Effects of roundabout on growth cone dynamics, filopodial length, and growth cone morphology at the midline and throughout the neuropile

roundabout (robo) encodes an axon guidance receptor that controls midline c rossing in the Drosophila CNS. In robo mutants, axons that normally project ipsilaterally can cross and recross the midline. Growth cones expressing R obo are believed to be repelled from the midline by the interaction of Robo and its ligand Slit, an extracellular protein expressed by the midline gli a. To help understand the cellular basis for the midline repulsion mediated by Robo, we used time-lapse observations to compare the growth cone behavi or of the ipsilaterally projecting motorneuron RP2 in robo and wild-type em byros. In wild-type embryos, filopodia can project across the midline but a re quickly retracted. In robo mutants, medial filopodia can remain extended for longer periods and can develop into contralateral branches. In many ca ses RP2 produces both ipsilateral and contralateral branches, both of which can extend into the periphery. The growth cone also exhibits longer filopo dia and more extensive branching both at the midline and throughout the neu ropile. Cell injections in fixed stage 13 embryos confirmed and quantified these results for both RP2 and the interneuron pCC. The results suggest tha t Robo both repels growth cones at the midline and inhibits branching throu ghout the neuropile by promoting filopodial retraction.