of Physical Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35/I, 72076 Tübingen, Germany 2Division of Developmental Neurobiology, Department of Neuroscience and Immunology, Kumamoto University Graduate School of Medical Sciences, Kuhonji, Kumamoto, 862 Japan 3The Burnham Institute, 10901 N. Torrey Pines Road, La Jolla, CA, 92037 USA 4Department of Microbiology and Immunology, The Pennsylvania State University, College of Medicine, Hershey, PA, 17033 USA The EMBO Journal (1997)16:1258-1267https://doi.org/10.1093/emboj/16.6.1258 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Two ligands for Eph-related receptor tyrosine kinases, RAGS and ELF-1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF-1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons. RAGS on the other hand is repellent for both types of axons, though to different degrees. Thus, RAGS and ELF-1 share some and differ in other properties. The biological activities of these molecules correlate with the strength of interaction with their receptors expressed on RGC axons. The meaning of these findings for guidance of retinal axons in the tectum is discussed. Introduction The processes by which retinal ganglion cell axons find their correct position within the target area, the optic tectum, are a matter of long-standing investigation. The retinotectal projection represents a popular model system for the study of topographic projections, which are numerous in the nervous system and of central importance for brain functioning (Udin and Fawcett, 1988; Holt and Harris, 1993; Roskies et al., 1995; Tessier-Lavigne, 1995; Goodman, 1996). The principle of topographic projections is to transfer faithfully spatially organized information from one group of neurons, the projecting area, onto another group of neurons, the target area. A number of hypotheses have been put forward to explain the development of these projections involving either fibre–fibre interactions (Willshaw and Malsburg, 1979), time of arrival at the target (Rager, 1976) or synaptic stabilization due to functional validation (Willshaw and Malsburg, 1976; Whitelaw and Cowan, 1981). The currently most favoured hypothesis is based on the chemoaffinity theory proposed by Sperry some decades ago (Sperry, 1963). He suggested the presence of cytochemical tags on cells in both the projecting area and the target area. These tags would provide each cell with positional information, allowing a 'matching' of corresponding cells in the projecting and target areas, so that invading retinal axons carrying certain receptor molecules interact specifically with their corresponding counter-receptors on the tectum. He excluded a mosaic of many different molecules on both the retina and the tectum (mosaic theory), arguing that this would require too much genetic information as well as extensive random searching by invading axons for target positions, at least for misrouted fibres. Instead, he proposed quantitative differences, i.e. a graded expression of these cytochemical tags, meaning that positional information would be encoded in the form of relative amounts of a few molecules providing directional as well as positional cues. In order to specify internal target positions in the tectum, 'two antagonistic gradients—or at least two spatially antagonistic effects arising from the same graded distribution' (Gierer, 1988)—have been postulated for both the dorsoventral and nasotemporal axes, whereby the combined effect of these gradients leads to a local maximum or minimum of a guiding parameter (Gierer, 1983, 1988). For the retina, it is proposed that invading axon populations express cell surface receptors in a graded manner specifying the position of origin. In recent years, a number of molecules have been identified which are expressed in a graded manner in either the retina or the tectum, or both, such as TOPAP and TOPDV (Savitt et al., 1995), TRAP (McLoon, 1991) and a 33 kDa protein (Stahl et al., 1990). In the retinotectal projection, axons from the temporal retina project to the anterior tectum, and axons from the nasal retina to the posterior tectum. In the perpendicular axis, dorsal retina is connected to ventral tectum and ventral retina to dorsal tectum (Mey and Thanos, 1992; Holt and Harris, 1993). In vitro assays established in recent years have provided an insight into the nature of guidance cues along the anteroposterior axis. In the stripe assay—where RGC axons are allowed to grow on