GABA and development of the Xenopus optic projection

In the developing visual system of Xenopus laevis retinal ganglion cell (RGC) axons extend through the brain towards their major target in the midbrain, the optic tectum. Enroute, the axons are guided along their pathway by cues in the environment. In vitro, neurotransmitters have been shown to act chemotropically to influence the trajectory of extending axons and regulate the outgrowth of developing neurites, suggesting that they may act to guide or modulate the growth of axons in vivo. Previous work by Roberts and colleagues (1987) showed that populations of cells within the developing Xenopus diencephalon and mid-brain express the neurotransmitter gamma amino butyric acid (GABA). Here we show that Xenopus RGC axons in the midoptic tract grow alongside the GABAergic cells and cross their GABA immunopositive nerve processes. Moreover, RGC axons and growth cones express GABA-A and GABA-B receptors, and GABA and the GABA-B receptor agonist baclofen both stimulate RGC neurite outgrowth in culture. Finally, the GABA-B receptor antagonist CGP54626 applied to the developing optic projection in vivo causes a dose-dependent shortening of the optic projection. These data indicate that GABA may act in vivo to stimulate the outgrowth of Xenopus RGC axons along the optic tract.     

Neurotrophin-independent attraction of growing sensory and motor axons towards developing Xenopus limb buds in vitro

The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.     

Mean velocity of optically detected intraaxonal particles measured by a cross-correlation method.

A method which uses by the cross correlation of optical signals is described for the determination of the mean velocity of somatopetally moving particles within nerve fibers. The method was validated by simulation experiments and by comparing the results with those obtained by averaging collections of velocities of individual particles. The significant contribution of the method is that it allows objective and rapid serial evaluation of mean particle velocity within individual nerve fibers with good accuracy and precision. A series of results from normal myelinated nerve fibers from Xenopus laevis is presented. Considerable variation (up to 50%) in mean velocity was found between individual nerve fibers. The mean of all determinations indicates that the mean velocity of somatopetally moving particles in axons with diameters greater than 10mum is in the region of 1.14 mum/s at a temperature of 22-24 degrees C. The findings are compared with small collection of such determinations which have been reported in the literature.     

Ephrin-B regulates the Ipsilateral routing of retinal axons at the optic chiasm

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.     

Neuroanatomical and functional analysis of neural tube formation in notochordless Xenopus embryos; laterality of the ventral spinal cord is lost.

Notochordless Xenopus embryos were produced by u.v. irradiation of the uncleaved fertilized egg. The spinal cords were examined using intermediate filament staining for glial cells, retrograde HRP staining for neuronal morphology and an anti-glycinergic antibody to reveal commissural cells and axons. The floorplate cells of the normal cord appear to be absent and their position along the ventral midline of the cord is occupied by motor neurones, Kolmer-Agduhr cells, radial glial cells and a ventrally placed marginal zone containing the longitudinal axons. Motor neurone number is reduced to 15% of control values, and the sensory extramedullary cell number is increased twentyfold. Commissural axons are still able to cross the ventral cord but do so at abnormal angles and some commissural axons continue to grow circumferentially up the contralateral side of the cord rather than turning to grow longitudinally. Extracellular electrophysiological recordings from motor axons reveal that the normal alternation of locomotor activity on the left and right side of the embryo is lost in notochordless animals. These results suggest that the notochord and/or the normal floor plate structure are important for the development of the laterality of spinal cord connections and may influence motor neurone proliferation or differentiation.     

Microtubule-associated proteins and the determination of neuronal form

1. The assembly of microtubules is essential for the maintenance of both the extension and the radial symmetry of axons and dendrites. Microtubule-associated proteins (MAPs) are implicated in this function because they promote tubulin polymerization and because they appear to be involved in cross-linking microtubules in the neuritic cytoplasm. 2. In a variety of species high molecular weight MAP2 is found only in dendrites and MAP tau is found only is axons, indicating that certain MAPs are associated with specific aspects of neuronal morphology. 3. All neuronal MAPs that have been studied are under strong developmental regulation with either their form or abundance changing between developing and adult brain. In both rat and Xenopus the change from "early" to "late" MAP forms occurs concurrently with the cessation of axon and dendrite growth and the maturation of neuronal morphology. 4. In situations where neuronal growth persists in the adult, such as retinal photoreceptor cells and the olfactory system, "early" MAPs continue to be expressed in the adult brain. 5. These results implicate MAPs in neuronal morphogenesis and suggest that "early" MAPs are involved in axon and dendrite growth whereas the "late" MAPs are involved in the stabilization of their mature form.     

