Forced notch signaling inhibits commissural axon outgrowth in the developing chick central nerve system

Background

A collection of in vitro evidence has demonstrated that Notch signaling plays a key role in the growth of neurites in differentiated neurons. However, the effects of Notch signaling on axon outgrowth in an in vivo condition remain largely unknown.

Methodology/Principal Findings

In this study, the neural tubes of HH10-11 chick embryos were in ovo electroporated with various Notch transgenes of activating or inhibiting Notch signaling, and then their effects on commissural axon outgrowth across the floor plate midline in the chick developing central nerve system were investigated. Our results showed that forced expression of Notch intracellular domain, constitutively active form of RBPJ, or full-length Hes1 in the rostral hindbrain, diencephalon and spinal cord at stage HH10-11 significantly inhibited commissural axon outgrowth. On the other hand, inhibition of Notch signaling by ectopically expressing a dominant-negative form of RBPJ promoted commissural axonal growth along the circumferential axis. Further results revealed that these Notch signaling-mediated axon outgrowth defects may be not due to the alteration of axon guidance since commissural axon marker TAG1 was present in the axons in floor plate midline, and also not result from the changes in cell fate determination of commissural neurons since the expression of postmitotic neuron marker Tuj1 and specific commissural markers TAG1 and Pax7 was unchanged.

Conclusions/Significance

We first used an in vivo system to provide evidence that forced Notch signaling negatively regulates commissural axon outgrowth.     

A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

Background

Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons.

Principal Findings

We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, βIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1.

Conclusions

Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.     

Experience-Driven Axon Retraction in the Pharmacologically Inactivated Visual Cortex Does Not Require Synaptic Transmission

Background

Experience during early postnatal development plays an important role in the refinement of specific neural connections in the brain. In the mammalian visual system, altered visual experiences induce plastic adaptation of visual cortical responses and guide rearrangements of afferent axons from the lateral geniculate nucleus. Previous studies using visual deprivation demonstrated that the afferents serving an open eye significantly retract when cortical neurons are pharmacologically inhibited by applying a γ-aminobutyric acid type A receptor agonist, muscimol, whereas those serving a deprived eye are rescued from retraction, suggesting that presynaptic activity can lead to the retraction of geniculocortical axons in the absence of postsynaptic activity. Because muscimol application suppresses the spike activity of cortical neurons leaving transmitter release intact at geniculocortical synapses, local synaptic interaction may underlie the retraction of active axons in the inhibited cortex.

Method and Findings

New studies reported here determined whether experience-driven axon retraction can occur in the visual cortex inactivated by blocking synaptic inputs. We inactivated the primary visual cortex of kittens by suppressing synaptic transmission with cortical injections of botulinum neurotoxin type E, which cleaves a synaptic protein, SNAP-25, and blocks transmitter release, and examined the geniculocortical axon morphology in the animals with normal vision and those deprived of vision binocularly. We found that afferent axons in the animals with normal vision showed a significant retraction in the inactivated cortex, as similarly observed in the muscimol-treated cortex, whereas the axons in the binocularly deprived animals were preserved.

Conclusions

Therefore, the experience-driven axon retraction in the inactivated cortex can proceed in the absence of synaptic transmission. These results suggest that presynaptic mechanisms play an important role in the experience-driven refinement of geniculocortical axons.     

The NC1 domain of type IV collagen promotes axonal growth in sympathetic neurons through interaction with the alpha 1 beta 1 integrin

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.     

Perturbations of MicroRNA Function in Mouse Dicer Mutants Produce Retinal Defects and Lead to Aberrant Axon Pathfinding at the Optic Chiasm

Background

During development axons encounter a variety of choice points where they have to make appropriate pathfinding decisions. The optic chiasm is a major decision point for retinal ganglion cell (RGC) axons en route to their target in order to ensure the correct wiring of the visual system. MicroRNAs (miRNAs) belong to the class of small non-coding RNA molecules and have been identified as important regulators of a variety of processes during embryonic development. However, their involvement in axon guidance decisions is less clear.

