Recognition of position-specific properties of tectal cell membranes by retinal axons in vitro

In order to test the preference of growing axons for membrane-associated positional specificity a new in vitro assay was developed. In this assay, membrane fragments of two different sources are arranged as a carpet of very narrow alternating strips. Axons growing on such striped carpets are simultaneously confronted with the two substrates at the stripe borders. If there is a preference of axons for one or the other substrate they become oriented by the stripes and grow within the lanes of the preferred substrate. Such preferential growth could, in principle, be due to affinity to attractive factors on the preferred stripes or avoidance of repulsive factors on the alternate stripes. This assay system was used to investigate growth of chick retinal axons on tectal membranes. Tissue strips cut from various areas of the retina were explanted and the extending axons were confronted with stripes of cell membranes from various areas within the optic tectum. Tectal cell membranes prove to be an excellent substrate for the growth of retinal axons. Nasal and temporal axons can grow well on membranes of both posterior and anterior tectal cells. If, however, temporal axons are given a choice and encounter the border between anterior and posterior membranes they show a marked preference for growth on membranes of the anterior tectum, their natural target area. Nasal axons do not show a preference in this assay system. The transition from nasal to temporal properties within the retina is abrupt. In contrast, the transition from anterior to posterior properties of the tectal cell membranes occurs as a smooth gradient. Significantly, the positional differences of tectal membrane properties are only seen during the period of development of the retinotectal projection and are independent of tectal innervation by retinal axons. These anterior-posterior differences disappear by embryonic day 14.     

Rotation of the tectal primordium reveals plasticity of target recognition in retinotectal projection

Retinotectal projection is precisely organized in a retinotopic manner. In normal projection, temporal retinal axons project to the rostral part of the tectum, and nasal axons to the caudal part of the tectum. The two-dimensional relationship between the retina and the tectum offers a useful experimental system for analysis of neuronal target recognition. We carried out rotation of the tectal primordium in birds at an early stage of development, around the 10-somite stage, to achieve a better understanding of the characteristics of target recognition, especially the rostrocaudal specificity of the tectum. Our results showed that temporal retinal axons projected to the rostral part of the rotated tectum, which was originally caudal, and that nasal axons projected to the caudal part of the rotated tectum, which was originally rostral. Therefore, the tectum that had been rotated at the 10-somite stage received normal topographic projection from the retinal ganglion cells. Rostrocaudal specificity of the tectum for target recognition is not determined by the 10-somite stage and is acquired through interactions between the tectal primordium and its surrounding structures.     

Xenopus temporal retinal neurites collapse on contact with glial cells from caudal tectum in vitro.

Nasal and temporal retinal neurites were confronted in culture with glial cells from the rostral and caudal parts of the optic tectum and with glial cells from the diencephalon. Twenty of each of the six classes of encounter between individual growth cones and isolated glial cells were analysed by time-lapse videorecording. The results show that growth cones from the temporal retina collapse when they contact glial cells from the caudal tectum, but do not collapse when they contact glia from other areas. Growth cones of nasal retinal fibres do not collapse on contact with any of the glial types examined. This suggests that the inhibitory phenomena described by others are in part due to the cell surface characteristics of glial cells, and that there are differences between glia from the front and back of the optic tectum.     

Topographic targeting and pathfinding errors of retinal axons following overexpression of ephrinA ligands on retinal ganglion cell axons

In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, we have shown that retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal tectum. The behavior of nasal axons, however, was only marginally affected. Here, we show that overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, we found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection.     

Axonal guidance in the chick visual system: posterior tectal membranes 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.     