On the turning of Xenopus retinal axons induced by ephrin-A5

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.     

Proteinase yscE of yeast shows homology with the 20 S cylinder particles of Xenopus laevis

Proteinase yscE of the yeast Saccharomyces cerevisiae has been compared with the 20 S cylinder particles of Xenopus laevis. Both proteins are characterized by a similar group of 10-12 polypeptides with molecular masses ranging between 21 and 38 kDa. Antibodies generated against the 20 S Xenopus cylinder particles show cross-reactivity with yeast proteinase yscE subunits. The Xenopus particles and yeast proteinase yscE exhibit an identical image in electron microscopy. Both proteins appear as hollow cylinders mostly composed of four stacked annuli. The Xenopus 20 S particles exhibit proteolytic activity against the three peptide derivatives known to be substrates of proteinase yscE. The pH optimum for activity and the inhibition spectrum of the proteolytic activities of Xenopus 20 S particles and of yeast proteinase yscE are identical. The RNA content of the cylinder particles and of proteinase yscE is below 0.1 RNA chain per molecule. Our data suggest that proteinase yscE from yeast and the 20 S cylinder particles of X. laevis are homologous, highly conserved proteins carrying the catalytic character of a peptidase.     

Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.     

MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.     

Stochastic simulation of neurofilament transport in axons: the "stop-and-go" hypothesis

According to the "stop-and-go" hypothesis of slow axonal transport, cytoskeletal and cytosolic proteins are transported along axons at fast rates but the average velocity is slow because the movements are infrequent and bidirectional. To test whether this hypothesis can explain the kinetics of slow axonal transport in vivo, we have developed a stochastic model of neurofilament transport in axons. We propose that neurofilaments move in both anterograde and retrograde directions along cytoskeletal tracks, alternating between short bouts of rapid movement and short "on-track" pauses, and that they can also temporarily disengage from these tracks, resulting in more prolonged "off-track" pauses. We derive the kinetic parameters of the model from a detailed analysis of the moving and pausing behavior of single neurofilaments in axons of cultured neurons. We show that the model can match the shape, velocity, and spreading of the neurofilament transport waves obtained by radioisotopic pulse labeling in vivo. The model predicts that axonal neurofilaments spend approximately 8% of their time on track and approximately 97% of their time pausing during their journey along the axon.     

Pathways of Retinotectal Projection in Developing Xenopus Tadpoles Revealed by Selectively Labeling of Retinal Axons with Horseradish Peroxidase (HRP)

Anatomical mapping was made of the retinal central pathways from the chiasm to the targets within the tectum in the developing Xenopus tadpoles, after labeling a specific regional population of retinal axons with horseradish peroxidase (HRP). In the tadpoles at stage 50, pathway sorting of retinal axons within the optic tract was clear for the dorsoventral axis of the retina, but not for the nasotemporal axis. Most nasal retinal axons and some dorsal and ventral retinal axons invaded the tectum directly at the diencephalotectal junction, and arrived at their correct sites of innervation after running through ectopic parts of the tectum. These findings indicate that the pathway orientation before targets is not a prerequisite factor for establishment of the orderly map of the retinotectal projection. Rather, a direct interaction between ingrowing retinal axons and tectal cells seems to be a predominant factor for specification of retinal central connections.     

The development of retinal ganglion cells deprived of their targets

The influence of central targets on the morphological differentiation of retinal ganglion cells was investigated in Xenopus laevis. Since the ganglion cells mature into distinct morphological subtypes after their axons have reached their central targets, it is possible that the target tissues may influence or specify this aspect of neuronal cell development. To test this idea, Xenopus eyebuds were target-deprived by transplantation to the flank region of host embryos where they developed ectopically. The grafted eyes grew at normal rates, but could not make any projections into the central nervous system. To examine the morphological differentiation of the retinal ganglion cells their structures were revealed using an in vitro retinal preparation and intracellular injections of the dye Lucifer yellow. The elaboration and maturation of ganglion cell dendrites were found to be indistinguishable between control and transplanted eyes throughout development. Thus, the development of retinal ganglion cells into distinct morphological classes can occur even when their axons do not interact with the appropriate central targets.     

Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.     

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.     

