Prox1 ablation in hepatic progenitors causes defective hepatocyte specification and increases biliary cell commitment

Abstract

The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver's architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.

Keywords: Hepatic precursors; Liver; Mouse; Prox1; TGFβ.

Fig. 1.

Prox1 is expressed in epithelial cells of the fetal and adult mouse liver. (A) Prox1 is highly expressed in hepatoblasts (arrows) of E10.0 livers. Prox1 levels are lower in the adjacent gut epithelium (arrowhead). (B) Prox1 is broadly expressed in epithelial cords [Ecadherin⁺ (Ecad), arrows] of E12.5 livers. (C) Prox1 co-expresses with Hnf4α in all hepatocytes (arrows) of E15.5 livers. (D) Prox1 also co-expresses with Sox9 in primitive ductal structures (PDSs; arrows) of E15.5 livers. Yellow arrow points to the nidogen-rich (Nid) basal membrane, and an arrowhead points to a Prox1⁺ hepatocyte. (E) Cells co-expressing Prox1 and Sox9 localize to PDSs (arrow on the left) and developing bile ducts (arrow on the right; yellow arrow points to the Nid⁺ basal membrane) of E18.5 livers (arrowhead points to a Prox1⁺ hepatocyte). (F) Hepatocytes express high Prox1 (arrowheads), and bile ducts express low Prox1 (arrows) in adult livers. Cell nuclei were stained with DAPI (A, blue) or hematoxylin (F). Scale bars: 25 μm (F); 50 μm (A-E).

Fig. 2.

Prox1-depleted livers have severely disrupted architecture. (A,B) E18.5 Prox1^ΔLIV (mut) livers have both significantly reduced weight (A) and smaller size (B) in comparison with E18.5 control (ctrl) livers. (C,D) Hematoxylin and Eosin staining reveals that the tissue architecture of E18.5 Prox1^ΔLIV livers (D) is severely disrupted compared with control livers (C). Asterisks indicate cysts or ducat structures in the mutant liver parenchyma. (E-H) Compared with control livers (E,G), E18.5-P0 Prox1^ΔLIV livers display extensive fibronectin deposition (F, arrows) and abundance of mesenchymal cells (vimentin⁺; H, arrows) in the parenchyma. Arrow in G indicates the periportal mesenchyme (vimentin⁺); arrowhead in E indicates laminin expression in sinusoids. V, portal vein. (E-H) Cell nuclei were stained with DAPI (E,F) or Methyl Green (G,H). P<0.001; n=3-4. Scale bars: 100 μm.

Fig. 3.

Prox1 ablation in hepatic precursors increases biliary gene expression and affects hepatocyte metabolic gene expression. (A) qRT-PCR data show reduced expression of Hnf4a and increased expression of Hnf6, Hnf1b and Nr2f2 in Prox1^ΔLIV livers dissected at E12.5. (B-D) qRT-PCR data also show increased expression of the biliary markers Krt19, Sox9, Lamb1 and Cldn7 (B) and defective expression of various hepatocyte metabolic transcripts (C,D) in Prox1^ΔLIV livers dissected at E12.5-E15.5 compared with control livers (n=3-4 liver specimens; ^*P<0.05, ^**P<0.01, ^***P<0.001).

Fig. 4.

