Axon guidance at the Drosophila midline

The correct wiring of the nervous system relies on the uncanny ability of axons to locate and recognize their appropriate synaptic partners. To help them find their way in the developing embryo, axons are tipped with a highly motile and exquisitely sensitive structure, the growth cone. Extracellular guidance cues can either attract or repel growth cones, and can operate either at close range or over a distance. Much progress has recently been made in the identification of guidance cues and their receptors, and in defining their functions in the developing nervous system. By contrast, we still have only a limited understanding of how these guidance cues are distributed extracellularly. Similarly, it is important to learn how the receptors for these molecules are distributed intracellularly, so that different axons can respond differently to such cues. The midline of the Drosophila central nervous system (CNS) provides an excellent opportunity to explore these issues. Cells at the midline secrete two cues that guide CNS axons. One of these, Slit, is the focus of this work. Slit is thought to act at short range to control which axons cross the midline and which do not, and at long range to control the pathways chosen by axons as their grow alongside the midline. Although Slit is a secreted molecule, there is no direct evidence that it spreads away from the midline in order to exert its long range guidance function. Here, we propose to obtain direct evidence for the long range distribution and function of Slit, and to investigate how proteolytic processing of Slit contributes to its function. In regulating midline crossing, Slit acts through the receptor Robo. Although Robo protein is made in all CNS neurons, it is trafficked to the growth cone in some neurons, but to endosomes and lysosomes in others. If Robo is delivered to the growth cone, then the axon is repelled by Slit and so cannot cross the midline. If Robo is instead delivered to lysosomes, very little of it reaches the growth cone, and so this axons can grow across the Slit- expressing midline cells. This differential trafficking of Robo is controlled by a sorting receptor, Comm, expressed specifically in neurons that extend axons across the midline. Here, we propose to investigate in detail how Comm controls the trafficking of Robo, using both in vitro and in vivo assays.

The correct wiring of the nervous system relies on the uncanny ability of axons to locate and recognize their appropriate synaptic partners. To help them find their way in the developing embryo, axons are tipped with a highly motile and exquisitely sensitive structure, the growth cone. Extracellular guidance cues can either attract or repel growth cones, and can operate either at close range or over a distance. Much progress has recently been made in the identification of guidance cues and their receptors, and in defining their functions in the developing nervous system. By contrast, we still have only a limited understanding of how these guidance cues are distributed extracellularly. Similarly, it is important to learn how the receptors for these molecules are distributed intracellularly, so that different axons can respond differently to such cues. The midline of the Drosophila central nervous system (CNS) provides an excellent opportunity to explore these issues. Cells at the midline secrete two cues that guide CNS axons. One of these, Slit, is the focus of this work. Slit is thought to act at short range to control which axons cross the midline and which do not, and at long range to control the pathways chosen by axons as their grow alongside the midline. Although Slit is a secreted molecule, there is no direct evidence that it spreads away from the midline in order to exert its long range guidance function. Here, we propose to obtain direct evidence for the long range distribution and function of Slit, and to investigate how proteolytic processing of Slit contributes to its function. In regulating midline crossing, Slit acts through the receptor Robo. Although Robo protein is made in all CNS neurons, it is trafficked to the growth cone in some neurons, but to endosomes and lysosomes in others. If Robo is delivered to the growth cone, then the axon is repelled by Slit and so cannot cross the midline. If Robo is instead delivered to lysosomes, very little of it reaches the growth cone, and so this axons can grow across the Slit- expressing midline cells. This differential trafficking of Robo is controlled by a sorting receptor, Comm, expressed specifically in neurons that extend axons across the midline. Here, we propose to investigate in detail how Comm controls the trafficking of Robo, using both in vitro and in vivo assays.