Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons
- University of Pennsylvania, United States
- University of Arkansas, United States
Figures
Figure 1
Robo2 commissural guidance defects are rescued by a Robo2 BAC transgene.
(A–E) Stage 17 Drosophila embryos of the indicated genotypes stained with anti-HRP antibodies to label all CNS axons. (F–J) Stage 15–16 embryos of the indicated genotypes carrying eg-GAL4 and UAS-Tau…
Figure 2
Robo2 can promote midline crossing independent of its cytoplasmic domain.
(A–E) Stage 17 embryos carrying elav-GAL4 and the indicated UAS-Robo transgenes, stained with anti-HRP (magenta) and the longitudinal pathway marker anti-FasciclinII (FasII; green). (A) Embryos …
Figure 3
Comparison of Robo1∆C and Robo2∆C gain of function activities.
Since the effects of expressing ∆C transgenes in the 86Fb insertion site are too potent to allow quantitative comparison, we used traditional UAS insertion lines that are expressed at lower and …
Figure 4 with 2 supplements
Slit binding and Robo gain of function.
(A–E) Slit-conditioned media was collected and used to treat cells expressing the indicated HA-tagged receptors. Receptor expression is shown with anti-HA in the top panels (magenta) and anti-Slit …
Figure 4—figure supplement 1
Quantification of relative fluorescence intensity of HA and Slit antibody staining in S2R+ cells transfected with HA-tagged Robo2 proteins, and treated with Slit-conditioned media.
Fluorescence intensities are normalized to the average pixel intensity of untransfected cells from the same slide. The values from three images per Robo2 variant were averaged; error bars represent …
Figure 4—figure supplement 2
Robo2 transgenes are localized to axons and expressed at equivalent levels in vivo and are present at the surface of S2R+ cells in vitro.
(A–E) Embryos carrying elav-GAL4 and the indicated UAS-Robo2 transgenes were stained with anti-HA antibodies and imaged via confocal microscopy. Staining and imaging conditions were identical for …
Figure 5
Robo2's pro-crossing activity depends on its Ig2 domain.
(A–F) Stage 17 embryos carrying elav-GAL4 and the indicated UAS-Robo transgenes, stained with anti-HRP and anti-FasII. (A) Embryos carrying elav-GAL4 alone exhibit a wild-type arrangement of axon …
Figure 6
Robo2 acts cell non-autonomously to promote midline crossing in ipsilateral neurons.
(A) Stage 17 embryos stained with anti-HRP (magenta) and anti-GFP (green) antibodies. Anti-GFP labels the apterous (ap) cell bodies and axons, which normally project ipsilaterally. Mis-expression of …
Figure 7
Robo2 can promote crossing non cell-autonomously.
(A–D) Stage 17 embryos stained with anti-HRP (magenta) and anti-FasII (green). (A and B) Mis-expression of Robo1 (A) in midline cells using slit-GAL4 results in a mild ectopic crossing phenotype. In …
Figure 8 with 1 supplement
robo2 is expressed in midline cells during commissural axon path finding, and over-expressing robo2 with slit-GAL4 restores midline crossing in robo2, fra double mutants.
(A) A robo2-GAL4 enhancer trap that recapitulates robo2's endogenous expression pattern drives UAS-TauMycGFP expression (green) in midline cells at stages 12–13, when many commissural axons cross …
Figure 8—figure supplement 1
robo2 mRNA is transiently expressed in midline cells.
Fluorescent in situ for robo2 mRNA (green). robo2 is transiently expressed in midline glia (magenta) during Stages 12 and 13 but is no longer detected there by Stage 14. Midline glia are labeled …
Figure 9
Robo2 cannot rescue midline crossing cell autonomously.
(A–D) Stage 16 embryos of the indicated genotypes stained with anti-HRP (magenta) and anti-GFP (green) antibodies. Anti-GFP labels the EG and EW cell bodies and axons. EW crossing defects in robo2, f…
Figure 10
Robo2 receptors that promote midline crossing suppress comm mutants.
(A) Schematic diagram of the two chimeric receptors shown in (B and C). Robo1 sequences are depicted in blue and Robo2 sequences are depicted in yellow. (B and C) Stage 16 embryos of the indicated …
Figure 11 with 1 supplement
Robo2 binds to the Robo1 receptor in vitro and in vivo.
(A–C) Protein extracts from embryos expressing Robo1-Myc and various HA-tagged receptors in all neurons were immunoprecipitated with anti-Myc antibodies and analyzed by western blot. …
Figure 11—figure supplement 1
Robo2 binding to Robo1 does not depend on its cytoplasmic domain or on Robo1's Ig1 domain.
(A–C) Protein extracts from embryos expressing Robo1-Myc and various HA-tagged receptors in all neurons were immunoprecipitated with anti-Myc antibodies and analyzed by western blot. …
Figure 12
Robo2's endogenous activity in promoting midline crossing depends on Ig2.
(A–D) Stage 16 embryos stained with anti-HRP (magenta) and anti-GFP (green) antibodies. Anti-GFP labels the EG and EW cell bodies and axons. Arrowheads indicate EW axons that have crossed the …
Figure 13
A model of Robo2's pro-crossing function.
In contralateral neurons, the endosomal sorting receptor Comm is expressed, and it prevents the majority of Robo1 from reaching the growth cone surface. We propose that Robo2 acts non-autonomously …
