To investigate which cells activate Notch signaling in ciBECs, we examined the expression levels of Notch receptors and ligands in ECs, pericytes, astrocytes, and neurons. We first purified CD31+/PDGFRβ− ECs and PDGFRβ+/CD31− pericytes at 12 days after differentiation and collected CD44+/PSA−NCAM− astrocytes and PSA−NCAM+/CD44−neurons from 90 to 120 days after differentiation by FACS (Figures 1B and S1C). We checked lineage-specific gene expressions by qPCR. CD31, SM22α, MAP2, and GFAP were highly expressed in ECs, pericytes, neurons, and astrocytes, respectively (Figure 4A). Next, we investigated mRNA expressions of Notch receptors and ligands in the four lineages. Notch1 and 4 were highly expressed in ECs and Notch2 and 3 were enriched in astrocytes. Notch ligands, Dll4 and Jagged2, were highly expressed in ECs and Jagged1 was enriched in pericytes. We focused on Dll1, which was highly expressed in neurons, to analyze the generation of ciBECs (Figure 4B).
