A new set of classifications for ductal plate malformations
Authors: Stacey S. Huppert
Journal: Hepatology (2011)

Jørgensen first coined the term ductal plate malformation (DPM) in 1977,1 referring to a common pathology observed in many human congenital liver diseases involving the intrahepatic bile duct (IHBD) system. The congenital diseases associated with DPM include, but are not limited to, congenital hepatic fibrosis, autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), Caroli's disease, Caroli's syndrome, and von Meyenburg complexes.2 It is proposed that the condition of DPM arises from a persistence or lack of remodeling of the embryonic ductal plate normally observed during IHBD formation. ARPKD, autosomal recessive polycystic kidney disease; DPM, ductal plate malformation; HNF, hepatocyte nuclear factor; IHBD, intrahepatic bile duct; PDS, primitive ductal structure; SOX9, SRY-related HMG box transcription factor 9; TβRII, transforming growth factor receptor type II; ZO-1, zonula occludens-1. From a developmental point of view, the cells that contribute to the IHBD system are a subpopulation of hepatoblast bipotential progenitors located near portal veins. This subpopulation of hepatoblasts forms a band of potential cholangiocytes, termed a ductal plate, encircling the portal veins. Remodeling of ductal plates into IHBDs start at the oldest ductal plates surrounding the larger portal veins near the hilum and is thought to move toward the periphery of liver following the portal vein system. The ductal plate cells that remain unincorporated into an IHBD then involute. If the unincorporated ductal plate cells do not receive or are deaf to the proper signals, they may contribute to DPM. Thus, there is a level of coordination that must regulate sequential tubulogenesis and regression of the ductal plates along portal veins within the three-dimensional space of the liver to connect the entire IHBD system to the extrahepatic ductal system. This indicates that careful orchestration of signals between epithelial and mesenchymal cells is required to guide IHBD formation.3 In this issue of HEPATOLOGY, the report by Raynaud et al.4 gives the general term DPM a new set of classifications according to distinct defects in biliary tubulogenesis. This article reassesses how DPM observed in human congenital liver disease might result from various tubulogenesis defects in light of a defined transient asymmetry step identified during the process of mouse IHBD maturation.5 This step delineates the structure surrounding a forming lumen as either a primitive ductal structure (PDS) or a mature duct. PDS is composed of two cell types as distinguished by the presence or absence of marker expression (SRY-related HMG box transcription factor 9 [SOX9], hepatocyte nuclear factor 4 [HNF4], and transforming growth factor receptor type II [TβRII]) compared to a mature duct. The PDS is asymmetrical; cells on the portal vein side of the lumen express the marker SOX9, compared to cells on the parenchymal side that express HNF4 and TβRII. A mature duct is symmetrical, composed of cells expressing SOX9. To evaluate DPM, the authors focused their investigation on differentiation, polarity, and ciliogenesis in mouse models and human cases of DPM. Raynaud et al. employed three mouse models of DPM in this report, Hnf6−/−,6 Hnf1bloxP/loxP; Alfp-Cre,7 and congenital polycystic kidney−/− (cpk−/−),8 focusing on embryonic day 17.5 and postnatal day 7. These time points allowed them to examine hilar IHBD formation of the ductal plate, PDS, and mature duct. All three of the mouse models in this study highlight defects at different steps of bile duct tubulogenesis. The absence of HNF6 caused a somewhat resolvable early defect in biliary cell differentiation, whereas liver-specific loss of HNF1β produced a defect in PDS maturation. A third type of defect is observed in the absence of cystin-1, where an abnormal expansion of ducts was observed even though the biliary cells differentiate normally. All three specific biliary tubulogenesis defects result in an endpoint of DPM. Specifically, examination of differentiation in Hnf6−/− mice revealed that cells lining lumens did not express the cholangiocyte marker SOX9, but did express the hepatocyte marker HNF4 at embryonic day 17.5. These data are in agreement with earlier studies that established deficient HNF6 generates hybrid hepatobiliary cells and indicates that HNF6 is required for differentiation of biliary cells at embryonic stages.9 Postnatally, the biliary differentiation was resolved, supported by the presence of SOX9+/HNF4− cholangiocytes. However, normal tubulogenesis did not proceed and resulted in DPM observed in hilar IHBDs at postnatal day 7. A different result was observed for Hnf1bloxP/loxP Alfp-Cre mice. Absence of HNF1β resulted in normal embryonic SOX9+/HNF4− expression around the forming bile duct lumen on the portal side, but the parenchymal side of the bile duct lumen remained SOX9−/HNF4+ throughout the liver. This is inconsistent with the progressive nature of bile duct tubulogenesis, asserting that the bile duct structures at the hilar region should be more mature than the ductal structures at the peripheral regions. The subsequent postnatal phenotype displayed differentiation of SOX9+/HNF4− cholangiocytes, but with observed DPM and dysplastic ducts. This phenotype was consistent with the patient samples analyzed that carried HNF1B (TCF2; Mendelian Inheritance in Man #137920) mutations. These results support an early differentiation role of HNF6 and a PDS maturation role for HNF1β, providing two different defects in tubulogenesis prior to the endpoint of DPM. Deficient cystin-1 (cpk−/−) does not lead to defects in differentiation at any stage of biliary tubulogenesis, but does cause DPM as also observed in human patients with ARPKD. These data demonstrate that deficient maturation during tubulogenesis is a cause of DPM. Cholangiocyte polarity was also disrupted at varying degrees of severity in each of the three mouse models. The apical-basal polarity in cases of deficient HNF6 and HNF1β was strongly affected. Apical expression of osteopontin was absent, in addition to abnormal localization of centrioles and Golgi apparatus. However, a deficiency in HNF6 and HNF1β differ in the localization of the tight junction marker zonula occludens-1 (ZO-1) and therefore the establishment of the boundary separating the apical pole from the basolateral pole of cholangiocytes. Loss of HNF6 results in normal apical-lateral localization of ZO-1, whereas loss of HNF1β results in very low levels of ZO-1 on the parenchymal side of forming bile duct lumen and improper apical localization of ZO-1 on the portal side. The postnatal consequences of these embryonic phenotypes that suggest loss of a cholangiocyte apical pole also differ between these two mouse models. Partial restoration of apical-basal polarity is observed when HNF6 is absent, but is not the case with HNF1β deficiency. Indeed, in patients with HNF1β mutations, ZO-1 was irregularly expressed in dysplastic ducts, and the observed DPM did not express ZO-1. Absence of cystin-1 did not influence the apical marker osteopontin, but ZO-1 expanded to the apical surface of cholangiocytes, indicating that the basal and lateral poles were not established correctly. These phenotypes correspond to liver samples examined from ARPKD fetuses. Because of the polarity defects observed during biliary tubulogenesis, the authors investigated whether cholangiocyte ciliogenesis was disrupted in these mouse models. Previously, HNF6 and HNF1β were implicated in either control of cilia formation or regulation of genes involved in cilia function in the pancreas and kidney, respectively.10, 11 Because of the random distribution of centrioles observed in the absence of HNF6 or HNF1β, it is not surprising that embryonic cilia formation on cholangiocytes was significantly disrupted. The postnatal partial restoration of the apical-basal polarity in deficient HNF6 mice correlates with a few cilia present on cholangiocytes. However, the lack of cilia present on cholangiocytes remains as a postnatal defect in liver deficient for HNF1β. To determine if either HNF6 or HNF1β are involved in regulating the formation or function of cholangiocyte cilia, expression levels of candidate genes were examined in these two mouse models. Cystin-1 was the only gene with reduced expression in both mouse models. Interestingly, in the cystin-1–deficient (cpk−/−) mouse model, the presence of a cilium is observed on some cholangiocytes. Therefore, reduced expression of cystin-1 in liver deficient in HNF6 and HNF1β is not the explanation for the reduced or absence of cilia in these two mouse models. Notably, and an avenue for further research, is the observation that the HNF1β targets in the kidney (Pkhd1, polycystic kidney and hepatic disease 1; Pkd2, polycystic kidney disease 2; Nphp1, nephronophthisis 1; IFT88, intraflagellar transport 88 homolog; and Kif12, kinesin family member 12) were not changed in HNF1β deficient liver, which indicates that HNF1β regulates a divergent transcriptional landscape for cilia in cholangiocytes versus kidney. Importantly, these three mouse models of DPM demonstrate that DPM can be classified into three distinct defects: (1) differentiation of biliary precursor cells, (2) maturation of PDS, or (3) duct expansion during development. In addition, these models illustrate that the processes of cholangiocyte differentiation and polarity are separable. Furthermore, these results reveal that epistatic relationships between HNF6, HNF1β, and cystin-1 are even more complicated at the transcriptional and translational levels than previously reported. Published work indicates that Hnf6 and Hnf1b are involved in the same transcriptional program.6, 7 However, in their report, Raynaud et al. use defined steps in biliary tubulogenesis to expose that these genes are not exclusively regulated by each other, based on the observed phenotypic differences. Astonishingly, bile duct lumina still form in all cases. These data indicate that bile duct lumen formation is a very early event in tubulogenesis and occurs independent of cholangiocyte polarity. At present, the proteins and their localization required to drive lumen formation remain unknown. This initial study will allow investigators to more explicitly define the requirement of additional genes and provide a more in-depth understanding of the mechanism behind biliary tubulogenesis. The therapeutic implication of delineating DPM classifications in clinical medicine, based on initial biopsies, will hopefully begin to improve diagnostic and prognostic stratification of patients with DPM in the diverse group of congenital and acquired cholangiopathies. In the future, this may allow for more individualized monitoring and intervention strategies.