Retinotectal Projection Research Articles

Retinotopic order in the absence of axon competition

The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.

Regulation of RALDH-1, RALDH-3 and CYP26A1 by transcription factors cVax/Vax2 and Tbx5 in the embryonic chick retina

In vertebrate development, polarity of the dorsal–ventral eye axis is determined by the spatially restricted expression of transcription factors cVax/Vax2 in the ventral retina and Tbx5 in the dorsal retina. In addition, an early gradient of retinoic acid (RA) has been suggested to be involved in this process. Using retroviral transfection of the early eye anlage of chick embryos we investigated whether cVax/Vax2 and Tbx5 regulate the expression of enzymes that determine the retinal distribution of RA. Transfection of cVax/Vax2 completely abolished the spatial expression pattern of the RA synthesizing enzyme RALDH-1, of the RA degrading enzyme Cyp26A1 and caused an ectopic expression of RALDH-3 in the dorsal retina. Transfection of Tbx5 did not significantly alter the distribution of RALDH-1 and RALDH-3 but caused additional patches of Cyp26 outside of its normal domain of expression. The distribution of RA was experimentally changed either by ectopic expression of Cyp26A1, or by applications of RA. These manipulations did not affect the distribution of cVax/Vax2 or interfere with development of the retinotectal projection from dorsal retinal ganglion cells. The results show that in the chick retina the compartmentalized expression of RA metabolizing enzymes requires the spatially restricted expression of the transcription factors cVax/Vax2 and Tbx5. On the other hand, RA may not be decisive for the correct retinal projection toward the optic tectum.

Neonatal tryptophan dietary restriction alters development of retinotectal projections in rats

Abstract The specification of sensory neural circuits includes the elimination of transitory axon collaterals/synapses that takes place during early post natal life, an important step for the acquisition of topographical order of sensory systems. Serotonin has been implicated in the patterning of connections in subcortical and cortical circuits. We investigated the effects of the dietary restriction of the only serotonin precursor, tryptophan, on the development of the uncrossed retinotectal pathway in pigmented rats. Litters were fed through their mothers with either a tryptophan restricted, corn and gelatin based diet or a similar control diet complemented with tryptophan during the lactation period. The developmental status of the uncrossed retinotectal terminal fields was studied after the anterograde transport of horseradish peroxidase injected into one eye. We also studied the effects of tryptophan restriction on 5-HT immunoreactivity of raphe neurons, on cAMP levels in the visual layers of the superior colliculus and on protein synthesis among retinal neurons. We found that tryptophan restriction resulted in reduced weight gain among tryptophan restricted rats, without differences in protein synthesis between tryptophan complemented and restricted groups. Tryptophan restriction was also associated with a reduction of serotonin immunoreactive cells in the raphe nuclei and increased cAMP levels in the superior colliculus. Finally we found that neonatal tryptophan restriction resulted in an abnormal patterning of retinotectal topography, which was consistent with a developmental delay in axonal elimination and fine tuning of central connections. These results suggest, therefore, that dietary tryptophan is crucial for the influence of serotonin in the maturation of central visual connections.

Intraretinal RGMa is involved in retino-tectal mapping

The repulsive guidance molecule (RGMa) is involved in controlling the topography of retinal ganglion cell axons along the anterioposterior axis of the tectum. Here, we generated a new RGMa-monoclonal antibody and show that it is expressed in the developing retina, suggesting that it may regulate retinal axon pathfinding. We tested this hypothesis by using in ovo electroporation to either overexpress or downregulate RGMa in the eye. Anterograde labeling of retinal axons entering the optic tecta revealed abnormal phenotypes when RGMa expression is perturbed. These included the absence of terminal zone, the premature stalling of arborization of fibers, overshooting of terminal zone, aberrant axonal turns in the optic tectum and abnormal projections into deeper tectal layers. Moreover, RGMa overexpression frequently leads to intraretinal pathfinding errors. Thus, these data suggest that RGMa expression on retinal axons is a major determinant of topographic targeting in the retino-tectal projection and in the retina.