An aberrant retinal pathway and visual centers in Xenopus tadpoles share a common cell surface molecule, A5 antigen

A monoclonal antibody A5 (MAb-A5), which was raised against Xenopus tadpole tectal cells, recognizes a cell surface-related protein molecule (A5 antigen) expressed on the visual centers of Xenopus tadpoles (S. Takagi, T. Tsuji, T. Amagai, T. Takamatsu, and H. Fujisawa, 1987, Dev. Biol. 122, 90-100). The present immunohistochemistry using MAb-A5 indicated that, in addition to the visual centers, A5 antigen was expressed on the general somatic sensory tract in the medulla and spinal cord of Xenopus tadpoles. As the general somatic sensory tract has been shown to be a pathway for ectopically transplanted retinal axons (M. Constantine-Paton and R. R. Capranica, 1976, J. Comp. Neurol. 170, 17-32; M. J. Katz and R. J. Lasek, 1979, J. Comp. Neurol. 183, 817-832), we examined whether retinal axons transplanted close to the spinal cord or medulla preferentially grow into the A5 antigen-positive general somatic sensory tract. We performed eye transplantation at embryonic stages and detected precise locations and trajectories of transplanted retinal axons within the medulla and spinal cord in tadpoles after filling retinal axons with horseradish peroxidase (HRP). HRP histochemistry in combination with MAb-A5 immunohistochemistry indicated that almost all HRP-filled transplanted retinal axons joined the A5 antigen-positive general somatic sensory tract. These findings suggest the involvement of A5 antigen in specific cell-cell recognition between retinal axons and their targets.     

Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice

The septal organ represents one of the three chemosensory subsystems found in most vertebrate species. Analyzing the projection pattern of septal organ neurons using the OMP-GFP transgenic mouse line revealed that axons navigate in highly variable fiber tracks across the main olfactory epithelium toward the main olfactory bulb. All septal organ axons cross through the cribriform plate at a spatially defined site and terminate exclusively in the posterior, ventromedial aspect of the bulb. Here, one portion of axons forms a dense network on the medial side where they apparently enter glomeruli which are mainly innervated by axons of olfactory sensory neurons from the main olfactory epithelium. Another significant portion of the axons targets a few glomeruli which appear to receive input exclusively from the septal organ neurons.     

Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing

Commissural interneurons (CI) of the vertebrate spinal cord are guided ventrally toward the floor plate, but subsequently cross the midline and select a longitudinal fascicle at specific dorsal-ventral (D-V) positions. We examined at high resolution the detailed behaviors of individual pathfinding CI growth cones on the ipsilateral and contralateral sides of the spinal cord of living Xenopus embryos. We find that pre-crossing CI growth cones exhibit distinct pathfinding behaviors compared to post-crossing axons and that the behavioral switch occurs immediately upon crossing to the contralateral side. Groups of pioneer commissural axons typically extend simultaneously toward the ventral midline following discrete paths with separation between adjacent commissurals apparently maintained through contact inhibition. In contrast, shortly after crossing the midline, commissural axons turn longitudinally and begin to fasciculate with other crossed CIs. However, growth cones of crossed commissurals often select their final D-V longitudinal track through a series of rapid step-like dorsal adjustments that may be due to differential fasciculation with longitudinal axons. Together, our results suggest that guidance of commissural axons is controlled in part through interactions among CIs that switch rapidly from avoidance to fasciculation after midline crossing.     

Fibroblast growth factors redirect retinal axons in vitro and in vivo

Growth factors have been shown previously to participate in the process of axon target recognition. We showed that fibroblast growth factor receptor (FGFR) signaling is required for Xenopus laevis retinal ganglion cell (RGC) axons to recognize their major midbrain target, the optic tectum [neuron 17 (1996), 245]. Therefore, we have hypothesized that a change in expression of a fibroblast growth factor (FGF) at the entrance of the optic tectum, the border between the diencephalon and mesencephalon, may serve as a signal to RGC axons that they have reached their target. To determine whether RGC axons can sense changes in FGF levels, we asked whether they altered their behavior upon encountering an ectopic source of FGF. We found that in vivo RGC growth cones avoided FGF-misexpressing cells along their path, and that FGF-2 directly repelled RGC growth cones in an in vitro growth cone turning assay. These data support the idea that RGC axons can sense changes in FGF levels, and as such provide a mechanism by which FGFR signaling is involved in RGC axon target recognition.