Retinotectal Projection Research Articles

Eph receptors are negatively controlled by protein tyrosine phosphatase receptor type O

Eph receptors are activated by the autophosphorylation of tyrosine residues upon the binding of their ligands, the ephrins; however, the protein tyrosine phosphatases (PTPs) responsible for the negative regulation of Eph receptors have not been elucidated. Here, we identified protein tyrosine phosphatase receptor type O (Ptpro) as a specific PTP that efficiently dephosphorylates both EphA and EphB receptors as substrates. Biochemical analyses revealed that Ptpro dephosphorylates a phosphotyrosine residue conserved in the juxtamembrane region, which is required for the activation and signal transmission of Eph receptors. Ptpro thus seems to moderate the amount of maximal activation of Eph receptors. Using the chick retinotectal projection system, we show that Ptpro controls the sensitivity of retinal axons to ephrins and thereby has a crucial role in the establishment of topographic projections. Our findings explain the molecular mechanism that determines the threshold of the response of Eph receptors to ephrins in vivo.

Retroviral misexpression of cVax disturbs retinal ganglion cell axon fasciculation and intraretinal pathfinding in vivo and guidance of nasal ganglion cell axons in vitro

The transcription factor cVax (Vax2) is expressed in the ventral neural retina and restricted expression is a prerequisite for at least three prominent aspects of retinal dorsal–ventral patterning: polarized expression of EphB/B-ephrin molecules, the retinotectal projection and the distribution of rod photoreceptors across the retina. In the chick retina, the fasciculation pattern of ganglion cell axons also differs between the dorsal and ventral eye. To investigate the molecular mechanisms involved, the nerve fiber layer was analyzed after retroviral misexpression of several factors known to regulate the positional specification of retinal ganglion cells. Forced cVax expression ventralized the fasciculation pattern and caused axon pathfinding errors near the optic disc. Ectopic expression of different ephrin molecules indicated that axon fasciculation is, at least in part, mediated by the EphB system. Finally, we report that retroviral misexpression of cVax increased the pool of EphA4 receptors phosphorylated on tyrosine residues and altered the guidance preference of nasal axons in vitro. These results identify novel functions for cVax in intraretinal axon fasciculation and pathfinding as well as suggest a mechanism to explain how restricted cVax expression may influence map formation along the dorso-ventral and antero-posterior axes of the optic tectum.

Regulation of GDNF and its receptor components GFR-α1, -α2 and Ret during development and in the mature retino-collicular pathway

The development of the retino-tectal projection as part of the central visual pathway is accomplished around postnatal day (P) 10–14 in rodents, and trophic factors are important for topographic refinement of this projection. Emerging data indicate that GDNF may influence synaptic plasticity of this projection. To date, maturation-dependent kinetics of GDNF release and expression and biological function of single GDNF receptors along the retino-collicular pathway are ill-defined. Here, we examined mRNA and protein expression of GDNF and its multicomponent receptor complex in the retina and superior colliculus (SC) during postnatal development of the rat visual system, and after optic nerve (ON) injury by RT-PCR, immunoblotting and immunofluorescence. Stable mRNA transcription of GDNF and its receptors GFR-α1, -α2 and Ret was found in retina and SC throughout development into adulthood and after ON transection. Expression of GDNF protein increased during retinal development, declined in adulthood and was further reduced in injured retina. In the SC, GDNF peaked at P0, continuously declined with maturation, and was undetectable in the deafferentiated SC. GFR-α1 was abundant in retina and SC throughout, while GFR-α2 was not expressed. Since Ret was localized primarily to the vascular compartment, the receptor tyrosine kinase may play a minor role in neuronal GDNF signaling. In summary, we provide evidence for GDNF as survival and guidance factor during development of the retino-tectal projection with differential regulation in early and premature retina and SC. Postlesionally, midbrain targets do not induce GDNF, suggesting that retrograde GDNF is not essential for rescue of adult injured retinal ganglion cells (RGCs).

