Skin-derived cues control arborization of sensory dendrites in Caenorhabditis elegans.

Sensory dendrites depend on cues from their environment to pattern their growth and direct them toward their correct target tissues. Yet, little is known about dendrite-substrate interactions during dendrite morphogenesis. Here, we describe MNR-1/menorin, which is part of the conserved Fam151 family of proteins and is expressed in the skin to control the elaboration of "menorah"-like dendrites of mechanosensory neurons in Caenorhabditis elegans. We provide biochemical and genetic evidence that MNR-1 acts as a contact-dependent or short-range cue in concert with the neural cell adhesion molecule SAX-7/L1CAM in the skin and through the neuronal leucine-rich repeat transmembrane receptor DMA-1 on sensory dendrites. Our data describe an unknown pathway that provides spatial information from the skin substrate to pattern sensory dendrite development nonautonomously.

Floor plate-derived neuropilin-2 functions as a secreted semaphorin sink to facilitate commissural axon midline crossing.

Commissural axon guidance depends on a myriad of cues expressed by intermediate targets. Secreted semaphorins signal through neuropilin-2/plexin-A1 receptor complexes on post-crossing commissural axons to mediate floor plate repulsion in the mouse spinal cord. Here, we show that neuropilin-2/plexin-A1 are also coexpressed on commissural axons prior to midline crossing and can mediate precrossing semaphorin-induced repulsion in vitro. How premature semaphorin-induced repulsion of precrossing axons is suppressed in vivo is not known. We discovered that a novel source of floor plate-derived, but not axon-derived, neuropilin-2 is required for precrossing axon pathfinding. Floor plate-specific deletion of neuropilin-2 significantly reduces the presence of precrossing axons in the ventral spinal cord, which can be rescued by inhibiting plexin-A1 signaling in vivo. Our results show that floor plate-derived neuropilin-2 is developmentally regulated, functioning as a molecular sink to sequester semaphorins, preventing premature repulsion of precrossing axons prior to subsequent down-regulation, and allowing for semaphorin-mediated repulsion of post-crossing axons.

Motor axon exit from the mammalian spinal cord is controlled by the homeodomain protein Nkx2.9 via Robo-Slit signaling.

Mammalian motor circuits control voluntary movements by transmitting signals from the central nervous system (CNS) to muscle targets. To form these circuits, motor neurons (MNs) must extend their axons out of the CNS. Although exit from the CNS is an indispensable phase of motor axon pathfinding, the underlying molecular mechanisms remain obscure. Here, we present the first identification of a genetic pathway that regulates motor axon exit from the vertebrate spinal cord, utilizing spinal accessory motor neurons (SACMNs) as a model system. SACMNs are a homogeneous population of spinal MNs with axons that leave the CNS through a discrete lateral exit point (LEP) and can be visualized by the expression of the cell surface protein BEN. We show that the homeodomain transcription factor Nkx2.9 is selectively required for SACMN axon exit and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9; loss of Nkx2.9 leads to a reduction in Robo2 mRNA and protein within SACMNs and SACMN axons fail to exit the spinal cord in Robo2-deficient mice. Consistent with short-range interactions between Robo2 and Slit ligands regulating SACMN axon exit, Robo2-expressing SACMN axons normally navigate through LEP-associated Slits as they emerge from the spinal cord, and fail to exit in Slit-deficient mice. Our studies support the view that Nkx2.9 controls SACMN axon exit from the mammalian spinal cord by regulating Robo-Slit signaling.

C. elegans bicd-1, homolog of the Drosophila dynein accessory factor Bicaudal D, regulates the branching of PVD sensory neuron dendrites.

