Netrin-guided accessory cell morphogenesis dictates the dendrite orientation and migration of a Drosophila sensory neuron.

Accessory cells, which include glia and other cell types that develop in close association with neurons, have been shown to play key roles in regulating neuron development. However, the underlying molecular and cellular mechanisms remain poorly understood. A particularly intimate association between accessory cells and neurons is found in insect chordotonal organs. We have found that the cap cell, one of two accessory cells of v'ch1, a chordotonal organ in the Drosophila embryo, strongly influences the development of its associated neuron. As it projects a long dorsally directed cellular extension, the cap cell reorients the dendrite of the v'ch1 neuron and tows its cell body dorsally. Cap cell morphogenesis is regulated by Netrin-A, which is produced by epidermal cells at the destination of the cap cell process. In Netrin-A mutant embryos, the cap cell forms an aberrant, ventrally directed process. As the cap cell maintains a close physical connection with the tip of the dendrite, the latter is dragged into an abnormal position and orientation, and the neuron fails to undergo its normal dorsal migration. Misexpression of Netrin-A in oenocytes, secretory cells that lie ventral to the cap cell, leads to aberrant cap cell morphogenesis, suggesting that Netrin-A acts as an instructive cue to direct the growth of the cap cell process. The netrin receptor Frazzled is required for normal cap cell morphogenesis, and mutant rescue experiments indicate that it acts in a cell-autonomous fashion.

Morphological characterization of the entire interneuron population reveals principles of neuromere organization in the ventral nerve cord of Drosophila.

Decisive contributions to our understanding of the mechanisms underlying the development of the nervous system have been made by studies performed at the level of single, identified cells in the fruit fly Drosophila. While all the motor neurons and glial cells in thoracic and abdominal segments of the Drosophila embryo have been individually identified, few of the interneurons, which comprise the vast majority of cells in the CNS, have been characterized at this level. We have applied a single cell labeling technique to carry out a detailed morphological characterization of the entire population of interneurons in abdominal segments A1-A7. Based on the definition of a set of spatial parameters specifying axonal projection patterns and cell body positions, we have identified 270 individual cell types as the complete hemisegmental set of interneurons and placed these in an interactive database. As well as facilitating analyses of developmental processes, this comprehensive set of data sheds light on the principles underlying the formation and organization of an entire segmental unit of the CNS.

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo.

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point - the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.

When are cellular oscillators sufficient for sequential segmentation?

Sequential segmentation during embryogenesis involves the generation of a repeated pattern along the embryo, which is concurrently undergoing axial elongation by cell division. Most mathematical models of sequential segmentation involve inherent cellular oscillators, acting as a segmentation clock. The cellular oscillation is assumed to be governed by the cell's physiological age or by its interaction with an external morphogen gradient. Here, we address the issue of when cellular oscillators alone are sufficient for predicting segmentation, and when a morphogen gradient is required. The key to resolving this issue lies in how cells determine positional information in the model--this is directly related to the distribution of cell divisions responsible for axial elongation. Mathematical models demonstrate that if axial elongation occurs through cell divisions restricted to the posterior end of the unsegmented region, a cell can obtain its positional information from its physiological age, and therefore cellular oscillators will suffice. Alternatively, if axial elongation occurs through cell divisions distributed throughout the unsegmented region, then positional information can be obtained through another mechanism, such as a morphogen gradient. Two alternative ways to establish a morphogen gradient in tissue with distributed cell divisions are presented--one with diffusion and the other without diffusion. Our model produces segment polarity and a distribution of segment size from the anterior-to-posterior ends, as observed in some systems. Furthermore, the model predicts segment deletions when there is an interruption in cell division, just as seen in heat shock experiments, as well as the growth and final shrinkage of the presomitic mesoderm during somitogenesis.

Neural development in Onychophora (velvet worms) suggests a step-wise evolution of segmentation in the nervous system of Panarthropoda.

