Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8

Abstract

Background & aims: Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms.

Methods: We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of β-catenin (Ctnnb1^flox/flox mice injected with Adeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n = 15).

Results: Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of β-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis.

Conclusions: Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Keywords: CDE Diet; Development; Hepatic; Signal Transduction.

Figure 1.. Identification of compounds that block LPC-to-hepatocyte differentiation during regeneration.

(A) Experimental scheme illustrating the stages of Mtz and testing compound treatment and analysis (arrow). (B) Single-optical section images showing the expression of Bhmt, fabp10a:rasGFP, and Tp1:H2B-mCherry in regenerating livers at R24h. Numbers indicate the proportion of larvae exhibiting the representative expression shown. (C) Single-optical section images showing the expression of Hdac1, Tp1:H2B-mCherry, and fabp10a:CFP-NTR in the normal liver at 5 dpf and the regenerating liver at R6h. Arrows point to BEC-derived cells expressing Hdac1. (D) Whole-mount in situ hybridization (WISH) images showing hdac1 and kdm1a expression in normal livers at 5 dpf and regenerating livers at R6h. Dashed lines outline control livers; arrows point to regenerating livers. (E) qPCR data showing the relative expression levels of hdac1 and kdm1a between 5-dpf control livers and R6h-regenerating livers. Scale bars: 100 µm; error bars: ±SEM.

Figure 2.. MS-275 treatment impairs LPC differentiation into either hepatocytes or BECs during regeneration.

(A) Experimental scheme illustrating the stages of Mtz and MS-275 treatment and analysis (arrow). (B) Single-optical section images showing the expression of Hnf4a, Tp1:VenusPEST, Tp1:H2B-mCherry, and fabp10a:CFP-NTR in regenerating livers at R24h. Among BEC-derived, H2B-mCherry⁺ cells, Hnf4a⁻/VenusPEST^strong cells are BECs (arrows) and Hnf4a⁺/VenusPEST⁻ cells are hepatocytes (open arrows). A third cell type, Hnf4a⁺/VenusPEST^weak cells, is present (arrowheads) in MS-275-treated, but not control, regenerating livers. Quantification of the percentage of hepatocytes, BECs, and undifferentiated or less differentiated cells among H2B-mCherry⁺ cells is shown. n indicates the number of larvae examined. (C) Single-optical section images showing the expression of Alcam, Tp1:VenusPEST, Tp1:H2B-mCherry, and fabp10a:CFP-NTR in regenerating livers at R24h. Arrows point to BECs; arrowheads point to Alcam⁺/VenusPEST⁻ cells derived from BECs. (D, E) Quantification of the percentage of BECs (D) or Alcam⁺/VenusPEST^{weak or −} cells (E) among H2B-mCherry⁺ (BEC-derived) cells, as shown in C. (F) WISH images showing cp and gc expression in regenerating livers at R24h. Arrows point to regenerating livers. Numbers indicate the proportion of larvae exhibiting the representative expression shown. Scale bars: 100 µm; error bars: ±SEM.

Figure 3.. Hdac1 is required for LPC differentiation during regeneration.

(A) Experimental scheme illustrating the stages of Mtz and MS-275 treatment and analysis (arrow). (B) Single-optical section images showing Bhmt and Tp1:H2B-mCherry expression in regenerating livers at R24h. (C) Quantification of the percentage of Bhmt⁺ hepatocytes among BEC-derived cells, as shown in B. (D) WISH images showing cp and gc expression in regenerating livers at R24h. Arrows point to regenerating livers. (E) Confocal projection images showing the expression of Alcam, Tp1:VenusPEST, and Tp1:H2B-mCherry in regenerating livers at R24h. Arrows point to VenusPEST⁺/Alcam⁺ cells (i.e., BECs); arrowheads point to VenusPEST⁻/Alcam⁺ cells. (F) Quantification of BEC number per area, as shown in E. Scale bars: 100 µm; error bars: ±SEM.

Figure 4.. Hdac1 regulates LPC-to-hepatocyte differentiation by repressing sox9b expression.