Stabilization of Axon Branch Dynamics by Synaptic Maturation

The developmental refinement of topographic projections in the brain is reflected in the dynamic sculpting of axonal arbors that takes place as connections between CNS structures form and mature. To examine the role of synaptogenesis and synaptic maturation in the structural development of axonal projections during the formation of the topographic retinotectal projection, we coexpressed cytosolic fluorescent protein (FP) and FP-tagged synaptophysin (SYP) in small numbers of retinal ganglion cells in living albino Xenopus laevis tadpoles to reveal the distribution and dynamics of presynaptic sites within labeled retinotectal axons. Two-photon time-lapse observations followed by quantitative analysis of tagged SYP levels at individual synapses demonstrated the time course of synaptogenesis: increases in presynaptic punctum intensity are detectable within minutes of punctum emergence and continue over many hours. Puncta lifetimes correlate with their intensities. Furthermore, we found that axon arbor dynamics are affected by synaptic contacts. Axon branches retract past faintly labeled puncta but are locally stabilized at intensely labeled SYP puncta. Visual stimulation for 4 h enhanced the stability of the arbor at intense presynaptic puncta while concurrently inducing the retraction of exploratory branches with only faintly labeled or no synaptic sites.

Tyrosine hydroxylase immunoreactivity in the developing visual pathway of the zebrafish

We analyzed the distribution of tyrosine hydroxylase immunoreactivity in the central nervous zones involved in the processing of visual information during zebrafish ontogeny, employing a segmental approach. In the retina, we observed immunolabeled cells in the inner nuclear layer after hatching. From the juvenile stages onwards, some of these cells presented two immunolabeled processes towards the inner and outer plexiform layers of the retina, which are identified as interplexiform cells. In the adult zebrafish retina, we have identified two cellular types displaying immunoreactivity for tyrosine hydroxylase: interplexiform and amacrine cells. In the optic tectum, derived from the mesencephalon, no immunolabeled neurons were observed in any of the stages analyzed. The periventricular gray zone and the superficial white zone display immunostained neuropile from the end of fry life onwards. At the 30-day postfertilization, the tyrosine hydroxylase immunoreactive neuropile in the optic tectum presents two bands located within the retinorecipient strata and deeper strata, respectively. All diencephalic regions, which receive direct retinal inputs, show immunolabeled cells in the preoptic area, in the pretectum, and in the ventral thalamus from embryonic stages onwards. During the fry development, the immunolabeled neurons can be observed in the periventricular pretectum from 15-days postfertilization and in both the ventrolateral thalamic nucleus and suprachiasmatic nucleus from 30-days postfertilization. The transient expression of tyrosine hydroxylase is observed in fibers of the optic tract during fry and juvenile development. The existence of immunolabeled neuropile in the zebrafish retinorecipient strata could be related to the turnover of retinotectal projections.

Blockade of arachidonic acid pathway induces sprouting in the adult but not in the neonatal uncrossed retinotectal projection

The uncrossed retinotectal projection of rats undergoes extensive axonal elimination and subsequent growth of axonal arbors in topographically appropriate territories within the first two/three postnatal weeks. Nitric oxide has been implicated in development and stabilization of synapses in the retinotectal pathway since blockade of nitric oxide synthesis disrupts the normal pattern of retinal innervation in subcortical nuclei. The present work investigated the role of arachidonic acid pathway in the development and maintenance of ipsilateral retinotectal axons. We also investigated the role of this retrograde messenger in the modulation of plasticity that follows retinal lesions in the opposite eye. Pigmented rats received systemic treatment with quinacrine, a phospholipase A 2 inhibitor, indomethacin, a cyclooxygenase inhibitor, nordihydroguaiaretic acid, a 5-lipoxygenase inhibitor or vehicle during 4–8 days at various postnatal ages. Rats given a unilateral temporal retinal lesion were treated with either quinacrine or vehicle during the same period. For anterograde tracing of ipsilateral retinal projections, animals received intraocular injections of horseradish peroxidase. Before the third postnatal week no difference was observed in the laminar or topographic organization of the ipsilateral retinotectal projection between vehicle and treated rats in either normal or lesion conditions. After the third postnatal week, however, systemic blockade of phospholipase A 2 or 5-lipoxygenase, but not cyclooxygenase induced sprouting of uncrossed axons throughout the collicular visual layers in unoperated rats. In retinal lesion groups, phospholipase A 2 blockade increased the sprouting of uncrossed intact axons to the collicular surface in the same period. The results suggest that arachidonic acid or lipoxygenase metabolites play a role in the maintenance of the retinotectal synapses after the critical period and that the blockade of the arachidonic acid pathway induces reactive sprouting of retinal axons late in development.