Matrix Metalloproteinase-9 Is Involved in the Development and Plasticity of Retinotectal Projections in Rats

Objective: During postnatal development, retinotectal projections undergo a process of misplaced axon elimination, leading to a topographical matching between the retinal surface and the superior colliculus. Matrix metalloproteinases (MMPs) have been implicated in the development and plasticity of the nervous system. We studied the expression and role of MMPs during normal development of retinotectal projections and after monocular enucleation-induced plasticity. Material and Methods: Lister hooded rats at different postnatal ages received subpial ethylene vinyl acetate 40W implants to deliver an MMP inhibitor or vehicle to the superior colliculus. Animals received intraocular injections of horseradish peroxidase for anterograde tracing of ipsilateral projections. For immunoblotting and zymography, colliculi were removed without fixation. Results: We observed the highest MMP activity in the first postnatal week, with decreasing activity thereafter. Monocular enucleation at postnatal day 10 yielded a rapid increase in MMP activity, 24 h following denervation of the contralateral colliculus. Importantly, inhibition of MMP activity in vivo induced a marked delay of axonal clustering along the medial aspect of colliculus. Conclusions: Our data indicate that MMPs are crucial in retinotectal development concurring to the fine tuning of topographical order and synaptic specificity of these connections.

Neuroprotection of retinal ganglion cell function and their central nervous system targets

Technological advances have enabled the observation of a large number of retinal ganglion cells (RGCs) in an objective manner. In animal models, it has been shown how retinal ischaemia induces profound functional and structural alterations of the inner retinal and RGC layers by 3 months. These findings reflect degeneration of the inner retinal layers, the RGC population and of the retinotectal projection. Functionally, this implies a permanent disconnection of the retina from its main retinorecipient target region in the brain. Brimonidine, a selective α-2 adrenergic agonist, has been shown to activate α-2 adrenergic receptors in the retina and promote the survival and function of RGCs post-injury. This agent may prevent or diminish ischaemia-induced alterations in the inner and RGC areas as well as in the main retinofugal projection. Understanding the pattern of degeneration that occurs in the major retinofugal pathway following retinal ischaemia will benefit ongoing studies conducted to develop neuroprotectant-based treatment strategies for progressive neuropathies such as glaucoma.

Rapid Homeostatic Plasticity in the Intact Adult Visual System

Neurons may possess activity-dependent homeostatic mechanisms that permit them to globally alter synaptic strength as activity varies. We used the retinotectal projection of goldfish to test this idea in the intact adult CNS. We first altered tectal neuron activity by selectively manipulating excitatory input. When excitatory synaptic drive to tectal neurons was eliminated by blocking optic fibers, current evoked at optic synapses increased by 183% within 90 min. With partial activity blockade, the increase in synaptic strength scaled with the magnitude of activity depression. This silence-induced potentiation was also rapidly reversible. Conversely, an increase in optic input was followed by a decrease in evoked synaptic current. When optic drive was not altered and tectal neuronal activity was instead increased or decreased pharmacologically via GABA(A) receptors, synaptic strength again changed inversely with activity, indicating that synaptic strength changed in response to neuronal activity and not excitatory drive. Furthermore, altered synaptic strength tended to return ongoing activity to baseline. Changes in synaptic strength could also be detected in heterosynaptic pathways, indicating a global response. Finally, changes in synaptic strength were associated with corresponding changes in ongoing and evoked firing rates, indicating that the responsivity of tectal neurons was altered. Thus, tectal neurons exhibit archetypical homeostasis, one of the first robust examples in the intact adult CNS.

The shape of the olfactory bulb influences axon targeting

Each primary olfactory neuron in the mouse expresses a single type of odorant receptor. All neurons expressing the same odorant receptor gene typically project to two topographically fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb. While topographic gradients of guidance receptors and their ligands help to establish the retinotectal projection, similar orthogonal distributions of cues have not yet been detected within the olfactory system. While odorant receptors are crucial for the final targeting of axons to glomeruli, it is unclear whether the olfactory bulb itself provides instructive cues for the establishment of the topographic map. To begin to understand the role of the olfactory bulb in the formation of the olfactory nerve pathway, we developed a model whereby the gross shape of the bulb in the P2-IRES-tau-LacZ line of mice was radically altered during postnatal development. We have shown here that the topography of axons expressing the P2 odorant receptor is dependent on the shape of the olfactory bulb. When the dorsoventral axis of the olfactory bulb was compressed during the early postnatal period, newly developing P2 axons projected to multiple inappropriate glomeruli surrounding their normal target site. These results suggest that the distribution of local guidance cues within the olfactory bulb is influenced by the shape of the olfactory bulb and that these cues contribute to the topographic positioning of glomeruli.

