Control of pattern duplication in the retinotectal system of Xenopus. Suppression of duplication by eye-fragment interactions.

Recombining right nasal half-eyes with left temporal half-eyes at embryonic stage 32 in Xenopus produces a double or “twinned” pattern of functional connections between retina and midbrain optic tectum. The left temporal half-eye is reprogrammed such that it projects to the tectum as a mirror-image duplicate pattern of the nasal right half-eye; both half-eyes project across the entire tectum. However, recombining a right nasal half-eye (in situ) with a right dorsal half-eye (grafted into the temporal position; N_RD_R eye) produces a single normal retinotectal projection. Where interactions between N_R and T_L involve both axial reprogramming and duplication of N_R positional values in T_L, N_RD_R interactions involve axial reprogramming in D_R without duplication of N_R values. In a second approach to interactions which suppress pattern duplication, both nasal and temporal one-third-sized eye fragments form approximate “NN” or “TT” duplicate pattern maps, respectively, when either is allowed to round up and form a whole eye. Allowing nasal and temporal thirds to permanently fuse (after removal of a one-third-sized vertical center strip of retina at stage 32) produces a normal projection; allowing the nasal and temporal thirds to interact (fuse) for 35–40 hr, followed by removal of one or the other third, suppresses pattern duplication (produces normal maps) in the remaining third in a majority of cases. Allowing the thirds to interact for 18–30 hr before removal of the temporal third produces a majority result of partial duplication in the remaining nasal third. Partial duplicates are apparent spatial intermediates with regard to interactions which suppress duplication in either fragment type.     

Specificity and retinotectal projections of quarter-eye fragments in Xenopus laevis

Three quarters of the eye anlage in Xenopus embryos of stage 33/34 were eliminated in three different sets of experiments. The remaining quadrant originated from the nasoventral part of the retina, from its ventral portion, or from the temporo-ventral area of the retina. All the fragments developed into small eyes of normal shape. The retinotectal connections did not deviate from those found in the control groups, even though mirror-image duplication was fairly frequent. For all fragments the tectal projection fields were rather limited. There was some indication of fragments retaining their original specificity. Irrespective, however, of their different origins, the optic projections always occupied the rostrolateral area of the tectum.     

Gradual appearance of a regulated retinotectal projection pattern in Xenopus laevis

The topographic projection pattern formed by the retinal ganglion cell axons in the tectum of the lower vertebrate appears to require positional cues that guide the optic nerve fibers to their appropriate targets. One approach to understanding these positional cues or "positional information" has been to investigate changes in the pattern of the retinotectal projection after surgical manipulation of the embryonic eyebud. Analysis of these apparent changes in the patterns of positional information in the eye, termed "pattern regulation," may provide clues to both the nature of positional information and the mechanisms by which it is assigned to cells in the eyebud. Here we examine pattern regulation in the Xenopus visual system following the replacement of the temporal half of a right eyebud with the temporal half of a left eyebud. This manipulation requires that the left half-eyebud be inverted along its dorsoventral axis. Electrophysiological maps of these compound eyes in postmetamorphic frogs reveal regulated maps; the cells in the temporal half of the NrTl eye project to the tectum with a dorsoventral polarity appropriate for their position in the host eye and not appropriate for the original positions of the grafted cells in the donor eyebud. Paradoxically, the regulated patterns are not apparent in the projections of the original grafted eyebud cells during early larval development. Using fiber-tracing and electrophysiological mapping techniques, we now show that the regulated patterns appear gradually in the projections made by peripheral retinal cells added during mid-larval development. Because the regulation occurs relatively late in development and probably only in the peripheral retinal cells, simple models of epimorphic or morphallactic regulation do not appear to fit this system. Thus, new or more complex models must be invoked to explain the phenomenon of pattern regulation in the developing visual system of Xenopus.     

