Ephrin-dependent growth and pruning of hippocampal axons.
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Neuronal connections are arranged topographically such that the spatial organization of neurons is preserved by their termini in the targets. During the development of topographic projections, axons initially explore areas much wider than the final targets, and mistargeted axons are pruned later. The molecules regulating these processes are not known. We report here that the ligands of the Eph family tyrosine kinase receptors may regulate both the initial outgrowth and the subsequent pruning of axons. In the presence of ephrins, the outgrowth and branching of the receptor-positive hippocampal axons are enhanced. However, these axons are induced later to degenerate. These observations suggest that the ephrins and their receptors may regulate topographic map formation by stimulating axonal arborization and by pruning mistargeted axons. (+info)
Requirement for the zebrafish mid-hindbrain boundary in midbrain polarisation, mapping and confinement of the retinotectal projection.
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The organizer at the midbrain-hindbrain boundary (MHB organizer) has been proposed to induce and polarize the midbrain during development. We investigate the requirement for the MHB organizer in acerebellar mutants, which lack a MHB and cerebellum, but retain a tectum, and are mutant for fgf8, a candidate inducer and polarizer. We examine the retinotectal projection in the mutants to assay polarity in the tectum. In mutant tecta, retinal ganglion cell (RGC) axons form overlapping termination fields, especially in the ventral tectum, and along both the anterior-posterior and dorsal-ventral axis of the tectum, consistent with a MHB requirement in generating midbrain polarity. However, polarity is not completely lost in the mutant tecta, in spite of the absence of the MHB. Moreover, graded expression of the ephrin family ligand Ephrin-A5b is eliminated, whereas Ephrin-A2 and Ephrin-A5a expression is leveled in acerebellar mutant tecta, showing that ephrins are differentially affected by the absence of the MHB. Some RGC axons overshoot beyond the mutant tectum, suggesting that the MHB also serves a barrier function for axonal growth. By transplanting whole eye primordia, we show that mapping defects and overshooting largely, but not exclusively, depend on tectal, but not retinal genotype, and thus demonstrate an independent function for Fgf8 in retinal development. The MHB organizer, possibly via Fgf8 itself, is thus required for midbrain polarisation and for restricting axonal growth, but other cell populations may also influence midbrain polarity. (+info)
Positionally selective growth of embryonic spinal cord neurites on muscle membranes.
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Motor neurons from distinct positions along the rostrocaudal axis generally innervate muscles or muscle fibers from corresponding axial levels. These topographic maps of connectivity are partially restored after denervation or transplantation under conditions in which factors of timing and proximity are eliminated. It is therefore likely that motor neurons and some intramuscular structures bear cues that bias synapse formation in favor of positionally matched partners. To localize these cues, we studied outgrowth of neurites from embryonic spinal cord explants on carpets of membranes isolated from perinatal rat muscles. Neurites from rostral (cervical) and caudal (lumbar) spinal cord slices exhibit distinct growth preferences. In many instances, rostrally derived neurites grew selectively on membranes from forelimb muscles or from a single thoracic muscle (the serratus anterior) when given a choice between these membranes and membranes from hindlimb muscles or laminin. Caudally derived neurites almost never exhibited such rostral preferences, but instead preferred membranes from hindlimb muscles or a single hindlimb muscle (the gluteus) to rostral muscles or laminin. Likewise, spinal neurites exhibited distinct position-related preferences for outgrowth on membranes of clonal myogenic cell lines derived from specific rostral and caudal muscles. Taken together these results suggest that the membranes of motor axons and myotubes bear complementary labels that vary with rostrocaudal position and regulate neuromuscular connectivity. (+info)
An early developmental role for eph-ephrin interaction during vertebrate gastrulation.
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Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals which regulate cytoskeletal organisation and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. In this report we show that soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organisation of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organisation of embryonic structures by coordinating the cellular movements of convergence during gastrulation. (+info)
Identification and characterization of splice variants of ephrin-A3 and ephrin-A5.
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Ephrins and Eph receptors have been implicated to play important roles in axon guidance. A variable spacer region exists that differs significantly among distinct ephrins. An ephrin-A5 isoform has previously been isolated which lacks 27 amino acids within the spacer region. The expression and biological activities of this isoform, as well as the existence of isoforms for other ephrins that show variation within the spacer region, remain unknown. We report here a novel alternatively spliced isoform of ephrin-A3 which lacks the corresponding variable region. When compared to the longer isoforms, the shorter isoforms of both ephrin-A3 and ephrin-A5 remained less prominent in the brain during development, though their expression increased at postnatal stages. In addition, they could inhibit neurite outgrowth of dorsal root ganglia (DRG) neurons, suggesting that the corresponding variable regions were not essential for their axon guidance activities. (+info)
Eph receptors and ephrins demarcate cerebellar lobules before and during their formation.
