Importantly, using simple co-culturing systems with hiPSC-derived ECs, pericytes, neurons, and astrocytes, we revealed a molecular mechanism for ciBEC specification. In particular, this technology allows us to investigate cell-cell interactions, which led to the discovery that Dll1 expression by neurons is crucial for activating the Notch signaling that leads to ciBEC specificity. Notch is a single-pass transmembrane receptor known for its function in controlling cell-fate decisions and creating boundaries through cell-cell communication. Notch signaling in ECs is known to specify arterial ECs (Yamamizu et al., 2010). Dll1, a Notch ligand in neurons, functions in neural differentiation and maintenance (Grandbarbe et al., 2003, Kageyama et al., 2008). Furthermore, it communicates with radial glial cells for the differentiation to astrocytes (Namihira et al., 2009). Our results showed that Dll1 was highly expressed in mature neurons (Figure S6A), and we demonstrated that it interacted with ECs for ciBEC specification (Figure 4C). Some reports have shown that the Wnt canonical pathway is crucial for proper neurovascular development. For example, the simultaneous ablation of Wnt7a/7b is embryonic lethal for neurovascular phenotypes (Daneman et al., 2009,