Prox1-depleted livers have defective expression of Sox9 and Hnf4α and abnormal parenchymal epithelial structures. (A,C,E) Cells expressing Hnf4α (arrows) are abundant throughout the parenchyma and are absent in periportal areas where bile ducts should develop (yellow arrows in A and arrowheads in C,E) in E12.5-E18.5 control livers. (B,D,F) The abundance of Hnf4α⁺ cells decline between E12.5 and E18.5 in the nidogen-rich epithelial aggregates (arrowheads) of Prox1^ΔLIV livers (asterisk in F indicates a cluster of Hnf4α⁺ cells lacking a basal membrane). Yellow arrow indicates nidogen expression at the periphery of the lobe. (G,I,K) A small population of cells expressing Sox9 (arrows) and restricted to periportal areas is observed in E12.5-E18.5 control livers [arrowheads indicate nidogen expression in the vein endothelium (G,I) or around the incipient bile ducts (K); yellow arrow in G indicates nidogen expression at the edge of the liver lobes]. (H,J,L) Sox9⁺ cells (arrows) are increasingly abundant and form aggregates surrounded by a nidogen-rich basal membrane (arrowheads) around the portal veins (H,J) and in the parenchyma (H,J,L) in E12.5-E18.5 Prox1^ΔLIV livers (yellow arrows in L indicate epithelial aggregates devoid of Sox9 expression). V, portal vein branches. Cell nuclei were stained with DAPI. Scale bars: 50 μm (E,K); 100 μm (A-D,F-J,L).

Fig. 5.

Intrahepatic bile ducts form prematurely and have defective morphology in the absence of Prox1. (A-I) Cholangiocytes expressing Sox9 (arrows) are not very numerous around the portal vein branches (V) in the hilar region of E12.5 control livers (A; yellow arrow indicates a Sox9⁺/Hnf4α⁺ periportal cell, and yellow arrowhead indicates an Hnf4α⁺/Sox9^- parenchymal cell). In control livers, cholangiocytes form ductal plates (arrows in B,C) or PDS displaying a basal membrane on the periportal side (arrowhead in C) at E15.5 and start forming bile ducts (arrows in G-I at ∼E18.5). In addition to Sox9 (A,G,H), cholangiocyes express Hnf1β (arrow in B), claudin 7 (arrows in C,I), osteopontin (arrowhead in H), but not Hnf4α (C,G,I), in E12.5-E18.5 control livers. (D) Large epithelial aggregates surrounded by a prominent nidogen-rich basal membrane (arrowheads) and containing abundant Sox9⁺ cells (arrows), Hnf4α⁺ cells (yellow arrowhead), and a few Sox9⁺/Hnf4α⁺ cells (yellow arrow) are seen around portal veins in the hilar region of E12.5 Prox1^ΔLIV liver. (E,F) Cholangiocytes form ductal structures (arrows) surrounded by a nidogen-rich basolateral membrane (arrowheads, blue staining), around the portal vein branches in E15.5 Prox1^ΔLIV livers. (J-L) E18.5 Prox1^ΔLIV livers have abnormally large intrahepatic bile ducts (arrows) surrounding the portal vein branches (arrowheads indicate the basolateral membrane). (E) Triple immunofluorescence staining for Hnf4α (red), Hnf1β (green) and nidogen (blue). Scale bars: 50 μm (A-K); 100 μm (L).

Fig. 6.

Ectopic bile ducts form in the liver parenchyma in the absence of Prox1. (A) Numerous hepatocytes (white arrow) expressing Hnf4α and C/EBPα and lacking a basal membrane colonize the E15.5 liver parenchyma [C/EBPα⁺/Hnf4α^- cells (yellow arrow) are not hepatocytes]. (B,C) Fewer Hnf4α⁺/C/EBPα⁺ cells (white arrows) are observed within the parenchymal aggregates of E15.5 (B) and E18.5 (C) Prox1^ΔLIV livers [yellow arrows point to C/EBPα⁺/Hnf4α^- cells (B) or C/EBPα⁺/Hnf4α⁺ cells located outside the epithelial aggregates (C); arrowheads point to the basal membrane]. (D-I) Two classes of parenchymal structures populate the Prox1^ΔLIV liver at E15.5-E18.5: aggregates (D,E) containing a mixture of cholangiocytes (Sox9⁺/Hnf4α^-, yellow arrowheads), hepatocytes (Hnf4α⁺/Sox9^-, white arrows), and hybrid cells (Hnf4α⁺/Sox9⁺, yellow arrows) and ductal structures (F-I) with prominent lumens (asterisks in F) and a basal membrane (arrowheads) expressing mostly cholangiocyte markers [e.g. Sox9 (arrow in F, arrowhead in G)], apical osteopontin (arrows in G-I), claudin7 (yellow arrows in H,I). The yellow arrow in F indicates an Hnf4α⁺/Sox9⁺ cell in a hybrid aggregate. Scale bars: 25 μm (B-F,H); 50 μm (A,G,I).