The establishment of cell type-specific dendritic arborization patterns is a key phase in the assembly of neuronal circuitry that facilitates the integration and processing of synaptic and sensory input. Although studies in Drosophila and vertebrate systems have identified a variety of factors that regulate dendrite branch formation, the molecular mechanisms that control this process remain poorly defined. Here, we introduce the use of the Caenorhabditis elegans PVD neurons, a pair of putative nociceptors that elaborate complex dendritic arbors, as a tractable model for conducting high-throughput RNAi screens aimed at identifying key regulators of dendritic branch formation. By carrying out two separate RNAi screens, a small-scale candidate-based screen and a large-scale screen of the ~3000 genes on chromosome IV, we retrieved 11 genes that either promote or suppress the formation of PVD-associated dendrites. We present a detailed functional characterization of one of the genes, bicd-1, which encodes a microtubule-associated protein previously shown to modulate the transport of mRNAs and organelles in a variety of organisms. Specifically, we describe a novel role for bicd-1 in regulating dendrite branch formation and show that bicd-1 is likely to be expressed, and primarily required, in PVD neurons to control dendritic branching. We also present evidence that bicd-1 operates in a conserved pathway with dhc-1 and unc-116, components of the dynein minus-end-directed and kinesin-1 plus-end-directed microtubule-based motor complexes, respectively, and interacts genetically with the repulsive guidance receptor unc-5.

Axon sorting within the spinal cord marginal zone via Robo-mediated inhibition of N-cadherin controls spinocerebellar tract formation.

The axons of spinal projection neurons transmit sensory information to the brain by ascending within highly organized longitudinal tracts. However, the molecular mechanisms that control the sorting of these axons within the spinal cord and their directed growth to poorly defined targets are not understood. Here, we show that an interplay between Robo and the cell adhesion molecule, N-cadherin, sorts spinal commissural axons into appropriate longitudinal tracts within the spinal cord, and thereby facilitates their brain targeting. Specifically, we show that d1 and d2 spinal commissural axons join the lateral funiculus within the spinal cord and target the cerebellum in chick embryos, and that these axons contribute to the spinocerebellar projection in transgenic reporter mice. Disabling Robo signaling or overexpressing N-cadherin on these axons prevents the formation of the lateral funiculus and the spinocerebellar tract, and simultaneously perturbing Robo and N-cadherin function rescues both phenotypes in chick embryos. Consistent with these observations, disabling Robo function in conditional N-cadherin knock-out mice results in a wild-type-like lateral funiculus. Together, these findings suggest that spinal projection axons must be sorted into distinct longitudinal tracts within the spinal cord proper to project to their brain targets.

Crossing the border: molecular control of motor axon exit.

Living organisms heavily rely on the function of motor circuits for their survival and for adapting to ever-changing environments. Unique among central nervous system (CNS) neurons, motor neurons (MNs) project their axons out of the CNS. Once in the periphery, motor axons navigate along highly stereotyped trajectories, often at considerable distances from their cell bodies, to innervate appropriate muscle targets. A key decision made by pathfinding motor axons is whether to exit the CNS through dorsal or ventral motor exit points (MEPs). In contrast to the major advances made in understanding the mechanisms that regulate the specification of MN subtypes and the innervation of limb muscles, remarkably little is known about how MN axons project out of the CNS. Nevertheless, a limited number of studies, mainly in Drosophila, have identified transcription factors, and in some cases candidate downstream effector molecules, that are required for motor axons to exit the spinal cord. Notably, specialized neural crest cell derivatives, referred to as Boundary Cap (BC) cells, pre-figure and demarcate MEPs in vertebrates. Surprisingly, however, BC cells are not required for MN axon exit, but rather restrict MN cell bodies from ectopically migrating along their axons out of the CNS. Here, we describe the small set of studies that have addressed motor axon exit in Drosophila and vertebrates, and discuss our fragmentary knowledge of the mechanisms, which guide motor axons out of the CNS.

UNC5A promotes neuronal apoptosis during spinal cord development independent of netrin-1.

In addition to their role as chemorepellent netrin-1 receptors, UNC5 proteins may mediate cell death because they induce apoptosis in cultured cells. To test this in vivo, we generated Unc5a (formerly Unc5h1) knockout mice and found that this deletion decreased apoptosis and increased the number of neurons in the spinal cord. In contrast, loss of netrin-1 (Ntn1) did not affect the amount of apoptosis, suggesting that NTN1 is not required for neuronal apoptosis in vivo.