Methodology/Principal Findings

We report here that the early loss of Dicer, an essential protein for the maturation of miRNAs, in all cells of the forming retina and optic chiasm leads to severe phenotypes of RGC axon pathfinding at the midline. Using a conditional deletion approach in mice, we find in homozygous Dicer mutants a marked increase of ipsilateral projections, RGC axons extending outside the optic chiasm, the formation of a secondary optic tract and a substantial number of RGC axons projecting aberrantly into the contralateral eye. In addition, the mutant mice display a microphthalmia phenotype.

Conclusions

Our work demonstrates an important role of Dicer controlling the extension of RGC axons to the brain proper. It indicates that miRNAs are essential regulatory elements for mechanisms that ensure correct axon guidance decisions at the midline and thus have a central function in the establishment of circuitry during the development of the nervous system.     

Ontogenetic changes in the regenerative ability of chick retinal ganglion cells as revealed by organ explants

Summary Whereas mature neurons in the central nervous system (CNS) of birds lack the capability of regenerating axons after injury, embryonic nerve cells are able to do so. The time course of this decline of regenerative ability was investigated in ganglion cells from embryonic chick retinae. Retinal strips from 7-to 19-day-old embryos (E7–E19) were explanted and cultured in vitro. The numbers of retinal ganglion cell (RGC) neurites that had extended during the first 22–23 h incubation, their elongation rates, and morphometric parameters of the growth cones were measured to characterize the regenerative behavior. We observed two periods of decline in neuritic growth: the first from E7 to E9, and another from E14 to E19. The first decrease may reflect a gradually disappearing portion of neurons which produced their axons de novo. The second decline coincides with the major period of synaptogenesis by ganglion cell axons in ovo. The time required for initiation of axonal outgrowth increased, accordingly, from less than 3 h in explants from younger retinae (E7–E16) to 10–12 h for E17 and E19 explants. Axonal elongation rates ranged between 36 m/h and 56 m/h (mean values) for E7–E13 explants, but were significantly lower for cells from E14–E19 retinae (13–21 m/h). Morphologically, neurites and growth cones for RGC explanted before E17 were characterized by their high variability. They possessed more filopodia than mature neurons (E17, E19), fasciculated to a higher degree and branched more frequently. In addition, older neurites appeared stiffer and were morphologically simpler. We propose that, during the embryogenesis of the chick retinofugal system, the changes that render mature ganglion cells incapable of axonal regeneration are mainly triggered in the period of establishing synaptic contacts in the optic tectum.     

Gogo Receptor Contributes to Retinotopic Map Formation and Prevents R1-6 Photoreceptor Axon Bundling

Background

Topographic maps form the basis of neural processing in sensory systems of both vertebrate and invertebrate species. In the Drosophila visual system, neighboring R1–R6 photoreceptor axons innervate adjacent positions in the first optic ganglion, the lamina, and thereby represent visual space as a continuous map in the brain. The mechanisms responsible for the establishment of retinotopic maps remain incompletely understood.

Results

Here, we show that the receptor Golden goal (Gogo) is required for R axon lamina targeting and cartridge elongation in a partially redundant fashion with local guidance cues provided by neighboring axons. Loss of function of Gogo in large clones of R axons results in aberrant R1–R6 fascicle spacing. Gogo affects target cartridge selection only indirectly as a consequence of the disordered lamina map. Interestingly, small clones of gogo deficient R axons perfectly integrate into a proper retinotopic map suggesting that surrounding R axons of the same or neighboring fascicles provide complementary spatial guidance. Using single photoreceptor type rescue, we show that Gogo expression exclusively in R8 cells is sufficient to mediate targeting of all photoreceptor types in the lamina. Upon lamina targeting and cartridge selection, R axons elongate within their individual cartridges. Interestingly, here Gogo prevents bundling of extending R1-6 axons.

