Excitatory and Inhibitory Descending Commissural Interneurons Differentially Integrate Supraspinal and Segmental Sensory Signals.

The limited information about how descending inputs from the brain and sensory inputs from the periphery use spinal cord interneurons (INs) is a major barrier to understanding how these inputs may contribute to motor functions under normal and pathologic conditions. Commissural interneurons (CINs) are a heterogeneous population of spinal INs that has been implicated in crossed motor responses and bilateral motor coordination (ability to use the right and left side of the body in a coordinated manner) and, therefore, are likely involved in many types of movement (e.g., dynamic posture stabilization, jumping, kicking, walking). In this study, we incorporate mouse genetics, anatomy, electrophysiology, and single-cell calcium imaging to investigate how a subset of CINs, those with descending axons called dCINs, are recruited by descending reticulospinal and segmental sensory signals independently and in combination. We focus on two groups of dCINs set apart by their principal neurotransmitter (glutamate and GABA) and identified as VGluT2+ dCINs and GAD2+ dCINs. We show that VGluT2+ and GAD2+ dCINs are both extensively recruited by reticulospinal and sensory input alone but that VGluT2+ and GAD2+ dCINs integrate these inputs differently. Critically, we find that when recruitment depends on the combined action of reticulospinal and sensory inputs (subthreshold inputs), VGluT2+ dCINs, but not GAD2+ dCINs, are recruited. This difference in the integrative capacity of VGluT2+ and GAD2+ dCINs represents a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.SIGNIFICANCE STATEMENT The way supraspinal and peripheral sensory inputs use spinal cord interneurons is fundamental to defining how motor functions are supported both in health and disease. This study, which focuses on dCINs, a heterogeneous population of spinal interneurons critical for crossed motor responses and bilateral motor coordination, shows that both glutamatergic (excitatory) and GABAergic (inhibitory) dCINs can be recruited by supraspinal (reticulospinal) or peripheral sensory inputs. Additionally, the study demonstrates that in conditions where the recruitment of dCINs depends on the combined action of reticulospinal and sensory inputs, only excitatory dCINs are recruited. The study uncovers a circuit mechanism that the reticulospinal and segmental sensory systems may avail themselves of to regulate motor behaviors both normally and after injury.

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.

The mammalian spinal commissural system: properties and functions.

Commissural systems are essential components of motor circuits that coordinate left-right activity of the skeletomuscular system. Commissural systems are found at many levels of the neuraxis including the cortex, brainstem, and spinal cord. In this review we will discuss aspects of the mammalian spinal commissural system. We will focus on commissural interneurons, which project from one side of the cord to the other and form axonal terminations that are confined to the cord itself. Commissural interneurons form heterogeneous populations and influence a variety of spinal circuits. They can be defined according to a variety of criteria including, location in the spinal gray matter, axonal projections and targets, neurotransmitter phenotype, activation properties, and embryological origin. At present, we do not have a comprehensive classification of these cells, but it is clear that cells located within different areas of the gray matter have characteristic properties and make particular contributions to motor circuits. The contribution of commissural interneurons to locomotor function and posture is well established and briefly discussed. However, their role in other goal-orientated behaviors such as grasping, reaching, and bimanual tasks is less clear. This is partly because we only have limited information about the organization and functional properties of commissural interneurons in the cervical spinal cord of primates, including humans. In this review we shall discuss these various issues. First, we will consider the properties of commissural interneurons and subsequently examine what is known about their functions. We then discuss how they may contribute to restoration of function following spinal injury and stroke.

WT1-Expressing Interneurons Regulate Left-Right Alternation during Mammalian Locomotor Activity.

The basic pattern of activity underlying stepping in mammals is generated by a neural network located in the caudal spinal cord. Within this network, the specific circuitry coordinating left-right alternation has been shown to involve several groups of molecularly defined interneurons. Here we characterize a population of spinal neurons that express the Wilms' tumor 1 (WT1) gene and investigate their role during locomotor activity in mice of both sexes. We demonstrate that WT1-expressing cells are located in the ventromedial region of the spinal cord of mice and are also present in the human spinal cord. In the mouse, these cells are inhibitory, project axons to the contralateral spinal cord, terminate in close proximity to other commissural interneuron subtypes, and are essential for appropriate left-right alternation during locomotion. In addition to identifying WT1-expressing interneurons as a key component of the locomotor circuitry, this study provides insight into the manner in which several populations of molecularly defined interneurons are interconnected to generate coordinated motor activity on either side of the body during stepping.SIGNIFICANCE STATEMENT In this study, we characterize WT1-expressing spinal interneurons in mice and demonstrate that they are commissurally projecting and inhibitory. Silencing of this neuronal population during a locomotor task results in a complete breakdown of left-right alternation, whereas flexor-extensor alternation was not significantly affected. Axons of WT1 neurons are shown to terminate nearby commissural interneurons, which coordinate motoneuron activity during locomotion, and presumably regulate their activity. Finally, the WT1 gene is shown to be present in the spinal cord of humans, raising the possibility of functional homology between these species. This study not only identifies a key component of the locomotor circuitry but also begins to unravel the connectivity among the growing number of molecularly defined interneurons that comprise this neural network.

