Fig 2: ANGPTL2 enhances CXCR4 and ETS1 expression in MDA-MB231 cells.(A) Representative image showing immunoblot of ETS1 and HSC70 in MB231/miANGPTL2 and MB231/miLacZ cells. Full-length blots are presented in Supplementary Fig. S7A. (B) Quantitative ETS1 protein levels relative to HSC70. Data from MB231/miLacZ was set at 1. Data are means ± SEM from three experiments; ?P < 0.05 (unpaired two-tailed Student's t-test). (C) Representative immunoblot of ETS1 and HSC70 in MB231/Control and MB231/ANGPTL2 cells. Full-length blots are shown in Supplementary Fig. S7D. (D) Quantitative ETS1 protein levels relative to HSC70. Data from MB231/Control was set at 1. Data are means ± SEM from three experiments; ?P < 0.05 (unpaired two-tailed Student's t-test). (E) Relative ANGPTL2 and CXCR4 expression in MB231/ANGPTL2 cells. Data are means ± SEM from three experiments; ??P < 0.01(unpaired two-tailed Student's t-test). (F) CXCR4 cell surface expression in MB231/ANGPTL2 and MB231/Control cells based on flow cytometry. Gray shaded area represents isotype control antibody group. MB231/ANGPTL2 cells, dotted-line; MB231/Control cells, solid line. (G) Representative image and analysis of immunoblotting for ETS1 relative to HSC70 in MB231/ANGPTL2 cells transduced with ETS1 siRNA. Full-length blots are presented in Supplementary Fig. S8. (H) Quantitative ETS1 protein levels relative to HSC70. Control siRNA cells, black bar; ETS1 siRNA-1 cells, blue; and ETS1 siRNA-2 cells, red. Data from Control was set at 1. Data are means ± SEM from three experiments; ?P < 0.05, ??P < 0.01 (unpaired two-tailed Student's t-test) (I) CXCR4 cell surface expression in Control siRNA, ETS1 siRNA-1, and ETS1 siRNA-2 cells based on flow cytometry. Gray shaded area represents isotype control antibody group. Control siRNA cells, black line; ETS1 siRNA-1, blue; and ETS1 siRNA-2, red.

Fig 3: Identification of Cxcr4 and Cxcr7 (Ackr3) and Integrin ß1 as candidate molecules that regulate the blood vessel-guided migration.a UMAP (uniform manifold approximation and projection) visualization of approximately 50,000 single cell RNA-seq data randomly sampled from the full dataset (1.3 million cells) of E18 mouse whole brains. 15 clusters were identified. b Annotation of cell types for the 15 clusters based on gene markers as indicated. Clusters 1, 6 and 11 represent migrating cortical neurons, glial progenitors (astrocyte progenitors and oligodendrocyte progenitors), and endothelial cells, respectively. c Identification of astrocyte-specific chemoattractant receptors (c1) and their candidate ligands secreted from endothelial cells (right). We identified Cxcr4 and Cxcr7 (Ackr3), which are receptors for Cxcl12 secreted from endothelial cells, as candidate molecules that regulate the blood vessel-guided migration. We also identified integrin ß family expressed in astrocyte progenitors (c2). logFC, log fold-change of the average expression of astrocyte progenitors versus Cluster 1 (c1, c2), or that of endothelial cells versus all cells (right). P, adjusted p-value. We applied two-sided Wilcoxon Rank Sum test with adjustments for multiple comparisons based on bonferroni correction. (d, e) Expression of Cxcr4 and Cxcr7 in cortical VZ-derived Olig2-positive cells. E15 mouse embryos were electroporated with PB-CAG-EGFP and fixed 3 days later (d, e, left panel). Some Olig2 (detected by immunohistochemistry (d) or HCR (e)) and GFP double-positive cells indicated by arrows were positive for Cxcr4 (HCR, 28.29 ± 3.36%, 131 cells/4 brains, mean ± SEM) and Cxcr7 (HCR, 35.28 ± 4.79%, 141 cells/4 brains, mean ± SEM). Scale bars, 10 µm (d), 20 µm (e).