Conclusion

Taken together, we propose that Gogo contributes to retinotopic map formation in the Drosophila lamina by controlling the distribution of R1–R6 axon fascicles. In a later developmental step, the regular position of R1–R6 axons along the lamina plexus is crucial for target cartridge selection. During cartridge elongation, Gogo allows R1–R6 axons to extend centrally in the lamina cartridge.     

Topological characterization of neuronal arbor morphology via sequence representation: II - global alignment

Background

The increasing abundance of neuromorphological data provides both the opportunity and the challenge to compare massive numbers of neurons from a wide diversity of sources efficiently and effectively. We implemented a modified global alignment algorithm representing axonal and dendritic bifurcations as strings of characters. Sequence alignment quantifies neuronal similarity by identifying branch-level correspondences between trees.

Results

The space generated from pairwise similarities is capable of classifying neuronal arbor types as well as, or better than, traditional topological metrics. Unsupervised cluster analysis produces groups that significantly correspond with known cell classes for axons, dendrites, and pyramidal apical dendrites. Furthermore, the distinguishing consensus topology generated by multiple sequence alignment of a group of neurons reveals their shared branching blueprint. Interestingly, the axons of dendritic-targeting interneurons in the rodent cortex associates with pyramidal axons but apart from the (more topologically symmetric) axons of perisomatic-targeting interneurons.

Conclusions

Global pairwise and multiple sequence alignment of neurite topologies enables detailed comparison of neurites and identification of conserved topological features in alignment-defined clusters. The methods presented also provide a framework for incorporation of additional branch-level morphological features. Moreover, comparison of multiple alignment with motif analysis shows that the two techniques provide complementary information respectively revealing global and local features.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0605-1) contains supplementary material, which is available to authorized users.     

Axons Amplify Somatic Incomplete Spikes into Uniform Amplitudes in Mouse Cortical Pyramidal Neurons

Background

Action potentials are the essential unit of neuronal encoding. Somatic sequential spikes in the central nervous system appear various in amplitudes. To be effective neuronal codes, these spikes should be propagated to axonal terminals where they activate the synapses and drive postsynaptic neurons. It remains unclear whether these effective neuronal codes are based on spike timing orders and/or amplitudes.

Methodology/Principal Findings

We investigated this fundamental issue by simultaneously recording the axon versus soma of identical neurons and presynaptic vs. postsynaptic neurons in the cortical slices. The axons enable somatic spikes in low amplitude be enlarged, which activate synaptic transmission in consistent patterns. This facilitation in the propagation of sequential spikes through the axons is mechanistically founded by the short refractory periods, large currents and high opening probability of axonal voltage-gated sodium channels.

Conclusion/Significance

An amplification of somatic incomplete spikes into axonal complete ones makes sequential spikes to activate consistent synaptic transmission. Therefore, neuronal encoding is likely based on spike timing order, instead of graded analogues.     

Why do mature CNS neurons of mammals fail to re-establish connections following injury--functions of bcl-2

Factors inside and outside neurons control the process of axonal growth and regeneration. Recently, it has become apparent that neurons are determined intrinsically for their ability to grow axons. In the mammalian CNS, the intrinsic machinery of neurons that triggers the growth of axons during early embryonic stages is shut down at a certain point in development; as a consequence, axon elongation and regeneration cannot occur in postnatal life. The proto-oncogene Bcl-2 has been recognized to act as a key regulator for the program of axon elongation inside neurons. However, expressing the gene Bcl-2 in CNS neurons is not sufficient to induce nerve regeneration in the adult CNS, eliminating the inhibitory mechanism in the mature CNS environment is still required. Recently, the formation of glia scar has been reported to be the major limiting factor in the CNS environment that blocks nerve regeneration. These new discoveries challenge the classical view of nerve regeneration in the mammalian CNS. It opens up a new dimension in the study of the cellular and molecular mechanisms underlying neurodevelopmental and neurodegenerative diseases.     