Signaling Mechanisms Underlying Slit2-Induced Collapse of Xenopus Retinal Growth Cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.

Ischemia Results 3 Months Later in Altered ERG, Degeneration of Inner Layers, and Deafferented Tectum: Neuroprotection with Brimonidine

To investigate the long-term effects of transient ligature of the ophthalmic vessels (LOV) on the inner and outer retina as well as on retinotectal projection, and whether brimonidine (BMD) has protective effects. In adult rats, the left eye was subjected to 90 minutes of LOV. One hour before ischemia, 2 drops of saline alone (vehicle group) or saline containing 0.5% brimonidine (BMD group) were instilled in the left eye. The effects of LOV on the inner and outer retina were assessed with ERG recordings of a- and b-wave amplitudes at 1, 8, and 12 weeks after LOV and with analysis of layer thickness in paraffin sections. The retinotectal projection was orthogradely labeled with cholera toxin subunit B (CTB) injected in the left eye and measured in serial coronal sections of the superior colliculus. There were significant reductions in the mean b-wave amplitudes of the ischemic eyes at 8 and 12 weeks after LOV in the vehicle-treated group of animals, but not in the BMD-treated group. The thickness of the inner nuclear and inner plexiform layers of the vehicle-treated group of retinas had decreased to approximately 71% of the thicknesses in the BMD-treated groups. Three months after LOV, the mean volume of the retinotectal projection in the vehicle- or BMD-treated group of animals had decreased to approximately 54% or 83%, respectively, of the mean values found in the control group of animals. LOV induces degeneration of the inner retinal layers and the retinotectal projection 3 months after the insult. BMD administration significantly protected against LOV-induced retinal damage and degeneration of retinal projection.

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.

Zebrafish R‐cadherin (Cdh4) controls visual system development and differentiation

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization.

Rapid and long-term plasticity in the neonatal and adult retinotectal pathways following a retinal lesion

The uncrossed retinotectal projection restricts its terminal fields to the ventral boundary of the visual layers at the rostral tectum during early post natal development. During this critical period, temporal retinal lesions in one eye induce laminar rearrangements in the uncrossed pathway of the intact eye toward the collicular surface previously occupied, almost exclusively, by the crossed retinal axon population. We have compared, using anterograde tracing techniques, the time course and magnitude of the axonal sprouting resulting from retinal lesions in neonates and adults. Early retinal lesions (within the first two post natal weeks) induced extensive and rapid plasticity of the ipsilateral projection 48 h after the lesions. On the third post natal week, similar retinal lesions induced a small reorganization of the intact eye's uncrossed projection within a 3-week survival time. Nevertheless, giving the animals a long-term survival, resulted in an increased plastic capability, suggesting that even after the critical period, intact retinal axons can respond efficiently to injury. The results suggest two phases of axonal reorganization within this subcortical pathway: a rapid plasticity within the critical period and a slow, but continuous plasticity in adulthood.

PAM May Inhibit TSC

D'Souza et al. report the identification of a relationship between PAM (protein associated with Myc) and tuberin, a subunit of the tuberous sclerosis complex (TSC) in axon guidance. D'Souza et al. analyzed zebrafish mutants for defects in axonal pathfinding in the retina and identified the gene esrom , encoding the zebrafish homolog of PAM. Esrom has multiple domains, including a RING finger domain, and the C-terminal portion of Esrom had autoubiquitinating activity in vitro. Esrom was present as punctate structures in retinal neurons, and retinal transplantation experiments confirmed that Esrom was required in the retinal neurons and not in the target brain region, the tectum. Analysis of the distal regions of retinal axons showed that phosphotuberin was enriched in the esr mutants--tuberin is inhibited by phosphorylation. Thus, D'Souza et al. propose that Esrom participates in a pathway involving TSC that interprets retinal growth cone cues. J. D'Souza, M. Hendricks, S. Le Guyader, S. Subburaju, B. Grunewald, K. Scholich, S. Jesuthasan, Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc). Development 132 , 247-256 (2005). [Abstract] [Full Text]

Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc)

Visual system development is dependent on correct interpretation of cues that direct growth cone migration and axon branching. Mutations in the zebrafish esrom gene disrupt bundling and targeting of retinal axons, and also cause ectopic arborization. By positional cloning, we establish that esrom encodes a very large protein orthologous to PAM (protein associated with Myc)/Highwire/RPM-1. Unlike motoneurons in Drosophila highwire mutants, retinal axons in esrom mutants do not arborize excessively, indicating that Esrom has different functions in the vertebrate visual system. We show here that Esrom has E3 ligase activity and modulates the amount of phosphorylated Tuberin, a tumor suppressor, in growth cones. These data identify a mediator of signal transduction in retinal growth cones, which is required for topographic map formation.

Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping

The mechanism of topographic mapping of retinal ganglion cells to the midbrain was previously elucidated by the servomechanism model, which is based on the fact that cells expressing Eph-receptors respond specifically to surface expressing membrane-bound ephrin-ligands at a critical level. The retina has increased nasal-to-temporal gradient of Eph receptor-density, and the optic tectum/superior colliculus has increased rostral-to-caudal gradient of membrane-bound ephrin-ligand. An axon from the retina has an identification tag of a certain level of Eph-receptor density depending on its retinal position, and adheres to the site on the tectum/superior colliculus expressing ephrin-ligands at a critical ligand-density level. The servomechanism model rigidly defines positions of axon terminals on the midbrain. However, optic nerve regeneration experiments combined with halved retina or tectum show a plastic or flexible mapping (expansion, compression and transposition of tectal projections). To reconcile the discrepancy between the rigid model and the plastic behavior, competition between retinal axon terminals for a target site was introduced to the servomechanism. The servomechanism/competition model succeeded in computer simulations of the plastic mapping of retinal axons on the tectum. Recent experiments of upregulated ligand-density on the tectum during nerve regeneration and the role of axonal competition are discussed.

Src Family Kinases Are Involved in EphA Receptor-Mediated Retinal Axon Guidance

EphA receptor tyrosine kinases and their ephrin ligands play important roles in wiring of the developing nervous system. We have investigated here the function of Src family kinases (SFKs) in the retinotectal projection to dissect the signaling pathways by which EphA receptors control actin/microtubule rearrangements that underlie growth cone guidance and collapse. Both EphAs and SFKs are expressed broadly in retinal growth cones, and SFKs are recruited to EphA receptors after ephrinA stimulation. In the stripe and growth cone collapse assays we observe an abolition of EphA-mediated repulsion after inhibiting SFKs, either pharmacologically or enzymatically via electroporation-mediated overexpression of the SFK inhibitor Csk. In addition, we identify cortactin and the RhoGEF ephexin, which interact with EphA receptors in retinal axons, as targets of SFK-dependent tyrosine phosphorylation. In sum, our data suggest an important role of SFKs as downstream signaling molecules in EphA receptor-mediated repulsive axon guidance.

Expression of MAP1B protein and its phosphorylated form MAP1B-P in the CNS of a continuously growing fish, the rainbow trout

