This study describes the modulation of postnatal IHBD development and cholestatic liver disease phenotype by HNF-6 and Notch signaling. RNA expression analysis of liver transcription factors presented in this study suggests that a direct in vivo genetic interaction between HNF-6 and Notch signaling exists. To date, no in vitro or in vivo studies have described the genetic interaction of these two factors in combination.
Independent genetic loss of HNF-1β or Sox9 leads to abnormalities IHBD development (17
). With loss of both HNF-6 and RBP-J, the expression of both HNF-1β and Sox9 was down regulated at E16.5 () and at P3 (). Alb-Cre
mediated recombination of the RBP-J locus begins at E14.5 (24
). HNF-6 mRNA expression was decreased in HNF-6 KO mice and reached significance in DKO animals at E16.5 () with a visible decrease in HNF-6 protein expression by E18.5 in HNF-6 KO mice (). During early postnatal time periods, DKO mice also demonstrated significant BEC paucity worse than that seen with RBP-J loss alone. This was not associated with changes in BEC apoptosis or proliferation (Supplemental Fig. 3
, data not shown). Thus, in the setting of diminished HNF-6 and Notch signaling, there is a decreased expression of both Sox9 and HNF-1β during continued hepatoblast specification and IHBD morphogenesis. The observed decrease in BECs in DKO mice may be secondary to these changes in genetic factors essential for normal IHBD development, leading to a phenotype of severe IHBD paucity and cholestatic liver disease.
The control of HNF-1β and Sox9 expression by HNF-6 and Notch signaling may occur either in a parallel or an epistatic relationship, however we propose that this interaction occurs largely along parallel pathways. HNF-1β expression was increased in both single KO models at E16.5 (Fig. ,), suggesting possible compensation from the alternate parallel arm of either HNF-6 or Notch signaling. Within our model, hepatoblast-specific deletion of RBP-J alone results in an increase in Sox9 expression at E16.5 (). This may be related to an observed 6-7 fold increase in HNF-6 mRNA and protein expression at E16.5 in RBP KO embryos (, Supplemental Fig. 4B
). With Notch signaling loss, this increase in HNF-6 is likely compensatory and may contribute to the observed increase in Sox9 expression. An alternate possibility would be an epistatic model in which Notch signaling occurs upstream of HNF-6, acting as an attenuator of HNF-6. However, previous experimental models have shown that constitutive Notch activation does not down-regulate expression of HNF-6 (12
). The possibility of HNF-6 occurring upstream of Notch signaling is also unlikely, given that Sox9 is a Notch target (12
) and isolated hepatoblast-specific loss of HNF-6 did not result in any changes in Sox9 at ages E16.5 and P3 (). The etiology of the decrease in Sox9 expression and increase in HNF-1β expression in HNF-6 KO mice compared to control at age P60 () is unknown. However, taken together these data suggest that control of factors essential for early IHBD development occurs along parallel mechanisms through HNF-6 and Notch signaling.
The pattern of HNF-1β and Sox9 expression in our model of conditional BHPC-specific loss of HNF-6 does not necessarily contradict previously published data describing a decrease in both Sox9 and HNF-1β expression with global HNF-6 loss. Initial regulation of both HNF-1β and Sox9 by HNF-6 appears to occur during early embryonic time points, with expression of both factors approaching or equaling control mice by E17.5 in a HNF-6 global loss model (14
). Given that HNF-6 protein expression is decreased compared to control by E18.5 (), conditional deletion of HNF-6 by Alb-Cre
may not occur early enough to affect the initial control of HNF-1β and Sox9 expression. However, our results do indicate a role for HNF-6, uncovered by the loss Notch signaling, in the continued control of downstream factor expression. We hypothesize that this role occurs in parallel with Notch signaling.
Interestingly, while Sox9 expression remains decreased in DKO animals at P60, the expression of HNF-1β is not decreased significantly compared to control mice at P60 (, ). A ductular proliferative response is seen as well at this age, with multiple disorganized CK19+ BECs seen throughout the peripheral periportal regions of DKO livers (, ). The etiology of this ductular response, as well as the restoration of HNF-1β during this adult time period, is unknown. Down-regulation of HNF-6 expression has been described to be an important factor in the ductular proliferative response seen in cholestatic liver injury resulting from bile duct ligation (25
), and HNF-6 protein expression continues to remain decreased in the DKO ductular response (). HNF-6 loss may thus contribute to the ductular response in the setting of cholestatic liver injury seen in DKO mice. However, this explanation by itself would not account for the restoration of HNF-1β expression. An alternate pathway or effector may thus drive the ductular proliferative response, and may or may not do so through improved expression of HNF-1β. Candidates include separate pathways such as Wnt or Hedgehog signaling (26
). Still, the observed abnormalities in both IHBD cast structure and cytokeratin-positive BECs indicate a severe defect in the formation of the communicating intrahepatic biliary system with loss of both HNF-6 and Notch signaling.
Associated with severe abnormalities in IHBD development in DKO mice, there was a worsening of cholestatic liver disease. This was evidenced by an increase in total bilirubin and alkaline phosphatase compared to RBP-J loss alone (), as well as extensive hepatic necrosis and bridging fibrosis (). To date, HNF-6 polymorphisms have not been described in AGS patients. However, given that loss of HNF-6 in the setting of Notch signaling loss causes a clear phenotypic worsening of cholestatic liver disease, HNF-6 or downstream effectors may contribute to the clinical variability in disease phenotype that can be seen in AGS patients (6
In summary, our data indicate that HNF-6 and Notch signaling interact to affect expression of similar downstream mediators that are important in normal intrahepatic biliary development. We suggest that this interaction occurs along a parallel course, and loss of both HNF-6 and Notch signaling leads to subsequent loss of HNF-1β and Sox9 expression. This continued requirement for HNF-6 in the postnatal expression of genetic effectors essential to IHBD development has not been described previously. The complex regulatory profile of HNF-1β and Sox9 presented in this study does not prove that alterations in expression of these molecules are responsible for the abnormalities in IHBD development in DKO mice. However, alterations in their expression during periods of continued BEC specification and morphogenesis suggest that the observed worsened BEC paucity in DKO mice compared to RBP-J loss alone may be related to genetic loss of these important factors. The cholestatic livery injury observed in the adult mouse occurs with an associated ductular response. We hypothesize this may represent the involvement of a yet undefined alternate signaling mechanism that leads to the appearance of a proliferative cytokeratin-positive cell population and improvement in HNF-1β expression. This may serve as a model to identify alternate modulators of IHBD development and to study factors contributing to the cholestatic injury response, as well as further our understanding of clinical variability in patients with chronic cholestatic liver disease such as AGS.