Modulation of NMDA-mediated intrinsic membrane properties of ascending commissural interneurons in neonatal rat spinal cord.

In this paper, the modulation of ascending commissural interneurons by N-methyl-D-aspartate was investigated in neonatal rats by using retrograde labeling and whole-cell patch clamp. Data shows these interneurons can be divided into three types (single spike, phasic, and tonic) based on their firing patterns. A hyperpolarization-activated nonselective cation current and persistent inward current are expressed in these interneurons. The parameters studied (n = 48) include: resting membrane potential (-59.2 ± 0.8 mV), input resistance (964.4 ± 49.3 MΩ), voltage threshold (-39.5 ± 0.6 mV), rheobase (13.5 ± 0.7 pA), action potential height (55.6 ± 2.2 mV), action potential half-width (2.8 ± 0.1 ms), afterhyperpolarization magnitude (16.1 ± 1.2 mV) and half-decay (217.9 ± 10.7 ms). 10 µM N-methyl-D-aspartate increases excitability of ascending commissural interneurons by depolarizing the membrane potential, hyperpolarizing voltage threshold, reducing rheobase, and shifting the frequency-current relationship to the left. N-methyl-Daspartate enhances persistent inward currents but reduces hyperpolarization-activated nonselective cation currents. This research uncovers unique ionic and intrinsic properties of ascending commissural interneurons which can be modulated by major excitatory neurotransmitters such as N-methyl-D-aspartate to potentially facilitate left-right alternation during locomotion.

Distinct roles of spinal commissural interneurons in transmission of contralateral sensory information.

Crossed reflexes are mediated by commissural pathways transmitting sensory information to the contralateral side of the body, but the underlying network is not fully understood. Commissural pathways coordinating the activities of spinal locomotor circuits during locomotion have been characterized in mice, but their relationship to crossed reflexes is unknown. We show the involvement of two genetically distinct groups of commissural interneurons (CINs) described in mice, V0 and V3 CINs, in the crossed reflex pathways. Our data suggest that the exclusively excitatory V3 CINs are directly involved in the excitatory crossed reflexes and show that they are essential for the inhibitory crossed reflexes. In contrast, the V0 CINs, a population that includes excitatory and inhibitory CINs, are not directly involved in excitatory or inhibitory crossed reflexes but downregulate the inhibitory crossed reflexes. Our data provide insights into the spinal circuitry underlying crossed reflexes in mice, describing the roles of V0 and V3 CINs in crossed reflexes.

Long ascending propriospinal neurons provide flexible, context-specific control of interlimb coordination.

Within the cervical and lumbar spinal enlargements, central pattern generator (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that inter-connect the lumbar and cervical CPGs disrupts left-right limb coupling of each limb pair in the adult rat during overground locomotion on a high-friction surface. These perturbations occurred independent of the locomotor rhythm, intralimb coordination, and speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. These data reveal surprising flexibility and context-dependence in the control of interlimb coordination during locomotion.

Orderly compartmental mapping of premotor inhibition in the developing zebrafish spinal cord.

In vertebrates, faster movements involve the orderly recruitment of different types of spinal motor neurons. However, it is not known how premotor inhibitory circuits are organized to ensure alternating motor output at different movement speeds. We found that different types of commissural inhibitory interneurons in zebrafish form compartmental microcircuits during development that align inhibitory strength and recruitment order. Axonal microcircuits develop first and provide the most potent premotor inhibition during the fastest movements, followed by perisomatic microcircuits, and then dendritic microcircuits that provide the weakest inhibition during the slowest movements. The conversion of a temporal sequence of neuronal development into a spatial pattern of inhibitory connections provides an "ontogenotopic" solution to the problem of shaping spinal motor output at different speeds of movement.

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate.

Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.

