Bile juice is produced by hepatocytes and enters the biliary tree, which is covered with its own epithelial cells named cholangiocytes. Biliary formation in the liver is important, as demonstrated by the end-stage liver disease in a human illness affecting this process (Alagille syndrome) (1). The components of Notch signaling (JAG1 and NOTCH2) are responsible for this syndrome (2) (3) (4). Indeed, loss-of function mutations or gain-of-function mutations of these components in mice results in decreased or increased biliary formation in the liver (5).
Notch signaling is a ligand‒receptor signaling pathway that is evolutionally conserved (6) and, in mammals, is composed of five DSL ligands (JAG1, JAG2, DLL1, DLL3 and DLL4) and four Notch receptors (NOTCH 1, 2, 3, 4) (7). For example, this signaling is essential for Drosophila neurogenesis, which shows a salt-and-pepper (fine-grained) pattern (Fig. 1a) (8). From this pattern, lateral inhibition with feedback mechanism was proposed, where the production rates of DSL ligands were reduced in Notch signaling-receiving cells. After iterations of cell‒cell communication via the ligands and receptors, the initial fluctuation of the amounts of ligands and receptors among the undifferentiated cells was augmented to show a salt-and-pepper (fine-grained) pattern (9). A computer simulation study suggested that lateral inhibition with feedback mechanism generates the salt-and-pepper pattern when the production rates of the ligands and receptors are high (10). The same study also suggested that spatially confined patterning via the Notch signaling pathway during biliary formation occurred when either the production rates of the ligands or receptors were low. In this case, the portal veins in the liver act as DSL ligand sources, while Notch signaling is prevented from spreading owing to the low production rates of the ligands or receptors among the undifferentiated cells, resembling the induction phenomenon (Fig. 1b). Indeed, an in vivo examination of DSL ligands and Notch receptors showed vasculature-confined expression of Jag1, supporting this mechanism (11). In addition, the upstream regulator (Slug) of this vasculature-confined expression of Jag1 was bioinformatically predicted (12). The biochemical foundation for spatially confined Notch signaling is, however, still unclear. Therefore, in this study, we aimed to examine the cellular capability to express Notch signaling-associated molecules by analyzing a publicly available single-cell ATAC-sequencing (scATACseq) dataset from human fetal liver (13).