Positioning sensory terminals in the olfactory lobe of Drosophila by Robo signaling

Olfactory receptor neurons and the interneurons of the olfactory lobe are organized in distinct units called glomeruli. We have used expression patterns and genetic analysis to demonstrate that a combinatorial code of Roundabout (Robo) receptors act to position sensory terminals within the olfactory lobe. Groups of sensory neurons possess distinct blends of Robo and Robo3 and disruption of levels by loss-of-function or ectopic expression results in aberrant targeting. In the wild type, most of the neurons send collateral branches to the contralateral lobe. Our data suggests that guidance of axons across brain hemispheres is mediated by Slit-dependent Robo2 signaling. The location of sensory arbors at distinct positions within the lobe allows short-range interactions with projection neurons leading to formation of the glomeruli.     

<Emphasis Type="Italic">Drosophila</Emphasis> central brain formation requires Robo proteins

The Robo proteins have been extensively studied in the Drosophila embryonic ventral nerve cord, in which their expression level controls the midline crossing and optic lobe formation, but nothing is known about their activities during adult central brain formation. We have analyzed how Robo guidance cues influence central complex (CX) and mushroom body (MB) formation. Mutations of robo2 and robo3 confer a series of strong MB and CX defects. We found that the Robo2 and Robo3 proteins are expressed in two structures of the developing CX, the fan-shaped body (FB) and the noduli (NO), and by fibers across the central neuropile. We conclude that the Robo2 and Robo3 receptors play postembryonic roles during central brain formation.     

Central projections of sensory cells of the midleg of the locust,Schistocerca gregaria

The central projections of trichoid hairs and of some scolopidial organs of the mesothoracic leg of the locust Schistocerca gregaria were studied by using nickel chloride backfilling and single cell recording. Trichoid hair sensilla on different parts of the legs project somatotopically in the ventral part of the ipsilateral neuropile of the mesothoracic ganglion. Generally, distally located receptors have their terminal arborizations in ventro-lateral areas of the neuropile, and proximally located receptors in ventro-medial areas. The axons of the subgenual organ and tarsal chordotonal organs project into the intermediate neuropile.     

Positional Cues in the Drosophila Nerve Cord: Semaphorins Pattern the Dorso-Ventral Axis

During the development of neural circuitry, neurons of different kinds establish specific synaptic connections by selecting appropriate targets from large numbers of alternatives. The range of alternative targets is reduced by well organised patterns of growth, termination, and branching that deliver the terminals of appropriate pre- and postsynaptic partners to restricted volumes of the developing nervous system. We use the axons of embryonic Drosophila sensory neurons as a model system in which to study the way in which growing neurons are guided to terminate in specific volumes of the developing nervous system. The mediolateral positions of sensory arbors are controlled by the response of Robo receptors to a Slit gradient. Here we make a genetic analysis of factors regulating position in the dorso-ventral axis. We find that dorso-ventral layers of neuropile contain different levels and combinations of Semaphorins. We demonstrate the existence of a central to dorsal and central to ventral gradient of Sema 2a, perpendicular to the Slit gradient. We show that a combination of Plexin A (Plex A) and Plexin B (Plex B) receptors specifies the ventral projection of sensory neurons by responding to high concentrations of Semaphorin 1a (Sema 1a) and Semaphorin 2a (Sema 2a). Together our findings support the idea that axons are delivered to particular regions of the neuropile by their responses to systems of positional cues in each dimension.     

