The Secreted Glycoprotein Reelin Suppresses the Proliferation and Regulates the Distribution of Oligodendrocyte Progenitor Cells in the Embryonic Neocortex

Reelin affects the proliferation of OPCs in a Dab1-dependent manner. A, Rat OPCs were incubated with vehicle only (control, left), WT-Reelin (middle), or KA₂-Reelin (right) for 19 h and a further 5 h in the presence of BrdU. OPCs were immunostained for BrdU (green) and Olig2 (red). Nuclei were stained using Hoechst 33342 (blue). Scale bar: 300 µm. B, Quantification of the percentage of BrdU⁺Olig2⁺ cells in rat Olig2⁺ OPCs. The data were analyzed using one-way ANOVA (F_(2,15) = 19.68, p < 0.0001) followed by Tukey–Kramer post hoc test; ***p < 0.001 and ****p < 0.0001. Control, N = 7; WT-Reelin, N = 6; KA₂-Reelin, N = 4. C, OPCs from Dab1^+/+ (upper panels) or Dab1^yot/yot (lower panels) mice were incubated with vehicle only (control, left) or WT-Reelin (middle) for 19 h and a further 5 h in the presence of BrdU. OPCs were immunostained for BrdU (green) and Olig2 (red). Nuclei were stained using Hoechst 33342 (blue). Scale bar: 300 µm. D, Quantification of the percentage of BrdU⁺Olig2⁺ cells in Dab1^+/+ Olig2⁺ OPCs. The data were analyzed using a two-tailed Student's t test. N = 4. E, Quantification of the percentage of BrdU⁺Olig2⁺ cells in Dab1^yot/yot Olig2⁺ OPCs. The data were analyzed using a two-tailed Student's t test. N = 7.

Reelin affects the migration of OPCs in a Dab1-dependent manner in vitro. A, Schematic diagram of the Boyden chamber assay. OPCs were cultured with vehicle only (Mock) or WT-Reelin (Reelin) in the upper or lower wells for 24 h. B, Rat OPCs were incubated for 24 h with mock or Reelin as described above the images of the Boyden chamber. Cells that migrated into the lower wells through the 8-µm pores of the membrane were immunostained for Olig2 (green) and PDGFRα (magenta). Scale bar: 50 µm. C, The number of PDGFRα⁺Olig2⁺ OPCs that migrated into the lower side of the membrane was counted. The culture media in the inserts and plates are indicated below the graph. The data were analyzed using one-way ANOVA (F_(2,9) = 54.89, p < 0.0001) followed by Tukey–Kramer post hoc test; *p < 0.05 and ***p < 0.001. N = 4. D, WB analysis of the culture medium placed in the inserts (upper wells, U) or the lower wells (L) using anti-Reelin antibody. The culture media in the inserts and plates are indicated below the figure. Positions of molecular mass markers (kDa) are shown on the left of the panel. FL: full-length Reelin. E, OPCs derived from Dab1^+/+ mice (upper panels) or Dab1^yot/yot mice (lower panels) were incubated for 24 h with mock or Reelin as described above the images of the Boyden chamber for 24 h. The cells that migrated into the lower wells through the 8-µm pores of the membrane were immunostained for Olig2 (green) and PDGFRα (magenta). Scale bar: 50 µm. F, The number of PDGFRα⁺Olig2⁺ OPCs derived from Dab1^+/+ mice that migrated into the lower side of the membrane was counted. The culture media in the inserts and plates are indicated below the graph. The data were analyzed using one-way ANOVA (F_(2,6) = 29.86, p = 0.0008) followed by Tukey–Kramer post hoc test; *p < 0.05 and ***p < 0.001. N = 3. G, The number of PDGFRα⁺Olig2⁺ OPCs derived from Dab1^yot/yot mice that migrated into the lower side of the membrane was counted. The culture media in the inserts and plates are indicated below the graph. The data were analyzed using one-way ANOVA (F_(2,9) = 0.1285, p = 0.8810) followed by Tukey–Kramer post hoc test. N.S., not significant. N = 4.