alternating stripes of membranes from the anterior and posterior tectum—temporal axons are found to grow only on anterior membranes, thus reproducing the in vivo situation. Preference for anterior membranes was found to be due to repulsion by posterior membranes. The collapse assay (Cox et al., 1990; Raper and Kapfhammer, 1990), in which retinal ganglion cells growing on a laminin-coated surface are exposed to membrane fragments of interest and their behaviour is documented by time-lapse video microscopy, suggests that repulsion is due to collapse-inducing molecules in the posterior tectum. The repulsive molecules appear to be glycosylphosphatidylinositol (GPI)-anchored to membranes and have a graded distribution in the tectum, with maximal concentration in the posterior part. These criteria were used to purify this activity and led to the cloning of a 25 kDa tectal protein named RAGS (for repellent axon guidance signal; Drescher et al., 1995). RAGS emerged as a ligand for Eph-related receptor tyrosine kinases (for reviews, see Brambilla and Klein, 1995; Pandey et al., 1995a). It was active in both the collapse and stripe assays, but had similar activity for temporal and nasal axons, in contradiction of its anticipated role, i.e. to be selectively repellent for temporal retinal axons. This was taken to indicate the existence of additional and/or modulatory activities conferring nasotemporal specificity (Drescher et al., 1995). Besides RAGS, another member of this family of Eph ligands, ELF-1 (for Eph ligand family-1), is known to be expressed in the tectum (Cheng and Flanagan, 1994). ELF-1 was cloned in a search for ligands of Eph-related receptors by using receptor alkaline phosphatase fusion proteins as probes. One of the receptors used, Mek4, and its corresponding ligand, ELF-1, were then found to be expressed in complementary gradients in the retina and tectum (Cheng et al., 1995), in agreement with Sperry's concept. Here, we show that ELF-1 and RAGS are expressed in the tectum in partially overlapping domains during the time of invasion of retinal axons. The present investigation concentrates on a comparative functional characterization of these two molecules. In a re-evaluation of RAGS function in the stripe assay, it transpires that this molecule can produce a concentration-dependent differential guidance of nasal and temporal axons. ELF-1, on the other hand, seems to have a bimodal effect in that it guides temporal axons but has apparently no effect on nasal axons. The activities of these molecules correlate with the strength of their interaction with the Cek4 receptor expressed on RGC axons. A preliminary model is presented that shows how RAGS and ELF-1 can account for the initial formation of the anteroposterior axis of the retinotectal projection. Results Identification of GPI-anchored Eph-related ligands in the tectum In order to clone additional members of the family of ligands for Eph-related receptors expressed in the tectum, we used a cocktail of different probes derived from various Eph ligands for a low-stringency hybridization of a posterior tectum cDNA library (see Materials and methods). However, the only other GPI-anchored ligand different from RAGS was identified as ELF-1. Comparative RNA expression analysis of RAGS and ELF-1 To study the functional significance of the simultaneous expression of two closely related Eph ligands in the tectum, we performed a detailed RNA expression analysis of both molecules at various developmental stages using DIG-labelled RNA probes (Figure 1A–E). Figure 1.RNA expression of RAGS and ELF-1 in the developing visual system. (A–E) Whole mounts or isolated tecta were hybridized with ELF-1- (A, C and E) and RAGS- (B, D and E) specific DIG-labelled antisense probes. (A and B) Day 4 embryo, viewed laterally; (C and D) day 7 embryo, viewed ventrally; posterior is to the left. (E) Day 9 embryo, viewed dorsally; the tecta on the left side were hybridized with an ELF-1-specific probe, those on the right side with a RAGS-specific probe. Posterior poles of the tecta are oriented toward the centre of the figure. Colour reactions were stopped usually after ∼1.5 h for ELF-1 and ∼6 h for RAGS. Prolonged reaction times do not lead to an obvious staining of the anterior tectum for RAGS, while for ELF-1 the whole tectum is stained. Owing to differences in GC content the ELF-1 probe was found to be ∼8-fold more sensitive than the RAGS probe (see Materials and