Expression of the SDF-1 protein in the meninges. (a) Anti-SDF-1 staining of the cerebellum at embryonic day 15 (E15) shown in red and nuclear staining with Hoechst 33258 dye shown in blue. (b) Anti-SDF-1 and nuclear staining of the cerebellum at birth (P0). Scale bar, 100 μm. M, meninges. EGL, external germinal layer. Protocols for animal experiments were approved by the institutional animal studies reviewing board of Washington University Medical School.

Responses of EGL cells to SDF-1. (a) Co-culture of an E17 EGL explant with control HEK cells. Scale bar, 100 μm. (b) Co-culture of an E17 EGL explant with HEK cells expressing SDF-1. (c) A higher-magnification view of the distal side of the explant shown in (b). (d) A higher-magnification view of the proximal side of the explant shown in (b). (e) Nuclear staining of the explants shown in (a). Green, staining with Hoechst 33258 dye. (f) Nuclear staining of the explants shown in (b). (g) Statistical analysis of EGL responses. The P/D ratio for control HEK cells differed significantly from that for SDF-1 cells (P < 0.0001, Mann-Whitney U-test), but not from that for RANTES cells (P > 0.5). (h–j) Co-culture of embryonic EGL explants with a control block (h), a collagen block containing recombinant SDF-1 (i) or a collagen block containing BDNF (j). SDF-1 (0.04 μM) was sufficient to attract embryonic EGL cells. BDNF did not attract EGL cells even at a concentration of 1.5 μM. (k–m) Co-culture of postnatal explants with a control block (k), a collagen block containing recombinant SDF-1 (l) or a collagen block containing BDNF (m). Postnatal EGL cells were not attracted or repelled by SDF-1 at concentrations as high as 1.0 μM. (n) Statistical analysis of EGL responses toward protein-containing collagen blocks. The P/D ratio for eEGL + SDF-1 differed significantly from that for eEGL + control (P < 0.01, Mann-Whitney U-test), whereas other comparisons did not show a significant difference from the controls (P = 1.000 for eEGL + BDNF, P = 0.107 for pEGL + SDF-1, P = 0.572 for pEGL + BDNF). The latter two were compared to pEGL + control. See Supplementary Methods online for details of these experiments.

Analysis of explants from mice lacking the SDF-1 gene. (a) Nuclear staining of a co-culture of an embryonic EGL explant with the meninges from a wild-type mouse (SDF ^+/+) at embryonic day (E) 14.5. Scale bar, 100 μm. (b) Nuclear staining of a co-culture of an embryonic EGL explant with the meninges from an SDF null mouse (SDF^−/−) at E14.5. Genotyping of SDF-1 knockout mice was done as previously described. (c) Statistical analysis (34 explants tested for SDF^+/+ and 41 for SDF^−/−). The difference between SDF^+/+ and SDF ^−/− was statistically significant (P < 0.0001, Mann-Whitney U-test).