BACKGROUND: The canonical Wnt signaling pathway controls the continuous renewal of the intestinal epithelium and the specification of epithelial cell lineages. Tcf4, a nuclear mediator of Wnt signaling, is essential for the differentiation and maintenance of Paneth cells in the small intestine. Its deficiency is associated with reduced expression of key α-defensins, highlighting its role in host-microbe interactions. However, the exact function of Tcf4 in specifying the secretory lineage and its contribution to antimicrobial peptide production remain incompletely understood. Remarkably, α-defensin expression has also been detected in human colon adenomas, where aberrant Wnt signaling is a hallmark. This raises important questions: What is the role of these Paneth-like cells in tumor biology, and how does Tcf4 influence their identity and function? METHODS: We investigated cell specification in small intestinal crypts and colon tumors using conditional Tcf7l2 deletion, cell type-specific Cre recombinases, and reporter alleles in mice. Transcriptomic (single-cell and bulk RNA sequencing) and histological analyses were performed and complemented by microbiome profiling, antibiotic treatment, and intestinal organoids to functionally validate the main findings. RESULTS: The inactivation of Tcf4 depletes Paneth cells and antimicrobial peptides, disrupting the gut microbiota balance. In secretory progenitors, loss of Tcf4 shifts differentiation toward goblet cells. In the small intestine, alternative secretory progenitors produce Wnt ligands to support stem cells and epithelial renewal in the absence of Paneth cells. In colon tumors, Paneth-like cells form a tumor cell population, express Wnt ligands, and require Tcf4 for their identity. Loss of Tcf4 redirects their differentiation toward goblet cells. CONCLUSIONS: Tcf4 controls the balance between Paneth and goblet cells and is essential for antimicrobial peptide production in the small intestine. In colon adenomas, Paneth-like tumor cells drive antimicrobial gene expression and provide Wnt3 ligands, which may have implications for cancer therapy.

• Keywords
• Antimicrobial peptides, Colorectal cancer, Intestinal cell lineage, Intestinal crypt, Paneth cells, Single-cell transcriptomics,
• MeSH
• alpha-Defensins metabolism   MeSH
• Cell Differentiation MeSH
• Humans MeSH
• Mice MeSH
• Colonic Neoplasms * pathology   genetics   microbiology   metabolism   MeSH
• Organoids metabolism   MeSH
• Paneth Cells metabolism   MeSH
• Goblet Cells metabolism   MeSH
• Wnt Signaling Pathway MeSH
• Gastrointestinal Microbiome * MeSH
• Intestine, Small * metabolism   pathology   microbiology   MeSH
• Transcription Factor 4 * metabolism   genetics   MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alpha-Defensins MeSH
• Tcf4 protein, mouse MeSH Browser
• Transcription Factor 4 * MeSH

The protective and absorptive functions of the intestinal epithelium rely on differentiated enterocytes in the villi. The differentiation of enterocytes is orchestrated by sub-epithelial mesenchymal cells producing distinct ligands along the villus axis, in particular Bmps and Tgfβ. Here, we show that individual Bmp ligands and Tgfβ drive distinct enterocytic programs specific to villus zonation. Bmp4 is expressed from the centre to the upper part of the villus and activates preferentially genes connected to lipid uptake and metabolism. In contrast, Bmp2 is produced by villus tip mesenchymal cells and it influences the adhesive properties of villus tip epithelial cells and the expression of immunomodulators. Additionally, Tgfβ induces epithelial gene expression programs similar to those triggered by Bmp2. Bmp2-driven villus tip program is activated by a canonical Bmp receptor type I/Smad-dependent mechanism. Finally, we establish an organoid cultivation system that enriches villus tip enterocytes and thereby better mimics the cellular composition of the intestinal epithelium. Our data suggest that not only a Bmp gradient but also the activity of individual Bmp drives specific enterocytic programs.

• Keywords
• Tgfβ / Bmp signalling, enterocytes, epithelial differentiation, intestinal mesenchymal cells, small intestine,
• MeSH
• Cell Differentiation MeSH
• Enterocytes * metabolism   MeSH
• Bone Morphogenetic Proteins metabolism   MeSH
• Ligands MeSH
• Intestinal Mucosa * metabolism   MeSH
• Transforming Growth Factor beta metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bone Morphogenetic Proteins MeSH
• Ligands MeSH
• Transforming Growth Factor beta MeSH