CrGAP RNAi enhances guidance defects in slit, robo/+ embryos. Stage 16 embryos are stained with the mAb1D4 to reveal the three FasII-positive longitudinal axon pathways. SRE indicates slit¹,robo⁵/+; elav-GAL4/+.(a) In WT embryos, the FasII-positive axon pathways never cross the midline. (b) In slit¹, robo⁵/+ transheterozygous embryos, the medial (innermost) longitudinal pathway occasionally crosses at the midline (arrows). (c) In slit¹,robo⁵/+ transheterozygotes, reducing CrGAP gene dose with the RNAi transgene significantly enhances the ectopic crossing defects. (d) Histogram showing the quantification of the midline crossing phenotypes in each of the indicated genotypes. The percentages of segments that showed ectopic crossing are shown. The number of segments scored in each genotype (n) was 275, 173, 297, 126, 136, 96, 159, and 136. *, P < 0.01, in Fisher's exact test. A second line of CrGAP^RNAi, UAS-CrGAP^RNAi-2/TM3 also shows significant enhancement of the crossing phenotype (53.3% crossing, n = 137) (data not shown).

CrGAP gain-of-function mimics Robo and Rac loss-of-function. Stage 16 filleted embryos of the indicated genotype stained with Abs that label a subset of noncrossing axons, anti-FasII (a–e), or all CNS axons, anti-BP102 (f–h). Anterior is at the top in all images. The genotypes of the embryos are UAS-CrGAP³⁷/elav (a and f); robo¹ (b and g); UAS-CrGAP²⁸/UAS-CrGAP²⁸; elav-GAL4/elav-GAL4 (c, e, and h); and rac1^J10mtl^Δ (d). (a and f) Embryos with low level of CrGAP overexpression show axon patterns identical to WT embryos. The longitudinal axon pathways run parallel to the midline and never cross (a). The axon scaffold has a characteristic ladder-like appearance with commissural axon bundles crossing the midline forming two commissures in each segment (f). (b and g) In the robo mutant, many axons cross and recross the midline. Regions that show ectopic midline crossing are indicated by arrows (b). The axon scaffold is severely altered with much thicker and fused commissures and thinner longitudinal connectives (g). (c and h) Embryos with a high level of CrGAP overexpression show defects similar to robo mutant in terms of both ectopic crossing (c) and altered axon scaffold (h). (d) The rac, mtl double mutant show multiple midline crossings reminiscent of robo mutants. (e) The axon outgrowth defects in CrGAP overexpression embryos. Arrows with asterisk indicate the sites where all of the three longitudinal pathways fail to extend and appear broken. (i) Percentage of segments in UAS-CrGAP²⁸/UAS-CrGAP²⁸; elav-GAL4/elav-GAL4 embryos showing the three classes of phenotypes. The “1D4 guidance defect” refers to the ectopic crossing as indicated by arrow heads in c. The “1D4 outgrowth defect” refers to the phenotype where all of the three longitudinal axon pathways show stalling, as indicated by the arrowheads with asterisk in e. The “BP102 guidance defect” refers to the robo-like axon pattern as shown in all of the four segments in h.

Efficiency of the RNAi method and the specificity of CrGAP. Scanning-electron photomicrographs of adult Drosophila eyes are shown. The pictures are representative of >20 flies examined for each genotype. (a) A WT eye. (b and c) UAS-Rac1, GMR-GAL4/+ (b) and UAS-RhoA,GMR-GAL4/+ (c) flies have rough eye phenotypes. (e) An eye from a UAS-Rac1,GMR-GAL4/+; UAS-RhoA^RNAi/+ fly. The phenotype caused by overexpressing Rac is unaffected by expression of RhoRNAi. (f) UAS-RhoA,GMR-GAL4/+; UAS-RhoA^RNAi/+ eyes show a marked suppression of the RhoA rough eye phenotype. (g and h) The gain-of-function phenotype of Rac (b) is strongly suppressed by the coexpression of UAS-CrGAP (g), but the gain-of-function phenotype of RhoA is not (h). Expression of UAS-CrGAP alone does not cause an observable phenotype (data not shown). The flies were raised at 25°C.

Dose-sensitive genetic interactions among CrGAP, robo, and rac. All shown embryos are at stage 16 and have been stained with either the BP102 (a–c) or 1D4 (e–g and i–k) mAb. (a–h) Antagonistic genetic interaction between CrGAP and Slit/Robo. In embryos that overexpress CrGAP in the SRE background, the ladder-like WT-looking axon scaffold (a and c) is severely disrupted and shows characteristics of the robo mutant phenotype (b). The ectopic crossing defect in SRE embryos (e) is also significantly enhanced (f). (g and i–l) Antagonistic genetic interaction between CrGAP and rac. Note the ectopic crossing defect is present only in embryos overexpressing both CrGAP and RacN17 (j) but not in embryos of the other genotypes (g, i, and k). The stars in l indicate the following complication in obtaining UAS-CrGAP²⁸, UAS-CrGAP³³/+; UAS-RacN17/elav-GAL4 embryos. UAS-CrGAP²⁸, UAS-CrGAP³³/+; elav-GAL4/+ flies were mated to UAS-RacN17/UAS-RacN17 flies. Statistically, only 25% of the progeny from these parents are expected to have the genotype of RacN17; UCrGAP shown in j. Therefore, the fact that close to 20% of the segments showed the crossing defect indicates that the penetrance of the crossing phenotype in embryos with the RacN17; UCrGAP genotype is close to 80% (l). A similar scheme was used to generate the Cdc42N17; UCrGAP embryos for the test with Cdc42. The number of segments scored in each genotype was 165, 164, and 98 (d); 275, 120, 243, and 165 (h); and 103, 220, 98, and 124 (l), respectively.

Biochemical Interactions between CrGAP and Robo. (a) The Robo cytoplasmic domain binds to the N-terminal part of CrGAP that contains the second WW domain. ³⁵S-labeled Robo was incubated with either GST-CrGAP or GST protein alone to test for binding. (b) Myc-tagged CrGAP amino acids 22–154 (myc-WWGAP) associates with HA-tagged Robo (HA-Robo) when coexpressed in HEK 293T cells. Cells expressing HA-Robo alone or in combination with myc-WWGAP were subjected to IP with anti-Robo Ab. To visualize protein levels, aliquots of the same lysates were directly run on a separate SDS/PAGE and probed with anti-HA to visualize Robo or anti-myc for CrGAP. (c) In vivo Co-IP of CrGAP and Robo. Embryos overexpressing CrGAP in the CNS were lysed. Soluble extract (left lane) was assessed by IP with anti-CrGAP Ab. The precipitated CrGAP and Robo were detected by using anti-CrGAP and anti-Robo, respectively (middle lane). Embryonic extract was also assessed by IP with anti-myc or preimmune serum (data not shown) as a negative control (right lane).