Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development

Abstract

BACKGROUND & AIMS: The stem/progenitor cells of the developing intestine arebiologically distinct from their adult counterparts. Here we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor, Delta-Like Protein 1 (DLK1). METHODS: mRNAseq analyses of intestinal mesenchymal cells (IMC) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel co-culture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR and immunocytochemistry. The intestinal phenotype of littermate Dlk1 +/+ and Dlk1 -/- mice was compared using immunohistochemistry. RESULTS: Using transcriptomic analyses we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate, DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth and shifted to the intestinal epithelium in adulthood. In co-culture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knock-down in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1 -/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1 +/+ embryos. CONCLUSIONS: Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.