Fig. 4. Overactivation of the Slit signaling pathway in the CNS can repel the ganglionic branches. (A-H) Stage 16 embryos stained to visualize the tracheal lumen in blue and the longitudinal axons (in A,B,E,F) in brown. (B) In comm mutants, GBs turn to migrate posteriorly before they reach the midline but after crossing the longitudinal tracts. The longitudinal axon tracts are further apart presumably due to the absence of commissures. (C) Ectopic expression of Robo in the GB1 cell (Robo staining in brown) using the SRF-GAL4 driver results in GBs turning posteriorly before they cross the longitudinal tracts (arrow). GBs that do not overexpress Robo migrate closer to the midline before turning posteriorly (arrowhead). (D) Early turning of GB1 before the longitudinal axons (arrow) can also be seen in embryos with ectopic expression of robo2 (embryos stained for a HA-tagged Robo2 in brown). Several of the Robo2 overexpressing GBs still reach the midline (arrowhead) in contrast to all the GBs that turn prematurely when they overexpress Robo in C. (E) Ectopic expression of Slit in the longitudinal glia using the C321c-GAL4 driver leads to premature turning (arrow) and stalling (arrowheads) of GBs. (F) Several GBs cross the ventral midline (arrows) in response to general tracheal misexpression of comm using the btl-GAL4 driver. (G) UAS-robo2 overexpression in GB1 using the SRF-GAL4 driver cannot rescue the GB midline crossing phenotype in robo mutants (arrows, embryos stained for HA-tagged Robo2 and the tracheal lumen). UAS-robo overexpression with the same driver can rescue the GB CNS entry phenotype in robo2 mutants (H; embryos stained for Robo-HA and tracheal lumen). Scale bar: 20 µm in A-H.