Semaphorin 3A‐Neuropilin‐1 signaling regulates peripheral axon fasciculation and pathfinding but not developmental cell death patterns

In early development, an excess of neurons is generated, of which later about half will be lost by cell death due to a limited supply of trophic support by their respective target areas. However, some of the neurons die when their axons have not yet reached their target, thus suggesting that additional causes of developmental cell death exist. Semaphorin 3A (Sema3A), in addition to its function as a guidance cue and mediator of timing and fasciculation of motor and sensory axon outgrowth, can also induce death of sensory neurons in vitro. However, it is unknown whether Neuropilin‐1 (Npn‐1), its binding receptor in axon guidance, also mediates the death‐inducing activity. We show here that abolished Sema3A‐Npn‐1 signaling does not influence the cell death patterns of motor or sensory neurons in mouse during the developmental wave of programmed cell death. The number of motor and sensory neurons was unchanged at embryonic day 15.5 when this wave is concluded. Interestingly, the defasciculation of early motor and sensory projections that is observed in the absence of Sema3A or Npn‐1 persists to postnatal stages. Thus, Sema3A‐Npn‐1 signaling plays an important role in the guidance and fasciculation of motor and sensory axons but does not contribute to the developmental elimination of these neurons.     

Defasciculation of neurites is mediated by tenascin-R and its neuronal receptor F3/11

Fasciculation and defasciculation of axons are major morphogenetic events in the formation of neuronal pathways during development. We have identified the extracellular matrix glycoprotein tenascin-R (TN-R) and its neuronal receptor, the immunoglobulin superfamily recognition molecule F3, as promoters of neurite defasciculation in cerebellar explant cultures. Perturbation of the interaction between these two molecules using both antibodies and an antisense oligonucleotide resulted in increased neurite fasciculation. The domains involved in defasciculation were identified as the N-terminal region of TN-R containing the cysteine-rich stretch and the 4.5 epidermal growth factor-like repeats and the immunoglobulin-like domains of F3. Fasciculation induced by antibodies and the antisense oligonucleotide could be reverted by a phorbol ester activator of protein kinase C, whereas the protein kinase inhibitor staurosporine increased fasciculation. Our observations indicate that defasciculated neurite outgrowth does not only depend on the reduction of the expression of fasciculation enhancing adhesion molecules, such as L1 and the neural cell adhesion molecule (NCAM), but also on recognition molecules that actively induce defasciculation by triggering second messenger systems. J. Neurosci. Res. 52:390–404, 1998. © 1998 Wiley-Liss, Inc.     

Requirement of cannabinoid CB(1) receptors in cortical pyramidal neurons for appropriate development of corticothalamic and thalamocortical projections

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.     

Axons crossing in the ventral commissure express L1 and GAD65 in the developing rat spinal cord

The neural cell adhesion molecule, L1, is thought to play a critical role in the formation and fasciculation of axon tracts during development. In the chick, the L1 cell adhesion molecule is expressed on both ipsi- and contralateral portions of commissural axons and perturbation studies produced a defasciculation of the ipsilateral commissural fibers. Yet in the rat, L1 is reported along commissural axons only after they have reached the contralateral marginal zone. When this species variation was reexamined, L1 was found to be expressed on rat commissural axons in a pattern similar to that observed in the chick. In addition, L1 is detected along commissural axons as early as embryonic day 12 in rats and maintained on both the ipsi- and contralateral surfaces during embryonic development. Other molecular markers that identify commissural axons in rats are TAG-1 (transiently expressed axonal glycoprotein) and DCC (deleted in colorectal cancer), and thus the pattern of L1 staining was compared with that of these other members of the immunoglobulin superfamily. Commissural axons emerging from dorsally located neurons are identified with TAG-1 and DCC, whereas L1 is detected only on ventrally located commissural axons. The pattern of L1 expression overlaps that of the more numerous laterally and ventromedially located GABAergic commissural axons. Furthermore, some of the GABAergic commissural axons express L1 on their surfaces. While commissural axons are often considered as a single population, differences in the combination of adhesion-type molecules on their surfaces and in their neurotransmitter phenotypes may signify distinctive neuronal subgroups.     

Secondary motoneuron axons localize DM-GRASP on their fasciculated segments

Cell surface adhesion molecules are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. We have studied a potential adhesion molecule using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. We show that zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion molecule of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. It is necessary for correct axon routing and fasciculation in the Drosophila visual system. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. We show that, of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: The protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets.     

