DEV023325 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Expression patterns of Slit1, Slit2 and Slit3 in E10.5 mouse brain. Whole-mount in situ hybridization, showing side views of E10.5 mouse embryos. All three Slits are expressed in the hindbrain floor plate, but Slit1 and Slit2 expression is also seen in the ventral midline in the midbrain and extending into the forebrain. Scale bar: 200 µm.

• Supplemental Figure S2 -

  Fig. S2. Increased variance in ILF trajectories in Slit double mutants. The trajectory of DiI-labeled axon segments with respect to the ventral midline was measured using ImageJ and the distribution was plotted. (A,B) DiI labels of Slit1^−/−;Slit2^+/− and Slit1^−/−; Slit2^−/− embryos showing examples of measurements. Anterior is to the left and the midline is marked by the dashed line. Dorsal-ward trajectories are positive angles, while ventral-ward trajectories are negative. (C) Combined distributions of axon trajectories (n=3 embryos/genotype, >100 segments/embryo) plotted as a percentage of total segments measured. V, sample variance.

• Supplemental Figure S3 -

  Fig. S3. Midline DiI sites label few MLF axons in control embryos. In control experiments for Fig 5M-O, DiI label sites were made in the floor plate of control embryos (Slit1^−/−;Slit2^+/−, n=4). These labeled a few MLF longitudinal axons, usually unilaterally but sometimes bilaterally. Thus, this labeling strategy largely avoids labeling axons lateral to the floor plate, i.e. in the normal MLF position. Scale bar: 200 µm.

• Supplemental Figure S4 -

  Fig. S4. MLF errors in Slit1^−/−;Slit2^−/− mutants visualized by Robo1 antibody labeling. Whole-mount labeling with Robo1 antibody, which labels MLF axons, as well as a subset of ventral ILF axons at lower levels. Robo1⁺ axon bundles project from the source of the MLF (dashed ovals, Robo⁻) into the floor plate of the midbrain and the anterior hindbrain. The Robo1⁺ axons then segregate out to either side of the floor plate in the hindbrain. Representative of n=3. Scale bar: 200 µm.

• Supplemental Figure S5 -

  Fig. S5. Increased variance in ILF trajectories in Robo double mutants. The trajectory of DiI labeled axon segments with respect to the ventral midline was measured using ImageJ and the distribution was plotted. (A,B) DiI labels of Robo1^+/−;Robo2^+/− and Robo1^−/−;Robo2^−/− embryos showing example measurements. Anterior is to the left and the midline is marked by the dashed line. Dorsal-ward trajectories are positive angles, while ventral-ward trajectories are negative. (C) Combined distributions of axon trajectories (n=3 embryos/genotype, >100 segments/embryo) plotted as a percentage of total segments measured. V, sample variance

• Supplemental Table S1 - Adobe PDF