Multiple influences on the migration of precerebellar neurons in the caudal medulla.

Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.     

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin^−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.     

Longitudinal axons are guided by Slit/Robo signals from the floor plate

Longitudinal axons grow long distances along precise pathways to connect major CNS regions. However, during embryonic development, it remains largely undefined how the first longitudinal axons choose specific positions and grow along them. Here, we review recent evidence identifying a critical role for Slit/Robo signals to guide pioneer longitudinal axons in the embryonic brain stem. These studies indicate that Slit/Robo signals from the floor plate have dual functions: to repel longitudinal axons away from the ventral midline, and also to maintain straight longitudinal growth. These dual functions likely cooperate with other guidance cues to establish the major longitudinal tracts in the brain.Key words: Slit, Robo, longitudinal axon, hindbrain, axon guidance     

Slit-mediated repulsion is a key regulator of motor axon pathfinding in the hindbrain

The floor plate is known to be a source of repellent signals for cranial motor axons, preventing them from crossing the midline of the hindbrain. However, it is unknown which molecules mediate this effect in vivo. We show that Slit and Robo proteins are candidate motor axon guidance molecules, as Robo proteins are expressed by cranial motoneurons, and Slit proteins are expressed by the tissues that delimit motor axon trajectories, i.e. the floor plate and the rhombic lip. We present in vitro evidence showing that Slit1 and Slit2 proteins are selective inhibitors and repellents for dorsally projecting, but not for ventrally projecting, cranial motor axons. Analysis of mice deficient in Slit and Robo function shows that cranial motor axons aberrantly enter the midline, while ectopic expression of Slit1 in chick embryos leads to specific motor axon projection errors. Expression of dominant-negative Robo receptors within cranial motoneurons in chick embryos strikingly perturbs their projections, causing some motor axons to enter the midline, and preventing dorsally projecting motor axons from exiting the hindbrain. These data suggest that Slit proteins play a key role in guiding dorsally projecting cranial motoneurons and in facilitating their neural tube exit.     

VEGF mediates commissural axon chemoattraction through its receptor Flk1

Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.     

Pioneer longitudinal axons navigate using floor plate and Slit/Robo signals

Longitudinal axons transmit all signals between the brain and spinal cord. Their axon tracts through the brain stem are established by a simple set of pioneer axons with precise trajectories parallel to the floor plate. To identify longitudinal guidance mechanisms in vivo, the overall role of floor plate tissue and the specific roles of Slit/Robo signals were tested. Ectopic induction or genetic deletion of the floor plate diverted longitudinal axons into abnormal trajectories. The expression patterns of the diffusible cues of the Slit family were altered in the floor plate experiments, suggesting their involvement in longitudinal guidance. Genetic tests of Slit1 and Slit2, and the Slit receptors Robo1 and Robo2 were carried out in mutant mice. Slit1;Slit2 double mutants had severe longitudinal errors, particularly for ventral axons, including midline crossing and wandering longitudinal trajectories. Robo1 and Robo2 were largely genetically redundant, and neither appeared to specify specific tract positions. However, combined Robo1 and Robo2 mutations strongly disrupted each pioneer tract. Thus, pioneer axons depend on long-range floor plate cues, with Slit/Robo signaling required for precise longitudinal trajectories.     

Fine structural analysis of calcitonin gene-related peptide in the mouse inferior olivary complex

Climbing fiber afferents to the cerebellum, from the inferior olivary complex, have a powerful excitatory effect on Purkinje cells. Changes in the responsiveness of olivary neurons to their afferent inputs, leading to changes in the firing rate or pattern of activation in climbing fibers, have a significant effect on the activation of cerebellar neurons and ultimately on cerebellar function. Several neuropeptides have been localized in both varicosities and cell bodies of the mouse inferior olivary complex, one of which, calcitonin gene related peptide (CGRP), has been shown to modulate the activity of olivary neurons. The purpose of the present study was to investigate the synaptic relationships of CGRP-containing components of the caudal medial accessory olive and the principal olive of adult mice, using immunohistochemistry and electron microscopy. The vast majority of immunoreactive profiles were dendrites and dendritic spines within and outside the glial boundaries of synaptic glomeruli (clusters). Both received synaptic inputs from non-CGRP labeled axon terminals. CGRP was also present within the somata of olivary neurons as well as in profiles that had cytological characteristics of axons, some of which were filled with synaptic vesicles. These swellings infrequently formed synaptic contacts. At the LM level, few, if any, CGRP-immunoreactive climbing fibers, were seen, suggesting that CGRP is compartmentalized within the somata and dendrites of olivary neurons and is not transported to their axon terminals. Thus, in addition to previously identified extrinsic sources of CGRP, the widespread distribution of CGRP within olivary somata and dendrites identifies an intrinsic source of the peptide suggesting the possibility of dendritic release and a subsequent autocrine or paracrine function for this peptide within olivary circuits.     