Alterations in the Xenopus retinotectal projection by antibodies to Xenopus N-CAM

The patterned neural projection from the eye to the optic tectum of lower vertebrates (the retinotectal projection) has been proposed to be ordered by interactions between the optic nerve fibers and their surrounding tissues. To investigate the role of one such defined cell interaction, agarose implants containing antibodies to the neural cell adhesion molecule, N-CAM, were inserted into the tectum of the African clawed frog, Xenopus laevis. Both monoclonal and polyclonal antibodies against N-CAM reversibly and specifically distorted the pattern of the retinotectal projection, decreasing the precision of the projection as determined by electrophysiological techniques as well as decreasing the density of retinal innervation of the tectum and the branching of single axons as determined by horseradish peroxidase tracing. The anatomical effects became maximal at 4 to 6 days after implantation and returned to undetectable levels by 2 weeks, whereas the physiological effects became maximal by 8 to 10 days and a normal physiological map was reestablished within 4 weeks. The results are consistent with the hypothesis that anti-N-CAM antibodies perturb the ongoing growth and retraction of the terminal arbors of the optic nerve fibers, such that a region of the tectum becomes largely denuded of fibers. The physiological defects may then be a consequence both of the initial retraction of optic nerve terminals and of the rapid ingrowth of the perturbed and neighboring optic nerve fibers into the denuded region after the antibodies were cleared from the tectum. These results support the concept of a major role for N-CAM-mediated adhesion during map regeneration and maintenance.     

Visuospatial information in the retinotectal system of xenopus before correct image formation by the developing eye

The retinotectal pathway of Xenopus laevis is a well-established experimental model for studying activity-dependent processes during visual system development. Such processes can be guided by stimulus-evoked activity patterns, which depend on the refractive characteristics of the eye. Previous work has shown that many animals are hyperopic at early developmental stages due to immature refractive properties. Whether this is also the case for Xenopus laevis is unknown. Here, we measure the focal length of the lens and the size of the eye of embryos at different stages and find that Xenopus laevis exhibits a similar shift from hyperopia to emmetropia. At early stages, immediately after innervation of the tectum by the optic nerve, Xenopus embryos are hyperopic. Soon afterwards the focal length of the lens decreases and the eye converges to a state of emmetropia. Despite being hyperopic we find that some visuospatial information is available to the young circuit. Calculations based on the optical properties of the eye show that even when the animals are hyperopic the blurred retinal image provides a crude spatial resolution. Furthermore, using whole-cell recordings in the optic tectum combined with visual stimulation through the intact eye, we show that tectal neurons in hyperopic embryos have spatially restricted glutamatergic receptive fields. Our data demonstrate that Xenopus laevis eyes undergo a process of developmental emmetropization, and suggest that despite an initial stage of suboptimal image formation there is potentially enough information to guide activity-dependent refinements of the retinotectal pathway from the onset of vision.     

Induction of a secondary body axis in Xenopus by antibodies to beta- catenin

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.     

Fiber optic mapping of the Xenopus visual system: shift in the retinotectal projection during development

Two new techniques for assaying the retina to tectum connections in the lower vertebrate visual system are presented. These techniques allow defined regions of the retina to be stimulated, thus circumventing some of the difficulties of the more conventional retinotectal mapping techniques. Applying these techniques to the Xenopus visual system demonstrates that the retina-to-tectum projection shifts during development. The central part of the retinotectal projection moves medially and caudally about 150 microns (10% of the size of the tectum) in two weeks. The presence of such plasticity in a normal developing animal indicates that the plasticity previously observed in experimentally altered animals probably reflects a normal developmental process.     