Neurobiology of the basal platyhelminth <Emphasis Type="Italic">Macrostomum lignano</Emphasis>: map and digital 3D model of the juvenile brain neuropile

We have analyzed brain structure in Macrostomum lignano, a representative of the basal platyhelminth taxon Macrostomida. Using confocal microscopy and digital 3D modeling software on specimens labeled with general markers for neurons (tyrTub), muscles (phalloidin), and nuclei (Sytox), an atlas and digital model of the juvenile Macrostomum brain was generated. The brain forms a ganglion with a central neuropile surrounded by a cortex of neuronal cell bodies. The neuropile contains a stereotypical array of compact axon bundles, as well as branched terminal axons and dendrites. Muscle fibers penetrate the flatworm brain horizontally and vertically at invariant positions. Beside the invariant pattern of neurite bundles, these “cerebral muscles” represent a convenient system of landmarks that help define discrete compartments in the juvenile brain. Commissural axon bundles define a dorsal and ventro-medial neuropile compartment, respectively. Longitudinal axons that enter the neuropile through an invariant set of anterior and posterior nerve roots define a ventro-basal and a central medial compartment in the neuropile. Flanking these “fibrous” compartments are neuropile domains that lack thick axon bundles and are composed of short collaterals and terminal arborizations of neurites. Two populations of neurons, visualized by antibodies against FMRFamide and serotonin, respectively, were mapped relative to compartment boundaries. This study will aid in the documentation and interpretation of patterns of gene expression, as well as functional studies, in the developing Macrostomum brain.     

Slit1a inhibits retinal ganglion cell arborization and synaptogenesis via Robo2-dependent and -independent pathways

Upon arriving at their targets, developing axons cease pathfinding and begin instead to arborize and form synapses. To test whether CNS arborization and synaptogenesis are controlled by Slit-Robo signaling, we followed single retinal ganglion cell (RGC) arbors over time. ast (robo2) mutant and slit1a morphant arbors had more branch tips and greater arbor area and complexity compared to wild-type and concomitantly more presumptive presynaptic sites labeled with YFP-Rab3. Increased arborization in ast was phenocopied by dominant-negative Robo2 expressed in single RGCs and rescued by full-length Robo2, indicating that Robo2 acts cell-autonomously. Time-lapse imaging revealed that ast and slit1a morphant arbors stabilized earlier than wild-type, suggesting a role for Slit-Robo signaling in preventing arbor maturation. Genetic analysis showed that Slit1a acts both through Robo2 and Robo2-independent mechanisms. Unlike previous PNS studies showing that Slits promote branching, our results show that Slits inhibit arborization and synaptogenesis in the CNS.     

Short-range and long-range guidance by Slit and its Robo receptors: a combinatorial code of Robo receptors controls lateral position

Slit is secreted by midline glia in Drosophila and functions as a short-range repellent to control midline crossing. Although most Slit stays near the midline, some diffuses laterally, functioning as a long-range chemorepellent. Here we show that a combinatorial code of Robo receptors controls lateral position in the CNS by responding to this presumptive Slit gradient. Medial axons express only Robo, intermediate axons express Robo3 and Robo, while lateral axons express Robo2, Robo3, and Robo. Removal of robo2 or robo3 causes lateral axons to extend medially; ectopic expression of Robo2 or Robo3 on medial axons drives them laterally. Precise topography of longitudinal pathways appears to be controlled by a combination of long-range guidance (the Robo code determining region) and short-range guidance (discrete local cues determining specific location within a region).     

Roundabout 2 regulates migration of sensory neurons by signaling in trans

BACKGROUND: Roundabout (Robo) receptors and their ligand Slit are important regulators of axon guidance and cell migration. The development of Drosophila embryonic sense organs provides a neuronal migration paradigm where the in vivo roles of Slit and Robo can be assayed using genetics. RESULTS: Here we show that Slit-Robo signaling controls migration of Drosophila larval sensory neurons that are part of the Chordotonal (Cho) stretch receptor organs. We used live imaging to show that abdominal Cho organs normally migrate ventrally during development, whereas thoracic Cho organs do not. Robo2 overexpression in cis (in the sensory neurons) or in trans (on neighboring visceral mesoderm) transforms abdominal organs to a thoracic morphology and position by blocking migration, while loss of Slit-Robo signaling produces a reverse transformation in which thoracic organs migrate ectopically. Rescue and tissue-specific knockout experiments indicate that trans signaling by Robo2 contributes to the normal positioning of the thoracic Cho organs. The differential positioning of Cho organs between the thorax and abdomen is known to be regulated by Hox genes, and we show that the essential Hox cofactor Homothorax, represses Robo2 expression in the abdominal visceral mesoderm. CONCLUSIONS: Our results suggest that segment-specific neuronal migration patterns are directed through a novel signaling complex (the "Slit sandwich") in which Robo2 on the thoracic visceral mesoderm binds to Slit and presents it to Robo receptors on Cho neurons. The differential positioning of Cho organs between thorax and abdomen may be determined by Hox gene-mediated repression of robo2.     