Neural cell adhesion molecule L1: Signaling pathways and growth cone motility

The neural cell adhesion molecule L1 plays a key role in nervous system development including neuronal migration, neurite growth, and axonal fasciculation. L1 is expressed on most developing axons, and homophilic binding of L1 molecules on adjacent axons is likely to play a key role in axon extension. It is now well documented that a number of second-messenger systems are involved in L1-stimulated neurite growth in vitro. However, it is unclear how L1 homophilic or heterophilic binding trigger signals that regulate the mechanical forces that produce axon extension. In this report, we will review recent advances in understanding L1-associated signals, L1 interactions with the cytoskeleton, and the molecular mechanisms underlying growth cone motility. J. Neurosci. Res. 49:1–8, 1997. © 1997 Wiley-Liss Inc.     

Monoclonal antibodies demonstrate the organization of axons in the leech

Monoclonal antibodies have been generated that bind to subsets of neurons within the leech central nervous system (Zipser, B., and R. McKay (1981) Nature 289: 549-554). In this report we describe the binding patterns of monoclonal antibodies to subsets of axons in the leech using HRP-immunohistochemistry. Each antibody has a characteristic staining pattern in the connective, the large bundle of axons that runs the length of the nerve cord connecting each ganglion to its rostral and caudal neighbors. These staining patterns are consistent along the rostrocaudal axis of each animal, between animals of the same species, and, in many cases, between animals of different species. These results show that axonal position, like neuron cell body position, is a consistent feature of the organization of the leech central nervous system. Two antibodies bind to all of the axons in particular fascicles that are delimited by glial cell processes; another binds to single axons in fascicles that contain other, unstained axons. The grouping of antibody-identified axons into fascicles does not correlate in a simple way with the grouping of neuron cell bodies identified with the same antibody. The presence of one of these antigens on the surface of axons suggests a possible role in axon fasciculation. This report shows that molecular heterogeneity is a property of axons as well as of neuron cell bodies and demonstrates the organization of specific antibody-identified groups of axons within the connective.     

Activation of epidermal growth factor receptor mediates receptor axon sorting and extension in the developing olfactory system of the moth Manduca sexta

During development of the adult olfactory system of the moth Manduca sexta, olfactory receptor neurons extend axons from the olfactory epithelium in the antenna into the brain. As they arrive at the brain, interactions with centrally derived glial cells cause axons to sort and fasciculate with other axons destined to innervate the same glomeruli. Here we report studies indicating that activation of the epidermal growth factor receptor (EGFR) is involved in axon ingrowth and targeting. Blocking the EGFR kinase domain pharmacologically leads to stalling of many axons in the sorting zone and nerve layer as well as abnormal axonal fasciculation in the sorting zone. We also find that neuroglian, an IgCAM known to activate the EGFR through homophilic interactions in other systems, is transiently present on olfactory receptor neuron axons and on glia during the critical stages of the sorting process. The neuroglian is resistant to extraction with Triton X-100 in the sorting zone and nerve layer, possibly indicating its stabilization by homophilic binding in these regions. Our results suggest a mechanism whereby neuroglian molecules on axons and possibly sorting zone glia bind homophilically, leading to activation of EGFRs, with subsequent effects on axon sorting, pathfinding, and extension, and glomerulus development.     

EphrinA6 on chick retinal axons is a key component for p75(NTR)-dependent axon repulsion and TrkB-dependent axon branching

A characteristic of the ephrin/Eph family is their capacity for bi-directional signalling. This means that an ephrin, for example, can function either as a ligand for an Eph 'receptor', or as a receptor for an Eph 'ligand'. A system in which this phenomenon is well studied is the retinotectal projection in which the guidance of retinal ganglion cell (RGC) axons to their target area in the tectum is controlled by both Ephs and ephrins expressed in gradients in both the retina and tectum. Here we have analysed the receptor function of ephrinAs on RGC axons in further detail by focussing on ephrinA6, which is the most strongly expressed ephrinA in the chick retina. EphrinAs are GPI-anchored proteins and therefore require the interaction with transmembrane proteins to exert this receptor function. Previous work has shown that ephrinAs interact on RGC axons in cis with the neurotrophin receptors p75(NTR) and TrkB. P75(NTR) then was shown to be necessary for the repulsion of ephrinA-expressing RGC axons from an EphA substrate and for the downregulation of axon branching. In turn, an interaction of ephrinAs with TrkB as well as an increase in axonal ephrinA expression augments the axon branch-promoting activity of TrkB. We now show that ephrinA6 is the necessary ephrinA component of the repulsive ephrinA/p75(NTR) receptor complex on chick RGC axons as axons lacking ephrinA6 no longer avoid an EphA matrix in stripe assay experiments. We also demonstrate that the branch-promoting activity of TrkB is dependent on ephrinA6 as a knockdown of ephrinA6 renders RGC axons insensitive to BDNF, the high affinity ligand for TrkB. In sum our data further strengthen the hypothesis that a fine-tuned interplay of ephrinAs with p75(NTR) and TrkB is important for the guidance and branching of RGC axons.     