Repulsive Guidance Molecule RGMa Alters Utilization of Bone Morphogenetic Protein (BMP) Type II Receptors by BMP2 and BMP4

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of multifunctional ligands that transduce their signals through type I and II serine/threonine kinase receptors and intracellular Smad proteins. Recently, we identified the glycosylphosphatidylinositol-anchored repulsive guidance molecules RGMa, DRAGON (RGMb), and hemojuvelin (RGMc) as coreceptors for BMP signaling (Babbit, J. L., Huang, F. W., Wrighting, D. W., Xia, Y., Sidis, Y., Samad, T. A., Campagna, J. A., Chung, R., Schneyer, A., Woolf, C. J., Andrews, N. C., and Lin, H. Y. (2006) Nat. Genet. 38, 531-539; Babbit, J. L., Zhang, Y., Samad, T. A., Xia, Y., Tang, J., Schneyer, A., Woolf, C. J., and Lin, H. Y. (2005) J. Biol. Chem. 280, 29820-29827; Samad, T. A., Rebbapragada, A., Bell, E., Zhang, Y., Sidis, Y., Jeong, S. J., Campagna, J. A., Perusini, S., Fabrizio, D. A., Schneyer, A. L., Lin, H. Y., Brivanlou, A. H., Attisano, L., and Woolf, C. J. (2005) J. Biol. Chem. 280, 14122-14129). However, the mechanism by which RGM family members enhance BMP signaling remains unknown. Here, we report that RGMa bound to radiolabeled BMP2 and BMP4 with Kd values of 2.4+/-0.2 and 1.4+/-0.1 nm, respectively. In KGN human ovarian granulosa cells and mouse pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling required BMP receptor type II (BMPRII), but not activin receptor type IIA (ActRIIA) or ActRIIB, based on changes in BMP signaling by small interfering RNA inhibition of receptor expression. In contrast, cells transfected with RGMa utilized both BMPRII and ActRIIA for BMP2 or BMP4 signaling. Furthermore, in BmpRII-null pulmonary artery smooth muscle cells, BMP2 and BMP4 signaling was reduced by inhibition of endogenous RGMa expression, and RGMa-mediated BMP signaling required ActRIIA expression. These findings suggest that RGMa facilitates the use of ActRIIA by endogenous BMP2 and BMP4 ligands that otherwise prefer signaling via BMPRII and that increased utilization of ActRIIA leads to generation of an enhanced BMP signal.

Genetic Single-Cell Mosaic Analysis Implicates ephrinB2 Reverse Signaling in Projections from the Posterior Tectum to the Hindbrain in Zebrafish

The optic tectum is a visual center in vertebrates. It receives topographically ordered visual inputs from the retina in the superficial layers and then sends motor outputs from the deeper layers to the premotor reticulospinal system in the hindbrain. Although the topographic patterns of the retinotectal projection are well known, it is not yet well understood how tectal efferents in the tectobulbar tract project to the hindbrain. The retinotectal and the tectobulbar projections were visualized in a zebrafish stable transgenic line Tg(brn3a-hsp70:GFP). Using a single-neuron labeling system in combination with the cre/loxP and Gal4/UAS systems, we showed that the tectal neurons that projected to rhombomeres 2 and 6 were distributed with distinctive patterns along the anterior-posterior axis. Furthermore, we found that ephrinB2a was critically involved in increasing the probability of neurons projecting to rhombomere 2 through a reverse signaling mechanism. These results may provide a neuroanatomical and molecular basis for the motor command map in the tectum.

