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Updated: Jun 27, 2026
Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes
Published on: January 12, 2020
Redundant mechanisms for regulation of midline crossing in Drosophila
1Research Institute of Molecular Pathology, Vienna, Austria. gilestro@wisc.edu
Abstract:
During development, all neurons have to decide on whether to cross the longitudinal midline to project on the contralateral side of the body. In vertebrates and invertebrates regulation of crossing is achieved by interfering with Robo signalling either through sorting and degradation of the receptor, in flies, or through silencing of its repulsive activity, in vertebrates. Here I show that in Drosophila a second mechanism of regulation exists that is independent from sorting. Using in vitro and in vivo assays I mapped the region of Robo that is sufficient and required for its interaction with Comm, its sorting receptor. By modifying that region, I generated new forms of Robo that are insensitive to Comm sorting in vitro and in vivo, yet still able to normally translate repulsive activity in vivo. Using gene targeting by homologous recombination I created new conditional alleles of robo that are sorting defective (robo(SD)). Surprisingly, expression of these modified proteins results in phenotypically normal flies, unveiling a sorting independent mechanism of regulation.
Insights
Neurons decide whether to cross the midline during development. In Drosophila, a new Robo receptor regulation mechanism, independent of Comm sorting, was discovered, revealing a novel developmental pathway.
Area of Science:
- Neuroscience
- Developmental Biology
- Molecular Biology
Background:
- Neuronal development requires precise guidance of axons.
- The longitudinal midline is a critical choice point for neuronal projection.
- Robo signaling and its interaction with Comm are known regulators of midline crossing in Drosophila.
Purpose of the Study:
- To investigate alternative regulatory mechanisms of Robo signaling in Drosophila axon guidance.
- To identify and characterize a novel mechanism of Robo regulation independent of Comm-mediated sorting.
Main Methods:
- In vitro and in vivo assays were used to map Robo interaction regions with Comm.
- Site-directed mutagenesis was employed to create Comm-insensitive Robo variants.
- Gene targeting via homologous recombination generated sorting-defective robo alleles (robo(SD)).
Main Results:
- A specific region of Robo sufficient and required for Comm interaction was identified.
- Comm-insensitive Robo variants retained normal repulsive signaling in vivo.
- Flies expressing sorting-defective Robo proteins exhibited normal phenotypes, indicating a sorting-independent regulatory pathway.
Conclusions:
- A novel, sorting-independent mechanism regulates Robo function in Drosophila midline crossing.
- This discovery expands our understanding of axon guidance regulation beyond receptor sorting.
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