Fig. 7.

TGFβ signaling is increased in Prox1-depleted livers. (A) E12.5 wild-type liver explants maintained in culture for 24 hours with 200 ng/ml recombinant TGFβ or activin A (ActA) express more Sox9 or Lamb1 transcripts than do vehicle-treated (veh) explants. TGFβ treatment also increases Krt19 expression in fetal liver explants (data represent the mean ± s.e.m. of three to five independent experiments). (B) Western blot results show increased levels of phospho-Smad3 (E13.5) and phospho-Smad2 (E14.5) proteins in extracts of individual mutant livers compared with control livers (notice that Sox9 expression also increases in Prox1-deficient extracts). (C) qRT-PCR results show increased expression of transcripts encoding TGFβ ligands (Tgfb1/2/3) and TGFβ/activin receptors (Tgbr2, Acvr2) in E15.5 Prox1^ΔLIV livers. (D) qRT-PCR results show reduced expression of the TGFβ/activin signaling inhibitors Fst and Ahsg in Prox1^ΔLIV livers compared with control livers at E12.5 and E14.5 (n=3-4 specimens per genotype). Data represent the mean ± s.e.m. of three to five independent experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Fig. 8.

Prox1-nullizygous livers, but not livers with Prox1 ablation in committed hepatic cells, have increased cholangiocyte formation. (A) Prox1 is highly expressed in hepatoblasts (white arrowhead) and poorly expressed (yellow arrowhead) in the gall bladder (gb) epithelium (Sox17⁺) in E11.5 wild-type embryos (arrows indicate the basal membrane surrounding the gall bladder). Yellow arrow indicates Sox17 expression in the gall bladder. (B) Hepatoblasts (GFP⁺, arrowhead) form an epithelium contiguous to the gall bladder in E11.5 Prox1-nullizygous (Prox1^GFP/GFP) embryos. Arrows indicate the basal membrane surrounding the mutant hepatic epithelium. (C,D) The E11.5 Prox1^GFP/GFP hepatic epithelium expresses Hnf4α (arrow in C) but not Sox17 (yellow arrow in D) indicates a single pair of Sox17⁺ cells located close to the gall bladder). (E) The E14.5 Prox1^GFP/GFP hepatic epithelium (GFP⁺) is covered with a nidogen⁺ basal membrane (arrows), displays prominent lumens (asterisks), and is confined to the hilar region (the broken line demarcates the liver periphery). (F) Numerous cells express Sox9 (yellow arrow), and fewer cells express Hnf4α (white arrow) in the E14.5 Prox1^GFP/GFP hepatic epithelium. At this stage, only a few Sox9⁺ cells are seen around the portal vein in E14.5 wild-type livers (inset). (G) Prox1 is expressed in both hepatocytes (arrows, Hnf4α⁺) and cholangiocytes (arrowhead, Hnf4α^-) of E18.5 wild-type livers. (J) Very few hepatocytes express Prox1 in E18.5 Prox1^f/f;AlbCre livers. (H,I,K,L) E18.5 wild-type (H,I) and Prox1^f/f;AlbCre (K,L) livers have similar expression of nidogen (H,K) and Sox9 (I,L) around periportal areas. Arrows indicate expression of nidogen (H,K) or Sox9 (I,L) restricted to periportal areas. V and asterisks indicate portal veins. Cell nuclei were stained with DAPI in B-E and G-L. Scale bars: 25 μm (C), 50 μm (A,B,D,F,G,I,J,L); 100 μm (E,H,K).