Cell Guidance on Nanogratings: A Computational Model of the Interplay between PC12 Growth Cones and Nanostructures

Background

Recently, the effects of nanogratings have been investigated on PC12 with respect to cell polarity, neuronal differentiation, migration, maturation of focal adhesions and alignment of neurites.

Methodology/Principal Findings

A synergistic procedure was used to study the mechanism of alignment of PC12 neurites with respect to the main direction of nanogratings. Finite Element simulations were used to qualitatively assess the distribution of stresses at the interface between non-spread growth cones and filopodia, and to study their dependence on filopodial length and orientation. After modelling all adhesions under non-spread growth cone and filopodial protrusions, the values of local stress maxima resulted from the length of filopodia. Since the stress was assumed to be the main triggering cause leading to the increase and stabilization of filopodia, the position of the local maxima was directly related to the orientation of neurites. An analytic closed form equation was then written to quantitatively assess the average ridge width needed to achieve a given neuritic alignment (R² = 0.96), and the alignment course, when the ridge depth varied (R² = 0.97). A computational framework was implemented within an improved free Java environment (CX3D) and in silico simulations were carried out to reproduce and predict biological experiments. No significant differences were found between biological experiments and in silico simulations (alignment, p = 0.3571; tortuosity, p = 0.2236) with a standard level of confidence (95%).

Conclusions/Significance

A mechanism involved in filopodial sensing of nanogratings is proposed and modelled through a synergistic use of FE models, theoretical equations and in silico simulations. This approach shows the importance of the neuritic terminal geometry, and the key role of the distribution of the adhesion constraints for the cell/substrate coupling process. Finally, the effects of the geometry of nanogratings were explicitly considered in cell/surface interactions thanks to the analytic framework presented in this work.     

Secreted amyloid precursor protein β and secreted amyloid precursor protein α induce axon outgrowth in vitro through Egr1 signaling pathway

Background

sAPPα released after α secretase cleavage of Amyloid Precursor Protein (APP) has several functions including the stimulation of neurite outgrowth although detailed morphometric analysis has not been done. Two domains involved in this function have been described and are present in sAPPβ released at the first step of amyloid peptide cleavage, raising the possibility that sAPPβ could also stimulate neurite outgrowth. We investigated the morphological effects of sAPPα and sAPPβ on primary neurons and identified a key signaling event required for the changes observed.

Methodology/Principal Findings

Final concentrations of 50 to 150 nM bacterial recombinant sAPPα or sAPPβ added to primary neuronal cultures after 1 day in vitro decreased cell adhesion 24 hours later and primary dendrite length 96 hours later. 150 nM sAPPα and sAPPβ induced a similar increase of axon outgrowth, although this increase was already significant at 100 nM sAPPα. These morphological changes induced by sAPPs were also observed when added to differentiated neurons at 5 days in vitro. Real time PCR and immunocytochemistry showed that sAPPα and sAPPβ stimulated Egr1 expression downstream of MAPK/ERK activation. Furthermore, in primary neurons from Egr1 −/− mice, sAPPs affected dendritic length but did not induce any increase of axon length.

Conclusion/Significance

sAPPα and sAPPβ decrease cell adhesion and increase axon elongation. These morphological changes are similar to what has been observed in response to heparan sulfate. The sAPPα/sAPPβ stimulated increase in axon growth requires Egr1 signaling. These data suggest that sAPPβ is not deleterious per se. Since sAPPβ and sAPPα are present in the embryonic brain, these two APP metabolites might play a role in axon outgrowth during development and in response to brain damage.     

