Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells

Abstract

BACKGROUND& AIMS: Embryonic biliary precursor cells form a periportal sheet called the ductal plate, which is progressively remodeled to generate intrahepatic bile ducts. A limited number of ductal plate cells participate in duct formation; those not involved in duct development are believed to involute by apoptosis. Moreover, cells that express the SRY-related HMG box transcription factor 9 (SOX9), which include the embryonic ductal plate cells, were proposed to continuously supply the liver with hepatic cells. We investigated the role of the ductal plate in hepatic morphogenesis.

Methods: Apoptosis and proliferation were investigated by immunostaining of mouse and human fetal liver tissue. The postnatal progeny of SOX9-expressing ductal plate cells was analyzed after genetic labeling, at the ductal plate stage, by Cre-mediated recombination of a ROSA26RYFP reporter allele. Inducible Cre expression was induced by SOX9 regulatory regions, inserted in a bacterial artificial chromosome. Livers were studied from mice under normal conditions and during diet-induced regeneration.

Results: Ductal plate cells did not undergo apoptosis and showed limited proliferation. They generated cholangiocytes lining interlobular bile ducts, bile ductules, and canals of Hering, as well as periportal hepatocytes. Oval cells that appeared during regeneration also derived from the ductal plate. We did not find that liver homeostasis required a continuous supply of cells from SOX9-expressing progenitors.

Conclusions: The ductal plate gives rise to cholangiocytes lining the intrahepatic bile ducts, including its most proximal segments. It also generates periportal hepatocytes and adult hepatic progenitor cells.

Figure 1

Lack of apoptosis and very low proliferation in ductal plate. (A-B) Mouse and human ductal plates were analyzed at the stages indicated. No evidence for apoptosis (activated caspase 3 and TUNEL) was found. (C) Only a few SOX9⁺ ductal plate cells were proliferating (Ki67; arrowhead). dp, ductal plate; pv, portal vein; *, lumen of developing duct; size bar, 20 μm.

Figure 2

Expression of CreER^T2 in developing liver is restricted to SOX9-expressing ductal plate cells. Pregnant females were injected with tamoxifen 18h prior to collection of the liver at the stages indicated. The livers were stained to detect CreER^T2 and biliary (SOX9) or hepatoblast (HNF4) markers. dp, ductal plate; pv, portal vein; size bar, 20 μm.

Figure 3

The ductal plate gives rise to cholangiocytes lining interlobular bile ducts and periportal hepatocytes. Pregnant females were injected with tamoxifen at E15.5, and the SOX9-CreER^T2;ROSA26R^YFP offspring were analyzed at the time points indicated. (A) The YFP⁺ progeny of the ductal plate consisted of interlobular ducts and of hepatocytes which persisted at least until 7 months of age. The biliary markers (SOX9, OPN, CK19) were expressed in the YFP⁺ ducts but not in YFP⁺ hepatocytes; the latter expressed the hepatocyte marker HNF4. (B) The YFP⁺ hepatocytes were restricted to the periportal zone and expressed periportal (CPS1) but not perivenous (GS) markers. cv, central vein; hc, hepatocyte; id, interlobular bile duct; pv, portal vein; size bar, 20 μm.

Figure 4

The YFP⁺ progeny of the ductal plate includes cholangiocytes lining the ductules and the canals of Hering. (A) Laminin (lam) fully surrounds ductules (yellow dotted line) and interlobular ducts (white dotted line). (B) Upper panels: Canals of Hering (yellow dotted line) were delineated by hepatocytes with a large nucleus and cholangiocytes expressing cytokeratin (pan-cytokeratin antibody); the white dotted line separates the cholangiocyte from the hepatocyte. Laminin is only found along the basal pole of the cholangiocytes. Middle and lower panels: canals of Hering are also identified by CEACAM staining which marks the bile canaliculi at the apical pole of hepatocytes. The middle pictures show a canal of Hering lined by a YFP⁺ hepatocyte and a YFP⁺ cholangiocyte. In A-B, the right three pictures correspond to magnified areas delineated in the left picture by a white box. CoH, canal of Hering; d, ductule; id, interlobular bile duct; pv, portal vein; size bar, 10 μm.

Figure 5

CDE diet-induced oval cells derive from the ductal plate. Pregnant females were injected with tamoxifen at E15.5, and 4 week-old SOX9-CreER^T2;ROSA26R^YFP offspring were fed a CDE diet. YFP⁺ oval cells express typical markers (CK19, SOX9, OPN, TROP2). The percentage of YFP⁺ oval cells [(YFP⁺SOX9⁺/SOX9⁺) × 100] per periportal area can reach close to 100%. 13,419 SOX9⁺ oval cells were counted over 146 periportal areas. OVc, oval cell; pv, portal vein; w, week after birth; size bar, 10 μm.

Figure 6

DDC diet-induced oval cells derive from the ductal plate. Pregnant females were injected with tamoxifen at E15.5, and 4 week-old SOX9-CreER^T2;ROSA26R^YFP offspring were fed a DDC diet for two weeks. Atypical ductular structures were induced. They co-express YFP and biliary/oval cells markers SOX9, OPN and CK (pan-cytokeratin antibody). The right pictures correspond to magnified areas delineated by a white box in the left panels. ads, atypical ductular struture; pv, portal vein; w, week after birth; size bar, 10 μm.