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Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo

Nature Neuroscience volume 8, pages 156–163 (2005)Cite this article

Abstract

Commissureless (Comm) controls axon guidance across the Drosophila melanogaster midline by regulating surface levels of Robo, the receptor for the midline repellent Slit. Two different models have been proposed for how Comm regulates Robo: a 'sorting' model and a 'clearance' model, both based on studies using heterologous cells in vitro. Here, we test these two models in vivo. We establish a genetic rescue assay for Comm, and use this assay to show that midline crossing does not require the presence of Comm in midline cells, as proposed by the clearance model. Moreover, by monitoring the trafficking of a Robo–green fluorescent protein (GFP) fusion in living embryos, we demonstrate that Comm prevents the delivery of Robo-GFP to the growth cone, as predicted by the sorting model. It has also been suggested that Comm must be ubiquitinated by the Nedd4 ubiquitin ligase. We show here, however, that ubiquitination of Comm is not required for its function in vitro or in vivo, and that Nedd4 is unlikely to function in axon guidance at the midline.

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Figure 1: Comm in midline cells does not contribute to midline crossing.
Figure 2: Structural requirements in the Comm lumenal and transmembrane domains.
Figure 3: Comm blocks Robo transport along the axon.
Figure 4: A Comm that cannot be ubiquitinated is still functional in vitro and in vivo.
Figure 5: No evidence for Nedd4 function in midline crossing.

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Acknowledgements

We thank G. Tear, M. Noll, C. Klämbt, R. Hiesinger, and the Drosdel consortium for fly stocks; A. Graf and K. Paiha for technical assistance; and F. Schnorrer and G. Gilestro for discussions and comments on the manuscript. This work was funded in part by a grant from the Austrian Science Foundation (FWF). C.R. is supported by an EMBO long-term postdoctoral fellowship.

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  1. Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr. Bohr-Gasse 3-5, Vienna, A-1030, Austria

    Krystyna Keleman, Carlos Ribeiro & Barry J Dickson

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  1. Krystyna Keleman
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Correspondence to Barry J Dickson.

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Supplementary information

Supplementary Fig. 1

Models for Comm function in midline crossing. (JPG 125 kb)

Supplementary Video 1

False-color time-lapse of a living stage 16 embryo expressing Robo-GFP under the control of the poxn-GAL4 driver. PNS axons were scanned at 2.2-second intervals. To improve the signal-to-noise ratio the axon was bleached after 8.8 seconds. Growth cones are to the right. (MOV 2663 kb)

Supplementary Video 2

False-color time-lapse of a living stage 16 embryo expressing Robo-GFP under the control of the poxn-GAL4 driver. PNS axons were scanned at 5.4-second intervals. Growth cones are to the right. (MOV 3946 kb)

Supplementary Video 3

False-color time-lapse of a living stage 16 embryo expressing Robo-GFP under the control of the poxn-GAL4 driver. PNS axons were scanned at 3-second intervals. To improve the signal-to-noise ratio the axon was bleached after 24 seconds and after 5 minutes and 27 seconds. Growth cones are to the right. (MOV 4180 kb)

Supplementary Video 4

False-color time-lapse of a living late-stage 15 embryo expressing Robo-GFP and Comm under the control of the poxn-GAL4 driver. PNS axons were scanned at 8-seconds intervals. Growth cones are to the right. (MOV 2118 kb)

Supplementary Video 5

False-color time-lapse of a living late-stage 15 embryo expressing Robo-GFP and Comm under the control of the poxn-GAL4 driver. PNS axons were scanned at 8-seconds intervals. Growth cones are to the right. (MOV 2005 kb)

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Keleman, K., Ribeiro, C. & Dickson, B. Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo. Nat Neurosci 8, 156–163 (2005). https://doi.org/10.1038/nn1388

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