Protein synthesis in distal axons is not required for axon growth in the embryonic spinal cord

It is now well established that new proteins are synthesized in the distal segments of elongating axons, where they may play an essential role in some guidance decisions. It remains unclear, however, whether distal protein synthesis also plays an essential role in axon growth per se. Previous in vitro experiments have shown that blocking protein synthesis in distal axons has no effect on the rate of axonal advance. However, because these experiments were performed in vitro and over a relatively short time period, the role of distal protein synthesis over longer periods and in a native tissue environment remained untested. Here, we tested whether protein synthesis in distal axons plays an essential role in the elongation of descending axons in the embryonic spinal cord. We developed an in situ model of the brainstem-spinal projection of the embryonic chick, and developed a split-chamber method in which inhibitors of proteins synthesis could be applied independently to cell bodies in the brainstem or to distal axons in the spinal cord. When protein synthesis was blocked in distal axons, axon growth remained robust for 2 days, which is the length of the experiment. However, when protein synthesis was blocked only in the brainstem, axonal elongation in the spinal cord ceased within 6 h. These data showed that protein synthesis in the distal axon is not essential to continue the advance of axons. Rather, essential proteins are synthesized more proximally and then transported rapidly to the distal axon.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.     

Netrin signaling breaks the equivalence between two identified zebrafish motoneurons revealing a new role of intermediate targets

Background

We previously showed that equivalence between two identified zebrafish motoneurons is broken by interactions with identified muscle fibers that act as an intermediate target for the axons of these motoneurons. Here we investigate the molecular basis of the signaling interaction between the intermediate target and the motoneurons.

Principal Findings

We provide evidence that Netrin 1a is an intermediate target-derived signal that causes two equivalent motoneurons to adopt distinct fates. We show that although these two motoneurons express the same Netrin receptors, their axons respond differently to Netrin 1a encountered at the intermediate target. Furthermore, we demonstrate that when Netrin 1a is knocked down, more distal intermediate targets that express other Netrins can also function to break equivalence between these motoneurons.

Significance

Our results suggest a new role for intermediate targets in breaking neuronal equivalence. The data we present reveal that signals encountered during axon pathfinding can cause equivalent neurons to adopt distinct fates. Such signals may be key in diversifying a neuronal population and leading to correct circuit formation.     

A chondroitin sulfate proteoglycan may influence the direction of retinal ganglion cell outgrowth.

In the developing retina, retinal ganglion cell (RGC) axons elongate toward the optic fissure, even though no obvious directional restrictions exist. Previous studies indicate that axon-matrix interactions are important for retinal ganglion cell axon elongation, but the factors that direct elongation are unknown. Chondroitin sulfate proteoglycan (CS-PG), a component of the extracellular matrix, repels elongating dorsal root ganglion (DRG) axons in vitro and is present in vivo in the roof plate of the spinal cord, a structure that acts as a barrier to DRG axons during development. In this study, we examined whether CS-PG may regulate the pattern of retinal ganglion cell outgrowth in the developing retina. Immunocytochemical analysis showed that CS-PG was present in the innermost layers of the developing rat retina. The expression of CS-PG moved peripherally with retinal development, always remaining at the outer edge of the front of the developing axons. CS-PG was no longer detectable with immunocytochemical techniques when RGC axon elongation in the retina is complete. Results of studies in vitro showed that CS-PG, isolated from bovine nasal cartilage and chick limb, was inhibitory to elongating RGC axons and that RGC growth cones were more sensitive to CS-PG than were DRG neurites tested at the same concentrations of CS-PG. The behavior of retinal growth cones as they encounter CS-PG was characterized using time-lapse video microscopy. Filopodia of the RGC growth cones extended to and sampled the CS-PG repeatedly. With time, the growth cones turned to avoid outgrowth on the CS-PG and grew only on laminin. While numerous studies have shown the presence of positive factors within the retina that may guide developing RGC axons, this is the first demonstration of an inhibitory or repelling molecule in the retina that may regulate axon elongation. Taken together, these data suggest that the direction of RGC outgrowth in the retina may be regulated by the proper ratio of growth-promoting molecules, such as laminin, to growth-inhibiting molecules, like CS-PG, present in the correct pattern and concentrations along the retinal ganglion cell pathway.     

