Fig. 6. Gain and loss of S1P signalling cause axon pathfinding errors in vivo. (A-H) HRP-filled optic projection of RGC axons in a lateral view of wholemount brain. Brains were exposed to S1P, DMS or FTY720 for 16 hours from stage 35/36 to 40/41. E-H are higher magnification views of A-D. (A,E) Control projection formed a defined optic tract in the diencephalon and crossed into the tectum. (B,F) An example of retinal axons exposed to S1P (5 µM) that failed to reach the tectum causing a bypass phenotype. (C,D,G,H) Projections exposed to DMS (12 µM; C,G) or to FTY720 (5 µM; D,H) failed to enter the tectum and axons bifurcated dorsally around the target area (black arrow in C and G). (I-M) Transverse sections of brains treated with FTY720 (J,L,M) showing the trajectory of HRP-labelled axons (brown) through the diencephalon/tectum compared with a control brain (SphK1 ISH; I,K). (M) Higher magnification view of L (black dashed box in L). Axons exposed to FTY720 penetrated deeper into the neuroepithelium (white dashed lines and arrows). (N) Quantitative analysis of the axon projection width (superficial-deep dimension). Three measurements were made: at the optic chiasm (OC), ventral diencephalon (Vd) and dorsal diencephalon/tectum (Dd/t) (see white arrows in I-L). FTY720 treatment increased the spread of axons by about twofold in the Vd and Dd/t. ^**P<0.01, ^***P<0.005; Mann-Whitney U test. n=5 brains analyzed. Tel, Telencephalon; Di, Diencephalon; Tec, Tectum. Scale bars: in A, 100 µm for A-D,I-L; in E, 50 µm for E-H,M.