Fig. 2. S1P₅ and heparan sulfates mediate S1P-elicited growth cone collapse. (A,B) Western blot from E18 embryonic mice brain extracts, and Xenopus brain, head or eye lysates of stage 32 and 40 embryos, probed with S1P₅ antibodies (S1P₅-EC and S1P₅-IC). One band of 43 kDa corresponding to the molecular weight of S1P₅ was detected. In stage 40 eye lysate, the S1P₅-IC antibody detected an additional band of 30 kDa that may correspond to a degradation product. (C) Western blot from E18 mice brain extracts and Xenopus stage 40 head and eye lysates using the S1P5-IC antibody incubated with the corresponding blocking peptide. No band of 43 kDa was detected. (D) Transverse cryostat sections (12 µm) of a stage 39 eye immunostained with the S1P₅-IC antibody (red) together with DAPI (nuclei dye, blue). S1P₅ expression was detected in all the layers, including the retinal ganglion cell layer, which was intensively labelled. (E) Expression of S1P₅ in growth cone from stage 32 embryos cultured for 24 hours using the S1P₅-IC antibody. Pre-incubation of the antibody with the corresponding peptide blocked the signal expression (white dashed box). (F) The S1P₅-EC antibody inhibited S1P-induced retinal growth cone collapse. S1P₅-EC antibody was bath-applied in culture for 30 minutes before the addition of S1P. (G) S1P₅-EC antibody did not affect LPA-induced growth cone collapse. (H) S1P-induced retinal growth cone collapse is blocked by the addition of heparan sulfate (HS), heparin or heparinase treatment. HS and heparin were added immediately prior to the S1P application in stage 32 retinal explants cultured for 24 hours. Retinal explant cultures were treated with heparinase for 3 hours before S1P application. (I) LPA-elicited growth cone collapse was not affected by HS, heparin or heparinase. LPA concentration: 1 µM. Numbers inside bars indicate growth cones tested. ^*P<0.05, ^**P<0.01, Mann-Whitney U test. Scale bars: D, 20 µm; E, 10 µm. GCL, ganglion cell layer; INL, inner nuclear layer; Ph, photoreceptor layer.