Short- and long-range repulsion by the Drosophila Unc5 netrin receptor.

Netrins are bifunctional guidance molecules, attracting some axons and repelling others. They act through receptors of the DCC and UNC5 families. DCC receptors have been implicated in both attraction and repulsion by Netrins. UNC5 receptors are required only for repulsion. In Drosophila, Netrins are expressed by midline cells of the CNS and by specific muscles in the periphery. They attract commissural and motor axons expressing the DCC family receptor Frazzled. Here we report the identification of the Drosophila Unc5 receptor, and show that it is a repulsive Netrin receptor likely to contribute to motor axon guidance. Ectopic expression of Unc5 on CNS axons can elicit either short- or long-range repulsion from the midline. Both short- and long-range repulsion require Netrin function, but only long-range repulsion requires Frazzled.     

Localized netrins act as positional cues to control layer-specific targeting of photoreceptor axons in Drosophila

A shared feature of many neural circuits is their organization into synaptic layers. However, the mechanisms that direct neurites to distinct layers remain poorly understood. We identified a central role for Netrins and their receptor Frazzled in mediating layer-specific axon targeting in the Drosophila visual system. Frazzled is expressed and cell autonomously required in R8 photoreceptors for directing their axons to the medulla-neuropil layer M3. Netrin-B is specifically localized in this layer owing to axonal release by lamina neurons L3 and capture by target neuron-associated Frazzled. Ligand expression in L3 is sufficient to rescue R8 axon-targeting defects of Netrin mutants. R8 axons target normally despite replacement of diffusible Netrin-B by membrane-tethered ligands. Finally, Netrin localization is instructive because expression in ectopic layers can retarget R8 axons. We propose that provision of localized chemoattractants by intermediate target neurons represents a highly precise strategy to direct axons to a positionally defined layer.     

Dscam guides embryonic axons by Netrin-dependent and -independent functions

Developing axons are attracted to the CNS midline by Netrin proteins and other as yet unidentified signals. Netrin signals are transduced in part by Frazzled (Fra)/DCC receptors. Genetic analysis in Drosophila indicates that additional unidentified receptors are needed to mediate the attractive response to Netrin. Analysis of Bolwig's nerve reveals that Netrin mutants have a similar phenotype to Down Syndrome Cell Adhesion Molecule (Dscam) mutants. Netrin and Dscam mutants display dose sensitive interactions, suggesting that Dscam could act as a Netrin receptor. We show using cell overlay assays that Netrin binds to fly and vertebrate Dscam, and that Dscam binds Netrin with the same affinity as DCC. At the CNS midline, we find that Dscam and its paralog Dscam3 act redundantly to promote midline crossing. Simultaneous genetic knockout of the two Dscam genes and the Netrin receptor fra produces a midline crossing defect that is stronger than the removal of Netrin proteins, suggesting that Dscam proteins also function in a pathway parallel to Netrins. Additionally, overexpression of Dscam in axons that do not normally cross the midline is able to induce ectopic midline crossing, consistent with an attractive receptor function. Our results support the model that Dscam proteins function as attractive receptors for Netrin and also act in parallel to Frazzled/DCC. Furthermore, the results suggest that Dscam proteins have the ability to respond to multiple ligands and act as receptors for an unidentified midline attractive cue. These functions in axon guidance have implications for the pathogenesis of Down Syndrome.     

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.     

Conserved and novel functions for Netrin in the formation of the axonal scaffold and glial sheath cells in spiders

Netrins are well known for their function as long-range chemotropic guidance cues, in particular in the ventral midline of vertebrates and invertebrates. Over the past years, publications are accumulating that support an additional short-range function for Netrins in diverse developmental processes such as axonal pathfinding and cell adhesion. We describe here the formation of the axonal scaffold in the spiders Cupiennius salei and Achaearanea tepidariorum and show that axonal tract formation seems to follow the same sequence as in insects and crustaceans in both species. First, segmental neuropiles are established which then become connected by the longitudinal fascicles. Interestingly, the commissures are established at the same time as the longitudinal tracts despite the large gap between the corresponding hemi-neuromeres which results from the lateral movement of the germband halves during spider embryogenesis. We show that Netrin has a conserved function in the ventral midline in commissural axon guidance. This function is retained by an adaptation of the expression pattern to the specific morphology of the spider embryo. Furthermore, we demonstrate a novel function of netrin in the formation of glial sheath cells that has an impact on neural precursor differentiation. Loss of Netrin function leads to the absence of glial sheath cells which in turn results in premature segregation of neural precursors and overexpression of the early motor- and interneuronal marker islet. We suggest that Netrin is required in the differentiated sheath cells for establishing and maintaining the interaction between NPGs and sheath cells. This short-range adhesive interaction ensures that the neural precursors maintain their epithelial character and remain attached to the NPGs. Both the conserved and novel functions of Netrin seem to be required for the proper formation of the axonal scaffold.     

