Liver Progenitor Cells Fold Up a Cell Monolayer into a Double-layered Structure during Tubular Morphogenesis

Abstract

Bile ducts are hepatic tubular structures that are lined by cholangiocytes, a type of liver epithelial cell. Cholangiocytes first form a single layer of cells, termed the ductal plate, surrounding the portal vein, which eventually remodels into the branching tubular network of bile ducts. The process of bile duct morphogenesis is not yet clear: a conventional model where cholangiocytes proliferate to duplicate a single layer of the ductal plate before lumen formation seems inconsistent with the observation that proliferation is dramatically reduced when hepatoblasts, liver progenitor cells, differentiate into cholangiocytes. Here, we developed a new culture system in which a liver progenitor cell line, HPPL, reorganizes from a monolayer to tubular structures in response to being overlaid with a gel containing type I collagen and Matrigel. We found that some of the HPPL in the monolayer depolarized and migrated to fold up the monolayer into a double-cell layer. These morphogenetic processes occurred without cell proliferation and required phosphatidylinositol 3-kinase and Akt activity. Later in morphogenesis, luminal space was generated between the two cell layers. This process, in particular enlargement of the apical lumen, involved transcriptional activity of HNF1␤. Thus, using this sandwich culture system, we could segregate tubulogenesis of bile ducts into distinct steps and found that the PI3K/Akt pathway and HNF1␤ regulated different steps of the morphogenesis. Although the process of tubulogenesis in culture specifically resembled early bile duct formation, involvement of these two key players suggests that the sandwich culture might help us to find common principles of tubulogenesis in general. INTRODUCTIONTubules are an essential structural unit for numerous epithelial organs, such as lung, liver, and kidney. Epithelial tubes in different organs form by a wide variety of mechanisms (Hogan and Kolodziej, 2002;Lubarsky and Krasnow, 2003;Bryant and Mostov, 2008). Understanding the cellular and molecular basis of these mechanisms and discovering common principles that may underlie these diverse mechanisms is an important goal.In the liver, bile ducts are tubular structures that consist of cholangiocytes, a type of liver epithelial cell. Bile ducts provide the excretory route for bile, which is synthesized by hepatocytes, another type of liver epithelial cell. Because bile is cytotoxic, abnormal development of bile ducts, which causes the accumulation of bile inside the liver, induces hepatic injury and ultimately results in severe liver fibrosis and cirrhosis (Schmucker et al., 1990;Yoon and Gores, 2002;Paumgartner, 2006).Cholangiocytes, the epithelial component of bile ducts, differentiate from liver progenitor cells called hepatoblasts around embryonic day 15 (E15) in mice (Lemaigre, 2003). Recent reports have identified the Notch and TGF␤ signaling pathways and a transcription factor Hex, which regulate differentiation of cholangiocytes from hepatoblasts (McCright et al., 2002;Kodama et al., 2004;Clotman ...

Wnt/β-Catenin Signaling in Liver Development, Homeostasis, and Pathobiology

“…The developing biliary cells form the ductal plate, or a cell monolayer encircling the periportal mesenchyme (129). During ductal morphogenesis, the ductal plate becomes bilayered to form the primitive ductal structure (129, 130). The cell layer adjacent to the portal vein expresses mature BEC markers, whereas the cells of the layer adjacent to the parenchyma resemble hepatoblasts because they express HNF4α (129).…”

Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome

“…The design of this 3D co-culture system allowed for the direct use of the Jag1-depleted PVM from J1 SMKO mice at specific stages of duct development. Other ductal culture systems require the use of Jag1-positive hepatic progenitor lines to differentiate into the distinct hepatic lineages for ductal development (Tanimizu et al, 2009).…”

Molecular mechanisms of bile duct development

“…Notably, an alternative model of biliary tubulogenesis was proposed in which some cholangiocytes in the first layer of the ductal plate fold up to form a double layer by cell migration (Tanimizu et al, 2009). This model was based on the performance of hepatoblasts in a 3-D cell culture system (Tanimizu et al, 2007; Tanimizu et al, 2009).…”

“…This model was based on the performance of hepatoblasts in a 3-D cell culture system (Tanimizu et al, 2007; Tanimizu et al, 2009). Time course movie of this culture system showed that hepatoblasts first formed a monolayer; later some cells moved vertically to form the second layer.…”