A fundamental question in biology is how animal segmentation arose during evolution. One particular challenge is to clarify whether segmental ganglia of the nervous system evolved once, twice, or several times within the Bilateria. As close relatives of arthropods, Onychophora play an important role in this debate since their nervous system displays a mixture of both segmental and non-segmental features. We present evidence that the onychophoran "ventral organs," previously interpreted as segmental anlagen of the nervous system, do not contribute to nerve cord formation and therefore cannot be regarded as vestiges of segmental ganglia. The early axonal pathways in the central nervous system arise by an anterior-to-posterior cascade of axonogenesis from neuronal cell bodies, which are distributed irregularly along each presumptive ventral cord. This pattern contrasts with the strictly segmental neuromeres present in arthropod embryos and makes the assumption of a secondary loss of segmentation in the nervous system during the evolution of the Onychophora less plausible. We discuss the implications of these findings for the evolution of neural segmentation in the Panarthropoda (Arthropoda+Onychophora+Tardigrada). Our data best support the hypothesis that the ancestral panarthropod had only a partially segmented nervous system, which evolved progressively into the segmental chain of ganglia seen in extant tardigrades and arthropods.

The atypical cadherin Flamingo is required for sensory axon advance beyond intermediate target cells.

The Drosophila atypical cadherin Flamingo plays key roles in a number of developmental processes. We have used the sensory nervous system of the Drosophila embryo to shed light on the mechanism by which Flamingo regulates axon growth. flamingo loss of function mutants display a highly penetrant sensory axon stall phenotype. The location of these axon stalls is stereotypic and corresponds to the position of intermediate target cells, with which sensory axons associate during normal development. This suggests that Flamingo mediates an interaction between the sensory neuron growth cones and these intermediate targets, which is required for continued axon advance. Mutant rescue experiments show that Flamingo expression is required only in sensory neurons for normal axon growth. The flamingo mutant phenotype can be partially rescued by expressing a Flamingo construct lacking most of the extracellular domain, suggesting that regulation of sensory axon advance by Flamingo does not absolutely depend upon a homophilic Flamingo-Flamingo interaction or its ability to mediate cell-cell adhesion. Loss of function mutants for a number of key genes that act together with Flamingo in the planar cell polarity pathway do not display the highly penetrant stalling phenotype seen in flamingo mutants.

A revision of brain composition in Onychophora (velvet worms) suggests that the tritocerebrum evolved in arthropods.

BACKGROUND: The composition of the arthropod head is one of the most contentious issues in animal evolution. In particular, controversy surrounds the homology and innervation of segmental cephalic appendages by the brain. Onychophora (velvet worms) play a crucial role in understanding the evolution of the arthropod brain, because they are close relatives of arthropods and have apparently changed little since the Early Cambrian. However, the segmental origins of their brain neuropils and the number of cephalic appendages innervated by the brain--key issues in clarifying brain composition in the last common ancestor of Onychophora and Arthropoda--remain unclear. RESULTS: Using immunolabelling and neuronal tracing techniques in the developing and adult onychophoran brain, we found that the major brain neuropils arise from only the anterior-most body segment, and that two pairs of segmental appendages are innervated by the brain. The region of the central nervous system corresponding to the arthropod tritocerebrum is not differentiated as part of the onychophoran brain but instead belongs to the ventral nerve cords. CONCLUSIONS: Our results contradict the assumptions of a tripartite (three-segmented) brain in Onychophora and instead confirm the hypothesis of bipartite (two-segmented) brain composition. They suggest that the last common ancestor of Onychophora and Arthropoda possessed a brain consisting of protocerebrum and deutocerebrum whereas the tritocerebrum evolved in arthropods.

Velvet worm development links myriapods with chelicerates.

Despite the advent of modern molecular and computational methods, the phylogeny of the four major arthropod groups (Chelicerata, Myriapoda, Crustacea and Hexapoda, including the insects) remains enigmatic. One particular challenge is the position of myriapods as either the closest relatives to chelicerates (Paradoxopoda/Myriochelata hypothesis), or to crustaceans and hexapods (Mandibulata hypothesis). While neither hypothesis receives conclusive support from molecular analyses, most morphological studies favour the Mandibulata concept, with the mandible being the most prominent feature of this group. Although no morphological evidence was initially available to support the Paradoxopoda hypothesis, a putative synapomorphy of chelicerates and myriapods has recently been put forward based on studies of neurogenesis. However, this and other morphological characters remain of limited use for phylogenetic systematics owing to the lack of data from an appropriate outgroup. Here, we show that several embryonic characters are synapomorphies uniting the chelicerates and myriapods, as revealed by an outgroup comparison with the Onychophora or velvet worms. Our findings, thus provide, to our knowledge, first morphological/embryological support for the monophyly of the Paradoxopoda and suggest that the mandible might have evolved twice within the arthropods.

Homeotic genes influence the axonal pathway of a Drosophila embryonic sensory neuron.

