14 However, most Hes1 null animals die by E18.5 from severe neural
tube defects and have gallbladder agenesis and hypoplasia of extrahepatic bile ducts never connecting with the IHBD system, possibly interfering BKM120 with proper ductal plate remodeling.27, 28 Moreover, those Hes1 null animals reaching birth all die within the first 24 hours and therefore are of limited informative value to study the impact of Hes1 on IHBD tubulogenesis as this process extends several days beyond birth. In addition to our observations in RbpjF/FAlbCre and Hes1F/FAlbCre animals we found that N2IC-induced morphogenetic effects in R26N2ICAlbCre animals could be reverted by the additional genetic deletion of Rbpj, but not Hes1. Although Hes1 has been clearly demonstrated to be expressed in developing bile ducts,6, 14 it is increasingly accepted that Hes1 may not be a perfect readout for Notch activity because Hes1 expression is also regulated independently of Notch.29 In support of this evidence, embryonic deletion of
Jagged1 in the portal mesenchyme resulted in severe IHBD morphogenesis defects without altered expression of Hes1.13 Furthermore, Hes1 may even function as a Notch suppressor30; in this context, we observed enhanced expression of Hey1 and Hes5 after genetic deletion of Hes1 in N2IC-expressing livers of R26N2ICHes1F/FAlbCre animals (Supporting Fig. 8), which might also argue for redundancy of these Notch targets as proposed in brain development.31 Roxadustat in vitro Inactivation of the Notch target gene Sox9 results in IHBD maturation defects32 and, therefore, Sox9 is a likely candidate to contribute to N2IC-expressing tubulogenesis in our model. However, because IHBD malformations are much more pronounced Fludarabine after genetic deletion of Jagged1, RBP-Jκ or Notch2 than after deletion of Sox9,6, 7, 10, 13, 32 additional Notch targets yet to be identified are likely involved to drive Notch-induced biliary tubulogenesis.
Taken together, our results underline the vital role of canonical Notch signaling but clearly argue against a pivotal role of Hes1 as the key Notch target in IHBD formation. It should be also kept in mind that besides Notch other signaling pathways such as the TGFβ or Wnt/β-Catenin pathway act in concert to induce biliary lineage defining proteins such as Sox9, HNF1β, CK19, or osteopontin.12 The observation that Sox9 expression is induced in periportal and interlobular hepatocytes of P10 RbpjF/FAlbCre livers that later acquire an intermediate phenotype (Fig. 4) underscores that induction of biliary proteins can take place in the absence of canonical Notch signaling. Remarkably, adult hepatocytes fully retained their susceptibility to N2IC-induced biliary reprogramming.