methods). Download figure Download PowerPoint At all time points analysed, RAGS and ELF-1 RNAs are expressed in gradients, with higher expression in the posterior part of the tectum. The ELF-1 expression domain at E4 covers the entire tectum (Figure 1A), whereas later expression in the anterior part seems to be reduced (Figure 1C and E). In contrast, the expression domain of RAGS is generally found more restricted to the posterior part of the tectum throughout the developmental time analysed (Figure 1B, D and E). A very strong expression at the posterior pole is apparent. In summary, both RAGS and ELF-1 are expressed in gradients in the tectum, but the RAGS gradient seems to be steeper and more confined to the posterior part of the tectum than ELF-1. Localization of RAGS and ELF-1 protein in the developing tectum If RAGS and ELF-1 function as axon guidance molecules, then retinal axons should co-localize with RAGS and ELF-1 protein during their ingrowth through the superficial layers of the tectum. RNA coding for RAGS is primarily located not in superficial, but in ventricular inner cell layers (Drescher et al., 1995). These layers contain radial glial cells possessing processes which span the tectum, ending in endfeet on the surface of the tectum. To localize RAGS and ELF-1 protein, an immunohistochemical analysis was performed using monoclonal antibodies specific for these two ligands (see Materials and methods). As shown in Figure 2B, RAGS protein can be detected not only in ventricular, but also in other layers of the tectum, including the superficial layers. At higher magnification, staining of processes which span the tectum from inner to outer layers can be identified (Figure 2E). This suggests that part of the observed expression pattern of RAGS protein reflects a process in which this ligand is produced in radial glial cells and then transported into the endfeet at the surface of the tectum, which is in contact with invading retinal axons. RAGS protein should also be expressed in other cell types, as corresponding RNA (at lower levels) can also be found in intermediate layers (Drescher et al., 1995). It is also conceivable that part of the observed protein distribution reflects other mechanisms such as migration of RAGS-expressing cells from inner to more superficial layers of the tectum. Figure 2.Expression of RAGS and ELF-1 protein in the developing tectum. Immunohistochemical analysis of the expression pattern of Eph ligands in para-sagittal sections of E9 chicken tecta using monoclonal antibodies specific for ELF-1 (A) and RAGS (B). Composite pictures are shown. In each case pictures were taken under manual control with the same exposure times (RAGS: 10 s; ELF-1: 5 s). Anterior is to the left, posterior to the right. (C and D) Measurements of fluorescence intensity of the outer surface (top) of these tecta (see Materials and methods). (E) Magnification of a staining of an E9 posterior tectal section with a RAGS-specific monoclonal antibody. Download figure Download PowerPoint ELF-1 protein can be detected in similar locations to RAGS and is therefore also accessible to contact by ingrowing axons (Figure 2A). A quantification of RAGS and ELF-1 immunofluorescence staining (shown in Figure 2C and D) is consistent with the corresponding RNA expression data (Figure 1), in that the gradient of RAGS protein appears to be steeper and more confined to the posterior part of the tectum compared with the ELF-1 expression pattern. Binding of RAGS and ELF-1 to Eph-related receptors expressed in the retina We set out to identify the cytochemical tags on RGC axons corresponding to these ligands. A characteristic of the Eph-related family is the promiscuity in the interaction of receptors and both GPI-anchored (Cheng and Flanagan, 1994; Davis et al., 1994; Kozlosky et al., 1995) and transmembrane ligands (Bergemann et al., 1995; Brambilla et al., 1995), which might also hold true for the two ligands RAGS and ELF-1. Therefore, we focused on Eph-related RTKs which are believed to interact specifically with GPI-anchored ligands, namely Eck (Lindberg and Hunter, 1990), Cek4 (Sajjadi et al., 1991), Cek7 (Siever and Verderame, 1994) and Cek8 (Sajjadi and Pasquale, 1993). As the Eck receptor is not expressed at relevant times in the visual system (Ganju et al., 1994; Ruiz and Robertson, 1994), we concentrated on the latter three. Cek7 was the prime candidate for the relevant RAGS receptor, as it was shown that a species