Each abdominal hemisegment of the Drosophila embryo has two sensory neurons intimately associated with a tracheal branch. During embryogenesis, the axons of these sensory neurons, termed the v'td2 neurons, enter the CNS and grow toward the brain with a distinctive pathway change in the third thoracic neuromere. We show that the axons use guidance cues that are under control of the bithorax gene complex (BX-C). Pathway defects in mutants suggest that a drop in Ultrabithorax expression permits the pathway change in the T3 neuromere, while combined Ultrabithorax and abdominal-A expression represses it in the abdominal neuromeres. We propose that the axons do not respond to a particular segmental identity in forming the pathway change; rather they respond to pathfinding cues that come about as a result of a drop in BX-C expression along the antero-posterior axis of the CNS.

Development of a polar morphology by identified embryonic motoneurons.

Motoneuron morphology arises through the coordinated growth of the motor axon and dendrites. In the Drosophila embryo the RP motoneurons have a contralaterally-extended motor axon, ipsilateral dendrites that extend a short distance in the ipsilateral connective, and a tuft of short dendrites in the contralateral connective. In the present study mechanical and genetic manipulations were utilized to test if (i) the ipsilateral dendrites can develop an axon morphology, (ii) the presence of the contralateral motor axon suppresses the development of an axon-like morphology by the ipsilateral dendrites and (iii) whether establishment of a contralateral motor axon can be genetically suppressed. It was found that an ipsilateral motor axon could develop-but only at the expense of the contralateral motor axon. Axotomy could overturn the normal polarity of the RP motoneurons in favor of the development of an ipsilateral motor axon, and this reversed morphology was also observed when the motor axon could not extend across the midline in the commissureless mutant. These findings show that the RP motoneurons have the plasticity for an alternative polarity, but that the extension of an ipsilateral axon is normally suppressed by the presence of the contralateral axon. The RP motoneurons now represent a genetically amenable in vivo system for analyzing the basis of polarity formation in neurons.

Necessity and redundancy of guidepost cells in the embryonic Drosophila CNS.

Guidepost cells are specific cellular cues in the embryonic environment utilized by axonal growth cones in pathfinding decisions. In the embryonic Drosophila CNS the RP motor axons make stereotypic pathways choices involving distinct cellular contacts: (i) extension across the midline via contact with the axon and cell body of the homologous contralateral RP motoneuron, (ii) extension down the contralateral longitudinal connective (CLC) through contact with connective axons and longitudinal glia, and (iii) growth into the intersegmental nerve (ISN) through contact with ISN axons and the segmental boundary glial cell (SBC). We have now ablated putative guidepost cells in each of the CNS pathway subsections and uncovered their impact on subsequent RP motor axon pathfinding. Removal of the longitudinal glia or the SBC did not adversely affect pathfinding. This suggests that the motor axons either utilized the alternative axonal substrates, or could still make filopodial contact with the next pathway section's cues. In contrast, RP motor axons did require contact with the axon and soma of their contralateral RP homologue. Absence of this neuronal substrate frequently impeded RP axon outgrowth, suggesting that the next cues were beyond filopodial reach. Together these are the first direct ablations of putative guidepost cells in the CNS of this model system, and have uncovered both pathfinding robustness and susceptibility by RP axons in the absence of specific contacts.

The origins of the arthropod nervous system: insights from the Onychophora.

A revision of evolutionary relationships of the Arthropoda has provided fresh impetus to tracing the origins of the nervous system of this group of animals: other members of the Ecdysozoa possess a markedly different type of nervous system from both the arthropods and the annelid worms, with which they were previously grouped. Given their status as favoured sister taxon of the arthropods, Onychophora (velvet worms) are a key group for understanding the evolutionary changes that have taken place in the panarthropod (Arthropoda + Onychophora + Tardigrada) lineage. This article reviews our current knowledge of the structure and development of the onychophoran nervous system. The picture that emerges from these studies is that the nervous system of the panarthropod ancestor was substantially different from that of modern arthropods: this animal probably possessed a bipartite, rather than a tripartite brain; its nerve cord displayed only a limited degree of segmentation; and neurons were more numerous but more uniform in morphology than in living arthropods. These observations suggest an evolutionary scenario, by which the arthropod nervous system evolved from a system of orthogonally crossing nerve tracts present in both a presumed protostome ancestor and many extant worm-like invertebrates, including the onychophorans.