Diverse spinal commissural neuron populations revealed by fate mapping and molecular profiling using a novel Robo3Cre mouse.

The two sides of the nervous system coordinate and integrate information via commissural neurons, which project axons across the midline. Commissural neurons in the spinal cord are a highly heterogeneous population of cells with respect to their birthplace, final cell body position, axonal trajectory, and neurotransmitter phenotype. Although commissural axon guidance during development has been studied in great detail, neither the developmental origins nor the mature phenotypes of commissural neurons have been characterized comprehensively, largely due to lack of selective genetic access to these neurons. Here, we generated mice expressing Cre recombinase from the Robo3 locus specifically in commissural neurons. We used Robo3 Cre mice to characterize the transcriptome and various origins of developing commissural neurons, revealing new details about their extensive heterogeneity in molecular makeup and developmental lineage. Further, we followed the fate of commissural neurons into adulthood, thereby elucidating their settling positions and molecular diversity and providing evidence for possible functions in various spinal cord circuits. Our studies establish an important genetic entry point for further analyses of commissural neuron development, connectivity, and function.

Post-crossing segment of dI1 commissural axons forms collateral branches to motor neurons in the developing spinal cord.

The dI1 commissural axons in the developing spinal cord, upon crossing the midline through the floor plate, make a sharp turn to grow rostrally. These post-crossing axons initially just extend adjacent to the floor plate without entering nearby motor columns. However, it remains poorly characterized how these post-crossing dI1 axons behave subsequently to this process. In the present study, to address this issue, we examined in detail the behavior of post-crossing dI1 axons in mice, using the Atoh1 enhancer-based conditional expression system that enables selective and sparse labeling of individual dI1 axons, together with Hb9 and ChAT immunohistochemistry for precise identification of spinal motor neurons (MNs). We found unexpectedly that the post-crossing segment of dI1 axons later gave off collateral branches that extended laterally to invade motor columns. Interestingly, these collateral branches emerged at around the time when their primary growth cones initiated invasion into motor columns. In addition, although the length of the laterally growing collateral branches increased with age, the majority of them remained within motor columns. Strikingly, these collateral branches further gave rise to multiple secondary branches in the region of MNs that innervate muscles close to the body axis. Moreover, these axonal branches formed presynaptic terminals on MNs. These observations demonstrate that dI1 commissural neurons develop axonal projection to spinal MNs via collateral branches arising later from the post-crossing segment of these axons. Our findings thus reveal a previously unrecognized projection of dI1 commissural axons that may contribute directly to generating proper motor output.

Factibilidad del alineamiento comisural y coronario precisos con TAVI balón-expandible / Feasibility of precise commissural and coronary alignment with balloon-expandable TAVI

Introducción y objetivos: Nuestro objetivo fue describir la factibilidad y resultados preliminares de una estrategia de alineamiento comisural preciso (ACP) con implante percutáneo de válvula aórtica balón-expandible. Métodos: Se analizó la relación entre las comisuras nativas y las neocomisuras en 10 pacientes consecutivos con estenosis aórtica grave trivalva y sintomática tras orientar el implante de TAVI basándose en la tomografía computarizada (TC) a través de un programa de análisis específicamente desarrollado. El ACP se predijo en base a modelos in silico que permitieron estimar cuantos grados había que girar la prótesis en el momento del crimpado. El grado de ACP y de solapamiento con los ostium coronarios se midió mediante TC al mes. Se recogieron gradientes transvalvulares y fuga perivalvular. Resultados: El mal alineamiento medio fue de 16,7±8°. Cuatro pacientes presentaron mal alineamiento ligero, pero ninguno moderado o grave. El análisis in silico predijo la posición final de las neocomisuras con un coeficiente de correlación de 0,983 (IC95%, 0,966-0,992), p <0,001. Se produjo solapamiento coronario severo con el ostium de la coronaria derecha en 3 casos en relación con excentricidad de su origen, pero en ningún caso con el ostium coronario izquierdo. El gradiente transaórtico medio fue de 6,1±3,3mmHg y no hubo casos de fuga perivalvular moderada o grave. Conclusiones: Es posible calcular una rotación paciente-específica de la prótesis balón-expandible en el momento del crimpado basándose en la TC preprocedimiento. De este modo, se logró evitar el mal alineamiento moderado o grave de las neo-comisuras y el solapamiento con el ostium coronario izquierdo en todos los casos.(AU)