Guidance of commissural growth cones at the floor plate in embryonic rat spinal cord

The floor plate of the embryonic rat spinal cord has been proposed to act as an intermediate target that plays a role in the pattern of extension of commissural axons. To begin to examine the role of the floor plate in axon guidance at the midline, we have studied the precision of the commissural axon projection to and across the floor plate during development. To delineate the pathway, the fluorescent carbocyanine dye, Di-I, has been used as a probe. We show that commissural axons traverse the floor plate and turn rostrally at its contralateral border with remarkable precision. Axons were not observed to turn ipsilaterally and turned only upon reaching the contralateral edge of the floor plate. Virtually all commissural axons follow this route. The morphology of commissural growth cones was also examined. As they encountered the floor plate, commissural growth cones became larger and increased in complexity. The reorientation of axons in register with the floor plate boundary and the change in the morphological properties of commissural growth cones as they traverse the midline suggest that the floor plate may act as a guidepost with functions similar to cells that have been implicated in axon guidance in invertebrates.     

Establishing the trochlear motor axon trajectory: role of the isthmic organiser and Fgf8

Formation of the trochlear nerve within the anterior hindbrain provides a model system to study a simple axonal projection within the vertebrate central nervous system. We show that trochlear motor neurons are born within the isthmic organiser and also immediately posterior to it in anterior rhombomere 1. Axons of the most anterior cells follow a dorsal projection, which circumnavigates the isthmus, while those of more posterior trochlear neurons project anterodorsally to enter the isthmus. Once within the isthmus, axons form large fascicles that extend to a dorsal exit point. We investigated the possibility that the projection of trochlear axons towards the isthmus and their subsequent growth within that tissue might depend upon chemoattraction. We demonstrate that both isthmic tissue and Fgf8 protein are attractants for trochlear axons in vitro, while ectopic Fgf8 causes turning of these axons away from their normal routes in vivo. Both inhibition of FGF receptor activation and inhibition of Fgf8 function in vitro affect formation of the trochlear projection within explants in a manner consistent with a guidance function of Fgf8 during trochlear axon navigation.     

Co-cultures of inferior olive and cerebellum: Electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca²⁺-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Co-cultures of inferior olive and cerebellum: electrophysiological evidence for multiple innervation of Purkinje cells by olivary axons.

Slices of inferior olive (IO) and cerebellum were co-cultured for several weeks by means of the roller tube technique. Recordings were carried out intracellularly from Purkinje cells (PCs) which were identified morphologically by intracellular injection of the fluorescent dye Lucifer yellow, or by immunohistochemical stainings with antibodies raised against the 28 kD Ca(2+)-binding protein calbindin. Following stimulation of olivary tissue, an all-or-none full complex spike response was recorded in some PCs consisting of a fast rising spike followed by a depolarizing potential. In other PCs, graded stimulation of the olivary explant induced synaptic potentials which were characterized by step-wise variation in their amplitude and resembled the ones occurring spontaneously. In contrast, only smoothly graded synaptic potentials were observed in cerebellar mono-cultures. These results indicate that some of the PCs in olivo-cerebellar co-cultures are innervated by several olivary neurons.     

Orientation of commissural axons in vitro in response to a floor plate-derived chemoattractant

Developing axons are guided to their targets by molecular cues in their local environment. Some cues are short-range, deriving from cells along axonal pathways. There is also increasing evidence for longer-range guidance cues, in the form of gradients of diffusible chemoattractant molecules, which originate from restricted populations of target cells. The guidance of developing commissural axons within the spinal cord depends on one of their intermediate cellular targets, the floor plate. We have shown previously that floor plate cells secrete a diffusible factor(s) that can alter the direction of commissural axon growth in vitro. Here we show that the factor is an effective chemoattractant for commissural axons. It can diffuse considerable distances through a collagen gel matrix and through dorsal and ventral neural epithelium in vitro to reorient the growth of virtually all commissural axons. The orientation of axons occurs in the absence of detectable effects on the survival of commissural neurons or on the rate of commissural axon extension. The regionally restricted expression of the factor suggests that it is present in the embryonic spinal cord in a gradient with its high point at the floor plate. These observations support the idea that the guidance of commissural axons to the ventral midline of the spinal cord results in part from the secretion of a chemoattractant by the floor plate.     