Fibre organization and reorganization in the retinotectal projection of Xenopus

This study concerns the retinotopic organization of the ganglion cell fibres in the visual system of the frog Xenopus laevis. HRP was used to trace the pathways taken by fibres from discrete retinal positions as they pass from the retina, along the optic nerve and into the chiasma. The ganglion cell fibres in the retina are arranged in fascicles which correspond with their circumferential positions of origin. Within the fascicles the fibres show little age-related layering and do not have a strict radial organization. As the fascicles of fibres pass into the optic nerve head there is some exchange of position resulting in some loss of the retinal circumferential organization. The poor radial organization of the fibres in the retinal fascicles persists as the fibres pass through the intraocular part of the nerve. At a position just behind the eye there is a major fibre reorganization in which fibres arising from cells of increasingly peripheral retinal locations are found to have passed into increasingly peripheral positions in the nerve. Thus, fibres from peripheral-most retina are located at the nerve perimeter, whilst fibres from central retina are located in the nerve core. It is at this point that the radial, chronotopic, ordering of the ganglion cell axons, found throughout the rest of the optic pathway, is established. This annular organization persists along the length of the nerve until a position just before the nerve enters the brain. Here, fibres from each annulus move to form layers as they pass into the optic chiasma. This change in the radial organization appears to be related to the pathway followed by all newly growing fibres, in the most superficial part of the optic tract, adjacent to the pia. Just behind the eye, where fibres become radially ordered, the circumferential organization of the projection is largely lost. Fibres from every circumferential retinal position, which are of similar radial position, are distributed within the same annulus of the nerve. At the nerve-chiasma junction where each annulus forms a single layer as it enters the optic tract, there is a further mixing of fibres from all circumferential positions. However, as the fibres pass through the chiasma some active pathway selection occurs, generating the circumferential organization of the fibres in the optic tract. Additional observations of the organization of fibres in the optic nerve of Rana pipiens confirm previous reports of a dual representation of fibres within the nerve. The difference in the organization of fibres in the optic nerve of Xenopus and Rana pipiens is discussed.     

Topographic mapping in dorsoventral axis of the Xenopus retinotectal system depends on signaling through ephrin-B ligands

Ephrin-B and EphB are distributed in matching dorsoventral gradients in the embryonic Xenopus visual system with retinal axons bearing high levels of ligand (dorsal) projecting to tectal regions with high receptor expression (ventral). In vitro stripe assays show that dorsal retinal axons prefer to grow on EphB receptor stripes supporting an attractive guidance mechanism. In vivo disruption of EphB/ephrin-B function by application of exogenous EphB or expression of dominant-negative ephrin-B ligand in dorsal retinal axons causes these axons to shift dorsally in the tectum, while misexpression of wild-type ephrin-B in ventral axons causes them to shift ventrally. These dorsoventral targeting errors are consistent with the hypothesis that an attractive mechanism that requires ephrin-B cytoplasmic domain is critical for retinotectal mapping in this axis.     

Demonstration of a polarizing signal that reverses future retinotectal patterns across Nuclepore filter barriers,in Xenopus embryonic eye

We have studied the interactions which occur in surgically disarranged eye rudiments by recombining a left anterior half-eye graft from a donor Xenopus embryo with a right host posterior half eye, across a variety of physical barriers, at embryonic stages 31 or 32. The anterior half-eye graft and barrier were removed 18 h later at stage 38, and the host posterior half-eye was allowed to reconstitute a whole eye whose visuotectal projection could be mapped electrophysiologically after metamorphosis. Anteroposteriorly reversed maps and double-anterior twinned maps that are characteristic of anterior half-eyes, were found in 50–65% of cases in each of the experimental series using no barrier (N = 16), or using Nuclepore filter barriers (N = 47), including 5 of 8 cases when a filter of 0.015 micrometer pore diameter was used. The latter cases are especially interesting, because the filter pores were much smaller than the minimum size known to permit cell-cell contact through the pores. No animals showed AP-reversed retinotectal maps or double-anterior twinned maps when the graft and host half-eyes were separated by a tantalum or plastic barrier (N = 21). Only a single case of AP-reversed mapping was found in 115 control animals including simple posterior half-eye preparations at stage 32 or 38 (N = 13), sham fusions (30 min) across Nuclepore filters (N = 35), or chronic application of a filter (or plastic or tantalum) barrier from stages 32–38 (N = 55) without a left anterior half-eye graft. We conclude that signals from an anterior half-eye can act to repolarize a posterior half-eye in the absence of cell transfer and under conditions which permit little or no direct cell-cell contact.     

Developmental expression of dynamin in the chick retinotectal system.