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The formation of the ten cerebellar lobules is an unsolved problem in brain development. We report a screen for the four subfamilies of Eph receptors and their ligands (ephrins) in developing mouse cerebellum, using soluble receptor-immunoglobulin and ligand-immunoglobulin fusion proteins, and antibodies against EphA and ephrin-B proteins. Our results identify Eph receptors and ephrins as the first molecules known to demarcate individual lobules during development. Staining for ephrin-A ligands is in lobule VIII as it forms, across the whole width of the cerebellum. Staining for three EphA receptors approximately coincides with presumptive lobules VI and/or VII before and just after birth, whereas a fourth EphA receptor (EphA4, which binds ligands of both subfamilies) has more widespread expression. Staining for EphB receptors is in lobules VII, VIII, and IX. Staining for ephrin-B ligands is much weaker, becomes detectable only after birth, and does not appear to be lobule-specific. Staining for all subfamilies spreads to at least some adjacent lobules as maturation proceeds. The lobule-specific patterns appear before the lobules form, and initially extend across the width of the cerebellum, in spite of the lesser conservation of the lateral extensions of the lobules. These expression patterns define previously unknown developmental units and suggest that Eph family proteins may contribute to cerebellar morphogenesis. (+info)
Regional differences in the developing cerebral cortex revealed by ephrin-A5 expression.
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The development of axonal connections between thalamic nuclei and their cortical target areas occurs in a highly specific manner. To explore the mechanisms of thalamocortical axon pathfinding, we investigated the expression of several members of the ephrin and Eph gene families in the forebrain. The Eph ligand ephrin-A5 was expressed in three distinct gradients during the development of the telencephalon. The first gradient occurred in the cortical ventricular zone and established ephrin-A5 as one of the earliest markers distinguishing cells residing in the anterior versus posterior cortical neuroepithelium. The second gradient was apparent in the subplate and occurred in spatial opposition to a distinct gradient for the low-affinity NGF receptor p75. This finding reveals that different regions of the early subplate are molecularly heterogeneous. Third, we confirmed that ephrin-A5 is expressed in a bi-directional gradient in the cortical plate, with highest levels in the somatomotor cortex. Three putative receptors for ephrin-A5 -- EphA3, EphA4 and EphA5 -- showed distinct expression patterns in the developing thalamus. The graded distributions of ephrin-A5 in the developing subplate and cortex and the expression of its receptors in the thalamus are consistent with the notion that the Eph ligands and their receptors may function in the topographic mapping of thalamic axons to specific cortical areas. (+info)
A role for the Eph ligand ephrin-A3 in entorhino-hippocampal axon targeting.
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Neurons of layers II and III of the entorhinal cortex constitute the major afferent connection of the hippocampus. The molecular mechanisms that target the entorhinal axons to specific layers in the hippocampus are not known. EphA5, a member of the Eph receptor family, which has been shown to play critical roles in axon guidance, is expressed in the entorhinal cortex, the origin of the perforant pathway. In addition, ligands that interact with EphA5 are expressed in distinct hippocampal regions during development of the entorhino-hippocampal projection. Of these ligands, ephrin-A3 mRNA is localized both in the granular cell layer of the dentate gyrus and in the pyramidal cell layer of the cornu ammonis, whereas ephrin-A5 mRNA is only expressed in the pyramidal cell layer of the cornu ammonis. In the dentate gyrus, the ligand protein is not present in the termination zone of the entorhinal efferents (the outer molecular layer of the dentate gyrus) but is concentrated in the inner molecular layer into which entorhinal efferents do not grow. We used outgrowth and stripe assays to test the effects of ephrin-A3 and ephrin-A5 on the outgrowth behavior of entorhinal axons. This functional analysis revealed that entorhinal neurites were repelled by ephrin-A3 but not by ephrin-A5. These observations suggest that ephrin-A3 plays an important role in the layer-specific termination of the perforant pathway and that this ligand may interact with the EphA5 receptor to restrict entorhinal axon terminals in the outer molecular layer of the dentate gyrus. (+info)