Toxicity in ALS: TDP-43 modifiers and C9orf72.

Amyotrophic Lateral Sclerosis (ALS) is a devastating and fatal neurodegenerative disease affecting approximately 30,000 individuals in the United States. The average age of onset is 55 years and progression of the disease is rapid with most patients dying of respiratory failure within 3-5 years. Currently available therapeutics have modest effects on patient survival, underscoring the immediate need for more effective medicines. Recent technological advances in next generation sequencing have led to a substantial uptick in the discovery of genes linked to ALS. Since 90 % of ALS cases are sporadic, risk genes identified in familial cases provide invaluable insights into the molecular pathogenesis of the disease. Most notably, TDP-43-expressing neuronal inclusions and C9orf72 mutations have emerged as the key pathological and genetic hallmarks, respectively, of ALS. In this review, we will discuss recent advances in modifiers of TDP-43 toxicity, with an emphasis on Ataxin-2, one of the most well-characterized TDP-43 modifiers. An understanding of Ataxin-2 function and related biological pathways could provide a framework for the discovery of other novel modifiers of TDP-43. We will also describe the pathogenic mechanisms underlying C9orf72 toxicity and how these impact the disease process. Finally, we will explore emerging therapeutic strategies for dampening TDP-43 and C9orf72 toxicity and, ultimately, slowing or halting the progression of ALS.

Manipulating Robo expression in vivo perturbs commissural axon pathfinding in the chick spinal cord.

In vertebrate embryos, most spinal commissural axons cross the ventral midline (VM) and project either alongside or significant distances away from the floor plate (FP). The upregulation of repulsive Robo1/2 receptors on postcrossing commissural axons, in mammals, presumably allows these axons to respond to the midline-associated repellents, Slit1-3, facilitating their expulsion from, and prohibiting their reentry into, the FP. Compelling data suggest that Robo3 represses Robo1/2 function on precrossing axons and that Robo1/2 inhibit attractive guidance receptors on postcrossing axons, thereby ensuring that decussated axons are selectively responsive to midline Slits. However, whether Robo1/2 expel decussated commissural axons from the VM and/or prevent their reentry into the FP has not been explicitly established in vivo. Furthermore, some commissural axons do not require Robo1/2 to elaborate appropriate contralateral projections in the mouse spinal cord. Here, we use unilateral in ovo electroporation together with Atoh1 and Neurog1 enhancer elements to visualize, and assess the consequences of manipulating Robo expression on, dl1 and dl2 chick commissural axons. In response to misexpressing a cytoplasmic truncation of Robo1 and/or Robo2, which should block all Robo-ligand interactions, postcrossing commissural axons extend alongside, but do not project away from or reenter the FP. In contrast, misexpression of full-length Robo2 prevents many commissural axons from crossing the VM. Together, these findings support key and selective in vivo roles for Robo receptors in presumably altering the responsiveness of decussated commissural axons and facilitating their expulsion from the VM within the chick spinal cord.

EphB receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord.

EphB receptors and their ephrin-B ligands are required for midline guidance decisions at several rostrocaudal levels of the developing CNS. In the embryonic vertebrate spinal cord, ephrin-B3 is localized to the floor plate (FP) at the ventral midline (VM), ephrin-B1 and ephrin-B2 are expressed in the dorsal spinal cord, and decussated EphB receptor-bearing commissural axons navigate between these ventral and dorsal ephrin-B domains. Despite these compelling expression patterns, the in vivo role(s) for EphB and ephrin-B proteins in regulating the guidance of spinal commissural axons has not been established. Here, we use DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeling to assess the pathfinding of commissural axons in the spinal cords of ephrin-B and EphB mutant mouse embryos. In mice lacking ephrin-B3 or multiple EphB receptors, a significant number of axons followed aberrant trajectories in the immediate vicinity of the VM. Furthermore, forked transverse commissural (FTC) axons, a unique class of commissural axons that continues to project in the transverse plane on the contralateral side of the FP, were present at a markedly higher frequency in ephrin-B3 and EphB mutants, compared with wild-type embryos. Neither the midline guidance errors nor excessive numbers of FTC axons were observed in the spinal cords of ephrin-B3(lacz) mice that express a truncated form of ephrin-B3, which is capable of forward but not reverse signaling. In contrast to the midline guidance defects observed in EphB and ephrin-B3 mutant embryos, wild-type-like contralateral projections were observed in mice lacking ephrin-B1 and/or ephrin-B2.

Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord.

Within the developing vertebrate spinal cord, motor neuron subtypes are distinguished by the settling positions of their cell bodies, patterns of gene expression, and the paths their axons follow to exit the CNS. The inclusive set of cues required to guide a given motor axon subtype from cell body to target has yet to be identified, in any species. This is attributable, in part, to the unavailability of markers that demarcate the complete trajectory followed by a specific class of spinal motor axons. Most spinal motor neurons extend axons out of the CNS through ventral exit points. In contrast, spinal accessory motor neurons (SACMNs) project dorsally directed axons through lateral exit points (LEPs), and these axons assemble into the spinal accessory nerve (SAN). Here we show that an antibody against BEN/ALCAM/SC1/DM-GRASP/MuSC selectively labels mouse SACMNs and can be used to trace the pathfinding of SACMN axons. We use this marker, together with a battery of transcription factor-deficient or guidance cue/receptor-deficient mice to identify molecules required for distinct stages of SACMN development. Specifically, we find that Gli2 is required for the initial extension of axons from SACMN cell bodies, and that netrin-1 and its receptor Dcc are required for the proper dorsal migration of these cells and the dorsally directed extension of SACMN axons toward the LEPs. Furthermore, in the absence of the transcription factor Nkx2.9, SACMN axons fail to exit the CNS. Together, these findings suggest molecular mechanisms that are likely to regulate key steps in SACMN development.

Distribution of EphB receptors and ephrin-B1 in the developing vertebrate spinal cord.

Contact-dependent interactions between EphB receptors and ephrin-B ligands mediate a variety of cell-cell communication events in the developing and mature central nervous system (CNS). These predominantly repulsive interactions occur at the interface between what are considered to be mutually exclusive EphB and ephrin-B expression domains. We previously used receptor and ligand affinity probes to show that ephrin-B ligands are expressed in the floor plate and within a dorsal region of the embryonic mouse spinal cord, while EphB receptors are present on decussated segments of commissural axons that navigate between these ephrin-B domains. Here we present the generation and characterization of two new monoclonal antibodies, mAb EfB1-3, which recognizes EphB1, EphB2, and EphB3, and mAb efrnB1, which is specific for ephrin-B1. We use these reagents and polyclonal antibodies specific for EphB1, EphB2, EphB3, or ephrin-B1 to describe the spatiotemporal expression patterns of EphB receptors and ephrin-B1 in the vertebrate spinal cord. Consistent with affinity probe binding, we show that EphB1, EphB2, and EphB3 are each preferentially expressed on decussated segments of commissural axons in vivo and in vitro, and that ephrin-B1 is expressed in a dorsal domain of the spinal cord that includes the roof plate. In contrast to affinity probe binding profiles, we show here that EphB1, EphB2, and EphB3 are present on the ventral commissure, and that EphB1 and EphB3 are expressed on axons that compose the dorsal funiculus. In addition, we unexpectedly find that mesenchymal cells, which surround the spinal cord and dorsal root ganglion, express ephrin-B1.

Muscle- and Skin-Derived Cues Jointly Orchestrate Patterning of Somatosensory Dendrites.