Behavior of fish retinal growth cones encountering chick caudal tectal membranes: A time-lapse study on growthcone collapse

In a cross species in vitro assay, growth cones from fish temporal retina elongating on laminin lanes were observed with time-lapse videomicroscopy as they encountered lanes and territories that carried membrane fragments from the chick caudal tectum. Caudal tectal membranes of adult fish and embryonic chick are known to possess a repellent guiding component for temporal retinal axons. The caudal membranes of chick exert a particularly strong influence on fish temporal axons. Contacts with chick caudal membranes by just a few filopodia and parts of the lamellipodia evoked a turning response away from the membrane lane of the entire growth cone. Contacts by filo- and lamellipodia over the entire circumference of the growth cone, however, caused invariably growth cone collapse and retraction. During growth cone turning and collapse and retraction, filopodia remained in contact with the tectal membrane fragments, suggesting strong membrane–filopodia adhesion simultaneous to growth cone repulsion by the repellent guiding component. © 1993 John Wiley & Sons, Inc.     

Establishment of rostrocaudal polarity in tectal primordium: engrailed expression and subsequent tectal polarity.

In the E4 (embryonic day 4) chick tectal primordium, engrailed expression is strong at the caudal end and gradually weakens toward the rostral end. We used quail-chick chimeric tecta to investigate how the caudorostral gradient of engrailed expression is established and whether it is correlated with the subsequent rostrocaudal polarity of tectal development. To examine the positional value of the tectal primordium, we produced ectopic tecta in the diencephalon by transplanting a part of the mesencephalic alar plate heterotopically. In the ectopic tectum, the gradient of the engrailed expression reversed and the strength of the expression was dependent on the distance from the mes-diencephalon junction; the nearer the ectopic tectum was to the junction, the weaker the expression was. Consequently, the pattern of the engrailed expression in the host and ectopic tecta was nearly a mirror image, suggesting the existence of a repressive influence around the mes-diencephalon junction on the engrailed expression. We examined cytoarchitectonic development in the ectopic tecta, which normally proceeds in a gradient along the rostrocaudal axis; the rostral shows more advanced lamination than the caudal. In contrast, the caudal part of the ectopic tecta (near to the mes-diencephalon junction) showed more advanced lamination than the rostral. In both the host and ectopic tecta, advanced lamination was observed where the engrailed expression was repressed, and vice versa. Next we studied the correlation between engrailed expression and retinotectal projection from a view of plasticity and rigidity of rostrocaudal polarity in the tectum. We produced ectopic tecta by anisochronal transplantations between E3 host and E2 donor, and showed that there is little repressive influence at E3 around the mes-diencephalon junction. We then made chimeric double-rostral tectum (caudal half of it was replaced by rostral half of the donor tectum) or double-caudal tectum at E3. The transplants kept their original staining pattern in hosts. Consequently, the chimeric tecta showed wholly negative or positive staining of engrailed protein on the grafted side. In such tecta retinotectal projection pattern was disturbed as if the transplants retained their original position-specific characters. We propose from these heterotopic and anisochronal experiments that the engrailed expression can be a marker for subsequent rostrocaudal polarity in the tectum, both as regards cytoarchitectonic development and retinotectal projection.     

Modulation of EphA receptor function by coexpressed ephrinA ligands on retinal ganglion cell axons

The Eph family is thought to exert its function through the complementary expression of receptors and ligands. Here, we show that EphA receptors colocalize on retinal ganglion cell (RGC) axons with EphA ligands, which are expressed in a high-nasal-to-low-temporal pattern. In the stripe assay, only temporal axons are normally sensitive for repellent axon guidance cues of the caudal tectum. However, overexpression of ephrinA ligands on temporal axons abolishes this sensitivity, whereas treatment with PI-PLC both removes ephrinA ligands from retinal axons and induces a striped outgrowth of formerly insensitive nasal axons. In vivo, retinal overexpression of ephrinA2 leads to topographic targeting errors of temporal axons. These data suggest that differential ligand expression on retinal axons is a major determinant of topographic targeting in the retinotectal projection.     

