DEV009563 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. S1P₅ and P-LIM-kinase are colocalized within retinal growth cones. (A-C) Double immunostaining of stage 32 retinal growth cones labeled with S1P₅-IC (A) and P-LIM-kinase (B) antibodies. (C) Resulting overlay. The phosphorylated (active) form of LIM-kinase is significantly colocalized with S1P₅. Quantitative analysis of the colocalization was done using the Intensity Correlation Analysis plugin. Pearson’s correlation coefficient: Rr=0.745±0.02; split Mander’s colocalization coefficients: M1=0.92±0.012 and M2=0.96±0.007; n=35 growth cones analysed. Scale bar: 10 µm.

• Supplemental Figure S2 -

  Fig. S2. S1P can rescue the effect of DMS treatment. (A,B) Bright field (A) and DiI-filled optic projection of RGC axons (B) in a lateral view of a wholemount brain. Brains were exposed to S1P (1µM) + DMS (12µM) at stage 33/34 and analyzed at stage 40. Most of the axons reached their final target without making axon pathfinding errors. (C) Quantitative analysis of the retinal axon pathfinding phenotypes. Percent of total embryos showing either a normal trajectory, or target recognition errors. Number in parenthesis indicates total number of embryos analyzed.

• Supplemental Figure S3 -

  Fig. S3. Gross organization of the neuroepithelium is unchanged by S1P treatment. (A-D) Embryos were exposed to control or S1P (5µM) solutions at stage 33/34 and analyzed at stage 40. (A,B) Anti-acetylated tubulin staining of control (A) or S1P-treated (B) wholemount brains. Major axonal tracts are not affected by S1P treatment. (C,D) Images of control (C) or S1P-treated (D) brain sections (12 µm) stained with anti-NCAM. NCAM is abundant in the neuropil (Np) and in the neuroepithelium. NCAM staining is not affected by S1P treatment. Exposed side is to the left. tAC, tract of the anterior commissure; tPC, tract of the posterior commissure; tPOC, tract of the post-optic commissure. Scale bar: 100 µm.