Ultrastructural observations on the expression of axonin-1: implications for the fasciculation of sensory axons during axonal outgrowth into the chick hindlimb.

To help understand how axons interact as they grow into the developing chick hindlimb, we used electron microscopy in conjunction with immunoperoxidase staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axons travel together in bundles, tightly apposed to one another. In contrast, motoneuron axons are more widely spaced, although motoneuron axons situated at the perimeter of sensory axon bundles are found in close contact with neighboring sensory axons. Sensory growth cones and lamellipodia tend to be located centrally within the bundles, with several lamellipodia typically being found stacked together. Strikingly, regions of close axonal apposition are accompanied by axonin-1 expression, suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow together, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axons. Axons situated at the periphery of sensory bundles are typically partly labelled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells. Thus, axonin-1 appears to become redistributed within the membranes of axons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically to axonin-1, appears uniformly distributed on even those axons that would have an asymmetric distribution of axonin-1. Thus, the localization of axonin-1 strongly suggests that it plays an important role in sensory axon fasciculation, but the relative contributions of its interactions with various potential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculation is important in sensory axon guidance, sensory axons may also explore the local environment.     

Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types

ABSTRACT: BACKGROUND: Understanding the cellular mechanisms regulating axon degeneration and regeneration is crucial for developing treatments for nerve injury and neurodegenerative disease. In neurons, axon degeneration is distinct from cell body death and often precedes or is associated with the onset of disease symptoms. In the peripheral nervous system of both vertebrates and invertebrates, after degeneration of detached fragments, axons can often regenerate to restore function. Many studies of axonal degeneration and regeneration have used in vitro approaches, but the influence of extrinsic cell types on these processes can only be fully addressed in live animals. Because of its simplicity and superficial location, the larval zebrafish posterior lateral line (pLL) nerve is an ideal model system for live studies of axon degeneration and regeneration. RESULTS: We used laser axotomy and time-lapse imaging of pLL axons to characterize the roles of leukocytes, Schwann cells and target sensory hair cells in axon degeneration and regeneration in vivo. Immune cells were essential for efficient removal of axonal debris after axotomy. Schwann cells were required for proper fasciculation and pathfinding of regenerating axons to their target cells. Intact target hair cells were not themselves required for regeneration, but chemical ablation of neuromasts caused axons to transiently deviate from their normal paths. CONCLUSIONS: Macrophages, Schwann cells, and target sensory organs are required for distinct aspects of pLL axon degeneration or regeneration in the zebrafish larva. Our work introduces a powerful vertebrate model for analyzing axonal degeneration and regeneration in the living animal and elucidating the role of extrinsic cell types in these processes.     

Local neurotrophin effects on central trigeminal axon growth patterns

In dissociated cell and wholemount explant cultures of the embryonic trigeminal pathway NGF promotes exuberant elongation of trigeminal ganglion (TG) axons, whereas NT-3 leads to precocious arborization [J. Comp. Neurol. 425 (2000) 202]. In the present study, we investigated the axonal effects of local applications of NGF and NT-3. We placed small sepharose beads loaded with either NGF or NT-3 along the lateral edge of the central trigeminal tract in TG-brainstem intact wholemount explant cultures prepared from embryonic day 15 rats. Labeling of the TG with carbocyanine dye, DiI, revealed that NGF induces local defasciculation and diversion of trigeminal axons. Numerous axons leave the tract, grow towards the bead and engulf it, while some axons grow away from the neurotrophin source. NT-3, on the other hand, induced localized interstitial branching and formation of neuritic tangles in the vicinity of the neurotrophin source. Double immunocytochemistry showed that axons responding to NGF were predominantly TrkA-positive, whereas both TrkA and TrkC-positive axons responded to NT-3. Our results indicate that localized neurotrophin sources along the routes of embryonic sensory axons in the central nervous system, far away from their parent cell bodies, can alter restricted axonal pathways and induce elongation, arborization responses.     