Acuity and contrast sensitivity of the bluegill sunfish and how they change during optic nerve regeneration

Spatial vision was studied in the bluegill sunfish, Lepomis macrochirus (9.5-14 cm standard length) to assess the limitations imposed by the optics of the eye, the retinal receptor spacing and the retinotectal projection during regeneration. Examination of images formed by the dioptric elements of the eye showed that spatial frequencies up to 29 c/ degrees could be imaged on the retina. Cone spacing was measured in the retina of fresh, intact eyes. The spacing of rows of double cones predicted 3.4 c/degrees as the cutoff spatial frequency; the spacing between rows of single and double cones predicted 6.7 c/degrees. Contrast sensitivity functions were obtained psychophysically in normals and fish with one regenerating optic nerve. Fish were trained to orient to gratings (mean luminance = 25 cd/m2) presented to either eye. In normals, contrast sensitivity functions were similar in shape and bandwidth to those of other species, peaking at 0.4 c/degrees with a minimum contrast threshold of 0.03 and a cutoff at about 5 c/degrees, which was within the range predicted by cone spacing. Given that the optical cutoff frequency exceeds that predicted by cone spacing, it is possible that gratings could be detected by aliasing with the bluegill's regular cone mosaic. However, tests with high contrast gratings up to 15 c/ degrees found no evidence of such detection. After crushing one optic nerve in three trained sunfish, recovery of visual avoidance, dorsal light reflex and orienting to gratings, were monitored over 315 days. At 64-69 days postcrush, responses to gratings reappeared, and within 2-5 days contrast sensitivity at low (0.15 c/degrees) and medium (1.0 c/ degrees) spatial frequencies had returned to normal. At a high spatial frequency (2.93 c/degrees) recovery was much slower, and complete only in one fish.

Enhanced Visual Activity In Vivo Forms Nascent Synapses in the Developing Retinotectal Projection

Patterned neural activity during development is critical for proper wiring of sensory circuits. Previous work demonstrated that exposing freely swimming Xenopus tadpoles to 4 h of enhanced visual stimulation accelerates the dendritic growth rate of optic tectal neurons in vivo. Here we test whether this same period of visual stimulation increased synaptic maturation and formation of new synapses in the retinotectal pathway. We assessed synaptic properties of stage 48 tadpoles that were exposed to a simulated-motion stimulus for 4-5 h. Based on our findings that immature retinotectal synapses have greater paired-pulse facilitation compared with more mature synapses, consistent with a lower release probability (Pr), we used a paired-pulse protocol to elicit responses selectively from nascent synapses with low Pr. Although AMPA/NMDA ratios for single and paired stimuli were the same in control tadpoles, visual stimulation caused a relative decrease in the AMPA/NMDA ratio of the paired response. We evoked retinotectal synaptic transmission in the presence of Sr(2+) to record asynchronous vesicle release. We compared evoked mEPSCs induced by single and paired stimuli and found that visual stimulation selectively enhances the amplitude and number of AMPA receptor (AMPAR)-mediated mEPSCs evoked by paired stimuli relative to those evoked by single stimuli. Together these results show that enhanced visual stimulation affects both AMPAR- and NMDAR-mediated responses in a population of synapses revealed by paired-pulse stimulation. This suggests that in vivo visual stimulation increases synapses that have a low Pr and that have properties consistent with immature synapses.

Vesicular Glutamate Transport at a Central Synapse Limits the Acuity of Visual Perception in Zebrafish

The neural circuitry that constrains visual acuity in the CNS has not been experimentally identified. We show here that zebrafish blumenkohl (blu) mutants are impaired in resolving rapid movements and fine spatial detail. The blu gene encodes a vesicular glutamate transporter expressed by retinal ganglion cells. Mutant retinotectal synapses release less glutamate, per vesicle and per terminal, and fatigue more quickly than wild-type in response to high-frequency stimulation. In addition, mutant axons arborize more extensively, thus increasing the number of synaptic terminals and effectively normalizing the combined input to postsynaptic cells in the tectum. This presumably homeostatic response results in larger receptive fields of tectal cells and a degradation of the retinotopic map. As predicted, mutants have a selective deficit in the capture of small prey objects, a behavior dependent on the tectum. Our studies successfully link the disruption of a synaptic protein to complex changes in neural circuitry and behavior.

