New Roundabouts Send Axons into the Fas Lane Jannette Rusch, David Van Vactor  Neuron  Volume 28, Issue 3, Pages 637-640 (December 2000) DOI: 10.1016/S0896-6273(00)00143-4

Figure 1 Events and Molecules at the Drosophila Midline (A) Cartoon of the Drosophila ventral nerve cord in a dorsal view. Shown are three representative neurons: two noncrossing neurons, one of which is medially located (red) and one that is more lateral (green), and a crossing neuron (blue) that enters one of the longitudinal fascicles after it has crossed the midline (ML, shown in orange). Crossing neurons have to accomplish three tasks: (1) they have to turn towards the midline, i.e., respond to attractants such as Netrin, (2) they have to be pushed out of the midline once they have entered it, and (3), they have to choose the correct longitudinal pathway along which to extend. Noncrossing (ispsilateral) neurons just have to be repelled from the midline (4). The presumptive gradient of Slit protein emanating from the midline is involved in steps 2, 3, and 4. (B) Sequence motifs of Drosophila Robo protein family members and Slit protein. The extracellular domains of the Robo family members are highly conserved, consisting of five immunoglobulin and three fibronectin type III domains. Robo has four intracellular motifs (CC0, CC1, CC2, CC3). The new Robo family members Robo2 and Robo3 contain the tyrosine phosphorylation sites CC0 and CC1, but they lack both CC2 (a binding site for Enabled) and CC3 (interacting with the Abl tyrosine kinase). The Slit ligand is a large secreted molecule consisting of four tandem leucine-rich repeats (LRR), followed by seven EGF repeats and a cysteine knot (a dimerization motif). (C) Phenotypes of robo mutants. Left: in robo mutants, medially located neurons (red, blue) cross and recross the midline (the “meandering” phenotype), while more laterally located neurons (green) extend relatively normally. Middle: in robo2 (and to a lesser degree in robo3) mutants, lateral neurons (green) are sometimes found to ectopically cross the midline. Right: in robo,robo2 double mutants all axons collapse onto the midline and are unable to leave it. This phenotype is identical to the slit mutant phenotype. Neuron 2000 28, 637-640DOI: (10.1016/S0896-6273(00)00143-4)

Figure 2 The Robo Code and the Labeled Pathways Model (A) Schematic representation of a cross section through the Drosophila ventral nerve cord, showing from left to right the midline (ML), a commissure, and the area of the longitudinal fascicles. A presumptive Slit protein gradient (blue) emanates from the midline, with progressively lower levels in lateral regions. In the longitudinal tract area, axons express one of three combinations of robo family members (i.e., one of three “Robo codes,” indicated in different shades of gray): axons in the most medial location express robo only, intermediate axons express robo plus robo3, and the most laterally located axons express all three robo family members on their surfaces. Also indicated are the approximate locations of the three Fas II–expressing longitudinal fascicles (red ovals) (M, medial; I, intermediate: L, lateral Fas II fascicle). (B) Longitudinal fascicle phenotypes in robo family member mutants. Left: in late-stage wild-type embryos, three longitudinal Fas II–expressing fascicles are seen on either side of the midline (ML). They are located according to their robo codes (indicated in boxes above the panels), the medial fascicle expressing only robo, the intermediate expressing robo and robo3, and the lateral fascicle expressing all three robos. Middle: in robo3 loss-of-function mutants, the intermediate fascicle disappears and its axons are now found in the medial fascicle that appears thicker. Right: if robo2 is overexpressed in all neurons, all axons converge in a single fascicle in a lateral position. (C) A combinatorial code for pathfinding at the midline. Neurons find their general target area according to which robo code they express on their cell surfaces (indicated in different shades of gray), which determines how far they are pushed laterally by the gradient of Slit repellent (blue). To arrive at their precise destination within this area, they rely on local cues, or pathway labels (indicated by colored circles, X, Y, Z), e.g., cell adhesion molecules such as Fas II or Connectin. Neuron 2000 28, 637-640DOI: (10.1016/S0896-6273(00)00143-4)