Stromal Dlk1 promotes epithelial proliferation and inhibits epithelial differentiation during the small intestine development
Background: Mesenchyme offers microenvironment for epithelial maturation during development of the small intestine. Molecular signature of immature mesenchyme has not yet been elucidated. DLK1 is known as a Notch ligand and has been reported to be highly expressed during embryogenesis. Although it has been investigated in other branching structure organs such as lung and salivary gland, DLK1's role in intestinal development remains unknown. Methods: Intestinal mesenchymal cells (IMCs) were collected from small intestines of embryonic day 14.5 (E14.5) and adult mice, respectively. Intestinal organoids collected from embryonic small intestines were co-cultured with IMCs. The effect of embryonic and adult IMCs on epithelial morphology and differentiation was compared using qPCR and immunocytochemistry. Organoid sustainability was compared to assess the mesenchymal effect on stemness. A global mRNA sequence analysis of differentially expressed transcripts in embryonic and adult IMCs was performed. Given DLK1 was among the morphogen genes that were differentially expressed, DLK1 expression was manipulated with recombinant protein or siRNA and its effect on organoids was investigated. The histology of DLK1 knock out mice and wild type littermates were compared with proliferation marker, Ki67 and secretory lineage staining. Results: Embryonic IMCs induced cystic change of organoids and reduced budding structure when compared with adult IMCs. Secretory lineage differentiation of epithelium was found to be reduced and secondary organoids formation was increased when co-cultured with embryonic IMCs. 23 differentially expressed morphogen genes were identified with GSEA analysis. Genes expressed in cell membranes and extracellular space were filtered and DLK1 was identified as a key mesenchymal gene for morphogenesis. Immunohistochemistry showed that DLK1 was expressed exclusively in the stroma at the top of emerging villi and co-localized with PDGFRa in E14.5 small intestine. DLK1 expression in mesenchyme decreased after birth and shifted to epithelium in adulthood. Addition of recombinant DLK1 to adult IMCs inhibited organoid differentiation, whereas siDLK1 in embryonic IMCs increased epithelial differentiation to secretory lineage cells. DLK1 knock out mice showed restricted distribution of Ki67 positive cells in the base of villi compared with wild type littermate embryos. Secretory lineage cells were increased in DLK1 knock out mice at E19.5. Conclusion: DLK1 is expressed in the mesenchyme of small intestines during embryogenesis and decreases with age. Our data suggest stromal DLK1 promotes epithelial proliferation and suppresses secretory lineage differentiation. Overall design: Three of Intestinal mesenchymal cells (IMCs) were collected from small intestines of embryonic day 14.5 (E14.5) and adult mice, respectively. The primary culture were used for RNA extraction at passage number 2-3.