Dataset Information

Sox9^EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

ABSTRACT:

Background & aims

Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9^EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types.

Methods

Sox9^EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9^EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence.

Results

BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9^EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP^high BECs. Single Sox9^EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9^EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation.

Conclusions

Our data demonstrate that Sox9^EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

SUBMITTER: Tulasi DY

PROVIDER: S-EPMC8024983 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Sox9<sup>EGFP</sup> Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Tulasi Deepthi Y DY Castaneda Diego Martinez DM Wager Kortney K Hogan Connor B CB Alcedo Karel P KP Raab Jesse R JR Gracz Adam D AD

Cellular and molecular gastroenterology and hepatology 20210123 5

<h4>Background & aims</h4>Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9<sup>EGFP</sup> transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types.<h4>Methods</h4>Sox9<sup>EGFP</sup> BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expr ...[more]

PMID: 33497866

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Project description:Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic. We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium ± Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this.BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy.

Project description:Background & aimsPrimary liver tumors comprise distinct subtypes. A subset of intrahepatic cholangiocarcinoma (iCCA) can arise from cell fate reprogramming of mature hepatocytes in mouse models. However, the underpinning of cell fate plasticity during hepatocarcinogenesis is still poorly understood, hampering therapeutic development for primary liver cancer. As YAP activation induces liver tumor formation and cell fate plasticity, we investigated the role of Sox9, a transcription factor downstream of Yap activation that is expressed in biliary epithelial cells (BECs), in Yap-induced cell fate plasticity during hepatocarcinogenesis.MethodsTo evaluate the function of Sox9 in YAP-induced hepatocarcinogenesis in vivo, we used several genetic mouse models of inducible hepatocyte-specific YAP activation with simultaneous Sox9 removal. Cell fate reprogramming was determined by lineage tracing and immunohistochemistry. The molecular mechanism underlying Yap and Sox9 function in hepatocyte plasticity was investigated by transcription and transcriptomic analyses of mouse and human liver tumors.ResultsSox9, a marker of liver progenitor cells (LPCs) and BECs, is differentially required in YAP-induced stepwise hepatocyte programming. While Sox9 has a limited role in hepatocyte dedifferentiation to LPCs, it is required for BEC differentiation from LPCs. YAP activation in Sox9-deficient hepatocytes resulted in more aggressive HCC with enhanced Yap activity at the expense of iCCA-like tumors. Furthermore, we showed that 20% of primary human liver tumors were associated with a YAP activation signature, and tumor plasticity is highly correlated with YAP activation and SOX9 expression.ConclusionOur data demonstrated that Yap-Sox9 signaling determines hepatocyte plasticity and tumor heterogeneity in hepatocarcinogenesis in both mouse and human liver tumors. We identified Sox9 as a critical transcription factor required for Yap-induced hepatocyte cell fate reprogramming during hepatocarcinogenesis.Lay summarySox9, a marker of liver progenitor cells and bile duct lining cells, is a downstream target of YAP protein activation. Herein, we found that YAP activation in hepatocytes leads to a transition from mature hepatocytes to liver progenitor cells and then to bile duct lining cells. Sox9 is required in the second step during mouse hepatocarcinogenesis. We also found that human YAP and SOX9 may play similar roles in liver cancers.

Dataset Information

Sox9^EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Background & aims

Methods

Results

Conclusions

Publications

Sox9<sup>EGFP</sup> Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

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Dataset Information

Sox9EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Background & aims

Methods

Results

Conclusions

Publications

Sox9<sup>EGFP</sup> Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Sox9^EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.