The number and distribution of OPCs are altered in Reelin heterozygote-deficient and Dab1 heterozygote-deficient mutant mice. A, Coronal sections of mouse brain at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. The magnified image of the part of primary somatosensory cortex (yellow box, left) is shown on the right. Primary somatosensory cortex was divided into four bins from the VZ to the MZ. Scale bar: 500 µm. B, Coronal sections of the primary somatosensory cortex of Reln^+/+ and Reln^+/– mice at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. C, D, Quantification of the density of OPCs in the primary somatosensory cortex (C) and in each of four equal-size bins (D). The data were analyzed using a two-tailed Student's t test and FDR-adjusted q values are shown. n = 5 each. E, Coronal sections of the primary somatosensory cortex of Reln^+/+ and Reln^+/– mice at E18.5 were immunostained with antibodies against Ki67 (green) and PDGFRα (red). Nuclei were stained using Hoechst 33342. Arrows indicate Ki67⁺PDGFRα⁺ cells. Scale bar: 100 µm. F, Quantification of the percentage of Ki67⁺PDGFRα⁺ cells of total PDGFRα⁺ cells. The data were analyzed using a two-tailed Student's t test. n = 5 each. G, Coronal sections of the primary somatosensory cortex of Dab1^+/+ and Dab1^+/yot mice at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. H, I, Quantification of the density of OPCs in the primary somatosensory cortex (H) and in each of four equal-size bins (I). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 6 each. J, Coronal sections of the primary somatosensory cortex of Dab1^+/+ and Dab1^+/yot mice at E18.5 were immunostained with antibodies against Ki67 (green) and PDGFRα (red). Nuclei were stained using Hoechst 33342. Arrows indicate Ki67⁺PDGFRα⁺ cells. Scale bar: 100 µm. K, Quantification of the percentage of Ki67⁺PDGFRα⁺ cells of total PDGFRα⁺ cells. The data were analyzed using a two-tailed Student's t test. n = 3 each.

The number and distribution of OPCs are altered in VLDLR-deficient mice. A, Coronal sections of the primary somatosensory cortex of Vldlr^+/+ and Vldlr^–/– mice at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. B, C, Quantification of the density of OPCs in the primary somatosensory cortex (B) and in each of four equal-size bins (C). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 6 each. D, Coronal sections of the primary somatosensory cortex of Vldlr^+/+ and Vldlr^–/– mice at E18.5 were immunostained with antibodies against Ki67 (green) and PDGFRα (red). Nuclei were stained using Hoechst 33342. Arrows indicate Ki67⁺PDGFRα⁺ cells. Scale bar: 100 µm. E, Quantification of the percentage of Ki67⁺PDGFRα⁺ cells of total PDGFRα⁺ cells. The data were analyzed using a two-tailed Student's t test. n = 5 each.

The number and distribution of OPCs are altered in mice with abrogated Reelin cleavage. A, Coronal sections of the primary somatosensory cortex of Adamts3^+/+ and Adamts3^–/– mice at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. B, C, Quantification of the density of OPCs in the primary somatosensory cortex (B) and in each of four equal-size bins (C). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 4 each. D, Coronal sections of the primary somatosensory cortex of Adamts3^+/+ and Adamts3^–/– mice at E18.5 were immunostained with antibodies against Ki67 (green) and PDGFRα (red). Nuclei were stained using Hoechst 33342. Arrows indicate Ki67⁺PDGFRα⁺ cells. Scale bar: 100 µm. E, Quantification of the percentage of Ki67⁺PDGFRα⁺ cells of total PDGFRα⁺ cells. The data were analyzed using a two-tailed Student's t test. n = 4 each. F, Coronal sections of the primary somatosensory cortex of Reln^+/+ and Reln^PA-DV/PA-DV mice at E18.5 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. G, H, Quantification of the density of OPCs in the primary somatosensory cortex (G) and in each of four equal-size bins (H). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 6 each. I, Coronal sections of the primary somatosensory cortex of Reln^+/+ and Reln^PA-DV/PA-DV mice at E18.5 were immunostained with antibodies against Ki67 (green) and PDGFRα (red). Nuclei were stained using Hoechst 33342. Arrows indicate Ki67⁺PDGFRα⁺ cells. Scale bar: 100 µm. J, Quantification of Ki67⁺PDGFRα⁺ cells of total PDGFRα⁺ cells. The data were analyzed using a two-tailed Student's t test. n = 5 each.

The effect of Reelin–Dab1 signaling on the number and distribution of OPCs becomes small in the postnatal brain. A, Coronal sections of the primary somatosensory cortex of Reln^+/+ and Reln^+/– mice at P2 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. B, C, Quantification of the density of OPCs in the primary somatosensory cortex (B) and in each of four equal-size bins (C). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 7 each. D, Coronal sections of the primary somatosensory cortex of Dab1^+/+ and Dab1^+/yot mice at P2 were immunostained with antibodies against PDGFRα (green) and Olig2 (red). Nuclei were stained using Hoechst 33342. Scale bar: 100 µm. E, F, Quantification of the density of OPCs in the primary somatosensory cortex (E) and in each of four equal-size bins (F). The data were analyzed using a two-tailed Student's t test, and FDR-adjusted q values are shown. n = 7 and 8 for Dab1^+/+ and Dab1^+/yot mice, respectively.