Biomedical subjects

Sarah Guthrie

Publications and source records attributed to Sarah Guthrie.

16 recordsLinked to original sources

PubMed2006

Ephrin A/EphA controls the rostral turning polarity of a lateral commissural tract in chick hindbrain.

Most post-crossing commissural axons turn into longitudinal paths to make synaptic connections with their targets. Mechanisms that control their rostrocaudal turning polarity are still poorly understood. We used the hindbrain as a model system to investigate the rostral turning of a laterally located commissural tract, identified as the caudal group of contralateral cerebellar-projecting second-order vestibular neurons (cC-VC). We found that the caudal hindbrain possessed a graded non-permissive/repulsive activity for growing cC-VC axons. This non-permissiveness/repulsion was in part mediated by glycosyl-phosphatidylinositol (GPI)-anchored ephrin A. We further demonstrated that ephrin A2 was distributed in a caudal-high/rostral-low gradient in the caudolateral hindbrain and cC-VC axons expressed EphA receptors. Finally, perturbing ephrin A/EphA signalling both in vitro and in vivo led to rostrocaudal pathfinding errors of post-crossing cC-VC axons. These results suggest that ephrin A/EphA interactions play a key role in regulating the polarity of post-crossing cC-VC axons as they turn into the longitudinal axis.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

Ephrin-As play a rhombomere-specific role in trigeminal motor axon projections in the chick embryo.

In this study, we investigate the possible role of ephrin-Eph signaling in trigeminal motor axon projections. We find that EphA receptors are expressed at higher levels by rhombomere 2 (r2) trigeminal motor neurons than by r3 trigeminal motor neurons in the chick embryo. Mapping of rhombomere-specific axon projections shows that r2 and r3 trigeminal motor neurons project to different muscle targets, including the mandibular adductor and the intermandibularis muscles respectively. Ephrin-A5 is expressed in these muscles, especially in some regions of the intermandibularis muscle, and can cause growth cone collapse of both r2 and r3 motor axons in vitro. We demonstrate that in vivo overexpression of ephrin-A5 in the intermandibularis muscle, or overexpression of dominant-negative EphA receptors in trigeminal motor neurons leads to a reduction in branching of r3-derived motor axons specifically. Overexpression of full-length EphA receptors impairs the formation of r3 projections to the intermandibularis muscle. These findings indicate that ephrins and their Eph receptors play a role in trigeminal motor axon topographic mapping and in rhombomere 3-derived projections in particular.

Animals↗

PubMed2004

Axon guidance: mice and men need Rig and Robo.

For many growing axons, navigating across the midline of the nervous system is a crucial stage of their development. New studies on mice and humans show that the axon guidance receptor Robo3/Rig1 is indispensable for axons to accomplish this task.

Animals↗

PubMed2004

Development of oculomotor axon projections in the chick embryo.

The pattern of innervation of the extraocular muscles is highly conserved across higher vertebrate species and mediates sophisticated visuomotor processes. Defects in oculomotor development often lead to strabismus, a misalignment of the eyes that can cause partial blindness. Although it has been intensively studied from a clinical perspective, relatively little is known about how the system develops embryonically. We have therefore mapped the development of the oculomotor nerve (OMN) in chick embryos by using confocal microscopy. We show that OMN development follows a series of stereotyped steps that are tightly regulated in space and time. The OMN initially grows past three of its targets to innervate its distal target, the ventral oblique muscle, only later forming branches to the more proximal muscles. We have also investigated spatiotemporal aspects of the unusual contralateral migration of a subpopulation of oculomotor neurons by using molecular markers and have found the semaphorin axon guidance molecules and their receptors, the neuropilins, to be expressed in discrete subnuclei during this migration. Finally, we have created an embryological model of Duane retraction syndrome (DRS), a form of strabismus in which the OMN is believed to innervate aberrantly the lateral rectus, the normal target of the abducens nerve. By ablating rhombomeres 5 and 6 and hence the abducens, we have mimicked a proposed oculomotor deficit occurring in DRS. We find that the absence of the abducens nerve is not sufficient to produce this inappropriate innervation, so other factors are required to explain DRS.

Abducens Nerve↗

PubMed2004

Neuronal development: putting motor neurons in their place.

In the developing spinal cord, motor neurons occupy discrete columns with different identities and axon projections. This organisation has now been shown to depend crucially on sequential phases of expression of Hox-c proteins, generated in response to fibroblast growth factor signals.

Animals↗

PubMed2003

On the topographic targeting of basal vomeronasal axons through Slit-mediated chemorepulsion.

The vomeronasal projection conveys information provided by pheromones and detected by neurones in the vomeronasal organ (VNO) to the accessory olfactory bulb (AOB) and thence to other regions of the brain such as the amygdala. The VNO-AOB projection is topographically organised such that axons from apical and basal parts of the VNO terminate in the anterior and posterior AOB respectively. We provide evidence that the Slit family of axon guidance molecules and their Robo receptors contribute to the topographic targeting of basal vomeronasal axons. Robo receptor expression is confined largely to basal VNO axons, while Slits are differentially expressed in the AOB with a higher concentration in the anterior part, which basal axons do not invade. Immunohistochemistry using a Robo-specific antibody reveals a zone-specific targeting of VNO axons in the AOB well before cell bodies of these neurones in the VNO acquire their final zonal position. In vitro assays show that Slit1-Slit3 chemorepel VNO axons, suggesting that basal axons are guided to the posterior AOB due to chemorepulsive activity of Slits in the anterior AOB. These data in combination with recently obtained other data suggest a model for the topographic targeting in the vomeronasal projection where ephrin-As and neuropilins guide apical VNO axons, while Robo/Slit interactions are important components in the targeting of basal VNO axons.