The superficial layers of the superior colliculus are cytoarchitectually and myeloarchitectually disorganized in the reelin-deficient mouse, reeler

The causative gene for the reeler mouse is reelin which encodes Reelin protein, an extracellular molecule. In the present study, we have examined the cytoarchitecture, myeloarchitecture, and afferent/efferent systems of the superior colliculus (SC) of the reeler mouse. In the reeler, the laminar structures of the superficial three layers of the SC were disorganized and intermingled into a single layer, i.e., the superficial fused layer (SuF), as previously reported in the reelin-deficient SRK rat (Sakakibara et al., Develop. Brain Res. 141:1–13). Next, we have investigated the course and terminals of visual corticotectal and retinotectal projections with an injection of biocytin into the visual cortex or an injection of cholera toxin subunit B into the retina, respectively. In the reeler, anterogradely labeled visual corticotectal and retinotectal fibers took an aberrant course within the SuF, resulting in abnormal myeloarchitecture of the superficial SC of the reeler. Retrograde labeling of tectospinal tract neurons could not show any differences between the normal and reeler mice, suggesting that the deep layers of the reeler SC are cytoarchitectually normal. In situ hybridization and immunohistochemical studies have shown that reelin mRNA and Reelin protein were both recognized in the normal SC. These results suggest that Reelin protein plays some roles in histogenesis of the superficial layers of the SC.

In Vivo Imaging Reveals Dendritic Targeting of Laminated Afferents by Zebrafish Retinal Ganglion Cells

Targeting of axons and dendrites to particular synaptic laminae is an important mechanism by which precise patterns of neuronal connectivity are established. Although axons target specific laminae during development, dendritic lamination has been thought to occur largely by pruning of inappropriately placed arbors. We discovered by in vivo time-lapse imaging that retinal ganglion cell (RGC) dendrites in zebrafish show growth patterns implicating dendritic targeting as a mechanism for contacting appropriate synaptic partners. Populations of RGCs labeled in transgenic animals establish distinct dendritic strata sequentially, predominantly from the inner to outer retina. Imaging individual cells over successive days confirmed that multistratified RGCs generate strata sequentially, each arbor elaborating within a specific lamina. Simultaneous imaging of RGCs and subpopulations of presynaptic amacrine interneurons revealed that RGC dendrites appear to target amacrine plexuses that had already laminated. Dendritic targeting of prepatterned afferents may thus be a novel mechanism for establishing proper synaptic connectivity.

Role of Bone Morphogenic Protein 2 in Retinal Patterning and Retinotectal Projection

It has been long believed that the anteroposterior (A-P) and dorsoventral (D-V) axes in the developing retina are determined independently and also that the retinotectal projection along the two axes is controlled independently. However, we recently demonstrated that misexpression of Ventroptin, a bone morphogenic protein (BMP) antagonist, in the developing chick retina alters the retinotectal projection not only along the D-V (or mediolateral) axis but also along the A-P axis. Moreover, the dorsal-high expression of BMP4 is relieved by the dorsotemporal-high expression of BMP2 at embryonic day 5 (E5) in the retina, during which Ventroptin continuously counteracts the two BMPs keeping on the countergradient expression pattern, respectively. Here, we show that the topographic molecules so far reported to have a gradient only along the D-V axis and ephrin-A2 so far only along the A-P axis are both controlled by the BMP signal, and that they are expressed in a gradient manner along the tilted axis from E6 on in the developing chick retina: the expression patterns of these oblique-gradient molecules are all changed, when BMP2 expression is manipulated in the developing retina. Furthermore, in both BMP2 knockdown embryos and ephrin-A2-misexpressed embryos, the retinotectal projection is altered along the two orthogonal axes. The expressional switching from BMP4 to BMP2 thus appears to play a key role in the retinal patterning and topographic retinotectal projection by tilting the D-V axis toward the posterior side during retinal development. Our results also indicate that BMP2 expression is essential for the maintenance of regional specificity along the revised D-V axis.

