The migratory behavior of immature enteric neurons.

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.

Dynamics of neural crest-derived cell migration in the embryonic mouse gut.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Pathfinding by sensory axons in Drosophila: substrates and choice points in early lch5 axon outgrowth.

We have examined the pattern of axon growth from the lateral chordotonal (lch5) neurons in the body wall of the Drosophila embryo and identified cellular substrates and choice points involved in early axon pathfinding by these sensory neurons. At the first choice point (TP1), the lch5 growth cones contact the most distal cells of the spiracular branch (SB) of the trachea. The SB provides a substrate along which the axons extend internally to the level of the intersegmental nerve (ISN). In the absence of the SB, the lch5 axons often stall near TP1 or follow aberrant routes towards the CNS. At the second choice point (TP2), the lch5 growth cones make their first contact with other axons and turn ventrally toward the CNS, fasciculating specifically with the motor axons of the ISN.

short stop is allelic to kakapo, and encodes rod-like cytoskeletal-associated proteins required for axon extension.

short stop (shot) is required for sensory and motor axons to reach their targets in the Drosophila embryo. Growth cones in shot mutants initiate at the normal times, and they appear normal with respect to overall morphology and their abilities to orient and fasciculate. However, sensory axons are unable to extend beyond a short distance from the cell body, and motor axons are unable to reach target muscles. The shot gene encodes novel actin binding proteins that are related to plakins and dystrophin and expressed in axons during development. The longer isoforms identified are predicted to contain an N-terminal actin binding domain, a long central triple helical coiled-coil domain, and a C-terminal domain that contains two EF-hand Ca(2+) binding motifs and a short stretch of homology to the growth arrest-specific 2 protein. Other isoforms lack all or part of the actin binding domains or are truncated and contain a different C-terminal domain. Only the isoforms containing full-length actin binding domains are detectably expressed in the nervous system. shot is allelic to kakapo, a gene that may function in integrin-mediated adhesion in the wing and embryo. We propose that Shot's interactions with the actin cytoskeleton allow sensory and motor axons to extend.

Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile.

roundabout (robo) encodes an axon guidance receptor that controls midline crossing in the Drosophila CNS. In robo mutants, axons that normally project ipsilaterally can cross and recross the midline. Growth cones expressing Robo are believed to be repelled from the midline by the interaction of Robo and its ligand Slit, an extracellular protein expressed by the midline glia. To help understand the cellular basis for the midline repulsion mediated by Robo, we used time-lapse observations to compare the growth cone behavior of the ipsilaterally projecting motorneuron RP2 in robo and wild-type embyros. In wild-type embryos, filopodia can project across the midline but are quickly retracted. In robo mutants, medial filopodia can remain extended for longer periods and can develop into contralateral branches. In many cases RP2 produces both ipsilateral and contralateral branches, both of which can extend into the periphery. The growth cone also exhibits longer filopodia and more extensive branching both at the midline and throughout the neuropile. Cell injections in fixed stage 13 embryos confirmed and quantified these results for both RP2 and the interneuron pCC. The results suggest that Robo both repels growth cones at the midline and inhibits branching throughout the neuropile by promoting filopodial retraction.

In vivo dynamics of axon pathfinding in the Drosophilia CNS: a time-lapse study of an identified motorneuron.

We developed a system for time-lapse observation of identified neurons in the central nervous system (CNS) of the Drosophila embryo. Using this system, we characterize the dynamics of filopodia and axon growth of the motorneuron RP2 as it navigates anteriorly through the CNS and then laterally along the intersegmental nerve (ISN) into the periphery. We find that both axonal extension and turning occur primarily through the process of filopodial dilation. In addition, we used the GAL4-UAS system to express the fusion protein Tau-GFP in a subset of neurons, allowing us to correlate RP2's patterns of growth with a subset of axons in its environment. In particular, we show that RP2's sharp lateral turn is coincident with the nascent ISN.

Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade.