Microtubule-associated protein-1B (MAP1B), and particularly its phosphorylated isoform MAP1B-P, play an important role in axonal outgrowth during development of the mammalian nervous system and have also been shown to be associated with axonal plasticity in the adult. Here, we used antibodies and mRNA probes directed against mammalian MAP1B to extend our analysis to fish species, trout ( Oncorhynchus mykiss), at different stages of development. The specificity of the cross-reaction of our anti-total-MAP1B/MAP1B-P antibodies was confirmed by Western blotting. Trout MAP1B-like proteins exhibited about the same apparent molecular weight (320 kDa) as rat-MAP1B. Immunohistochemistry and in situ hybridization analysis performed on hindbrain and spinal cord revealed the presence of MAP1B in neurons and some glial subpopulations. Primary sensory neurons and motoneurons maintain high levels of MAP1B expression from early stages throughout adulthood, as has been shown for mammals. Unlike mammals, however, MAP1B and axon-specific MAP1B-P continue to be strongly expressed by hindbrain neurons projecting into spinal cord, with the important exception of Mauthner cells. MAP1B/MAP1B-P immunostaining were also detected elsewhere within the brain, including axons of the retino-tectal projection. This obvious difference between adult fish and mammals is likely to reflect the capacity of fish for continued growth and regeneration. Our results suggest that MAP1B/MAP1B-P expression is generally maintained in neurons known to regenerate after axotomy. The regenerative potential of the adult nervous system may in fact depend on continued expression of neuron-intrinsic growth related proteins, a feature of MAP1B that appears phylogenetically conserved.

Aber Lenken Sie Auch? (But do they also guide?).

One of the most intriguing problems in developmental neurobiology is that of how growing axons find the correct way to their proper target cells. Often axonal connections are organized in topographic maps, where neighboring cells of the projecting area are connected to neighboring cells in the target area, thus allowing a faithful transfer of positionally stored information from one area to another. The retinotectal projection is the classical model system for studying topographic projections.

Growth and guidance cues for regenerating axons: where have they gone?

Both attractive and repellent cues are required to guide developing axons to their targets in the central nervous system. Critical guidance molecules in the developing brain include the semaphorins, netrins, slits, and ephrins. Current research indicates that many of these molecules and their receptors are expressed in the adult central nervous system (CNS), and that injury can alter the levels of these ligands/receptors. Recent studies have begun the process of elucidating the functions of these receptors in adult mammals, and the effects that they have on the regeneration of adult neurons. This review addresses our current knowledge with respect to the response of adult CNS neurons to axonal injury, interventions for enhancing the survival and regeneration of injured neurons, and the expression of developmental axon guidance cues in the injured mature CNS, with specific focus on the retino-tectal projection.

Insights into activity-dependent map formation from the retinotectal system: a middle-of-the-brain perspective.

The development of orderly topographic maps in the central nervous system (CNS) results from a collaboration of chemoaffinity cues that establish the coarse organization of the projection and activity-dependent mechanisms that fine-tune the map. Using the retinotectal projection as a model system, we describe evidence that biochemical tags and patterned neural activity work in parallel to produce topographically ordered axonal projections. Finally, we review recent experiments in other CNS projections that support the proposition that cooperation between molecular guidance cues and activity-dependent processes constitutes a general paradigm for CNS map formation.

Activity-driven sharpening of the retinotectal projection: the search for retrograde synaptic signaling pathways.

Patterned visual activity, acting via NMDA receptors, refines developing retinotectal maps by shaping individual retinal arbors. Because NMDA receptors are postsynaptic but the retinal arbors are presynaptic, there must be retrograde signals generated downstream of Ca(++) entry through NMDA receptors that direct the presynaptic retinal terminals to stabilize and grow or to withdraw. This review defines criteria for retrograde synaptic messengers, and then applies them to the leading candidates: nitric oxide (NO), brain-derived neurotrophic factor (BDNF), and arachidonic acid (AA). NO is not likely to be a general mechanism, as it operates only in selected projections of warm blooded vertebrates to speed up synaptic refinement, but is not essential. BDNF is a neurotrophin with strong growth promoting properties and complex interactions with activity both in its release and receptor signaling, but may modulate rather than mediate the retrograde signaling. AA promotes growth and stabilization of synaptic terminals by tapping into a pre-existing axonal growth-promoting pathway that is utilized by L1, NCAM, N-cadherin, and FGF and acts via PKC, GAP43, and F-actin stabilization, and it shares some overlap with BDNF pathways. The actions of both are consistent with recent demonstrations that activity-driven stabilization includes directed growth of new synaptic contacts. Certain nondiffusible factors (synapse-specific CAMs, ephrins, neurexin/neuroligin, and matrix molecules) may also play a role in activity-driven synapse stabilization. Interactions between these pathways are discussed.