Given the observed cholestatic phenotype and early postnatal IHBD

Given the observed cholestatic phenotype and early postnatal IHBD

paucity in DKO mice, we determined the effect of alterations in both HNF-6 and RBP-J on the intact communicating intrahepatic biliary system. To do this, we used an IHBD resin-casting approach with tissue clearance to allow for direct visual analysis of the biliary cast within the left hepatic lobe. Figure 4 represents data obtained for all separate genotypes at age P60. Loss of RBP-J causes LDK378 a decrease in the density of cast branches arising from major intrahepatic bile ducts (Fig. 4C), which correlates to a reduction in the number of bile ducts per portal vein (Fig. 4G) as previously quantified.24 Although loss of HNF-6 alone failed to show an appreciable IHBD defect (Fig. 4B,F), loss of HNF-6 in the setting of RBP-J loss resulted in a further decrease in peripheral IHBD cast

density as compared to RBP-J loss alone, with near complete loss of cast branching off main intrahepatic ducts (Fig. 4D). This phenotype was consistent among DKO mice (n = 8). Analysis of histopathology at this age revealed an unexpected yet consistent ductular reaction in DKO mice (Fig. 4H, selleck chemicals n = 10), defined as disorganized CK19+ BECs surrounding portal veins. These cells were present throughout peripheral periportal regions of the liver parenchyma and were not communicating with the intrahepatic biliary system, based on resin-cast analysis (Fig. 4D). CK-positive BECs in P60 DKO animals had a higher proliferative index compared to BECs in age-matched control Bay 11-7085 mice (Fig. 5A), as determined by proliferation analysis costaining with CK19 and Ki67 (Fig. 5B,C). Importantly, these reactive CK19+ cells did not represent

BECs that escaped Alb-Cre–mediated HNF-6 deletion. HNF-6 protein expression was not visible in reactive peripheral ductular cells on immunostain analysis compared to control (Fig. 5D,E). In DKO mice at P60, only limited hilar BECs remained positive for HNF-6 protein compared to control (Fig. 5F,G), a pattern similar to that seen at earlier time points in HNF-6 KO mice (Fig. 1F,H). Loss of HNF-6 in the setting of RBP-J loss leads to more severe cholestatic liver injury and IHBD abnormalities compared to RBP-J loss alone. These findings indicate a possible genetic interaction between HNF-6 and Notch signaling during IHBD development. To molecularly characterize this interaction, we analyzed the expression of hepatic transcription factors by quantitative real-time RT-PCR analysis of total liver mRNA. Given that both HNF-6 and Notch signaling modulate expression of HNF-1β and Sox912, 14-16 and that IHBD defects are observed with conditional loss of these genetic factors,17, 18 we hypothesized that either HNF-1β, Sox9, or both may be a common downstream mediator lost in DKO mice. Indeed, HNF-1β and Sox9 mRNA expression was decreased at E16.

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