BACKGROUND: Dramatic changes in body size and pattern occurred during the radiation of many taxa in the Cambrian, and these changes are best documented for the arthropods. The sudden appearance of such diverse body plans raises the fundamental question of when the genes and the developmental control systems that regulate these designs evolved. As Hox genes regulate arthropod body patterns, the evolution of these genes may have played a role in the origin and diversification of the arthropod body plan from a homonomous ancestor. To trace the origin of arthropod Hox genes, we examined their distribution in a myriapod and in the Onychophora, a sister group to the arthropods. RESULTS: Despite the limited segmental diversity within myriapods and Onychophora, all insect Hox genes are present in both taxa, including the trunk Hox genes Ultrabithorax and abdominal-A as well as an ortholog of the fushi tarazu gene. Comparative analysis of Hox gene deployment revealed that the anterior boundary of expression of trunk Hox genes has shifted dramatically along the anteroposterior axis between Onychophora and different arthropod classes. Furthermore, we found that repression of expression of the Hox target gene Distal-less is unique to the insect lineage. CONCLUSIONS: A complete arthropod Hox gene family existed in the ancestor of the onychophoran/arthropod clade. No new Hox genes were therefore required to catalyze the arthropod radiation; instead, arthropod body-plan diversity arose through changes in the regulation of Hox genes and their downstream targets.

Central projections of sensory neurons in the Drosophila embryo correlate with sensory modality, soma position, and proneural gene function.

The peripheral nervous system (PNS) of the Drosophila embryo is especially suited for investigating the specification of neuronal identity: the PNS consists of a relatively simple but diverse set of individually identified sensory neurons; mutants, including embryonic lethals, can be readily generated and analyzed; and axon growth can potentially be followed from the earliest stages. We have developed a staining method to reveal the central projections of the full set of sensory neurons in the preterminal abdominal segments of the embryo. The sensory neurons exhibit modality-specific axonal projections in the CNS. The axons of external sense (es) organ neurons, primarily tactile in function, are restricted to a particular region within each neuromere and exhibit a somatotopic mapping within the CNS. The axons of stretch-receptive chordotonal (ch) organs project into a discrete longitudinal fascicle. Sensory neurons with multiple-branched dendrites (md neurons) project into a separate fascicle. A small number of md neurons have distinctive dorsal-projecting axonal processes in the CNS. A classification of sensory neurons based on their axon morphology correlates closely with the identity of the proneural gene responsible for their generation, suggesting that proneural genes play a central role in determining neuronal identity in the PNS of the embryo.

Evolutionary change in neural development within the arthropods: axonogenesis in the embryos of two crustaceans.

It has been previously suggested that there is a conservative program for neural development amongst the arthropods, on the basis that a stereotyped set of cells involved in establishing the axon tracts in the CNS of insect embryos is also present in crayfish embryos. We have examined the spatiotemporal pattern of axon growth from a set of early differentiating central neurons in the embryo of two crustaceans, the woodlouse Porcellio scaber and the freshwater crayfish Cherax destructor, and drawn comparisons with insect neurons whose somata lie in corresponding positions within the CNS. While many of the woodlouse and crayfish neurons show a similar pattern of axon growth to their insect counterparts, the axon trajectories taken by others differ from those seen in insects. We conclude that this aspect of early neural development has not been rigidly conserved during the evolution of the crustaceans and insects. However, the extent of similarity between the insects and the crustaceans is consistent with the idea that these groups of arthropods share a common evolutionary 'Bauplan' for the construction of their nervous systems. While the pattern of early axon growth in the woodlouse and crayfish embryos is sufficiently similar that many neurons could be confidently recognised as homologues, several differences were noted in both the relative order of axon outgrowth and axon morphologies of individual neurons.

Axon guidance factors in invertebrate development.

The contribution that studies in the invertebrates have made to our understanding of the factors responsible for directing axon growth is reviewed. Cellular mechanisms for axon guidance are considered, particularly the question of the accuracy of initial axon growth, and the implications of these observations for models of growth cone turning. The cellular substrates followed by growing axons during embryogenesis are identified, together with the experimental evidence that each is essential for reliable axon navigation. The significance of these studies for investigations into the molecular nature of axon guidance factors is discussed, and the likely cellular roles of putative axon guidance molecules considered.

The role of the cut gene in the specification of central projections by sensory axons in Drosophila.

