Fig. 7.

Expression of an uncleavable form of Robo does not rescue robo mutants. (A) Schematic depicting the receptors used in the rescue experiment. In Robo-U the Fn domains of Robo are swapped for the first three Fn domains of Fra. The Fra-Ro receptor has the ectodomain of Fra and the transmembrane and intracellular domains of Robo. (B) The media and total lysates of S2 cells expressing the receptors depicted in A, with or without added UASkuzHA, were subjected to PAGE and subsequent western blotting as previously described. No ectodomain shedding was detected from cells expressing Fra-RoMyc, Robo-UMyc, or FraMyc. Western blotting of the total lysates with antibodies directed against Myc, HA and tubulin show the relative levels of Myc-tagged receptor, KuzHA and tubulin, respectively. (C) Quantification of the number of segments in which ap axons ectopically cross the midline in robo mutants expressing different forms of the Robo receptor under the control of apGal4. Shown is the percentage of segments in which the ap axons cross the midline. *P<0.0001 (unpaired Student's t-test) compared to ap axons expressing wild-type Robo. Error bars indicate s.d. (D-H) Stage 16 embryos were stained with a MAb to FasII (1D4). Anterior is up. (D) A wild-type embryo stained with mAb 1D4. (E) In robo mutants, the medial-most fascicles cross the midline (arrows with asterisks). In robo embryos expressing Robo-UMyc (F) or Fra-RoMyc (G) under the control of the elavGal4 driver, the medial-most fascicles still cross the midline in many segments (arrows with asterisks). (H) Pan-neural (elavGal4) expression of wild-type Robo provides significant rescue of the robo phenotype, although one can still observe infrequent ectopic crossing of FasII-positive axons (arrow with asterisk). The small panels below the FasII panels indicate the expression levels and patterns of the Myc-tagged receptors. Arrowheads indicate the midline; asterisks indicate Myc-tagged receptor expression on crossing axons. (I-M) Stage 16 embryos were stained with a polyclonal antibody directed against GFP to detect the ap neurons. Anterior is up. (I) In wild-type embryos the ap neurons never cross the midline. (J) In robo mutants, the ap neurons collapse upon the midline. In robo mutants expressing Robo-U (K) or Fra-Ro (L) driven by apGal4, the ap axons from either side of the midline are fused and collapsed on the midline in some segments (<*), but are correctly routed on either side of the midline in others (<). (M) Expression of wild-type Robo in ap neurons in a robo mutant background rescues the ectopic crossing phenotype.