Chondroitin sulfate proteoglycan phosphacan associates with parallel fibers and modulates axonal extension and fasciculation of cerebellar granule cells

Phosphacan is a nervous system-specific chondroitin sulfate proteoglycan and one of the major components of extracellular matrix in the brain. In the present study, we examined its spatiotemporal expression, ultrastructural localization, binding manner, and in vitro analysis on cell adhesion, axonal extension, and fasciculation in rat cerebellum. The present light microscopic immunohistochemistry showed that phosphacan immunoreactivity was localized mainly at the molecular layer in the cerebellum, but not at the external granular layer. Further double labeling immunohistochemical and immunoelectron microscopic studies revealed that phosphacan was localized around parallel fibers, but not at synapses. The binding of phosphacan to membrane and/or extracellular matrix partly required Ca2+ and was mediated through its core glycoprotein. Phosphacan inhibited adhesion and axonal extension of cerebellar granule cells in dissociated culture, while it promoted axonal fasciculation of their aggregated culture. These results indicate that phosphacan around parallel fibers may be the repulsive substratum for adhesion and extension of granule cells and promote the fasciculation of parallel fibers.     

Eye‐specific segregation and differential fasciculation of developing retinal ganglion cell axons in the mouse visual pathway

Prior to forming and refining synaptic connections, axons of projection neurons navigate long distances to their targets. While much is known about guidance cues for axon navigation through intermediate choice points, whether and how axons are organized within tracts is less clear. Here we analyze the organization of retinal ganglion cell (RGC) axons in the developing mouse retinogeniculate pathway. RGC axons are organized by both eye‐specificity and topography in the optic nerve and tract: ipsilateral RGC axons are segregated from contralateral axons and are offset laterally in the tract relative to contralateral axon topographic position. To identify potential cell‐autonomous factors contributing to the segregation of ipsilateral and contralateral RGC axons in the visual pathway, we assessed their fasciculation behavior in a retinal explant assay. Ipsilateral RGC neurites self‐fasciculate more than contralateral neurites in vitro and maintain this difference in the presence of extrinsic chiasm cues. To further probe the role of axon self‐association in circuit formation in vivo, we examined RGC axon organization and fasciculation in an EphB1^?/? mutant, in which a subset of ipsilateral RGC axons aberrantly crosses the midline but targets the ipsilateral zone in the dorsal lateral geniculate nucleus on the opposite side. Aberrantly crossing axons retain their association with ipsilateral axons in the contralateral tract, indicating that cohort‐specific axon affinity is maintained independently of guidance signals present at the midline. Our results provide a comprehensive assessment of RGC axon organization in the retinogeniculate pathway and suggest that axon self‐association contributes to pre‐target axon organization.     

Sprouting adult CNS cholinergic axons express nile and associate with astrocytic surfaces expressing neural cell adhesion molecule

To assess the cellular and molecular substrates for cholinergic axon growth in the adult central nervous system (CNS), we implanted grafts of control and nerve growth factor (NGF)-producing genetically modified fibroblasts within the striatum of rats. Sprouting cholinergic axonal processes that grew into grafts of NGF-producing fibroblasts were fasciculated and followed the surface of astrocytic processes for long distances within the grafts. The close and long distance anatomical relationship between the sprouted axons and the astrocytes supported previous ultrastructural evidence that astrocytes may serve as a cellular substrate for sprouting cholinergic axons in vivo. The sprouted axon processes were associated with the expression of nerve growth factor-inducible large external (NILE) glycoprotein on their surfaces. NILE expression was not seen in control grafts where there was an absence of cholinergic ingrowth. NILE has been demonstrated to play a role in axon fasciculation in a number of other neural systems. The astrocytic processes in both control and NGF-producing fibroblast grafts expressed neural cell adhesion molecule (NCAM), suggesting that NCAM-mediated adhesion may be responsible for the close relationship between the axons and astrocytes within the grafts. NGF-induced heterotypic interactions between neuronal NILE and astroglial NCAM may also be required for adult cholinergic axonal sprouting. © 1996 Wiley-Liss, Inc.     