Introduction and objectives: We aimed to describe the feasibility and preliminary outcomes of commissural alignment (CA) for the balloon-expandable transcatheter heart valve. Methods: The relationship among native commissures and transcatheter aortic valve implantation neocommissures was analyzed in 10 consecutive patients with tricuspid severe aortic stenosis undergoing transcatheter aortic valve implantation after guided implantation based on computed tomography analysis with a self-developed software. CA was predicted by in silico bio-modelling in the 10 patients and the calculated rotation was applied during crimping. Degrees of CA and coronary overlap (CO) were measured through 1-month follow up computed tomography. Transvalvular residual gradients and the rate of paravalvular leak were also analyzed. Results: Mean commissural misalignment was 16.7±8°. Four patients showed mild misalignment but none of them showed a moderate or severe degree of misalignment. The in silico model accurately predicted the final in vivo position with a correlation coefficient of 0.983 (95%CI, 0.966-0.992), P <.001. Severe CO with right coronary ostium occurred in 3 patients likely due to ostial eccentricity, and CO was not present with the left coronary artery in any of the patients. Mean transaortic gradient was 6.1±3.3mmHg and there were no moderate-severe paravalvular leaks. Conclusions: Patient-specific rotation during valve crimping based on preprocedural computed tomography is feasible with balloon-expandable devices and is associated with the absence of moderate or severe commissural misalignment and left main CO.(AU)

Pax3 overexpression induces cell aggregation and perturbs commissural axon projection during embryonic spinal cord development.

Pax3 is a transcription factor that belongs to the paired box family. In the developing spinal cord it is expressed in the dorsal commissural neurons, which project ascending axons contralaterally to form proper spinal cord-brain circuitry. While it has been shown that Pax3 induces cell aggregation in vitro, little is known about the role of Pax3 in cell aggregation and spinal circuit formation in vivo. We have reported that Pax3 is involved in neuron differentiation and that its overexpression induces ectopic cadherin-7 expression. In this study we report that Pax3 overexpression also induces cell aggregation in vivo. Tissue sections and open book preparations revealed that Pax3 overexpression prevents commissural axons from projecting to the contralateral side of the spinal cord. Cells overexpressing Pax3 aggregated in cell clusters that contained shortened neurites with perturbed axon growth and elongation. Pax3-specific shRNA partially rescued the morphological change induced by Pax3 overexpression in vivo. Our results indicate that the normal expression of Pax3 is necessary for proper axonal pathway finding and commissural axon projection. In conclusion, Pax3 regulates neural circuit formation during embryonic development. J. Comp. Neurol. 525:1618-1632, 2017. © 2016 Wiley Periodicals, Inc.

Corticospinal and Reticulospinal Contacts on Cervical Commissural and Long Descending Propriospinal Neurons in the Adult Rat Spinal Cord; Evidence for Powerful Reticulospinal Connections.

Descending systems have a crucial role in the selection of motor output patterns by influencing the activity of interneuronal networks in the spinal cord. Commissural interneurons that project to the contralateral grey matter are key components of such networks as they coordinate left-right motor activity of fore and hind-limbs. The aim of this study was to determine if corticospinal (CST) and reticulospinal (RST) neurons make significant numbers of axonal contacts with cervical commissural interneurons. Two classes of commissural neurons were analysed: 1) local commissural interneurons (LCINs) in segments C4-5; 2) long descending propriospinal neurons (LDPNs) projecting from C4 to the rostral lumbar cord. Commissural interneurons were labelled with Fluorogold and CST and RST axons were labelled by injecting the b subunit of cholera toxin in the forelimb area of the primary somatosensory cortex or the medial longitudinal fasciculus respectively. The results show that LCINs and LDPNs receive few contacts from CST terminals but large numbers of contacts are formed by RST terminals. Use of vesicular glutamate and vesicular GABA transporters revealed that both types of cell received about 80% excitatory and 20% inhibitory RST contacts. Therefore the CST appears to have a minimal influence on LCINs and LDPNs but the RST has a powerful influence. This suggests that left-right activity in the rat spinal cord is not influenced directly via CST systems but is strongly controlled by the RST pathway. Many RST neurons have monosynaptic input from corticobulbar pathways therefore this pathway may provide an indirect route from the cortex to commissural systems. The cortico-reticulospinal-commissural system may also contribute to functional recovery following damage to the CST as it has the capacity to deliver information from the cortex to the spinal cord in the absence of direct CST input.

Interstitial branch formation within the red nucleus by deep cerebellar nuclei-derived commissural axons during target recognition.

