Development of the bile ducts: essentials for the clinical hepatologist

Abstract

Several cholangiopathies result from a perturbation of developmental processes. Most of these cholangiopathies are characterised by the persistence of biliary structures with foetal configuration. Developmental processes are also relevant in acquired liver diseases, as liver repair mechanisms exploit a range of autocrine and paracrine signals transiently expressed in embryonic life. We briefly review the ontogenesis of the intra- and extrahepatic biliary tree, highlighting the morphogens, growth factors, and transcription factors that regulate biliary development, and the relationships between developing bile ducts and other branching biliary structures. Then, we discuss the ontogenetic mechanisms involved in liver repair, and how these mechanisms are recapitulated in ductular reaction, a common reparative response to many forms of biliary and hepatocellular damage. Finally, we discuss the pathogenic aspects of the most important primary cholangiopathies related to altered biliary development, i.e. polycystic and fibropolycystic liver diseases, Alagille syndrome.

Figure 1. Embryological stages of intrahepatic bile duct development

The development of intrahepatic bile ducts starts when the periportal hepatoblasts, in close contact with the portal mesenchyme surrounding a portal vein branch, begin to organise into a single layered sheath of small flat epithelial cells, called ductal plate (“ductal plate stage”, A). In the following weeks, discreet portions of the ductal plates are duplicated by a second layer of cells (double layered ductal plate, B), which then dilate to form tubular structures in the process of being incorporated into the mesenchyma of the nascent portal space (incorporating bile duct, “migratory stage”, C). Once incorporated into the portal space, the immature tubules are remodeled into individualised bile ducts (incorporated bile duct, “bile duct stage”, D).

Figure 2. Notch signaling

Jagged binding to a Notch receptor leads to the proteolytic processing and subsequent translocation into the nucleus of the Notch intracellular domain (NICD) of the receptor. Cleavage of NICD is an essential step in this process and is mediated by a γ-Secretase enzyme in the cytoplasm. Once delivered into the nucleus, NICD forms a complex with its DNA-binding partner, the recombination signal binding protein for immunoglobulin kappa J (RBP-Jk). The formation of this complex leads to the up-regulation of cholangiocyte-specific transcription factors, such as HNF1β and the SRY-related HGM box transcription factor 9 (Sox9), and to the down-regulation of hepatocyte-specific transcription factors such as HNF1α and HNF4. Sox9 in particular, is the most specific and earliest marker of biliary cells in developing liver, as it controls the timing and maturation of primitive ductal structures in tubulogenesis.

Figure 3. Wnt signaling (canonical and non-canonical)

Wnt signals in two different ways depending upon the activation (canonical) or the non-activation (non canonical) of β-catenin. In the canonical Wnt pathway (A), the binding of Wnt to Frizzled (Fzd) receptors activates Dishevelled (Dvl), which prevents the phosphorylation and the following ubiquitination of β-catenin. If β-catenin is not phosphorylated, it can thus accumulate in the cytoplasm and then translocate to the nucleus, where it activates Wnt target genes by interacting with the TCF/LEF family of transcription factors. In the non-canonical Wnt pathway (B), the binding of specific Wnt isoforms (Wnt 4, 5a, 11) to Fzd can activate Dvl but the downstream signal pathways involve small GTPases and the C-Jun N-terminal kinase (JNK) instead of β-catenin. Once activated, Dvl leads to increased intracellular Ca²⁺ levels that activate a number of proteins, including PKA, CaMK and NFAT. Acting as transcription factors, these proteins may activate downstream effectors that are crucial regulators of different cellular responses, such as planar cell polarity and cytoskeletal rearrangement.