Restoration of retino-tectal connections in frogs during regeneration of the optic nerve

At various times after unilateral division of the optic nerve in the frogRana temporaria L. evoked potentials in response to electrical stimulation of the optic nerve were investigated in a segment distal to the site of operation, spike activity was recorded from endings of regenerating and intertectal axons when stimuli of different shapes were placed in the field of vision, and the distribution of axonal bulbs of growth by depth in the tectum mesencephal was studied electron-microscopically. During regeneration of the axons the responses of the retinal ganglionic cells to visual stimuli retained most of their individual features. Myelinated axons of the retinal ganglionic cells regenerate first (starting on the 21st day after operation). Myelination of these fibers lags significantly behind their growth and is complete more than 100 days after the operation. Unmyelinated axons of the retinal ganglionic cells grow up toward the tectum mesencephali after myelinated axons (80 or more days after the operation). Axonal bulbs of growth in the initial periods after the operation are located close to the pial surface and the level of spread of the myelinated axons of the retinal ganglionic cells differs significantly from their normal level of localization. Intertectal connections persist after division of the nerve and are activated by visual stimuli during regeneration of the axons of the retinal ganglionic cells. Connections were found mainly between intertectal fibers terminating superficially and retinal ganglionic cells belonging to class 1 and 2 detectors. Axons of the retinal ganglionic cells grow up toward the caudal region of the tectum mesencephali later than toward the rostral region.A. N. Severtsov Institute of Evolutionary Morphology and Ecology of Animals, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 5, No. 6, pp. 611–620, November–December, 1973.     

In vitro experiments on axon guidance demonstrating an anterior-posterior gradient on the tectum.

Axonal growth cones originating from explants of embryonic chick retina were simultaneously exposed to two different cell monolayers and their preference for particular monolayers as a substrate for growth was determined. These experiments show that: (1) nasal retinal axons can distinguish between retinal and tectal cells; (2) temporal retinal axons can distinguish between tectal cells that originated from different positions within the tectum along the antero-posterior axis; (3) axons originating from nasal parts of the retina have different recognizing capabilities from temporal axons; (4) the property of the tectal cells, which is attractive for temporal axons, has a graded distribution along the antero-posterior axis of the tectum; and (5) this gradient also exists in non-innervated tecta.     

Changes in retinal arbors in compressed projections to half tecta in goldfish

In adult goldfish, electrophysiological studies have shown that the retinotectal projection reorganizes, following removal of half of the tectum, to form a complete but compressed projection over the remaining half tectum. As a result, each fiber terminates more rostrally than normal. Electron microscopic studies suggest a competition between retinal fibers for a fixed number of synaptic sites. The current study examines whether retinal arbors in the compressed projection are smaller than normal in extent or branching and whether the fiber paths in the tectum show the rostral movements and the search strategy that the retinal fibers use. The caudal half tectum was removed without cutting retinal fibers except those at the cut edge. At 3 to 19 months afterward, retinal fibers were labeled with horseradish peroxidase. In whole-mounted tecta, fibers and terminals were drawn under camera lucida and compared with normal arbors. The axonal paths were also traced across the tectum to their termination sites. At 3 to 6 months (early stages of compression), the arbors were rather normal in appearance, although they were actually significantly larger (23%) than normal in linear extent, arborized somewhat deeper and had fewer branches (18%). The fibers normally terminating in the rostral tectum followed normal stereotyped paths, whereas those cut at the edge had grown back and forth loops (apparent searching behavior) with little branching. By 10 months when compression is complete, arbors were significantly smaller than normal (19%), were arborizing significantly deeper, and had significantly fewer branches (19%). The differences were more pronounced in arbors of coarse and medium caliber than in fine caliber axons. The axons still ran in stereotyped fascicles, but included an extrafascicular portion that, unlike any axons in normals, turned back in a rostral direction before branching. This striking effect, present even in far rostral tectum, indicated that arbors had been forced to move rostrally to accomodate those from the ablated half. The small effect on arbor extent suggests that this is influenced by factors other than the magnification factor of the map, perhaps postsynaptic dendritic extent. The increased depth of termination is consistent with the increased thickness of the retinal terminal layer. The decreased number of branches is consistent with the conclusion that the remaining fixed number of synaptic sites shared among the full complement of retinal fibers should result in fewer synapses per retinal fiber. © 1995 John Wiley & Sons, Inc.     

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.     

