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 |

REPOSITORIES: biostudies-literature

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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:Purpose: SOX9Y440X is the most commonly reported SOX9 mutation identified in human patients with Campomelic dysplasia, a dominant semi-lethal skeletal disease characterized by congenital shortening and angulation of long bones (campomelia). To understand how the SOX9Y440X mutant cause the abnormal limb development and compare with previously heterozygous Sox9 null mutant, a mouse model with Sox9Y440X mutation has been generated. The goal of this study is to carry out transcriptome analyses (RNA-seq) of E13.5 limbs collected from Sox9+/IRES-EGFP and Sox9+/Y440X-IRES-EGFP mice. GFP+ and GFP- cells were collected from each genotype by FACS. Comparing transcriptome profiles of cells from these two genotypes aims to reveal how the SOX9Y440X mutant causes abnormal limb development. Method: Forelimbs (FL) and hindlimbs (HL) mRNA profiles of E13.5 Sox9+/IRES-EGFP (WT) and Sox9+/Y440X-IRES-EGFP (CD) mice were generated by sequencing. 4-6 pairs of FL and HL buds were collected separately for each genotype. Limb tissues were then digested into single cells, then EGFP positive and negative cells were collected by FACS for each sample. Total RNA from each sample was prepared using Ambion RNA isolation kit (Cat. AM1580). 100ng of total RNA was used from each sample for RNA-sequencing. cDNA libraries were prepared by TruSeq Stranded mRNA Sample Prep Kit (Illumina). Pair-End sequencing of 101bp was done using HiSeq 1500 Sequencer Model. Data processing: Reads was pre-processed and aligned to the mouse GRCm38 (mm10) reference genome and expression levels were quantified using the nfcore/rnaseq workflow. The count matrix from featureCounts was used in the downstream analyses. The Nextflow pipeline for downstream analysis is available at https://github.com/fuxialexander/sox9. With R library edgeR 3.36.0, read counts were normalized using the ‘TMM’ method. We identified differentially expressed genes between the WT and CD groups in GFP+ or GFP- samples with the ‘glmQLFTest’ function and a Benjamini & Hochberg adjusted P-value cutoff of 0.05. Conclusions: Our study represents the first detailed analysis of mouse foetal limbs transcriptomes between Sox9Y440X mutant and wild type. Our work contributes to gain insight into the molecular signalling pathways affected by SOX9Y440X in vivo.

Dataset Information

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

Background & aims

Methods

Results

Conclusions

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Sox9EGFP Defines Biliary Epithelial Heterogeneity Downstream of Yap Activity.

Background & aims

Methods

Results

Conclusions

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

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