Notch signaling regulates formation of the three‐dimensional architecture of intrahepatic bile ducts in mice

Notch signaling is an evolutionarily conserved intercellular signaling pathway required for cell specification, lineage commitment and maintenance of stem/progenitor cells, both during development and in adults (1). Altered Notch signaling may lead to impairment of tissue development and function (2) due to precocious differentiation of progenitor cell populations. In humans, mutations in both JAGGED1 (JAG1) and NOTCH2, a Notch pathway ligand and receptor respectively, cause Alagille syndrome (AGS) (3–5). AGS is an autosomal dominant disorder that is primarily characterized by neonatal jaundice, cholestasis and paucity of IHBDs, although AGS patients also may have characteristic facies along with abnormalities of heart, eye, skeleton, vasculature, kidney and pancreas (6). In mammals, four Notch receptors (Notch1 [N1], N2, N3 and N4) transduce signals from two families of ligands (Jagged1 [Jag1], Jag2, Delta-like-1 [Dll1], Dll3, and Dll4) expressed on the surface of neighboring cells. Previously it has been shown that both N1 and N2 are essential for embryonic development (7–9), since mice that globally lack these receptors die early during embryonic development. In contrast, N3 and N4 are not essential, since mice lacking these receptors are viable (10,11). However, when either N1 or N2 are deleted, N3 and N4 exhibit various degrees of redundant function (12). It has previously been observed that mice that are heterozygous for both a null Jag1dDSL allele and for a hypomorphic N2swl1 mutation partially recapitulate AGS (13). These mice exhibit liver defects (jaundice and bile duct paucity) as well as other abnormalities that characterize AGS in humans. More recent experiments have begun to explore the effects of changing Notch signaling specifically in liver. These studies suggest that mutations in either Jag1 or N2, but not in N1, are the cause of IHBD morphogenetic defects (14–16). Additionally, loss of RBP-JK (DNA-binding partner for all Notch receptors) or activation of either N1 or N2 elicits reduced or excessive ductal plate structures, respectively (17,18). While the specific Notch ligands and receptors that are responsible for defects in AGS patients is becoming clear, our understanding of how reduction of Notch signaling below a critical threshold causes defects in IHBD structure is not understood. For instance, it is not clear whether levels of Notch activity influence IHBD defects due to change in cell fate determination, altered cellular proliferation or maintenance of the bile duct structure. It is also unresolved as to whether different sensitivities to Notch signaling levels exist for specific IHBDs in the three-dimensional IHBD architecture. To systematically analyze contribution of Notch signaling to formation and patterning of mouse biliary system we used a Cre-LoxP strategy to specifically inactivate and activate Notch signaling within BHPCs. The ability to view and assess regional differences within the IHBD system was enhanced by use of a resin casting method. We observed 1) dose-dependent reductions of peripheral IHBD branches in BHPC-specific deletion of N2 alone, both N2 and N1 receptors, and RBP-JK, suggesting redundant roles for N1, N3 and/or N4, 2) increased density of IHBDs when N1 is activated in BHPCs and 3) different underlying two-dimensional cytokeratin phenotypes as a result of varying Notch signaling dosage. Together, these findings indicate that Notch signaling is required at different thresholds for multiple steps of peripheral IHBD formation.