homologue of this receptor, Rek7, interacts specifically with the human homologue of RAGS, AL-1 (Winslow et al., 1995). An immunohistochemical analysis performed between E9 and E13 showed expression of Cek7 in various layers of the retina, including the RGC layer, with no obvious gradient along the anteroposterior axis (D.A.Siever and M.F. Verderame, manuscript in preparation). As shown by Cheng et al. (1995), Cek4 and Cek8 are expressed at E8 in the retina. Cek4 is expressed differentially in the RGC layer, with higher expression in the temporal half than in the nasal half, while Cek8 is expressed uniformly. However, on the basis of Northern blot analyses from E7 retina, Cek8 RNA seems to be slightly more abundant in the nasal half of the retina (data not shown). This finding correlates with the time of differentiation of retinal ganglion cells of temporal and nasal retina (Rager et al., 1993). All three receptors were shown immunohistochemically, by using specific antibodies, to be located on RGC axons (Figure 3). An analysis of the binding affinities of these three receptors to RAGS and ELF-1 is therefore essential for dissecting their biological function. Figure 3.Expression of Cek4, Cek7 and Cek8 on E6 retinal ganglion cell axons and growth cones. Axons grown from retinal explants in vitro were immunostained with (C and D) Cek4, (E) Cek7 and (F) Cek8 antisera. Controls were done without primary antibody, visualized by (A) phase-contrast and (B) fluorescence microscopy. Cek4 staining was stronger on axons grown from (C) temporal compared with (D) nasal retinal explants. No such difference was seen for Cek7 and Cek8 staining. Download figure Download PowerPoint For a precise quantification of binding affinities, the receptor alkaline phosphatase (RAP) technique (Flanagan and Leder, 1990) was used. Various fusion proteins containing the extracellular domain of individual receptors linked to the coding region of alkaline phosphatase (AP) were generated. These are soluble tags and were used to probe Cos cells expressing the ligands. Dissociation constants for receptor–ligand pairs were then determined on the basis of a Scatchard analysis (Scatchard, 1949). As shown in Figure 4 and illustrated diagrammatically in Figure 5, the strongest interaction was seen between Cek4 and RAGS, with a dissociation constant of 1.44×10−10 M. ELF-1, in contrast, bound to Cek4 with a Kd of 8.60×10−10 M. The interaction of ELF-1 with Cek4 was in the same range as the interaction of RAGS with the Cek7 and Cek8 receptors (6.16×10−10 M and 6.22× 10−10 M). The interaction of ELF-1 with Cek7 and Cek8 was weakest with Kds of 8.62×10−9 M and 1.27×10−8 M, respectively. In summary, three different categories of interactions with respect to Kd values are evident: a very strong binding of RAGS to Cek4, a strong interaction of RAGS with Cek7 and Cek8, very similar to the binding of ELF-1 to Cek4, and a weak binding of ELF-1 to Cek7 and Cek8. Figure 4.Binding of RAGS and ELF-1 to Eph-related receptors. Scatchard analysis of the binding of Cek4-AP, Cek7-AP and Cek8-AP to membrane-bound RAGS and ELF-1. Hyperbolic representations are shown as insets. The binding characteristics calculated from these experiments are shown schematically in Figure 5. Download figure Download PowerPoint Figure 5.Logarithmic representation of dissociation constants for Cek4-AP, Cek7-AP, and Cek8-AP, and RAGS and ELF-1. After regression analysis of the binding data shown in Figure 4, the negative reciprocal slope from the Scatchard equation was taken as the dissociation constant. Download figure Download PowerPoint In a further investigation of the interaction between receptors and ligands, it could be shown that ELF-1 and RAGS can induce the phosphorylation of both Cek4 and Cek8 (Ohta et al., 1996; K.Ohta, H.Iwamasa, U.Drescher, H.Terasaki and H.Tanaka, manuscript in preparation), as is true for Cek7 (Shao et al., 1995; Winslow et al., 1995). Very recent studies by Gale et al. (1996) have shown that RAGS and ELF-1 can also bind to the Eph-related receptors Ehk-2 (Maisonpierre et al., 1993) and Ehk3/Mdk1 (Ciossek et al., 1995; Valenzuela et al., 1995). Further investigations will be directed toward a possible expression of these receptors in the retinotectal system. Comparative functional analysis of ELF-1 and RAGS The expression of ELF-1, a member of the same ligand family as RAGS, in the tectum and its interaction with the same set of RGC-expressed, Eph-related