Sensory dendrite arbors are patterned through cell-autonomously and non-cell-autonomously functioning factors [1-3]. Yet, only a few non-cell-autonomously acting proteins have been identified, including semaphorins [4, 5], brain-derived neurotrophic factors (BDNFs) [6], UNC-6/Netrin [7], and the conserved MNR-1/Menorin-SAX-7/L1CAM cell adhesion complex [8, 9]. This complex acts from the skin to pattern the stereotypic dendritic arbors of PVD and FLP somatosensory neurons in Caenorhabditis elegans through the leucine-rich transmembrane receptor DMA-1/LRR-TM expressed on PVD neurons [8, 9]. Here we describe a role for the diffusible C. elegans protein LECT-2, which is homologous to vertebrate leukocyte cell-derived chemotaxin 2 (LECT2)/Chondromodulin II. LECT2/Chondromodulin II has been implicated in a variety of pathological conditions [10-13], but the developmental functions of LECT2 have remained elusive. We find that LECT-2/Chondromodulin II is required for development of PVD and FLP dendritic arbors and can act as a diffusible cue from a distance to shape dendritic arbors. Expressed in body-wall muscles, LECT-2 decorates neuronal processes and hypodermal cells in a pattern similar to the cell adhesion molecule SAX-7/L1CAM. LECT-2 functions genetically downstream of the MNR-1/Menorin-SAX-7/L1CAM adhesion complex and upstream of the DMA-1 receptor. LECT-2 localization is dependent on SAX-7/L1CAM, but not on MNR-1/Menorin or DMA-1/LRR-TM, suggesting that LECT-2 functions as part of the skin-derived MNR-1/Menorin-SAX-7/L1CAM adhesion complex. Collectively, our findings suggest that LECT-2/Chondromodulin II acts as a muscle-derived, diffusible cofactor together with a skin-derived cell adhesion complex to orchestrate the molecular interactions of three tissues during patterning of somatosensory dendrites.

Caenorhabditis elegans VEM-1, a novel membrane protein, regulates the guidance of ventral nerve cord-associated axons.

In the developing CNS, pathfinding growth cones use intermediate target- and pioneer axon-associated guidance cues to navigate along stereotypical trajectories. We previously showed that the novel membrane-associated protein Vema is localized to the floor plate and the optic chiasm, intermediate targets located at the ventral midline of the spinal cord and diencephalon in the developing rodent CNS, respectively. Here, we report that the Caenorhabditis elegans ortholog of vema, vem-1, is expressed by the AVG pioneer midline neuron and by several neurons that extend longitudinally projecting axons into the ventral nerve cord (VNC). In vem-1 mutants and vem-1 (RNAi) animals, a subset of posteriorly projecting interneuron axons either fail to extend ventrally to the VNC and, instead, assume aberrant lateral positions or are inappropriately located in the left tract of the VNC. In addition, ventral motor neuron axons exhibit pathfinding errors within the VNC and along the dorsoventral body axis. The conserved UNC-40/DCC and SAX-3-/Robo receptors mediate signaling events that regulate axon guidance in a wide variety of systems. Double-mutant analyses reveal that vem-1 genetically interacts with unc-40 and is likely to function in parallel with sax-3 to regulate the guidance of a subset of VNC-associated interneuron and motor neuron axons. Consistent with these genetic data, we also show that VEM-1 is capable of physically interacting with UNC-40 but not SAX-3.

Expression of Vema in the developing mouse spinal cord and optic chiasm.

A critical phase of nervous system development is the formation of connections between axons and their synaptic targets. Intermediate targets play important roles in axon pathfinding by supplying growing axons with long- and short- range guidance cues at decision points along their trajectory. We recently identified Vema as a novel membrane-associated protein that is expressed at the ventral midline of the developing vertebrate central nervous system (CNS). We report that Vema is expressed in the floor plate, an intermediate target for pathfinding commissural axons located at the ventral midline of the developing mouse spinal cord. Interestingly, Vema expression overlaps with the position of an unique population of neurons situated at the midline of the ventral diencephalon and that function as intermediate targets for pathfinding retinal ganglion cell axons. The distribution of Vema in the developing spinal cord and optic chiasm resembles the expression patterns of a variety of molecules known to play important roles in axon guidance, including Robo2, Neuropilin2, and SSEA. The expression of Vema at two key choice points for pathfinding axons suggests an important role for this protein in regulating axon guidance at the midline of the developing mouse central nervous system.