Two homeobox genes define the domain of EphA3 expression in the developing chick retina

Graded expression of the Eph receptor EphA3 in the retina and its two ligands, ephrin A2 and ephrin A5 in the optic tectum, the primary target of retinal axons, have been implicated in the formation of the retinotectal projection map. Two homeobox containing genes, SOHo1 and GH6, are expressed in a nasal-high, temporal-low pattern during early retinal development, and thus in opposing gradients to EphA3. Retroviral misexpression of SOHo1 or GH6 completely and specifically repressed EphA3 expression in the neural retina, but not in other parts of the central nervous system, such as the optic tectum. Under these conditions, some temporal ganglion cell axons overshot their expected termination zones in the rostral optic tectum, terminating aberrantly at more posterior locations. However, the majority of ganglion cell axons mapped to the appropriate rostrocaudal locations, although they formed somewhat more diffuse termination zones. These findings indicate that other mechanisms, in addition to differential EphA3 expression in the neural retina, are required for retinal ganglion axons to map to the appropriate rostrocaudal locations in the optic tectum. They further suggest that the control of topographic specificity along the retinal nasal-temporal axis is split into several independent pathways already at a very early time in development.     

Denervation and age modify neuromuscular positional selectivity

The rostrocaudal position of neurons within the spinal motor pool maps systematically onto the surface of several muscles in mammals. In an effort to understand the mechanisms that generate such maps, we have been studying choices made by embryonic spinal cord neurons on muscle membrane substrates in the in vitro stripe assay. In this report we explore the effects of postnatal age of the muscle on neurite choice, and how prior denervation modifies this choice. Our results further differentiate rostral from caudal motor neurons in preferring one substrate to another. First, caudal neurites prefer to grow on P6 neonatal caudal over rostral membranes, but lose this ability to distinguish axial position of origin in older muscles. Rostral neurites prefer growth on rostral membranes, but this preference also diminishes with age. Second, when adult muscles have been denervated, both rostral and caudal neurites regain their positional growth selectivity. Third, caudal neurites are particularly sensitive to substrate choice. When growing on a preferred substrate (gluteus) caudal neurites prefer neonatal over adult membranes. These results support the concept of fundamental differences in the growth preferences of rostral and caudal spinal neurites. These differences will assist in the identification of molecular guidance cues that determine the formation of neuromuscular positional maps.     

Response of retinal ganglion cell axons to striped linear gradients of repellent guidance molecules

Although molecular gradients have long been postulated to play a role in the development of topographic projections in the nervous system, relatively little is known about how axons evaluate gradients. Do growth cones respond to concentration or to slope? Do they react suddenly or gradually? Is there adaptation? In the developing retinotectal system, temporal retinal ganglion cell axons have previously been shown to avoid repellent cell-surface activities distributed in gradients across the optic tectum. We confronted temporal retinal axons with precisely formed striped linear gradients of repellent tectal membranes and of two candidate repellent molecules, ephrin-A2 and -A5. Axons entered gradient stripes independently of their slope and extended unhindered in the uphill direction until they suddenly avoided an apparent threshold concentration of repellent material that was independent of slope. This critical concentration was similar in both linear and nonlinear gradients, and hence independent of gradient shape. When gradients of identical slope were formed on different basal levels of repellent material, axons grew uphill for a fixed increment of concentration, possibly measured from the lowest point of the gradient, rather than up to a fixed absolute concentration. The speed of growth cones was not affected by repellent unstriped gradients below the critical concentration level. Similar results were found with membranes from cell lines stably transfected with either ephrin-A5 or ephrin-A2, two previously identified growth cone repellent cell-surface proteins. These data suggest that growth cones or axons can integrate guidance information over large distances, probably by a combined memory and adaptation mechanism. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 541–562, 1998     

Axo-axonal synapses in the frog tectum opticum

A quantitative electron-microscopic investigation of synaptic endings in large sections showed that about 50% of all axo-axonal synapses are located in the outer zone of the neuropil (layer 9) of the tectum opticum ofRana temporaria L. These synapses are more numerous in the rostral part of the tectum than the caudal. Hardly any axo-axonal synapses lie deeper than 50–60 µ Most axo-axonal synapses are located on axon endings of retinal ganglionic cells, for after degeneration of the optic nerve the number of these synapses is reduced by two-thirds. During ontogenetic differentiation and regeneration of the optic nerve axo-axonal synapses develop before axo-dendritic and their presynaptic processes have the normal structure and differ sharply from the bulbs of growth of the optic fibers. On this basis the central origin of most presynaptic processes forming these synapses is postulated. The results point to the possibility of presynaptic control over the effectiveness of action of the efferent axons, primarily optic, terminating in the outer zone of the frog tectum opticum.     

