Ectopic Expression in the Giant Fiber System ofDrosophila Reveals Distinct Roles for Roundabout (Robo), Robo2, and Robo3 in Dendritic Guidance and Synaptic Connectivity

Coexpression of wild-type Comm rescues Robo2-induced lateral displacement. A, Expression of dominant negativeUAS-robo^cΔ using the A307 driver results in a midline crossing of the GF axons in the target area (arrow). B, Expression ofUAS-comm^wt using the A307 driver results in a collapse of the GF at the midline. The GF axons wrap around each other and cross the midline (arrow). The GF bends appear to be normal (arrowheads). C, Coexpression of wild-type UAS-comm and UAS-robo2 using A307 results in GFs with a normal lateral position. In approximately one-third of the specimens, the GF is seen to cross the midline once in the target area, as seen in this specimen (arrow).D, Coexpression ofUAS-comm^cΔ, lacking the intracellular domain, and UAS-robo2 using A307 results in a lateral displacement of the GF, as seen whenUAS-robo2 alone is expressed in the GF (compare with Fig. 3C). Asterisks indicate that these extensions have been also seen in A307, UAS-lacZ/+ control flies (Allen et al., 1998) and are therefore probably attributable to a nonspecific effect in these recombinant flies. Scale bar, 20 μm.

Overexpression of Robo, Robo2, and Robo3 causes lateral displacement of the GF. A, Control adult (UAS-lacZ/+;A307/+) CNS whole-mount preparation stained for β-gal using immunohistochemistry. This specimen illustrates control GF axons projecting into T2, where they make a characteristic lateral bend (arrows). When UAS-robo(B), UAS-robo2(C), and UAS-robo3(D) were expressed in the GF, they revealed a differential strength in their ability to push the GF axons laterally. Note that expression of Robo3 can induce lateral displacement (asterisks) in the connective before reaching the thoracic ganglion. Additional terminal branches of the axon were occasionally observed when UAS-robo,UAS-robo2, or UAS-robo3 was ectopically expressed in the giant fiber using either driver (B,arrow). E1, ben-like termination in a specimen expressing Robo [A307/+;(2×)UAS-robo/+] exclusively presynaptically. The ending is swollen or tapered (arrows).E2, ben-like termination in a specimen expressing Robo [c17/+;(2×)UAS-robo/+]. In this case, the ending is swollen (arrows). F, Quantification of the lateral position of the axons. The location of the axon in each specimen was determined relative to the midline and the lateral edge of the ganglion. This distance was divided into 13 intervals, and each GF axon was scored for the relative position of the axon just anterior to the bend (white scale).Black scale bar, 20 μm.

Physiology of the GF circuit. A, Schematic depiction of the two methods of stimulation as well as the method for recording from the TTM muscle. Brain stimulation was used to activate the GF; thoracic stimulation was used to excite the TTMn directly. B, Schematic of the GF and the TTMn with approximate conduction times. The estimated response latency is shown for brain (0.8 msec) as well as thoracic stimulation (0.6 msec).C, Responses of control (C1) and Robo gain-of-function (C2–C5) flies to brain and thoracic stimulation. Note that c17/+;UAS-robo/+ and A307/+;UAS-robo/+ flies have an increased response latency (1.4 and 1.9 msec) and are not able to follow repetitive stimulation at 100 Hz (C2, C3). Some Robo gain-of-function flies (A307/+;UAS-robo/+) show no response to brain stimulation (C4, asterisk), but thoracic stimulation reveals that the neuromuscular junction of the TTMn is normal (C5).

Weak or laterally displaced GF→TTMn synapses are dye-coupled. A, Schematic of the morphology of the GF (black) and the TTMn (dark gray) within the thoracic portion of the CNS. The box indicates the regions of the thoracic ganglion depicted in B, C.B, Retrograde staining of the TTMn in a specimen expressing Robo [c17/+;(2×)UAS-robo] with a physiologically determined weakened GF→TTMn synapse. Note that the TTMn is dye-coupled to the GF. C, Specimen expressing Robo [c17/+;(2×)UAS-robo] with a physiologically determined wild-type GF→TTMn synapse. Note that the TTMn is dye-coupled to the laterally displaced GF. Scale bar, 20 μm.

Robo mediates dendritic repulsion of the TTMn, but Robo2 and Robo3 do not. Whole-mount preparations of the thoracic ganglion of late pupas (50–75%) were stained for β-gal using immunohistochemistry. A, Example of a control motor neuron (UAS-lacZ/shakB-Gal4). cb, Cell body; ax, axon; ld, lateral dendrite; md, medial dendrite. B, Overexpression of Robo disrupts dendrite formation in the TTMn. The medial dendrite does not reach the midline (arrows), and the lateral dendrite is often missing (asterisk). The genotype is UAS-lacZ/shakB-Gal4;UAS-robo-myc/+.C, Expression of Robo2 or Robo3 has no effect on the TTMn dendrites (example only shown forUAS-lacZ/shakB-Gal4;UAS-robo2-myc/+). As in wild type, the medial TTMn dendrite reaches the midline (arrow). Scale bar, 20 μm.

Ectopic GF→TTMn synapses in flies expressing Robo or Robo2 presynaptically and postsynaptically. A, Simultaneous expression of robo presynaptically and postsynaptically (shakB-Gal4/c17,UAS-lacZ;UAS-robo2-myc/+) at ∼70% of pupal development. On the right, the GF contacts the TTMn dendrite more lateral than usual where a thickening of the dendrite and the GF terminal can be seen, presumably representing the ectopic synapse (black arrow). The medial TTMn dendrite extends beyond the contact and reaches the midline (arrowheads). The left GF first grows laterally (white arrows), contacts the TTMn neurite (white arrowhead), and than grows along the medial dendrite from lateral to medial (black arrow). B, C, Examples of specimens expressing Robo presynaptically and postsynaptically. Specimens with the genotypeshakB-Gal4/c17,UAS-lacZ;(2×)UAS-robodissected at 50–80% of pupal development are shown. B, The GF turns at the midline and grows toward the stunted medial dendrite of the TTMn (black arrow). C, In this example, the right GF grows toward and contacts the stalled dendrite ∼30 μm lateral to the midline (black arrow). The left GF displays aben-like ending (black arrow) ∼10 μm from the midline and does not contact the TTMn dendrite (white arrow). Scale bars, 20 μm.