Fig. 4

Tribolium Robo2/3 can substitute for Drosophila Robo3 to promote axon pathway formation in Drosophila embryos. a–d Stage 16 Drosophila embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show anti-FasII channel alone from the same embryos. In wild-type embryos, FasII-positive axons form three distinct longitudinal pathways on either side of the midline, one each in the medial, intermediate, and lateral zones of the neuropile. The intermediate FasII pathway is distinct from the medial and lateral pathways in every hemisegment in wild-type embryos (a, arrow). In robo3 ¹ embryos, FasII-positive axons that normally form the intermediate pathway are displaced medially, and the intermediate pathway fails to form (b, arrow with asterisk). Intermediate pathways form correctly in embryos in which the robo3 gene is replaced with a robo3 cDNA (c, arrow). When robo3 is replaced with a TcRobo2/3 cDNA, intermediate pathways form correctly in over 88% of hemisegments (d, arrow), indicating that TcRobo2/3 can substitute for robo3 to promote axon pathway formation in the intermediate region of the neuropile. Bar graph shows quantification of intermediate FasII pathway defects in the genotypes shown in a–d. Error bars indicate standard error of the mean. Number of embryos scored for each genotype is indicated in parentheses. e–h Embryos carrying the sema2b-TauMyc transgene and stained with anti-HRP (blue), anti-FasII (red), and anti-Myc (green) antibodies. The sema2b-TauMyc transgene labels the cell bodies and axons of 2–3 neurons per hemisegment in abdominal segments A4–A8. These axons normally project across the midline and then extend anteriorly in the intermediate region of the neuropile (e, arrowhead). In robo3 ¹ embryos, these axons are displaced medially (f, arrowhead with asterisk), but their normal intermediate position is restored in both robo3 ^robo3 (g, arrowhead) and robo3 ^TcRobo2/3 embryos (h, arrowhead)