RNA-Seq analysis of colonic mesenchyme lacking tumor necrosis factor receptor 1

Our study reports a role for mesenchymal TNFR1 in maintaining colonic mesenchymal cell diversity and facilitation of a function epithelial stem cell niche. Through RNA-seq profiling, we show that a TNFR1-deficient mesenchyme has altered anchoring and cell substrate junctions, focal adhesions, extracellular matrix, and actin binding processes. We find specific reduction in the key stem cell niche factor, RSPO3, crucial for Lgr5+ stem cell maintenance, downregulated 5.1-fold in the TNFR1 deficient mesenchyme. Pathways classically associated with proliferation and differentiation, including Phosphoinositide 3-kinase (P13k) and Mitogen-activated protein kinase (MAPK), and those involved in migration, namely Ras-proximate-1 or Ras-related protein 1 (RAP1), TGF-ß and RAS, were significantly enriched in TNFR1-/- mesenchyme compared to controls. In particular, Itga6, representing the integrin alpha 6 subunit, was one of the most highly upregulated transcripts. Therefore, the transcriptome profiling of the TNFR1 deficient mesenchyme deletion suggests an important role for TNFR1 in the regulation of mesenchymal cell-matrix interactions and niche transcript expression. Overall design: To determine a functional role for TNFR1 signaling in the colonic mesenchyme and its interaction with the epithelial stem cell niche, we investigated sub-epithelial mesenchymal cell populations in control (TNFR1+/-) and TNFR1-/- mice through immunostaining, whole lysate cell preps, epithelial-mesenchyme co-cultures and through RNA-Seq of amplified primary control (TNFR1+/-) and TNFR1-/- colonic myofibroblasts (CMFs).