Cytoplasmic domain requirements for Frazzled-mediated attractive axon turning at the Drosophila midline

The conserved DCC ligand-receptor pair Netrin and Frazzled (Fra) has a well-established role in axon guidance. However, the specific sequence motifs required for orchestrating downstream signaling events are not well understood. Evidence from vertebrates suggests that P3 is important for transducing Netrin-mediated turning and outgrowth, whereas in C. elegans it was shown that the P1 and P2 conserved sequence motifs are required for a gain-of-function outgrowth response. Here, we demonstrate that Drosophila fra mutant embryos exhibit guidance defects in a specific subset of commissural axons and these defects can be rescued cell-autonomously by expressing wild-type Fra exclusively in these neurons. Furthermore, structure-function studies indicate that the conserved P3 motif (but not P1 or P2) is required for growth cone attraction at the Drosophila midline. Surprisingly, in contrast to vertebrate DCC, P3 does not mediate receptor self-association, and self-association is not sufficient to promote Fra-dependent attraction. We also show that in contrast to previous findings, the cytoplasmic domain of Fra is not required for axonal localization and that neuronal expression of a truncated Fra receptor lacking the entire cytoplasmic domain (Fra delta C) results in dose-dependent defects in commissural axon guidance. These findings represent the first systematic dissection of the cytoplasmic domains required for Fra-mediated axon attraction in the context of full-length receptors in an intact organism and provide important insights into attractive axon guidance at the midline.     

The Abelson tyrosine kinase, the Trio GEF and Enabled interact with the Netrin receptor Frazzled in Drosophila

The attractive Netrin receptor Frazzled (Fra), and the signaling molecules Abelson tyrosine kinase (Abl), the guanine nucleotide-exchange factor Trio, and the Abl substrate Enabled (Ena), all regulate axon pathfinding at the Drosophila embryonic CNS midline. We detect genetic and/or physical interactions between Fra and these effector molecules that suggest that they act in concert to guide axons across the midline. Mutations in Abl and trio dominantly enhance fra and Netrin mutant CNS phenotypes, and fra;Abl and fra;trio double mutants display a dramatic loss of axons in a majority of commissures. Conversely, heterozygosity for ena reduces the severity of the CNS phenotype in fra, Netrin and trio,Abl mutants. Consistent with an in vivo role for these molecules as effectors of Fra signaling, heterozygosity for Abl, trio or ena reduces the number of axons that inappropriately cross the midline in embryos expressing the chimeric Robo-Fra receptor. Fra interacts physically with Abl and Trio in GST-pulldown assays and in co-immunoprecipitation experiments. In addition, tyrosine phosphorylation of Trio and Fra is elevated in S2 cells when Abl levels are increased. Together, these data suggest that Abl, Trio, Ena and Fra are integrated into a complex signaling network that regulates axon guidance at the CNS midline.     

Dscam1-mediated self-avoidance counters netrin-dependent targeting of dendrites in Drosophila

Dendrites and axons show precise targeting and spacing patterns for proper reception and transmission of information in the nervous system. Self-avoidance promotes complete territory coverage and nonoverlapping spacing between processes from the same cell [1, 2]. Neurons that lack Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) show aberrant overlap, fasciculation, and accumulation of dendrites and axons, demonstrating a role in self-recognition and repulsion leading to self-avoidance [3-11]. Fasciculation and accumulation of processes suggested that Dscam1 might promote process spacing by counterbalancing developmental signals that otherwise promote self-association [9, 12]. Here we show that Dscam1 functions to counter Drosophila sensory neuron dendritic targeting signals provided by secreted Netrin-B and Frazzled, a netrin receptor. Loss of Dscam1 function resulted in aberrant dendrite accumulation at a Netrin-B-expressing target, whereas concomitant loss of Frazzled prevented accumulation and caused severe deficits in dendritic territory coverage. Netrin misexpression was sufficient to induce ectopic dendritic targeting in a Frazzled-dependent manner, whereas Dscam1 was required to prevent ectopic accumulation, consistent with separable roles for these receptors. Our results suggest that Dscam1-mediated self-avoidance counters extrinsic signals that are required for normal dendritic patterning, but whose action would otherwise favor neurite accumulation. Counterbalancing roles for Dscam1 may be deployed in diverse contexts during neural circuit formation.     