Oculomotor Instabilities in Zebrafish Mutantbelladonna: A Behavioral Model for Congenital Nystagmus Caused by Axonal Misrouting

A large fraction of homozygous zebrafish mutant belladonna (bel) larvae display a reversed optokinetic response (OKR) that correlates with failure of the retinal ganglion cells to cross the midline and form the optic chiasm. Some of these achiasmatic mutants display strong spontaneous eye oscillations (SOs) in the absence of motion in the surround. The presentation of a stationary grating was necessary and sufficient to evoke SO. Both OKR reversal and SO depend on vision and are contrast sensitive. We built a quantitative model derived from bel fwd (forward) eye behaviors. To mimic the achiasmatic condition, we reversed the sign of the retinal slip velocity in the model, thereby successfully reproducing both reversed OKR and SO. On the basis of the OKR data, and with the support of the quantitative model, we hypothesize that the reversed OKR and the SO can be completely attributed to RGC misrouting. The strong resemblance between the SO and congenital nystagmus (CN) seen in humans with defective retinotectal projections implies that CN, of so far unknown etiology, may be directly caused by a projection defect.

Getting into shape: Optimal ligand gradients for axonal guidance

During neural development, neurons from downstream, presynaptic regions of the nervous system (such as the retina) send spatially patterned axonal projections to upstream, target regions (the tectum or superior colliculus). A servomechanism model has been proposed to explain the pattern and time-course of axonal growth between these two regions [Honda, H., 1998. Topographic mapping in the retinotectal projection by means of complementary ligand and receptor gradients: a computer simulation study. J. Theor. Biol., 192, 235-246]. Here, we show that a modification of this model incorporating a different criterion for axonal decision-making, called the local optimum rule, is guaranteed to converge to a topographic map under a wide range of conditions encountered during neural development. A theoretical investigation of these conditions leads to new hypotheses regarding map formation.

A reporter-assisted mutagenesis screen using α1-tubulin-GFP transgenic zebrafish uncovers missteps during neuronal development and axonogenesis

α1-tubulin expression occurs in a neural-specific, temporally regulated, and regeneration-inducible fashion in zebrafish. A GFP reporter driven by the α1-tubulin promoter in transgenic zebrafish acts as a stable, in vivo molecular tag that follows neuronal development from birth/specification through postmitotic differentiation to axonal outgrowth and synaptogenesis. We exploited this transgenic system in a reporter expression-dependent (morphology-independent) mutagenesis screen to identify disruptions in genetic loci essential for neuronogenesis and axon elaboration, which would manifest as visually appreciable perturbations in GFP fluorescence. Thirty-two such recessive mutations were obtained, a subset of which was screened through a secondary RNA quantification-based assay to eliminate housekeeping gene defects. Three representative loci, when characterized in detail, were found to exhibit missteps in discrete, sequential stages of embryonic neuronal development. Mutation in sookshma panneurally diminishes the neural precursor pool by affecting cell proliferation in the developing embryo while patterning along the neuraxis remains unperturbed. Disruption of drishti on the other hand ameliorates the mitotic neural population by affecting cell cycle exit of progenitors and stalling their progression to the postmitotic neuronal stage, without impairing subsequent cell fate determination or differentiation. Finally, dhruva is required during neuronal differentiation for axonal branching and terminal innervation in spinal motoaxons and the retinotectal projection. Molecular identification of these loci and analysis of the remaining mutational repertoire will offer unique insights into the genetic inputs that go on to make a mature, differentiated neuron.

Repulsive Guidance Molecule Plays Multiple Roles in Neuronal Differentiation and Axon Guidance

Repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as a guidance molecule for retinal axons. Three RGM isoforms (RGMa-RGMc) exist in vertebrates. We showed previously that RGMa is a cell-survival factor in the neuroepithelium of chick embryos that suppresses the proapoptotic activity of its receptor neogenin. In the present study, we performed gain- and loss-of-function analysis of RGMa in chick embryos to further investigate RGMa function. We found that RGMa overexpression promotes neuronal differentiation, whereas RGMa small interference RNA represses it. Similar experiments conducted at later developmental stages using retroviral vectors reveal that perturbation of RGMa expression disturbs the retinotectal projection. Our work provides the first evidence for a role for RGMs in axon guidance in vivo. In addition, these results suggest that RGMa exerts multiple functions during neural development.