Biomedical subjects
Publications and source records attributed to F Bonhoeffer.
Most monoclonal antibodies do not functionally inactivate the antigen they recognize. This limitation can be overcome by localized chromophore-assisted laser inactivation (CALI) of specific antibody-linked proteins.
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.
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In the CNS of mammals, axonal regeneration is limited by inhibitory influences of the glial and extracellular environment. Myelin-associated inhibitors of neurite growth, as well as some properties of so-called 'reactive astrocytes' which make the environment non-permissive for axonal growth, contribute to the inhibitory nature of the mammalian CNS. Furthermore, mechanisms for effective removal or neutralization of inhibitory components of cell debris are lacking in the mature mammalian CNS. However, in a permissive environment, mammalian CNS axons are able to regrow, to recognize target areas and to re-establish functional synapses with target neurones. Moreover, recent observations suggest that guiding molecules, like those required for axon guidance in the developing CNS, become expressed after lesions. Regenerating CNS axons seem to be able to recognize such guidance cues. Thus, even regenerating CNS axons of mammals might ultimately succeed in re-establishing topographically ordered functional synapses in their target regions.
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.
In the rat, a small subpopulation of retinal ganglion cell axons forms a persistent projection to the ipsilateral half of the brain. These fibres originate almost exclusively from the ventrotemporal margin of the retina. In contrast to all other retinal axons they seem to be deflected from the midline of the optic chiasm and thereby led into the ipsilateral optic tract. In order to analyse the interactions between growing fibres and chiasm midline, we have developed the following in vitro model. Axons of the embryonic rat retina are grown on a carpet of tectal cell membranes used as a general growth-permissive substratum. At a certain distance from the explant (200-450 microns), the advancing fibres are confronted with two stripes of cell membranes prepared from the chiasm midline. Such chiasm membranes are shown to act as a barrier for the presumptive non-crossing axons, while they do not influence growth of fibres originating from any other regions of the retina, including the dorsotemporal part. The repulsion of non-crossing fibres by chiasm membranes is observed in vitro only when retinal explants from embryonic day (E) 17/18 and chiasm preparations from E14/15 are used. Fibres and tissue from different regions of the brain as well as from different developmental ages, and even from different species, can be combined in this assay system. In a first attempt to characterize the molecular basis of the repulsive effect of chiasm membranes on ventrotemporal fibres, similar assays were performed with membranes derived from other regions of the central nervous system midline, some of which are known to have repulsive properties against certain axon populations. Since these cell membranes did not act as a barrier for the ventrotemporal retinal axons, we suggest that the guidance cues at the chiasm are very specific. Our results are consistent with the hypothesis that certain cells at the chiasm midline (very likely radial glial cells) express 'repulsive or inhibitory' molecules, which act in a specific way on ipsilaterally projecting axons.
Spatial gradients of axon guiding molecules have long been suspected to provide positional and directional cues for retinal ganglion cell axons growing within the optic tectum. With the identification of a guiding activity from tectal cell membranes, it has become possible to investigate the potential physiological significance of molecular gradients for retinal growth cone behavior in vitro. A subset of retinal growth cones, those from the temporal half, were highly sensitive to small concentration changes of the guiding component. The degree of response was correlated with the strength of the gradient. These findings demonstrate that the neural growth cone can read gradients of surface-associated information.
Rhodamine-labeled explants of embryonic chick retinae were placed on a substratum consisting of alternating lanes of cell membranes derived from anterior and posterior chick optic tectum. Extending axons from temporal retinae prefer to grow in vitro on anterior tectal membranes because of a repulsive component within posterior membranes obtained from a tectum area which is not innervated by these axons in vivo. None of 16 protease inhibitors, specific for all known protease classes, when added to the culture medium, neutralized the repulsive activity of posterior membranes suggesting that the repulsive activity is not a protease. However, 2 metalloprotease inhibitors affect growth cone morphology and axon-axon interactions.