Evidence for the coevolution of axon guidance molecule Netrin and its receptor Frazzled

Coevolution of a ligand and its receptor is critical for maintaining their function in different species, but how ligand and its receptor coevolve is poorly understood. The axon guidance molecule Netrin and its receptor Frazzled (Fra) are useful to study the mechanisms of ligand–receptor coevolution. Here, we have applied codon substitution models to identify positive selection of the netrin and fra genes. The sites under positive selection in netrin and fra were detected in same lineage, such as nematode, dipteran, hymenopteran, hemichordate, and teleost. Several amino acid residues that are under positive selection were identified in the interaction domains. Here we provide evidence that positive selection is essential for the coevolution of Netrin and Fra during central nervous system evolution.     

Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion.

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, we created chimeras carrying the ectodomain of one receptor and the cytoplasmic domain of the other and tested their function in transgenic Drosophila. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline.     

Cross-species recognition of tectal cues by retinal fibers in vitro

The retinae of vertebrates project in a topographic manner to several visual centers of the brain. The formation of these projections could depend on the existence of position-specific properties of retinal and target cells. In this study, we have tested the in vitro growth of mouse retinal fibers on membranes derived from various regions of the embryonic superior colliculus, a main target of the retina in this species. Fibers had the choice of elongating on membranes taken from either the anterior or the posterior half of the superior colliculus. Fibers from temporal areas of the retina prefer to elongate on anterior collicular membranes, while fibers from nasal areas do not show a preference. These phenomena are observed with membranes from embryonic (E15-E18) or young postnatal mice. In interspecies cultures where mouse retinal fibers had to grow on chick tectal membranes, or vice versa, the same preference for anterior tectal or collicular membranes in growth of temporal retinal fibers is observed, suggesting some similarities in the cues used in both species.     

Presenilin-dependent receptor processing is required for axon guidance

The Alzheimer's disease-linked gene presenilin is required for intramembrane proteolysis of amyloid-β precursor protein, contributing to the pathogenesis of neurodegeneration that is characterized by loss of neuronal connections, but the role of Presenilin in establishing neuronal connections is less clear. Through a forward genetic screen in mice for recessive genes affecting motor neurons, we identified the Columbus allele, which disrupts motor axon projections from the spinal cord. We mapped this mutation to the Presenilin-1 gene. Motor neurons and commissural interneurons in Columbus mutants lacking Presenilin-1 acquire an inappropriate attraction to Netrin produced by the floor plate because of an accumulation of DCC receptor fragments within the membrane that are insensitive to Slit/Robo silencing. Our findings reveal that Presenilin-dependent DCC receptor processing coordinates the interplay between Netrin/DCC and Slit/Robo signaling. Thus, Presenilin is a key neural circuit builder that gates the spatiotemporal pattern of guidance signaling, thereby ensuring neural projections occur with high fidelity.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.     

Limited topographic specificity in the targeting and branching of mammalian retinal axons.

We have studied in rats the topographic targeting of retinocollicular axons anterogradely labeled by focal retinal injections of the axon tracer DiI. We find that developing retinal axons widely mistarget along both the medial-lateral and the rostral-caudal axes of the superior colliculus (SC). In neonatal rats, labeled axons originating from injection sites in the temporal periphery covering less than 1% of the retina grow over most of the contralateral SC, suggesting that the growth cones of many axons initially fail to recognize their appropriate target region at the rostral SC border. Some of these axons correct their targeting errors and are retained; most do not and are eliminated. In neonates, peripheral nasal axons transiently develop branches throughout the SC. Branches formed by nasal axons are later restricted to a discrete terminal zone at the topographically appropriate, caudal SC border. At the neonatal stage, injections in temporal or nasal retina do result in a zone of increased labeling in the topographically correct region of the SC, but this zone is considerably larger than that labeled by a similar injection at a later stage. Thus, although the early projection is very diffuse, there is some bias for the correct region of the SC. Our findings indicate that in rats, developing retinal axons show only a limited specificity in their topographic targeting and branching. We conclude that mechanisms in addition to directed axon growth are required to establish the order characteristic of mature mammalian retinal projections.     