Mutations in the cut gene transform sense organs in Drosophila embryos from external sensory (es) receptors to chordotonal (ch) organs. We have investigated whether their central axonal projections are also transformed. Following Lucifer yellow injection of the sensory neuron, wild-type es and ch organs show characteristic, different projection patterns in the CNS. Transformed es neurons in cut embryos are variable in their projection patterns: some resemble wild-type es neurons, others ch neurons, while yet others are unlike either of these. We conclude that the cut gene influences axonal projections, although its action as a simple modality switch is open to question. Additional genes could be involved in the specification of the central axonal projection of the transformed neurons.

From growth cone to synapse: the life history of the RP3 motor neuron.

In Drosophila, the ability to analyze the development of individually identified neurons with a variety of imaging and biophysical techniques can be complemented by sophisticated genetics and molecular biology. This powerful combination is allowing the development and function of single neurons and their synaptic connections to be unraveled at an unparalleled level of resolution. In this article, we focus on a single, identified motoneuron--RP3--arguably the best understood neuron in the fruitfly. Many events in the life history of RP3 are well characterized, including cell migration, axon outgrowth and pathfinding within the central nervous system, pathfinding in the periphery to its appropriate muscle target domain, the specific recognition of its muscle targets, the events of synapse formation and maturation, and its mature function in the locomotion of the fly larva. Genetic analysis has revealed mutations in a number of different genes which affect specific aspects of RP3 development from axon outgrowth to synapse formation.

Early ablation of target muscles modulates the arborisation pattern of an identified embryonic Drosophila motor axon.

The Drosophila RP3 motor axon establishes a stereotypic arborisation along the adjoining edges of muscles 6 and 7 by the end of embryogenesis. The present study has examined the role of the target muscles in determining this axonal arborisation pattern. Target muscles were surgically ablated prior to the arrival of the RP3 axon. Following further development of the embryo in culture medium, the morphology of target-deprived RP3 motor axons was assayed by intracellular injection with the dye Lucifer Yellow. Axonal arborisations were formed on a variety of non-target muscles when muscles 6 and 7 were removed and these contacts were maintained into stage 16. The pattern of axonal arborisations over non-target muscles varied between preparations in terms of the number of muscles contacted, and the distribution of arborisations on individual muscles. Following removal of muscle 6, the RP3 motor axon frequently contacted muscle 7, and axonal arborisations were present along the distal edge of the muscle. In the absence of muscle 7, the RP3 axon often did not contact muscle 6 and when muscle 6 was contacted, the arborisation of RP3 was poorly developed. Axonal processes were retained on non-target muscles when only one target muscle was present. Therefore, the establishment of a stereotypic arborisation by the RP3 motor axon is apparently dependent on growth cone contact with both target muscles.

Pathfinding in the central nervous system and periphery by identified embryonic Drosophila motor axons.

We have studied the pattern of axon outgrowth from the identified embryonic Drosophila motorneurons, RP1, RP3, RP4 and RP5, from the onset of axonogenesis to the time of arborization over target muscles. Lucifer Yellow was intracellularly injected into each of these neurons to obtain a detailed description of the morphology of their growth cones and of the pathways that they follow. We have divided the sequence of axon growth from these neurons into five major phases. In the first phase, the growth cone of each RP axon grows medially along its contralateral homologue along the anterior commissure. Each RP axon follows a separate path across the midline in the anterior commissure. After crossing the ventral midline, the axons wrap around specific contralateral RP somata. In the second phase, each axon grows posteriorly and dorsally down the contralateral longitudinal connective, fasciculating with the other RP axons. In the third phase, the axons turn into the intersegmental nerve via the anterior nerve root, then cross over to the segmental nerve, before contacting the external surfaces of intermediate muscles 15/16. They do not fasciculate with the pioneering aCC and RP2 axons at this time. In the fourth phase, the axons advance laterally across the ventral muscle group. During this phase, each axon extends processes over a number of inappropriate muscles as well as contacting its correct, target muscle. In the final phase, the processes to inappropriate muscles are withdrawn, generating the mature pattern of motor axon projections. There is no consistent, clear difference between the RP motorneurons in the relative timing of axon outgrowth.

Location and connectivity of abdominal motoneurons in the embryo and larva of Drosophila melanogaster.

