Evolutionary conservation of midline repulsive signaling by Robo family receptors in flies and mice

The Roundabout (Robo) family is an evolutionarily conserved group of axon guidance receptors that regulate midline crossing in a wide range of animal groups by signaling midline repulsion in response to Slit ligands. However, it is not known whether Robo receptors from different species signal midline repulsion via equivalent mechanisms. To examine the evolutionary conservation of midline repulsive signaling, here we express Robo family proteins from mouse in neurons of the fruit fly, Drosophila melanogaster . We show that Robo proteins which normally function in canonical Slit-dependent midline repulsion in the mouse (mRobo1 and mRobo2) can also repel Drosophila axons from the midline in the fly embryonic ventral nerve cord, and can partially rescue the ectopic midline crossing defects caused by loss of Drosophila robo1 , though less efficiently as Drosophila Robo1 itself. In contrast, mouse Robo3 isoforms (mRobo3.1 and mRobo3.2) which do not normally respond to Slit have no detectable effect on axon guidance when expressed in fly embryos. We further show that the differences in midline repulsive signaling effectiveness between fly and mouse Robos is not due to a simple difference in Slit affinity conferred by the Slit-binding Ig1 domain. Together, our results support the idea that the core signaling mechanisms employed by Robo family receptors are conserved across bilaterians, though some degree of evolutionary divergence has decreased the ability of receptors from different bilaterian clades to directly substitute for one another.