An in vitro assay is presented in which different soluble substrates are arranged in narrow alternating stripes which forces growing axons and migratory cells to choose between them. The usefulness of this assay is exemplified by offering goldfish retinal axons and glial cells of the optic nerve a variety of substrates in stripes. Given a choice between substrates of unequal growth supporting activities axons and migratory cells grow in stripes, thus expressing their preference for one of the substrates. Growth in stripes was observed 1. when a substrate with growth promoting properties was next to one which did not possess these properties, 2. when the growth promoting activity of a substrate applied to both stripes was in one stripe blocked by an antibody, 3. when two different growth promoting substrates were offered.
Axonal guidance in the retinotectal system and in spinal nerve segmentation is based on repulsion or inhibition. In both systems the membrane glycoprotein responsible for the guiding activity is capable of inducing growth cone collapse. We discuss two models of axonal guidance that correlate axonal guidance and growth cone collapse. The models are applicable to axon guidance by membrane-associated or diffusible stimuli, and are not based on preferential adhesion of axons to certain substrata.
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.
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.
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In the retinotectal projection, nasal retinal axons project to posterior tectum, while temporal axons project to the anterior part of the tectum. In in vitro experiments, a similar specificity can be observed: the nasal and temporal retinal axons can be guided by tectal membrane components so that, for example, temporal retinal axons, when growing on a striped substratum consisting of anterior and posterior tectal membranes, express a very strong preference for the anterior stripes. This preference is not due to attractivity of anterior membranes but rather to avoidance of posterior material, although the pure posterior membranes are a very good substratum for growth of temporal axons. The repellent guidance molecule has been identified. Interestingly, besides guidance this molecule causes another reaction: when growing temporal axons are exposed to medium containing either posterior membranes or artificial lipid vesicles containing the repellent guidance molecule, the axonal growth cones collapse. As in guidance, there is a clear regional specificity: e.g. the repellent guidance molecule derived from posterior tectum induces collapse of temporal but not of nasal axons. Since the guiding and the collapse-inducing activity are expressed by one and the same glycoprotein molecule (Mr 33 x 10(3), linked to the membrane by phosphatidylinositol) and since another molecule has been identified by Keynes' group which also expresses both guiding and collapse-inducing activity, one might speculate that axonal guidance and axonal collapse have something in common. Models of axonal guidance will be discussed.
1. On a substrate consisting of alternating lanes of anterior and posterior tectal membranes, temporal retinal axons have a strong tendency to grow on the lanes of anterior membranes and to avoid the lanes of posterior membranes. 2. Temporal axons do extend neurites on posterior material, and in equivalent numbers and lengths to that of anterior membranes if the substrate consists of pure anterior or posterior membranes. 3. Inactivation of posterior membranes by heat or the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) abolishes their ability to induce the avoidance reaction of temporal axons. It is concluded that the posterior membranes contain a repulsive component for temporal retinal axons. 4. Growth cones growing on anterior membranes, which encounter posterior membranes at the strip boundary, in general do not become reduced in their growth rate. 5. These results are most easily explained by a "gradient-reading model" similar to chemotaxis where the steering of a growth cone is independent on the growth rate. 6. According to the model, a gradient of a guiding component outside the growth cone is transformed into an internal gradient which gives the growth cone its directionality. 7. Other models like growth inhibition cannot be ruled out but need at least two additional assumptions like habituation for growth on the putative posterior inhibitory substrate and a strong local restriction of the inhibitory effect within the growth cone which contacts the posterior material.
Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection.
The retinae of vertebrates project in a topographic manner to several visual centers of the brain. The formation of these projections could depend on the existence of position-specific properties of retinal and target cells. In this study, we have tested the in vitro growth of mouse retinal fibers on membranes derived from various regions of the embryonic superior colliculus, a main target of the retina in this species. Fibers had the choice of elongating on membranes taken from either the anterior or the posterior half of the superior colliculus. Fibers from temporal areas of the retina prefer to elongate on anterior collicular membranes, while fibers from nasal areas do not show a preference. These phenomena are observed with membranes from embryonic (E15-E18) or young postnatal mice. In interspecies cultures where mouse retinal fibers had to grow on chick tectal membranes, or vice versa, the same preference for anterior tectal or collicular membranes in growth of temporal retinal fibers is observed, suggesting some similarities in the cues used in both species.
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.