Commissural Axon Pathfinding Research Articles

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.

Use of lipophilic dyes in studies of axonal pathfinding in vivo.

Fluorescent lipophilic dyes are an ideal tool to study axonal pathfinding. Because these dyes do not require active axonal transport for their spreading, they can be used in fixed tissue. Here, we describe the method we have used to study the molecular mechanisms of commissural axon pathfinding in the embryonic chicken spinal cord in vivo. Based on in vitro studies, different families of molecules had been suggested to play a role in the guidance of developing axons. In order to test their function in vivo, we used the commissural neurons that are located at the dorsolateral border of the chicken spinal cord as a model system [Stoeckli and Landmesser (1995) Neuron 14:1165-1179]. Axonin-1, NgCAM, and NrCAM, three members of the immunoglobulin (Ig) superfamily of cell adhesion molecules (CAMs), were shown to be important for the correct growth pattern of commissural axons. We studied the effect of perturbations of specific CAM/CAM interactions by injection of function-blocking antibodies into the central canal of the spinal cord in ovo. After 2 days, the embryos were sacrificed and fluorescent tracers, such as Fast-DiI, were used to visualize commissural axons, and thus, to analyze their response to these perturbations in two different types of fixed preparations: transverse vibratome sections and whole-mount preparations of the spinal cord. Both pathfinding errors and defasciculation of axons were observed as a result of the perturbation of CAM/CAM interactions.

Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord

Early-forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E12). Patterns of GAD65 localization were compared with those of TAG-1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG-1–positive commissural axons were GAD65-negative. These results suggest that commissural neurons have both γ-aminobutyric acid (GABA)ergic and non-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14–15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding. J. Comp. Neurol. 409:285–298, 1999. © 1999 Wiley-Liss, Inc.

Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord

Early-forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E 12). Patterns of GAD65 localization were compared with those of TAG-1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG-1-positive commissural axons were GAD65-negative. These results suggest that commissural neurons have both gamma-aminobutyric acid (GABA)ergic and non-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14-15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding.

Chapter 10 Molecular mechanisms of commissural axon pathfinding

Correct axonal path finding is dependent on the continuous integration of signals derived from the interactions between molecules on the growth cone surface and molecules presented by the environment of the elongating axon. This chapter shows that the guidance cues provided by the surrounding tissue can be divided in long-range and short-range guidance cues. The long-range guidance cues outline the target, which is then reached by selecting appropriate pathways laid down by short-range guidance cues, such as CAMs. Distinctive interactions of CAMs are responsible for pathway decisions of growth cones at choice points by generating preferences. Path finding is not determined by attractive cues only, but rather by a balance between positive and negative cues. The involvement of multiple factors in any pathway decision allows for a fine tuning of the growth cone's response and also for the distinctive behavior of growth cones of different neuronal subpopulations encountering the same choice points. Interestingly, the mechanisms of axonal pathfinding are comparable in vertebrates and invertebrates, and the same classes of molecules are used as guidance cues.