(A) qPCR data showing the relative expression levels of fabp10a, hnf4a, sox9b foxa1/2/3, epcam, and prox1a between DMSO- and MS-275-treated regenerating livers at R6h. (B) Single-optical section images showing Bhmt and Tp1:H2B-mCherry expression in regenerating livers at R24h. Quantification of the percentage of Bhmt⁺ hepatocytes among BEC-derived cells is shown. (C) Single-optical section images showing Bhmt and Tp1:H2B-mCherry expression in regenerating livers of adult zebrafish at R4d. (D) qPCR data showing the relative expression levels of bhmt and sox9b among uninjured control livers, DMSO- and MS-275-treated regenerating livers at R4d. (E) ChIP-qPCR data showing the relative enrichment of the selected sox9b promoter regions among uninjured control livers, DMSO- and MS-275-treated regenerating livers at R4d, following immunoprecipitation with H3K9ac antibody. Scheme illustrates the sox9b genomic locus. Arrows point to the regions amplified by qPCR. Green and yellow boxes denote untranslated and coding regions, respectively. Scale bars: 100 µm; error bars: ±SEM.

Figure 5.. Hdac1 regulates LPC-to-BEC differentiation via Cdk8, Fbxw7 and Notch3.

(A) Confocal projection images showing the expression of Tp1:H2B-mCherry, Tp1:VenusPEST, and Alcam in regenerating livers (dashed lines) at R24h. Quantification of VenusPEST⁺/Alcam⁺ cell (i.e., BEC) number is shown. (B) Single-optical section images showing Tp1:VenusPEST and Tp1:H2B-mCherry expression in regenerating livers (dashed lines) at R30h. hs:N3ICD expression was induced by multiple heat-shocks at A24h, R3h, and R24h. (C) WISH images showing fbxw7, cdk8, and skp1 expression in normal livers (dashed lines) at 5 dpf and regenerating livers (arrows) at R6h. (D) qPCR data showing the relative expression levels of fbxw7, skp1, cdk8, her9, her2, and her15.1 between DMSO- and MS-275-treated regenerating livers at R6h. (E-G) Quantification of BEC number per area, as shown in Fig. S5A, S5B and S6A, respectively. Scale bars: 100 µm; error bars: ±SEM.

Figure 6.. Kdm1a regulates LPC differentiation during regeneration.

(A, C) Confocal projection images showing the expression of Alcam, Tp1:VenusPEST, and Tp1:H2B-mCherry in regenerating livers (dashed lines) at R24h. Quantification of BEC number per area is shown. (B) Single-optical section images showing Bhmt and Tp1:H2B-mCherry expression in regenerating livers at R24h. Quantification of the percentage of Bhmt⁺ hepatocytes among BEC-derived cells is shown; both strong and weak Bhmt expression were considered Bhmt⁺. (D) Cartoon illustrating the process of BEC-driven liver regeneration upon massive hepatocyte ablation in zebrafish larvae, focusing on the role of Hdac1 in LPC differentiation into hepatocytes and BECs. Scale bars: 100 µm; error bars: ±SEM.

Figure 7.. Evidence in mammals that supports the role of Hdac1 in LPC-to-hepatocyte differentiation.

(A) Experimental scheme illustrating the period of a CDE diet, AAV8 and MS-275 injection stages, and analysis stage. (B) Section confocal images showing CK19, HNF4A and β-catenin expression in regenerating mouse livers at R7d. Arrows point to BEC-derived hepatocytes (CK19⁻/HNF4A⁺/β-catenin⁺); arrowheads to BECs (CK19⁺/HNF4A⁻/β-catenin⁺). (C) Section confocal images showing PCNA, HNF4A and β-catenin expression with DAPI staining in regenerating livers at R7d. Arrows point to PCNA⁺/HNF4A⁺/β-catenin⁺ hepatocytes. Quantification of the percentage of PCNA⁺ among BEC-derived hepatocytes is shown. (D) Section images showing anti-pan-cytokeratin (panCK) immunostaining. Quantification of panCK⁺ area is shown. (E) Western blot images of whole liver lysate from DMSO- or MS-275-injected regenerating livers at R7d. (F) Human liver section images showing SOX9 and HDAC1 expression. Arrows point to SOX9⁺ hepatocytes; arrowheads to HDAC1⁺ reactive ducts; white arrows to HDAC1⁺ hepatocytes. Scale bars: 100 µm (B–D), 50 µm (F); error bars: ±SEM.