Target recognition by developing axons is one of the fundamental steps for establishing the proper pattern of neuronal connectivity during development. However, knowledge of the mechanisms that underlie this critical event is still limited. In this study, to examine how commissural axons in vertebrates recognize their targets after crossing the midline, we analyzed in detail the behavior of postcrossing commissural axons derived from the deep cerebellar nuclei (DCN) in the developing mouse cerebellum. For this, we employed a cell-type-specific genetic labeling approach to selectively visualize DCN axons during the time when these axons project to the red nucleus (RN), one of the well-characterized targets of DCN axons. We found that, when DCN axons initially entered the RN at its caudal end, these axons continued to grow rostrally through the RN without showing noticeable morphological signs of axon branching. Interestingly, after a delay, DCN axons started forming interstitial branches from the portion of the axon shaft selectively within the RN. Because commissural axons acquire responsiveness to several guidance cues when they cross the midline, we further addressed whether midline crossing is a prerequisite for subsequent targeting by using a Robo3 knockdown strategy. We found that DCN axons were still capable of forming interstitial branches within the RN even in the absence of midline crossing. These results therefore suggest that the mechanism of RN recognition by DCN axons involves a delayed interstitial branching, and that these axons possess an intrinsic ability to respond to the target-derived cues irrespective of midline crossing.

Long-Distance Descending Spinal Neurons Ensure Quadrupedal Locomotor Stability.

Locomotion is an essential animal behavior used for translocation. The spinal cord acts as key executing center, but how it coordinates many body parts located across distance remains poorly understood. Here we employed mouse genetic and viral approaches to reveal organizational principles of long-projecting spinal circuits and their role in quadrupedal locomotion. Using neurotransmitter identity, developmental origin, and projection patterns as criteria, we uncover that spinal segments controlling forelimbs and hindlimbs are bidirectionally connected by symmetrically organized direct synaptic pathways that encompass multiple genetically tractable neuronal subpopulations. We demonstrate that selective ablation of descending spinal neurons linking cervical to lumbar segments impairs coherent locomotion, by reducing postural stability and speed during exploratory locomotion, as well as perturbing interlimb coordination during reinforced high-speed stepping. Together, our results implicate a highly organized long-distance projection system of spinal origin in the control of postural body stabilization and reliability during quadrupedal locomotion.

NOVA regulates Dcc alternative splicing during neuronal migration and axon guidance in the spinal cord.

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development.

Vertebrate spinal commissural neurons: a model system for studying axon guidance beyond the midline.

For bilaterally symmetric organisms, the transfer of information between the left and right side of the nervous system is mediated by commissures formed by neurons that project their axons across the body midline to the contralateral side of the central nervous system (CNS). After crossing the midline, many of these axons must travel long distances to reach their targets, including those that extend from spinal commissural neurons. Owing to the highly stereotyped trajectories of spinal commissural neurons that can be divided into several segments as these axons project to their targets, it is an ideal system for investigators to ask fundamental questions related to mechanisms of short- and long-range axon guidance, fasciculation, and choice point decisions at the midline intermediate target. In addition, studies of patterning genes of the nervous system have revealed complex transcription factor codes that function in a combinatorial fashion to specify individual classes of spinal neurons including commissural neurons. Despite these advances and the functional importance of spinal commissural neurons in mediating the transfer of external sensory information from the peripheral nervous system (PNS) to the CNS, only a handful of studies have begun to elucidate the mechanistic logic underlying their long-range pathfinding and the characterization of their synaptic targets. Using in vitro assays, in vivo labeling methodologies, in combination with both loss- and gain-of-function experiments, several studies have revealed that the molecular mechanisms of long-range spinal commissural axon pathfinding involve an interplay between classical axon guidance cues, morphogens and cell adhesion molecules. For further resources related to this article, please visit the WIREs website.

Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons.

During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). In this study, we describe a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. We show that the extracellular domains required for binding to Robo1 are also required for Robo2's ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

Crossing the embryonic midline: molecular mechanisms regulating axon responsiveness at an intermediate target.

In bilaterally symmetric animals, the precise assembly of neural circuitry at the midline is essential for coordination of the left and right sides of the body. Commissural axons must first be directed across the midline and then be prevented from re-crossing in order to ensure proper midline connectivity. Here, we review the attractants and repellents that direct axonal navigation at the ventral midline and the receptors on commissural neurons through which they signal. In addition, we discuss the mechanisms that commissural axons use to switch their responsiveness to midline-derived cues, so that they are initially responsive to midline attractants and subsequently responsive to midline repellents.