Fig 4: The final distribution of astrocytes is affected by functional blocking of the Cxcr4/7-Integrin ß1 signaling axis.a–c CRISPR vectors for Cxcr4 and 7 disrupted the positioning of astrocytes in the CP at P8. a E15 brains were electroporated with the indicated combinations of CRISPR vectors for Cxcr4 and 7, and were fixed at P8. b Resulting brains. Astrocytes and neurons were labeled with GFP and RFP, respectively. c The astrocytes electroporated with CRISPR-vectors for Cxcr4 and Cxcr7 were reduced in bin 1 (horizontal bars represent mean ± SD, two-sided Dunnett’s multiple comparison test, control (11 brains) vs. Cxcr4 CR#2 + Cxcr7 CR#1 (9 brains), P = 0.0108; control vs. Cxcr4 CR#1 + Cxcr7 CR#2 (8 brains), P = 0.0298 in bin 1). d–f Knockdown of Itgb1 also disrupted the distribution of astrocytes within the CP. d E15 embryos were electroporated with indicated plasmids, and fixed at P8. In the rescue experiment, KD#1 resistant Itgb1 expressing vector (pPB-CAG-Itgb1-R) was added in the above plasmid mixture. e RFP labels a subset of transfected astrocytes. f The radial distributions of RFP+/Aldh1l1+ cells are shown in violin plots. Statistical analysis revealed significant shift of Itgb1 KD cells toward deep CP, which could be rescued by pPB-CAG-Itgb1-R (two-sided Dunnett’s multiple comparison test, 891 cells/8 brains control, 764 cells/11 brains Itgb1 KD#1, 497 cells/8 brains Itgb1KD#2, 521 cells/7 brains Itgb1 KD#1 + Itgb1-R, control vs. Itgb1 KD#1, P = 0.000244, control vs. Itgb1 KD#2, P = 0.002399, control vs. Itgb1 KD#1 + Itgb1-R, P = 0.999476). For detailed information of box plots, see “Statistical analysis” section in Methods. *p < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 µm. Source data are provided as a Source Data file.

Fig 5: CXCL12 mediates ATF4 upregulation via CXCR4.a Cultured DRG neurons were treated with CXCL12 (1 µg/mL) for 120, 240 or 360 min, and then ATF4 expression was measured. n = 6. F(3,20) = 4.642, P = 0.0462 in 120 min, P = 0.0154 in 240 min, P = 0.0128 in 360 min. *P < 0.05 versus 0 min. b CXCL12 (1 µg in 10 µL PBS + 0.5% BSA) was intrathecally injected three times every 3 h, and ATF4 expression was measured in the DRG 2 h after the last injection. n = 6 mice per group. t10 = 5.151, P = 0.0004. **P < 0.01. c ATF4 siRNA significantly relieved CXCL12-induced thermal hyperalgesia. n = 12 mice per group. F(2,33) = 15.68, P < 0.0001 in vehicle vs. CXCL12, P = 0.0101 in CXCL12 vs. CXCL12 + ATF4-siRNA. *P < 0.05, **P < 0.01. d CXCR4 siRNA but not CXCR7 siRNA abolished the increase in ATF4 expression in the DRG induced by CXCL12 in vivo. n = 6 mice per group. F(3,20) = 54.47, P < 0.0001 in CXCL12, CXCL12 + CXCR4-siRNA and CXCL12 + CXCR7-siRNA. **P < 0.01 versus the vehicle group, ##P < 0.01 versus the CXCL12 group. e Hypothetical model illustrating that ATF4 interacts with TRPM3 and KIF17 to form a complex to regulate the membrane trafficking of TRPM3 in sensory neurons and thus contributes to thermal sensitivity. a, c, d One-way ANOVA followed by Tukey’s multiple comparisons test. b Two-tailed Independent Student’s t test. The error bars indicate the SEMs.