Dynamin I, a GTPase involved in the endocytic cycle of synaptic vesicle membranes, is believed to support axonal outgrowth and/or synaptogenesis. To explore the temporal and spatial patterns of dynamin I distribution in neuronal morphogenesis, we compared the developmental expression of dynamin with the expression of presynaptic membrane proteins such as SV2, synaptotagmin, and syntaxin in the chick primary visual pathway. Western blots of retina and tectum revealed a steady increase of synaptotagmin and syntaxin from embryonic Day 7 (E7) to E11, whereas for the same time frame no detectable increase of dynamin was found. Later stages showed increasing amounts of all tested proteins until the first postnatal week. Immunofluorescence revealed that SV2, synaptotagmin, and syntaxin are present in retinal ganglion cell axons from E4 on. In later stages, the staining pattern in the retina and along the visual pathway paralleled the formation and maturation of axons. In contrast, dynamin is not detectable by immunofluorescence in the developing retina and optic tectum before synapse formation. Our data indicate that, in contrast to the early expression of synaptotagmin, SV2, and syntaxin during axonal growth, dynamin is upregulated after synapse formation, suggesting its function predominantly during and after synaptogenesis but not in axonogenesis.(J Histochem Cytochem 47:1297-1306, 1999)     

Patterning of retinotectal connections in the vertebrate visual system.

Recent work on the retinotectal projection clearly establishes the roles of neuronal activity and position-based cues in the patterning of nerve connections. In some species, the high degree of spatial order has been shown to emerge from a continued process of terminal growth and refinement. The future challenge is now to determine how multiple cues work together to guide the sculpting of the final pattern.     

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.     

Expression patterns of Ephs and ephrins throughout retinotectal development in Xenopus laevis

The Eph family of receptor tyrosine kinases and their ligands the ephrins play an essential role in the targeting of retinal ganglion cell axons to topographically correct locations in the optic tectum during visual system development. The African claw-toed frog Xenopus laevis is a popular animal model for the study of retinotectal development because of its amenability to live imaging and electrophysiology. Its visual system undergoes protracted growth continuing beyond metamorphosis, yet little is known about ephrin and Eph expression patterns beyond stage 39 when retinal axons first arrive in the tectum. We used alkaline phosphatase fusion proteins of EphA3, ephrin-A5, EphB2, and ephrin-B1 as affinity probes to reveal the expression patterns of ephrin-As, EphAs, ephrin-Bs, and EphBs, respectively. Analysis of brains from stage 40 to adult frog revealed that ephrins and Eph receptors are expressed throughout development. As observed in other species, staining for ephrin-As displayed a high caudal to low rostral expression pattern across the tectum, roughly complementary to the expression of EphAs. In contrast with the prevailing model, EphBs were found to be expressed in the tectum in a high dorsal to low ventral gradient in young animals. In animals with induced binocular tectal innervation, ocular dominance bands of alternating input from the two eyes formed in the tectum; however, ephrin-A and EphA expression patterns were unmodulated and similar to those in normal frogs, confirming that the segregation of axons into eye-specific stripes is not the consequence of a respecification of molecular guidance cues in the tectum.     

EphrinB2a in the zebrafish retinotectal system

Members of the Eph-B family of receptors tyrosine kinase and their transmembrane ligands have been implicated in dorsoventral patterning of the vertebrate retinotectal projection. In the zebrafish retinotectal system, however, ephrinB2a is expressed strongly in the posterior tectum, in tectal neurons that form physical contacts with retinal ganglion cell (RGC) axons. In the gnarled mutant, where tectal neurons form ectopically in the pretectum, RGC axons stall before entering the tectum, or else are misrouted or branch aberrantly in the tectal neuropil. Ectopic expression of ephrinB2a in the anterior midbrain of wild-type embryos, with the aid of baculovirus, also inhibits RGC axon entry into the tectum. In vitro, zebrafish RGC axons are repelled by stripes of purified ephrinB2a. It is proposed that ephrinB2a may signal a subpopulation of RGC axons that they have reached their target neurons in the tectum.     

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.