C. elegans slit acts in midline, dorsal-ventral, and anterior-posterior guidance via the SAX-3/Robo receptor

Robo receptors interact with ligands of the Slit family. The nematode C. elegans has one Robo receptor (SAX-3) and one Slit protein (SLT-1), which direct ventral axon guidance and guidance at the midline. In larvae, slt-1 expression in dorsal muscles repels axons to promote ventral guidance. SLT-1 acts through the SAX-3 receptor, in parallel with the ventral attractant UNC-6 (Netrin). Removing both UNC-6 and SLT-1 eliminates all ventral guidance information for some axons, revealing an underlying longitudinal guidance pathway. In the embryo, slt-1 is expressed at high levels in anterior epidermis. Embryonic expression of SLT-1 provides anterior-posterior guidance information to migrating CAN neurons. Surprisingly, slt-1 mutants do not exhibit the nerve ring and epithelial defects of sax-3 mutants, suggesting that SAX-3 has both Slit-dependent and Slit-independent functions in development.     

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is due to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field.     

Monosynaptic excitation of lateral giant fibres by proprioceptive afferents in the crayfish

Giant interneurones mediate a characteristic `tail flip' escape response of the crayfish, Procambarus clarkii, which move it rapidly away from the source of stimulation. We have analysed the synaptic connections of proprioceptive sensory neurones with one type of giant interneurone, the lateral giant. Spikes in sensory neurones innervating an exopodite-endopodite chordotonal organ in the tailfan, which monitors the position and movements of the exopodite, are followed at a short and constant latency by excitatory postsynaptic potentials in a lateral giant interneurone (LG) recorded in the terminal abdominal ganglion. These potentials are unaffected by manipulation of the membrane potential of LG, by bath application of saline with a low calcium concentration, or by one containing the nicotinic antagonist, curare. The potentials evoked in LG by chordotonal organ stimulation are thus thought to be monosynaptic and electrically mediated. This is the first demonstration that LG receives input from sensory receptors other than exteroceptors in the terminal abdominal ganglion. Accepted: 7 April 1997     

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3’s ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3’s ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.     

Patterns of growth, axonal extension and axonal arborization of neuronal lineages in the developing Drosophila brain

The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, we distinguish between three types of lineages: PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; C lineages with arborizations distributed continuously along the entire length of their tract; D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. We propose that compartments are “scaffolded” by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments.     

Slit and robo: expression patterns in lung development

First described as an axonal guidance cue through its repulsive effect on neurons expressing its receptor Roundabout (Robo), the Slit ligand has effects on cell migration, axon branching and elongation. Indirect evidence implicates Slit and Robo in lung development. We now demonstrate that Slit-2 and Slit-3 are developmentally regulated in embryonic murine lung. Immunohistochemistry demonstrates Slit-2 and Slit-3 expression by the pulmonary mesenchyme and airway epithelium. Robo-1 and Robo-2 are also expressed by the developing mesenchyme and airway epithelium. As lung development progresses, Robo-1 and Robo-2 expression localizes to only the airway epithelium. We conclude Slit/Robo are expressed in temporo-spatially adjacent domains suggesting interactive roles in pulmonary bronchiolar development.     