The L1-type cell adhesion molecule Neuroglian is necessary for maintenance of sensory axon advance in the Drosophila embryo.

BACKGROUND: Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth. RESULTS: We have found a highly penetrant sensory axon stalling phenotype in neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth. CONCLUSION: We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion.

Semaphorin 2a secreted by oenocytes signals through plexin B and plexin A to guide sensory axons in the Drosophila embryo.

The semaphorin gene family has been shown to play important roles in axonal guidance in both vertebrates and invertebrates. Both transmembrane (Sema1a, Sema1b, Sema5c) and secreted (Sema2a, Sema2b) forms of semaphorins exist in Drosophila. Two Sema receptors, plexins (Plex) A and B, have also been identified. Many questions remain concerning the axon guidance functions of the secreted semaphorins, including the identity of their receptors. We have used the well-characterized sensory system of the Drosophila embryo to address these problems. We find novel sensory axon defects in sema2a loss-of-function mutants in which particular axons misproject and follow inappropriate pathways to the CNS. plexB loss-of-function mutants show similar phenotypes to sema2a mutants and sema2a interacts genetically with plexB, supporting the hypothesis that Sema2a signals through PlexB receptors. Sema2a protein is expressed by larval oenocytes, a cluster of secretory cells in the lateral region of the embryo and the sema2a mutant phenotype can be rescued by driving Sema2a in these cells. Ablation of oenocytes results in sensory axon defects similar to the sema2a mutant phenotype. These data support a model in which Sema2a, while being secreted from oenocytes, acts in a highly localized fashion: It represses axon extension from the sensory neuron cell body, but only in regions in direct contact with oenocytes.

Segmentation, neurogenesis and formation of early axonal pathways in the centipede,Ethmostigmus rubripes (Brandt).

We have examined the embryo of the centipedeEthmostigmus rubripes to determine the degree of evolutionary conservatism in the developmental processes of segmentation, neurogenesis and axon formation between the insects and the myriapods. A conspicuous feature of centipede embryogenesis is the early separation of the left and right sides of the ganglionic primordia by extra-embryonic ectoderm. An antibody to the protein encoded by theDrosophila segmentation geneengrailed binds to cells in the posterior margin of the limb buds in the centipede embryo, in common with insect and crustacean embryos. However, whereas in insects and crustaceans this protein is also expressed in a subset of cells in the neuroectoderm, the anti-engrailed antibody did not bind to cells in the ganglionic primordia of the centipede embryo. Use of the BrdU labelling technique to mark mitotically active cells revealed that neuroblasts, the ubiquitous neuron stem cell type in insects, are not present in the centipede. The earliest central axon pathways in the centipede embryo do not arise from segmentally repeated neurons, as is the case in insects, but rather by the posteriorly directed growth of axons originating from neurons located in the brain. Axonogenesis by segmental neurons begins later in development; the pattern of neurons involved is not obviously homologous to the conservative set of central pioneering neurons found in insects. Our observations point to considerable differences between the insects and the myriapods in mechanisms for neurogenesis and the formation of central axon pathways, suggesting that these developmental processes have not been strongly conserved during arthropod evolution.

Early axonogenesis in the embryo of a primitive insect, the silverfish Ctenolepisma longicaudata.

The pattern of axon growth from the population of neurons that pioneers the major axon pathways in the central nervous system is highly conserved in winged insects. This study sought to determine whether the same pattern of axon growth is shared by an apterygotic insect, the silverfish. We have found that homologues to at least nine early differentiating winged insect neurons are present in the silverfish. The axon trajectories and the sequence of axon outgrowth from these neurons are very similar in silverfish and winged insects, suggesting that the pterygotic and apterygotic insects share a common developmental Bauplan for the construction of the central nervous system. Some of these neurons do show differences in several aspects of axon growth, including the relative timing of axonogenesis, the polarity of axon growth and the pattern of axon fasciculation. In addition, a major, early-appearing fascicle in the posterior commissure of the silverfish is pioneered by a neuron which does not appear to have an equivalent in the winged insects. These differences are similar in character to, albeit more pronounced than, differences previously reported between two winged insects, the fruitfly Drosophila and the grasshopper. Some of the features of early central axon growth, that set the silverfish embryo apart from the winged insects, are shared by crustacean embryos, providing support for the claim that insects and crustaceans share a common developmental Bauplan for the construction of central axonal pathways.

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms.

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.