To define the biological effect of the high Dll1 expression, we performed a loss-of-function study using Dll1 small interfering RNA (siRNA) (siDll1). Approximately 81% inhibition of Dll1 mRNA expression in neurons and astrocytes at 120 days after differentiation was induced with the transfection of siDll1#2 (Figure S4A). We co-cultured the siRNA-treated neurons and astrocytes (siDll1 or control siRNA [siCon]) on ECs and pericytes for 5 days. siCon-treated neurons and astrocytes significantly induced the six BBB-specific transporters compared with ECs. siDll1 treatment tended to inhibit the six BBB-specific transporters and resulted in significant inhibition of ABCA1, MFSD2A, BCRP, and MRP5 compared with siCon treatment (Figure 4C). Taken together, these results indicated that Dll1 in neurons is crucial for the generation of ciBECs by activating Notch signaling.