Formation of the transverse nerve in moth embryos. II. Stereotyped growth by the axons of identified neuroendocrine neurons

We are interested in the cellular mechanisms that guide neuroendocrine axons to their neurohaemal target regions and that regulate the extent and positioning of their terminal arbor. The neurohaemal organ we have studied is the segmentally repeated transverse nerve of the moth Manduca. In the mature animal, two motor neurons and a heterogeneous set of identified neuroendocrine neurons project to this nerve; the latter release hormonal peptides from along its length. In the preceding report, we demonstrated that during embryogenesis, the position, trajectory and extent of the transverse nerve are anticipated by two sets of nonneuronal cells, the strap and the bridge. In this paper we show that four identified neuroendocrine neurons (L1 and B1-3), like the identified motor neurons before them, elaborate growth cones that use this preexisting scaffolding as a substrate for axonal elongation. Moreover, growth cone navigation by these neuroendocrine neurons is as precise and invariant as that displayed by the motor neurons. One feature that differentiates the behavior of the developing neuroendocrine cells from that of the motor neurons is a stereotyped interaction that the L1 and B1-3 axons undergo with an identified syncytial cell that lies in close proximity to the strap. Each neuroendocrine neuron specifically adheres to the syncytium by extending numerous filopodia, and an occasional large lamellopodium, over its surface. These contacts are maintained by the neuroendocrine axons after their growth cones have left the vicinity of the syncytium and proceeded into the strap/bridge complex. Adhesion to the syncytium is transient and specific to the neuroendocrine neurons: although motor neuron axons are present at this same time and place, they display no affinity for the syncytium. This distinction correlates with the fact that the neuroendocrine neurons go on to elaborate arbor within the confines of the transverse nerve, while the motor neurons do not. We suggest that the syncytium may act as a "fictive target" for these neurons to aid in the differentiation of features that are specific to their cellular phenotype.     

Physiology of vibration-sensitive afferents in the femoral chordotonal organ of the stick insect

The femoral chordotonal organ in orthopterans signals proprioceptive sensory information concerning the femur-tibia joint to the central nervous system. In the stick insect, 80 out of 500 afferents sense tibial position, velocity, or acceleration. It has been assumed that the other sensory cells in the chordotonal organ would serve as vibration detectors. Extracellular recordings from the femoral chordotonal organ nerve in fact revealed a sensitivity of the sense organ for vibrations with frequencies ranging from 10 Hz to 4 kHz, with a maximum sensitivity between 200 and 800 Hz. Single vibration-sensitive afferents responded to the same range of frequencies. Their spike activity depended on acceleration amplitude and displacement amplitude of the vibration stimulus. Additionally, 80% of the vibration-sensitive afferents received indirect presynaptic inputs from themselves or from other afferents of the femoral chordotonal organ, the amplitude of which depended on stimulus frequency and displacement amplitude. They were associated with a decrease of input resistance in the afferent terminal. From the present investigation we conclude that the femoral chordotonal organ of the stick insect is a bifunctional sensory organ that, on the one hand, measures position and movement of the tibia and, on the other hand, detects vibration of the tibia. Accepted: 6 November 1998     

Sensorimotor pathways processing vibratory signals from the femoral chordotonal organ of the stick insect

The femoral chordotonal organ of stick insects senses position and velocity of movements in the femur-tibia joint, as well as tibial vibration. While sensory information about large-scale tibial movements is processed by a well-known neuronal network and elicits resistance reflexes in extensor and flexor tibiae motoneurons, it is not yet known how sensory information about vibration of the tibia is processed. We investigated the transmission of vibration stimuli to tibial extensor motoneurons and their premotor interneurons. Vibration stimuli applied to the femoral chordotonal organ evoked responses in tibial extensor and flexor muscles. During ongoing vibration this response adapted rapidly. This adaptation had no effect on the motoneuronal response to large-scale tibial movements. Recording from premotor interneurons revealed that vibratory signals were processed in part by the same interneuronal pathways as (large-scale) velocity and position information. While only certain parts of the interneuronal reflex pathways showed little or no response during vibration stimuli, most neurons responded to both position or velocity stimuli and vibration at the femoral chordotonal organ. We conclude that sensory information about vibration of the tibia shares part of the interneuronal pathways that transmit sensory information about large-scale tibial movements to the motoneurons. Accepted: 25 April 1999