receptors as RAGS suggests an involvement of this molecule in the formation of the retinotectal projection. To investigate this, an elf-1 cDNA-containing expression plasmid was transfected into Cos cells; 2 days later membranes from these cells were isolated and analysed in the stripe assay. These membranes were prepared in alternating lanes with mock-transfected Cos cell membranes. Strikingly, in this set of experiments, temporal axons avoided ELF-1-containing Cos cell membranes, while nasal axons grew equally well on both types of membranes, indicating a very clear in vitro guidance activity of ELF-1 for temporal but, at least under these experimental conditions, not for nasal axons (Figure 6A). This clear-cut difference in axon guidance is comparable with that seen in 'standard' stripe assays using anterior and posterior tectal membranes (Walter et al., 1987). Figure 6.Functional characterization of ELF-1 and RAGS in the stripe assay. Nasal axons (N) and temporal axons (T) were given the choice of growing on membranes derived either from mock-transfected Cos cells or from Cos cells transfected with (A) ELF-1 or (B and C) RAGS. The ELF-1/RAGS-transfected cell membranes are labelled with rhodamine isothiocyanate (RITC) fluorescent beads, visualized in the lower part of each figure. RAGS-transfected Cos cells were used at dilutions of 1:2 (B) and 1:4 (C). Retinal ganglion cells were stained with DiAsp during preparation of retinal tissue. Download figure Download PowerPoint To reinforce conclusions from the stripe assay, ELF-1 function was analysed in the collapse assay. ELF-1-containing membranes from transiently transfected Cos cells elicited only a weak response in the collapse assay (data not shown), whereas membranes derived from the same transfection led to a guidance of temporal axons in the stripe assay (see above). This result suggests that the stripe assay is more sensitive in detecting molecules with a potential axon guidance activity than is the collapse assay. In making this comparison it is assumed that both assays detect mechanistically similar activities (Walter et al., 1990; Fan and Raper, 1995). To increase the relative amounts of ELF-1 in the relevant membrane fractions, we established human 293 cell lines stably expressing high amounts of ELF-1. As estimated from a rough quantification of ELF-1 by determining Cek4-AP binding activity (see Materials and methods), membranes from selected cell lines contain ∼8-fold higher concentrations of ELF-1 than do transiently transfected Cos cells. With membranes containing higher amounts of ELF-1, a strong collapse-inducing activity was detected. As in the stripe assay, this transpired to be specific for temporal RGC growth cones. Here, 5 μg of 293/ELF-1 membranes induced 100% collapse of temporal (34/34), but only 16.7% collapse of nasal growth cones (4/24). In control experiments using the same amount of mock-transfected 293 cell membranes, retinal growth cones were barely affected [temporal growth cones: 6.3% (2/32); nasal growth cones: 14.8% (4/27)]. Even with very high amounts of ELF-1 membranes, no effect on nasal growth cones was seen. This indicates a broad concentration range in which ELF-1 shows a bimodal effect on temporal versus nasal axons. Further experiments using still higher amounts of membranes were not carried out because they caused severe non-specific growth cone collapse. The interaction of RAGS with the relevant Eph-related receptors expressed on RGC axons led us to functionally re-characterize RAGS itself. RAGS binds with high affinity to all three receptors, but owing to differences in binding affinity (Kd 1.44×10−10 for Cek4, Kd 6.16×10−10 for Cek7, Kd 6.22×10−10 for Cek8; Figures 4 and 5) it should be expected that at higher concentrations all three receptors will be activated, but at lower concentrations the Cek4 receptor, which is expressed more strongly on temporal axons, will be preferentially activated. In the collapse assay, it became apparent that RAGS at 10 μg of total membrane protein shows growth cone collapse-inducing activity with little topographic specificity (Figure 7). However, as predicted from the biochemical data, at lower amounts of membranes (e.g. 3 μg), temporal and nasal growth cones showed a distinct difference in their sensitivity to RAGS. Even at these low amounts of membrane, 50% of temporal growth cones collapsed, while nasal axons were no longer affected (Figure 7). Figure 7.Growth cone collapse-in