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Chromophore-assisted laser inactivation of a repulsive axonal guidance molecule
B K Müller1, D G Jay, F Bonhoeffer
1Max-Planck-Institut für Entwicklungsbiologie, Tübingen, Germany. bemue@mpib-tuebingen.mpg.de
Background:
The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons.
Results:
To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum.
Conclusions:
These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.
Insights
A novel repulsive guidance molecule (RGM) was identified, crucial for guiding retinal axons in the optic tectum. This molecule
Area of Science:
- Neuroscience
- Developmental Biology
- Molecular Biology
Background:
- Retinal ganglion cell axons form a topographic map in the optic tectum.
- Directional cues in the tectum are essential for guiding these axons.
- Temporal retinal axons avoid posterior tectal membranes due to repellent guidance activities.
Purpose of the Study:
- To identify the molecular basis of repellent guidance cues for retinal axons.
- To investigate the role of a putative 33 kDa GPI-anchored molecule in axon guidance.
Main Methods:
- Raised monoclonal antibodies against GPI-anchored molecules from tectal membranes.
- Used antibody F3D4 to target a 33 kDa molecule.
- Employed chromophore-assisted laser inactivation to assess functional impact.
Main Results:
- Monoclonal antibody F3D4 recognized the 33 kDa molecule, named RGM.
- Inactivation of RGM abolished repellent activity in posterior tectal membranes.
- Temporal retinal fibers were no longer repelled by posterior tectal membranes in vitro.
- RGM expression increases from anterior to posterior in the optic tectum in vivo.
Conclusions:
- RGM is involved in the in vitro guidance of retinal axons.
- Findings support guidance by gradients of repulsive molecules.
- Suggests the existence of multiple repellent guidance molecules with similar functions and expression patterns.