Friedrich Bonhoeffer

In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

The results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily.

RGM is a repulsive guidance molecule for retinal axons

Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.

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

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.

Biochemical characterization of a putative axonal guidance molecule of the chick visual system.

Temporal retinal axons growing in vitro on carpets of tectal membranes are deflected by cell membranes of posterior tectum. The activity responsible for this deflection can be abolished by antibodies raised against tectal membranes and the corresponding Fab fragments. Analysis of tectal membranes by two-dimensional gel electrophoresis and immunoblotting reveals a 33 kd glycoprotein that has a higher concentration in posterior than in anterior tectum. Its expression is developmentally regulated, and it is sensitive to phosphatidylinositol-specific phospholipase C. These are properties expected for a molecule responsible for the phenomena observed in experiments on in vitro guidance of retinal axons.

Axonal guidance in the chick visual system: Posterior tectal membanes induce collapse of growth cones from the temporal retina

Membranes from posterior and anterior thirds of the chick optic tectum were added to explants from nasal and temporal retina. Posterior membranes, and to a lesser extent anterior membranes, cause temporal growth cones to collapse and their axonal processes to retract. Neither tectal source has an effect on nasal growth cones. We interpret these results to mean that there is a tectal activity, stronger in the posterior than the anterior region of the tectum, which helps guide growth cones during the development of the retinotectal map. We believe that in vivo this activity helps to steer temporal growth cones away from the posterior tectum. Nasal growth cones, which must map to the posterior tectum, are resistant to it. In vitro, when posterior membranes contact temporal growth cones over their surface, filopodia and lamellipodia withdraw rapidly. This leads to loss of contact between the growth cone and the substrate, followed by collapse.

The Eph family in retinal axon guidance

The continued functional characterization of Eph-related receptors and ligands has provided further information toward an understanding of the mechanisms controlling the retinotectal projection. Recent in vivo analyses have strengthened the idea that Engrailed defines the positional identity of the tectum along the anteroposterior axis, possibly by regulating the expression of Eph family members.

Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression.

BACKGROUND Expression of the homeobox-containing gene Engrailed (En) in an increasing rostral-to-caudal gradient in the dorsal mesencephalon is the earliest known marker for polarity of the chick optic tectum. In heterotopic transplantation experiments, En protein expression correlates well with the subsequent gradient of cytoarchitecture as well as the pattern of retinotectal projections. The En gradient also correlates with the expression of two putative retinal axon-guidance molecules, RAGS and ELF-1, which are Eph-like receptor tyrosine kinase ligands that may function in the establishment of retinotopic projections by excluding temporal axons from the caudal tectum. RESULTS To examine the function of En in determining tectal polarity, we used the replication-competent retroviral vector RCAS to misexpress mouse En-1 throughout the chick tectal primordium. Our results show that the rostral portion of the tectum adopts a caudal phenotype: the gradient of cytoarchitectonic differentiation is abolished, and the molecular markers RAGS and ELF-1 are strongly expressed rostrally. In addition, cell membranes from rostral tectum of RCAS En-1-infected embryos preferentially repel temporal axons in in vitro membrane stripe assays. CONCLUSIONS These results are consistent with a role for En in determining rostrocaudal polarity of the developing tectum. The demonstration that both RAGS and ELF-1 are upregulated following En misexpression provides a molecular basis for understanding the previous observation, also based on retrovirus-mediated En misexpression, that nasal axons form ectopic connections in rostral tectum, from which temporal axons are excluded.

Appearance of target-specific guidance information for regenerating axons after CNS lesions

During development of the vertebrate visual system, an orderly projection of ganglion cells from the retina onto the superior colliculus (SC) is established. Mechanisms that might govern this process include the coordinated action of guidance and corresponding receptor molecules that are specifically distributed on the axons and their targets. In birds and mammals, information for axonal guidance and targeting appears to be confined to the time when the retinocollicular projection is being formed. Here we show that putative guidance activities for temporal and nasal retinal axons, which are not detectable in the normal adult SC, appear after optic nerve transection in adult rats. Both embryonic and adult retinal axons are able to respond to these guiding cues, although the guidance activities detectable in the deafferented adult rat SC might be different from those found during development. These findings imply that it might be possible to reestablish an ordered projection after lesions in the adult mammalian visual system.

Neurofascin: A novel chick cell-surface glycoprotein involved in neurite-neurite interactions

We have identified neurofascin, a novel chick cell-surface glycoprotein involved in neurite-neurite interactions. Neurofascin is defined by its reactivity with monoclonal antibody (MAb) F6, which detects two polypeptides (160 and 185 kd) in immunotransfers of brain plasma membrane proteins. Immunoaffinity chromatography using immobilized MAb F6 yields major molecular mass bands at 185, 160, 135-110, and 92 kd. Fingerprint analyses show that these polypeptides are related. Neurofascin is expressed primarily in fiber-rich areas of embryonic cerebellum, spinal cord, and retina. Fab fragments of polyclonal antibodies to neurofascin interfere with the outgrowth of retinal and sympathetic axons in two different in vitro bioassays. Neurofascin is immunologically distinct from other known neurite-associated surface glycoproteins.

Growth cone navigation in substrate-bound ephrin gradients

Graded distributions of ephrin ligands are involved in the formation of topographic maps. However, it is still poorly understood how growth cones read gradients of membrane-bound guidance molecules. We used microcontact printing to produce discontinuous gradients of substrate-bound ephrinA5. These consist of submicron-sized protein-covered spots, which vary with respect to their sizes and spacings. Growth cones of chick temporal retinal axons are able to integrate these discontinuous ephrin distributions and stop at a distinct zone in the gradient while still undergoing filopodial activity. The position of this stop zone depends on both the steepness of the gradient and on the amount of substrate-bound ephrin per unit surface area. Quantitative analysis of axon outgrowth shows that the stop reaction is controlled by a combination of the local ephrin concentration and the total amount of encountered ephrin, but cannot be attributed to one of these parameters alone.