Ten-m3 Is Required for the Development of Topography in the Ipsilateral Retinocollicular Pathway

Background

The alignment of ipsilaterally and contralaterally projecting retinal axons that view the same part of visual space is fundamental to binocular vision. While much progress has been made regarding the mechanisms which regulate contralateral topography, very little is known of the mechanisms which regulate the mapping of ipsilateral axons such that they align with their contralateral counterparts.

Results

Using the advantageous model provided by the mouse retinocollicular pathway, we have performed anterograde tracing experiments which demonstrate that ipsilateral retinal axons begin to form terminal zones (TZs) in the superior colliculus (SC), within the first few postnatal days. These appear mature by postnatal day 11. Importantly, TZs formed by ipsilaterally-projecting retinal axons are spatially offset from those of contralaterally-projecting axons arising from the same retinotopic location from the outset. This pattern is consistent with that required for adult visuotopy. We further demonstrate that a member of the Ten-m/Odz/Teneurin family of homophilic transmembrane glycoproteins, Ten-m3, is an essential regulator of ipsilateral retinocollicular topography. Ten-m3 mRNA is expressed in a high-medial to low-lateral gradient in the developing SC. This corresponds topographically with its high-ventral to low-dorsal retinal gradient. In Ten-m3 knockout mice, contralateral ventrotemporal axons appropriately target rostromedial SC, whereas ipsilateral axons exhibit dramatic targeting errors along both the mediolateral and rostrocaudal axes of the SC, with a caudal shift of the primary TZ, as well as the formation of secondary, caudolaterally displaced TZs. In addition to these dramatic ipsilateral-specific mapping errors, both contralateral and ipsilateral retinocollicular TZs exhibit more subtle changes in morphology.

Conclusions

We conclude that important aspects of adult visuotopy are established via the differential sensitivity of ipsilateral and contralateral axons to intrinsic guidance cues. Further, we show that Ten-m3 plays a critical role in this process and is particularly important for the mapping of the ipsilateral retinocollicular pathway.     

Unc-51/ATG1 controls axonal and dendritic development via kinesin-mediated vesicle transport in the Drosophila brain

Background

Members of the evolutionary conserved Ser/Thr kinase Unc-51 family are key regulatory proteins that control neural development in both vertebrates and invertebrates. Previous studies have suggested diverse functions for the Unc-51 protein, including axonal elongation, growth cone guidance, and synaptic vesicle transport.

Methodology/Principal Findings

In this work, we have investigated the functional significance of Unc-51-mediated vesicle transport in the development of complex brain structures in Drosophila. We show that Unc-51 preferentially accumulates in newly elongating axons of the mushroom body, a center of olfactory learning in flies. Mutations in unc-51 cause disintegration of the core of the developing mushroom body, with mislocalization of Fasciclin II (Fas II), an IgG-family cell adhesion molecule important for axonal guidance and fasciculation. In unc-51 mutants, Fas II accumulates in the cell bodies, calyx, and the proximal peduncle. Furthermore, we show that mutations in unc-51 cause aberrant overshooting of dendrites in the mushroom body and the antennal lobe. Loss of unc-51 function leads to marked accumulation of Rab5 and Golgi components, whereas the localization of dendrite-specific proteins, such as Down syndrome cell adhesion molecule (DSCAM) and No distributive disjunction (Nod), remains unaltered. Genetic analyses of kinesin light chain (Klc) and unc-51 double heterozygotes suggest the importance of kinesin-mediated membrane transport for axonal and dendritic development. Moreover, our data demonstrate that loss of Klc activity causes similar axonal and dendritic defects in mushroom body neurons, recapitulating the salient feature of the developmental abnormalities caused by unc-51 mutations.

Conclusions/Significance

Unc-51 plays pivotal roles in the axonal and dendritic development of the Drosophila brain. Unc-51-mediated membrane vesicle transport is important in targeted localization of guidance molecules and organelles that regulate elongation and compartmentalization of developing neurons.