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.     

Differential responsiveness to the chemorepellent Semaphorin 3A distinguishes Ipsi- and contralaterally projecting axons in the chick midbrain.

In the chick dorsal mesencephalon, the optic tectum, the developing axons must choose between remaining on the same side of the midline or growing across it. The ipsilaterally projecting axons, forming the tectobulbar tract, course circumferentially toward the ventrally situated floor plate but before reaching the basal mesencephalon, the tegmentum, gradually turn caudally. Here, they follow the course of the medial longitudinal fasciculus (MLF), located parallel to the floor plate. By in vivo labeling of tectal axons, we could demonstrate that these axons arise primarily in the dorsal tectum. To test the idea that chemorepellent molecules are involved in guidance of the nondecussating axons, we performed coculture experiments employing tectal explants from various positions along the dorso-ventral axis. Axons emanating from dorsal tectal explants were strongly repelled by diencephalic tissue containing the neurons that give rise to the MLF whereas ventral tectal axons showed only a moderate response. This inhibitory effect was substantially neutralized by the addition of anti-neuropilin-1 antibodies. A similar differential response of axons was observed when tectal explants were cocultured with cell aggregates secreting the chemorepellent Semaphorin 3A (Sema3A). Sema3B and Sema3C, respectively, did not inhibit growth of tectal axons. In addition, neither the floor plate nor Slit2-secreting cell aggregates influenced outgrowth of dorsal fibers. In Sema3A-deficient mice, DiI-labeling revealed that dorsal mesencephalic axons cross the MLF instead of turning posteriorly upon reaching the fiber tract, thus behaving like the ventrally originating contralaterally projecting axons. A differential responsiveness of tectal axons to Sema3A most likely released by the MLF thus contributes to pathfinding in the ventral mesencephalon.     

The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance

Developing axons are guided to their targets by attractive and repulsive guidance cues. In the embryonic spinal cord, the floor plate chemoattractant Netrin-1 is required to guide commissural neuron axons to the midline. However, genetic evidence suggests that other chemoattractant(s) are also involved. We show that the morphogen Sonic hedgehog (Shh) can mimic the additional chemoattractant activity of the floor plate in vitro and can act directly as a chemoattractant on isolated axons. Cyclopamine-mediated inhibition of the Shh signaling mediator Smoothened (Smo) or conditional inactivation of Smo in commissural neurons indicate that Smo activity is important for the additional chemoattractant activity of the floor plate in vitro and for the normal projection of commissural axons to the floor plate in vivo. These results provide evidence that Shh, acting via Smo, is a midline-derived chemoattractant for commissural axons and show that a morphogen can also act as an axonal chemoattractant.     

Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets.     

Comparison of the effects of HGF, BDNF, CT-1, CNTF, and the branchial arches on the growth of embryonic cranial motor neurons

In the developing embryo, axon growth and guidance depend on cues that include diffusible molecules. We have shown previously that the branchial arches and hepatocyte growth factor (HGF) are growth-promoting and chemoattractant for young embryonic cranial motor axons. HGF is produced in the branchial arches of the embryo, but a number of lines of evidence suggest that HGF is unlikely to be the only factor involved in the growth and guidance of these axons. Here we investigate whether other neurotrophic factors could be involved in the growth of young cranial motor neurons in explant cultures. We find that brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1) all promote the outgrowth of embryonic cranial motor neurons, while glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) fail to affect outgrowth. We next examined whether HGF and the branchial arches had similar effects on motor neuron subpopulations at different axial levels. Our results show that HGF acts as a generalized rather than a specific neurotrophic factor and guidance cue for cranial motor neurons. Although the branchial arches also had general growth-promoting effects on all motor neuron subpopulations, they chemoattracted different axial levels differentially, with motor neurons from the caudal hindbrain showing the most striking response.     

Bilaterally Symmetric Populations of Chicken dI1 (Commissural) Axons Cross the Floor Plate Independently of Each Other

Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1 (commissural) axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to “double” in ovo electroporate the embryonic chicken spinal cord to independently manipulate the rate of growth of the two bilateral populations of dI1 axons. Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.