Constitutively active myosin light chain kinase alters axon guidance decisions in Drosophila embryos

Conventional myosin II activity provides the motile force for axon outgrowth, but to achieve directional movement during axon pathway formation, myosin activity should be regulated by the attractive and repulsive guidance cues that guide an axon to its target. Here, evidence for this regulation is obtained by using a constitutively active Myosin Light Chain Kinase (ctMLCK) to selectively elevate myosin II activity in Drosophila CNS neurons. Expression of ctMLCK pan-neurally or in primarily pCC/MP2 neurons causes these axons to cross the midline incorrectly. This occurs without altering cell fates and is sensitive to mutations in the regulatory light chains. These results confirm the importance of regulating myosin II activity during axon pathway formation. Mutations in the midline repulsive ligand Slit, or its receptor Roundabout, enhance the number of ctMLCK-induced crossovers, but ctMLCK expression also partially rescues commissure formation in commissureless mutants, where repulsive signals remain high. Overexpression of Frazzled, the receptor for midline attractive Netrins, enhances ctMLCK-dependent crossovers, but crossovers are suppressed when Frazzled activity is reduced by using loss-of-function mutations. These results confirm that proper pathway formation requires careful regulation of MLCK and/or myosin II activity and suggest that regulation occurs in direct response to attractive and repulsive cues.     

Midline Signalling Systems Direct the Formation of a Neural Map by Dendritic Targeting in the Drosophila Motor System

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. We recently showed that in the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. In this study, we show that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. We demonstrate that this map is “hard-wired”; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. We show that the midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. Our results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. We further propose that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common “meeting regions.”     

Modeling activity and target-dependent developmental cell death of mouse retinal ganglion cells ex vivo

Programmed cell death is widespread during the development of the central nervous system and serves multiple purposes including the establishment of neural connections. In the mouse retina a substantial reduction of retinal ganglion cells (RGCs) occurs during the first postnatal week, coinciding with the formation of retinotopic maps in the superior colliculus (SC). We previously established a retino-collicular culture preparation which recapitulates the progressive topographic ordering of RGC projections during early post-natal life. Here, we questioned whether this model could also be suitable to examine the mechanisms underlying developmental cell death of RGCs. Brn3a was used as a marker of the RGCs. A developmental decline in the number of Brn3a-immunolabelled neurons was found in the retinal explant with a timing that paralleled that observed in vivo. In contrast, the density of photoreceptors or of starburst amacrine cells increased, mimicking the evolution of these cell populations in vivo. Blockade of neural activity with tetrodotoxin increased the number of surviving Brn3a-labelled neurons in the retinal explant, as did the increase in target availability when one retinal explant was confronted with 2 or 4 collicular slices. Thus, this ex vivo model reproduces the developmental reduction of RGCs and recapitulates its regulation by neural activity and target availability. It therefore offers a simple way to analyze developmental cell death in this classic system. Using this model, we show that ephrin-A signaling does not participate to the regulation of the Brn3a population size in the retina, indicating that eprhin-A-mediated elimination of exuberant projections does not involve developmental cell death.     

Apoptosis in the developing visual system

Programmed cellular death is a widespread phenomenon during development of the nervous system. Two classes of molecules are particularly important in the context of apoptosis control in the nervous system: intracellular effectors homologous to the Caenorhabditis elegans Ced-3, -4, and -9 proteins, which in mammals correspond to the proteases of the caspase family, Apaf-1, and the members of the Bcl-2 protein family, and neurotrophic factors. Retinal ganglion cells lend a convenient model system with which to investigate apoptosis in central neurons during development as well as after injury. In this review, we discuss the role of these molecules in the control of programmed cellular death in the retinotectal system. Transgenic animal models and expression studies have shown that caspases, Bcl-2, Bax, and possibly Bcl-X are necessary players for the control of programmed cellular death in retinal ganglion cells. Bax and caspase 3 expression in retinal ganglion cells is upregulated after injury, and inhibition of Bax or caspase 3 increases the survival of injured retinal ganglion cells. Neurotrophins can support the survival of injured retinal ganglion cells, but this effect is transient. The physiological role of neurotrophins in the development of the retinocollicular system seems more related to the topographic refinement of retinocollicular projections, a process that is mediated, at least partially, by selective elimination of retinal ganglion cells making inappropriate topographic projections.