Afferent Pathways↗

PubMed2003

Signals from the cerebellum guide the pathfinding of inferior olivary axons.

During development, inferior olivary axons cross the floor plate and project from the caudal to the rostral hindbrain, whence they grow into the cerebellar plate. We have investigated the axon guidance signals involved in the formation of this projection in vitro. When the cerebellar plate was grafted ectopically along the margin of the hindbrain in organotypic cultures, inferior olivary axons could pathfind to the ectopic cerebellum, establishing a topographically normal projection. Following rostrocaudal reversal of a region of tissue in the axon pathway between the inferior olive and the cerebellum, olivary axons still navigated towards the cerebellum. Moreover, olivary axons could cross a bridging tissue explant (spinal cord) to reach a cerebellar explant. In collagen gel cultures of inferior olive explants, olivary axon outgrowth increased significantly in the presence of cerebellar explants and axons deflected towards the cerebellar tissue. These results show that the cerebellum is a source of diffusible axon guidance signals for olivary axons. We also found that, in organotypic cultures, olivary axons which had crossed the floor plate showed an increased tendency to respond to cerebellar cues. Taken together, these results indicate that the cerebellum is the source of cues that are chemoattractant and growth-promoting for inferior olivary axons; prior exposure to the floor plate increases responsiveness to these cues.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

Cranial expression of class 3 secreted semaphorins and their neuropilin receptors.

The semaphorin family of chemorepellents and their receptors the neuropilins are implicated in a variety of cellular processes, including axon guidance and cell migration. Semaphorins may bind more than one neuropilin or a heterodimer of both, thus a detailed knowledge of their expression patterns may reveal possible cases of redundancy or mutual antagonism. To assess their involvement in cranial development, we cloned fragments of the chick orthologues of Sema3B and Sema3F. We then carried out mRNA in situ hybridisation of all six class 3 semaphorins and both neuropilins in the embryonic chick head. We present evidence for spatiotemporal regulation of these molecules in the brainstem and developing head, including the eye, ear, and branchial arches. These expression patterns provide a basis for functional analysis of semaphorins and neuropilins in the development of axon projections and the morphogenesis of cranial structures.

Animals↗

PubMed2003

Patterning of motor neurons by retinoic acid in the chick embryo hindbrain in vitro.

Motor neurons are found throughout the developing chick hindbrain, while somatic motor (SM) neurons develop only in rhombomeres 5 to 8 (r5-8), and in r1. In r2-8 neuroepithelial explants from stage 7-10 embryos cultured in collagen gels, we found that motor neurons were generated throughout r2-8, while SM neuron differentiation was restricted to r5-8, as in vivo. Exposure of such explants to retinoic acid (RA) resulted in SM neuron differentiation throughout r2-8, while inclusion of the mesoderm and endoderm suppressed this effect. In explants with mesoderm/endoderm, RA-dependent SM neuron differentiation in rostral rhombomeres was restored by the application of an inhibitor of the RA-degrading enzyme CYP26. We found that the mesoderm/endoderm (either with or without RA) induced Cyp26 expression in the neuroepithelium in vitro, suggesting that the modulatory effect of CYP26 on RA-dependent patterning might be dependent on local signals.

Animals↗

PubMed2003

Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3.

We have investigated the mechanisms involved in generating hindbrain motoneurone subtypes, focusing on somatic motoneurones, which are confined to the caudal hindbrain within rhombomeres 5-8. Following heterotopic transplantation of rhombomeres along the rostrocaudal axis at various developmental stages, we have found that the capacity of rhombomeres to generate somatic motoneurones is labile at the neural plate stage but becomes fixed just after neural tube closure, at stage 10-11. Grafting of somites or retinoic acid-loaded beads beneath the rostral hindbrain induced the formation of somatic motoneurones in rhombomere 4 only, and Hox genes normally expressed more caudally (Hoxa3, Hoxd4) were induced in this region. Targeted overexpression of Hoxa3 in the rostral hindbrain led to the generation of ectopic somatic motoneurones in ventral rhombomeres 1-4, and was accompanied by the repression of the dorsoventral patterning gene Irx3. Taken together, these observations suggest that the somites, retinoic acid and Hox genes play a role in patterning somatic motoneurones in vivo.

Animals↗

PubMed2002

Neuronal development: sorting out motor neurons.

In the developing spinal cord, motor neurons become segregated into important functional units termed motor pools. Now it has been discovered that repertoires of cadherin surface molecules play key roles in motor pool sorting.

Animals↗

PubMed2002

Expression of the ETS transcription factor ER81 in the developing chick and mouse hindbrain.

ER81 is an ETS domain-containing transcription factor, which is expressed in various developing tissues and organs of the embryo and in pools of developing spinal motor neurons and proprioceptive sensory neurons. Analysis of mice lacking ER81 function showed that this gene played an important role in the establishment of sensory-motor circuitry in the spinal cord. Here, we investigate the expression pattern of er81 in the hindbrain of both chick and mouse embryos. We find that er81 is expressed in a subpopulation of inferior olive neurons, which send their projections to the caudal cerebellum.

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

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.

Animals↗