Analysis of ephrin-A2 in the chick retinotectal projection using a function-blocking monoclonal antibody

Eph receptor tyrosine kinases and their ligands have been shown to be involved in processes of cell migration and axon guidance during embryonic development. Here we describe the development of a function-blocking monoclonal antibody against chick ephrin-A2, and its effect on retinal ganglion cell axons studied both in vitro and in vivo. In the stripe assay, the blocking antibody completely abolished the repulsive effect of posterior tectal membranes. In vivo, in a loss-of-function approach, hybridoma cells secreting the antiephrin-A2 antibody were applied to chick embryos from embryonic day 3 (E3) on, and the retinotectal projection was subsequently analyzed at E16. DiI tracing analyses showed that although the projection of both temporal and nasal retinal ganglion axons in the tectum was, overall, normal, occasionally diffuse and extra termination zones were observed, in addition to axons over-shooting their termination zones. These data support the idea that ephrin-A2 contributes to the establishment of the chick retinotectal projection.     

Surface fine structure of the subfornical organ in the Japanese quail,Coturnix coturnix japonica

The surface ultrastructure of the subfornical organ (SFO) was investigated in the Japanese quail. The SFO consists of a body and a stalk. The body of the SFO can be divided into rostral and caudal parts. On the rostral part, each ependymal cell possesses a short central solitary cilium; clustered cilia are also occasionally seen. Microvilli are abundant. On the caudal part, cells with a solitary cilium are fewer in number, and clustered cilia are rarely found. Microvilli are not as abundant as on the rostral part. In addition, large bulbous protrusions, tufts of small protrusions, deep funnel-shaped hollows, small pinocytotic invaginations and possible cerebrospinal fluid-contacting axons are sporadically observed on the surface of various regions of the body. Each ependymal cell of the stalk has a wide apical surface. A central solitary cilium, microvilli and other structures are observed more rarely on the stalk than on the body, while clustered cilia are not seen on the stalk. These structures are compared with those of the mammalian SFO and further discussed in relation to the possible dipsogenic receptor function for angiotensin II.     

Development of inhibition by ephrin-A5 on outgrowth of embryonic spinal motor neurites

The spinal motor pool maps systematically onto the surface of muscles. This map is detectable in rat embryonic muscles, and is partially restored after reinnervation. Recent evidence shows that either overexpression or deletion of the ephrin-A5 gene significantly disrupts the map, suggesting that ephrin-A5 plays a critical role in the formation of this topography. Several studies have demonstrated that ephrin-A5 is a repulsive molecule in the nervous system, including the neuromuscular system. To examine the development of sensitivity of ventral spinal axons to this inhibitory ligand, slices of E11 to E15 embryonic rat spinal cords were cocultured with membranes derived from ephrin-A5-expressing cell lines. We detected a progressive expression of inhibition by ephrin-A5 between E11 and E15. By E15, rostral and caudal spinal neurites showed clear differences in responsiveness to the ephrin-A5 ligand. Further, we found that at this age caudal neurites are more sensitive to changes of ephrin-A5 concentration along a gradient. In addition, growth cones of caudal, more than rostral, neurites tended to assume a collapsed shape in the presence of the ligand. These results demonstrate a progressive development of sensitivity to ephrin-A5, and suggest a divergence in this sensitivity between rostral and caudal spinal cord neurites. These results provide further insight into how subtle rostrocaudal differences in the development of sensitivity to ephrin-A5 may explain, in part, neuromuscular topography.