Figure 4. Sonic Hedgehog (Shh) signaling

In physiological conditions (A), Patched (Ptc) receptor binds to and consequently suppresses the function of its co-receptor Smoothened (Smo). This maintains the bonding of the downstream regulator Glioblastoma-3 (Gli3) to a tetramer complex encompassing its suppressor factors, Suppressor Fused (Su(Fu)), Costal2 (Cos2), and Fused (Fu). In this complex, Gli3 is proteolytically cleaved by Protein Kinase A (PKA), and converted into the repressor form (Gli3R), which exerts an inhibitory effect on nuclear transcription factors regulating Hh-responsive genes (Ptc, Gli1, Gli2). When Shh interacts with Ptc (B), it prevents its inhibitory action on Smo. Following Smo activation, the tetramer complex is disassembled so that Su(Fu) inhibitory effect is restricted to Cos2 and PKA, and thereby preventing the proteolytic cleavage of Gli3. Gli3 can thus enter the nucleus in the activated state (Gli3A) to promote activation of Hh target genes.

Figure 5. Epithelial phenotypes involved in liver repair driven by the activation of hepatic progenitor cells (“Hepatic Reparative Complex”)

In acute and chronic liver diseases, especially in fulminant hepatic failure, liver repair is driven by the activation of hepatic progenitor cells (HPC) to differentiate into hepatocytes and/or cholangiocytes. However, hepatocyte and cholangiocyte proliferation can be directly stimulated without exploiting HPC activation in experimental models, such as partial hepatectomy and acute biliary obstruction, respectively. HPC are small epithelial cells with an oval nucleus and scant cytoplasm, similar to oval cells in rodents treated with carcinogens. HPC originate from a niche located in the smaller branches of the biliary tree and in the canals of Hering. HPC behave as a bipotent, transit amplifying compartment. Differentiation of HPC towards hepatocytes occurs via intermediate hepatobiliary cells (IHBC), whilst differentiation towards the biliary lineage leads to the formation of reactive ductular cells (RDC). HPC, IHBC and RDC constitute the “hepatic reparative complex”, and can be distinguished by morphology and pattern of K7 expression. Whereas Wnt signaling is a key regulator of proliferation of HPC, Notch and Hh signaling are mostly involved in biliary differentiation through RDC generation, along with other cytokines released from the inflammatory microenvironment (TNF-α, TWEAK, TGF-β, HGF, VEGF, IL-6) (see text for details).

Figure 6. Reactive ductular cells acquire the ability to exchange a range of paracrine signals with mesenchymal, vascular and inflammatory cells

Owing to the de novo expression of a variety of cytokines, chemokines, growth factors, angiogenic factors, together with a rich expression of many of the respective cognate receptors, reactive ductular cells can establish an extensive cross-talk with other liver cell types, particularly with stellate cells, endothelial cells and inflammatory cells. In response to biliary damage, these interactions becomes functionally relevant leading to the generation of a fibro-vascular stroma able to sustain and feed the ductular reaction, and to the recruitment of a peribiliary inflammatory infiltrate which further enhances the bile duct damage.

Figure 7. Phenotypic changes of ductular reactive cells shared with ductal plate cells

An important feature of reactive cholangiocytes is the foetal reminiscence of their phenotype. Reactive ductular cells express neuroendocrine features, adhesion molecules, cytokines and chemokines, receptors and other metabolically active molecules, which are transiently expressed by ductal plate cells during embryonic development.

Figure 8. Main differences in liver phenotype between ARPKD and ADPKD

On magnetic resonance imaging, liver cysts appear as focal lesions with regular margins, which are of small size and in continuity with the biliary tree in ARPKD (A), whereas they are large, of different size and scattered throughout the hepatic parenchyma leading to extensive cyst substitution in ADPKD (D). At histological examination, irregularly shaped biliary structures (microhamartomas) surrounded by an extensive deposition of fibrotic tissue, are present in the portal tracts in ARPKD (B, H&E, M: 100x), while in ADPKD, biliary cysts appear as large, circular biliary structures, lined by cuboidal or flattened epithelium, with negligible amount of peribiliary fibrosis (E, H&E, M: 100x). The pathogenetic mechanism underlying cyst formation is characterised by progressive segmental dilation of biliary structures which maintain their connection to the biliary tree in ARPKD (C), whereas biliary cysts detach from the bile duct and then progressively increase in size in ADPKD (F). Alternatively, based on previous histopathological studies^–, liver cysts in ADPKD may also derive from dilatation of components of von Meyenburg complexes.