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Analyzing the effect of Lef1 deletion in mouse intestinal adenoma cells with single-cell RNA sequencing

ABSTRACT: Somatic mutations in APC or CTNNB1 genes lead to aberrant Wnt signaling and colorectal cancer (CRC) initiation and progression. Activation of Wnt pathway leads to the formation of beta-catenin-T-cell factor/Lymphoid enhancer binding factor 1 (Tcf/Lef1) complexes that activate transcription of oncogenic target genes. Lef1 is the only member of the Tcf gene family that is not expressed in the normal intestine, but is induced during intestinal tumorigenesis. Thus, we wanted to assess the role of Lef1 using genetic mouse models of intestinal adenomas and scRNA-seq technology. Tumorigenesis was initiated by inducing Apc mutation in Lgr5+ stem cells. Intestinal EpCAM+ epithelial cells of Lgr5-CreERT;Apc fl/fl (LApc) mouse and Lgr5-CreERT;Apc fl/fl; Lef1 fl/fl (LApcL) mouse were used to analyze the effects of Lef1 deletion in intestinal adenoma cells. We used WT mice as a control to distinguish adenoma cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE179483 | GEO | 2021/12/10

REPOSITORIES: GEO

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Project description:The adult stem cell marker Lgr5 and its close relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. Conditional deletion of the two genes in the gut impairs expression of Wnt target genes and results in rapid demise of intestinal crypts, thus phenocopying the effects of Wnt pathway inhibition. By mass-spectrometry, we find that Lgr4 and Lgr5 associate with the endogenous Wnt co-receptors Frizzled-5/7 and Lrp5/6 in HEK293 cells and in colorectal cancer cells. We also find that soluble Rspondin1, a Wnt pathway agonist, specifically binds to endogenous Lgr4 on HEK293 cells. In these cells, soluble Rspondin1 potently enhances canonical Wnt signals initiated by Wnt3A. Removal of Lgr4 does not affect Wnt3A-induced signals, but abrogates the Rspondin1-mediated enhancement of these signals, a phenomenon rescued by re-expression of Lgr4, -5 or -6. Rspondin1 and can be rescued by Wnt pathway activation. Lgr4/5/6 are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble Rspondin proteins. We used two different experimental setups to show that concomitant deletion of Lgr4 and Lgr5 affects the expression of Wnt pathway target genes. First, we determined the downregulated genes after Lgr4/5 deletion in AhCre_Lgr4fl/fl_Lgr5 fl/fl mice on day 1 post-deletion, before any histological changes are apparent in these mice (Lgr4/5 gene signature). Next, we acutely withheld the Wnt agonist Rspondin1 from in vitro crypt organoid cultures and performed differential gene expression before, and 1 day after withdrawal (Rspondin1 withdrawal signature). For better comparison we also deleted the Lgr4 and Lgr5 in vitro in organoids isolated from VillinCre_Lgr4fl/fl_Lgr5 fl/fl mice, and performed microarrays 1 day after deletion in vitro. Furthermore, we used published expression data from Apcfl/fl mice (Apc deletion signature). Deletion of Apc results in the immediate upregulation of Wnt pathway target genes. Comparison of the Lgr4/5 gene signature to both the Rspondin1 withdrawal signature and the Apc deletion signatures using Gene Set Enrichment Analysis (GSEA) showed that the genes deregulated after simultaneous deletion of Lgr4 and -5 are in the majority under the control of the Wnt pathway.

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Analyzing the effect of Lef1 deletion in mouse intestinal adenoma cells with single-cell RNA sequencing

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