Axonal fasciculation and the role of polysialic acid‐neural cell adhesion molecule in rat cortical neurons

Axonal fasciculation is a mechanism deployed by growing axons to reach their targets during development of the nervous system. Published data have suggested the involvement of neuronal cell adhesion molecules (NCAM) in axonal fasciculation. We have characterized the formation of axonal fascicles in serum‐free, primary cultures of cortical neurons from embryonic rat brains. Unlike the published data, axonal fascicles in our system have a unique morphology: they are waveform, are rarely thicker than 20 μm, and can reach up to several millimeters in length. We observed an age and time dependence in the formation of fascicles. They formed only in cultures from embryonic day 15–17 brain and only between 4 days in vitro (DIV) and 11 DIV. Electron microscopy showed that the fascicles consisted of mostly axonal processes. Immunocytochemical staining confirmed that the fascicles were positive for the 66‐kDa neurofilament protein, NF66, but they contained few, if any, microtubule‐associated protein‐2‐positive or glial fibrillary acidic protein‐positive processes. Polysialic acids appeared to be critical in the formation of fascicles. Neuraminidase treatment prevented the formation of fascicles when added before 5 DIV. Addition of a specific inhibitor blocked the effect of neuraminidase. The cortical neurons in our model shared several important features with axon fasciculation in vivo and may provide a unique system for studying the molecular mechanisms involved in the formation of axonal tracts in the brain. © 2013 Wiley Periodicals, Inc.     

Cadherin-8 and N-cadherin differentially regulate pre- and postsynaptic development of the hippocampal mossy fiber pathway

Cells sort into regions and groups in part by their selective surface expression of particular classic cadherins during development. In the nervous system, cadherin-based sorting can define axon tracts, restrict axonal and dendritic arbors to particular regions or layers, and may encode certain aspects of synapse specificity. The underlying model has been that afferents and their targets hold in common the expression of a particular cadherin, thereby providing a recognition code of homophilic cadherin binding. However, most neurons express multiple cadherins, and it is not clear whether multiple cadherins all act similarly in shaping neural circuitry. Here we asked how two such cadherins, cadherin-8 and N-cadherin, influence the guidance and differentiation of hippocampal mossy fibers. Using organotypic hippocampal cultures, we find that cadherin-8 regulates mossy fiber fasciculation and targeting, but has little effect on CA3 dendrites. In contrast, N-cadherin regulates mossy fiber fasciculation, but has little impact on axonal growth and targeting. However, N-cadherin is essential for CA3 dendrite arborization. Both cadherins are required for formation of proper numbers of presynaptic terminals. Mechanistically, such differential actions of these two cadherins could, in theory, reflect coupling to distinct intracellular binding partners. However, we find that both cadherins bind beta-catenin in dentate gyrus (DG). This suggests that cadherins may engage different intracellular signaling cascades downstream of beta-catenin, coopt different extracellular binding partners, or target distinct subcellular domains. Together our findings demonstrate that cadherin-8 and N-cadherin are critical for generating the mossy fiber pathway, but that each contributes differentially to afferent and target differentiation, thereby complementing one another in the assembly of a synaptic circuit.     

Axonal growth towards Xenopus skin in vitro is mediated by matrix metalloproteinase activity

We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)‐18 and also other MMPs, and that this long‐range neurotropic activity is inhibited by the broad‐spectrum MMP inhibitors BB‐94 and GM6001. We also show that forced expression of MMP‐18 in COS‐7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants. Nidogen is the target of MMPs released by cultured skin in matrigel, whereas other components remain intact. Our results suggest a novel link between MMP activity and extracellular matrix breakdown in the control of axonal growth.     

Axon fasciculation defects and retinal dysplasias in mice lacking the immunoglobulin superfamily adhesion molecule BEN/ALCAM/SC1

The immunoglobulin superfamily adhesion molecule BEN (other names include ALCAM, SC1, DM-GRASP, neurolin, and CD166) has been implicated in the control of numerous developmental and pathological processes, including the guidance of retinal and motor axons to their targets. To test hypotheses about BEN function, we disrupted its gene via homologous recombination and analyzed the resulting mutant mice. Mice lacking BEN are viable and fertile, and display no external morphological defects. Despite grossly normal trajectories, both motor and retinal ganglion cell axons fasciculated poorly and were occasionally misdirected. In addition, BEN mutant retinae exhibited evaginated or invaginated regions with photoreceptor ectopias that resembled the "retinal folds" observed in some human retinopathies. Together, these results demonstrate that BEN promotes fasciculation of multiple axonal populations and uncover an unexpected function for BEN in retinal histogenesis.