The soma location and peripheral connectivity of motoneurons in abdominal segments of the embryo and larva of the fruitfly, Drosophila melanogaster are described as an initial step in determining the mechanisms by which motoneurons make connections with their target muscles in a genetically accessible organism. Embryonic motoneuron somata were retrogradely labelled by application of the fluorescent dye, DiI, to the whole peripheral nerve or to its separate anterior or posterior fascicles in segments A5-A7 of late stage 15/early stage 16 embryos. This technique reveals a stereotyped, segmentally repeated population of 34 motoneurons per hemisegment, several of which can be individually identified from their soma position. The same set of motoneurons was revealed in third instar larvae of D. melanogaster by cobalt backfilling of abdominal peripheral nerves, although the positions of some of these neurons change during larval development. The peripheral connectivity and axon morphology of several of the abdominal motoneurons was determined by intracellular injection with Lucifer Yellow in stage 16 embryos. For the motoneurons with axons in the anterior fascicle there is no clear relationship between somata groupings and the muscle targets innervated: contrary to earlier claims, these motoneurons arborize over both ventral and dorsal muscles. Individual motoneurons possess a stereotyped pattern of terminal arborization.

Embryonic development of the innervation of the locust extensor tibiae muscle by identified neurons: formation and elimination of inappropriate axon branches.

Intracellular dye fills have been used to reveal the pattern of embryonic growth of each of the four neurons which innervate the extensor tibiae muscle (ETi) of the hind leg of the locust. The growth cone of the slow extensor tibiae motoneuron (SETi), the first of the four neurons to leave the central nervous system, pioneers nerve 3 (N3). The fast extensor motoneuron (FETi), the next neuron to grow out, follows earlier outgrowing motoneurons into the periphery in nerve 5 (N5) and then rejoins SETi in N3. As it transfers from N5 to N3, it is transiently dye-coupled to the Tr1 pioneer neuron which spans the gap between the two nerves. It then follows SETi onto the ETi muscle in the femur. The common inhibitory neuron and the dorsal unpaired median neuron (DUMETi) follow SETi and FETi in nerves 3B2 and 5B1, respectively. SETi's growth cone requires almost twice as long to reach ETi as those of the three later motoneurons, all of which follow preexisting neural pathways. At least three of the four developing motoneurons form one or more axon branches not found in the adult. These branches may occur (1) at segmental boundaries; (2) where the nerve, which the growth cone is following, itself branches or the growth cone encounters another nerve; or (3) when the axon continues to grow beyond its target muscle. These findings contrast with the apparent absence of inappropriate axon branches in another developing locust neuromuscular system and during the innervation of zebrafish myotomes, but resemble in some ways the transient production of inappropriate axonal branches reported for embryonic leech motoneurons.

Functional connections with foreign muscles made by a target-deprived insect motorneuron.

Metathoracic limb buds were removed unilaterally from Locusta migratoria embryos at 30% of embryonic development, thereby depriving limb-innervating neurons of the opportunity of innervating their normal target muscles. The operated embryos were allowed to hatch and develop to adulthood, and then the connections between the identified limb motorneuron Fast Extensor Tibiae (FETi) and body wall muscles on the operated side of the segment were determined electrophysiologically. FETi innervated a number of foreign muscles in the ipsilateral body wall in limb-ablated locusts, showing that this neuron is not programmed to exclusively innervate its normal target muscle.

Axon growth from limb motorneurons in the locust embryo: the effect of target limb removal on the pattern of axon branching in the periphery.

Metathoracic limb buds have been unilaterally ablated from locust embryos at 25 to 30% of embryonic development and the effect of this operation on the axon morphology of the motorneuron fast extensor tibiae (FETi) observed at later embryonic stages. In control embryos this neuron sends a single axon out the main leg nerve, nerve 5, to the extensor tibiae muscle in the femur. In limb ablated embryos the axon of FETi is found in a wide variety of aberrant peripheral nerve pathways and projects to a wide range of foreign muscles. There is a degree of apparent selectivity, but no rigid hierarchy, in the choice of pathway and muscle made by FETi. A high degree of variability is found between one embryo and another in the extent and pattern of axon branching. The axon of FETi is generally found in pathways that correspond to nerves in control embryos but on occasion grows along novel routes. An anteriorly directed dendritic branch, seldom seen in control FETi neurons, is frequently seen in experimental FETis. These findings are discussed in terms of the rules for specific axon growth in normal development.