Diversity of contralateral commissural projections in the embryonic rodent spinal cord.

In vertebrate embryos, the axons of spinal commissural neurons grow toward and across the floor plate, a specialized structure located at the ventral midline. Although the initial segment of this trajectory has been intensively studied, relatively little is known about commissural axon pathfinding on the contralateral side of the floor plate in higher vertebrates. We recently demonstrated that many embryonic mouse and chick spinal commissural axons follow a complex trajectory once they cross the ventral midline. Here we use focal applications of 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate (DiI) to identify four different contralateral commissural trajectories, two of which have not previously been described in the embryonic rodent spinal cord. Intermediate longitudinal commissural (ILC) axons travel away from the floor plate along an arcuate trajectory into intermediate regions of the spinal cord. In contrast, medial longitudinal commissural (MLC) axons grow alongside the floor plate, projecting primarily in the rostral direction. Bifurcating longitudinal commissural (BLC) axons branch into rostrally and caudally directed projections. Forked transverse commissural (FTC) axons either execute two orthogonal turns before crossing the floor plate or extend directly across the floor plate. We also show a variation in the relative frequencies of individual contralateral commissural projections along the dorsoventral and anteroposterior axes of the spinal cord. In addition, using a novel culture system, we demonstrate that commissural axons elaborate ILC-, MLC-, BLC-, and FTC-like trajectories in vitro. These results provide a basis for examining the mechanisms that regulate commissural axon pathfinding on the contralateral side of the floor plate in the embryonic rodent spinal cord.

Neuropilin2 regulates the guidance of post-crossing spinal commissural axons in a subtype-specific manner.

BACKGROUND: Spinal commissural axons represent a model system for deciphering the molecular logic that regulates the guidance of midline-crossing axons in the developing central nervous system (CNS). Whether the same or specific sets of guidance signals control the navigation of molecularly distinct subtypes of these axons remains an open and largely unexplored question. Although it is well established that post-crossing commissural axons alter their responsiveness to midline-associated guidance cues, our understanding of the repulsive mechanisms that drive the post-crossing segments of these axons away from the midline and whether the underlying guidance systems operate in a commissural axon subtype-specific manner, remains fragmentary at best. RESULTS: Here, we utilize axonally targeted transgenic reporter mice to visualize genetically distinct dorsal interneuron (dI)1 and dI4 commissural axons and show that the repulsive class 3 semaphorin (Sema3) guidance receptor Neuropilin 2 (Npn2), is selectively expressed on the dI1 population and is required for the guidance of post-crossing dI1, but not dI4, axons. Consistent with these observations, the midline-associated Npn2 ligands, Sema3F and Sema3B, promote the collapse of dI1, but not dI4, axon-associated growth cones in vitro. We also identify, for the first time, a discrete GABAergic population of ventral commissural neurons/axons in the embryonic mouse spinal cord that expresses Npn2, and show that Npn2 is required for the proper guidance of their post-crossing axons. CONCLUSIONS: Together, our findings indicate that Npn2 is selectively expressed in distinct populations of commissural neurons in both the dorsal and ventral spinal cord, and suggest that Sema3-Npn2 signaling regulates the guidance of post-crossing commissural axons in a population-specific manner.

Guidance of longitudinally projecting axons in the developing central nervous system.

The directed and stereotypical growth of axons to their synaptic targets is a crucial phase of neural circuit formation. Many axons in the developing vertebrate and invertebrate central nervous systems (CNSs), including those that remain on their own (ipsilateral), and those that cross over to the opposite (commissural), side of the midline project over long distances along the anterior-posterior (A-P) body axis within precisely positioned longitudinally oriented tracts to facilitate the transmission of information between CNS regions. Despite the widespread distribution and functional importance of these longitudinal tracts, the mechanisms that regulate their formation and projection to poorly characterized synaptic targets remain largely unknown. Nevertheless, recent studies carried out in a variety of invertebrate and vertebrate model systems have begun to elucidate the molecular logic that controls longitudinal axon guidance.