Summary

Wnt and Rspondin (RSPO) signaling drives proliferation, and bone morphogenetic protein inhibitors (BMPi) impede differentiation, of intestinal stem cells (ISCs). Here we identify the adult mouse ISC niche as a complex, multi-layered structure that encompasses distinct mesenchymal and smooth muscle cell populations. Diverse sub-cryptal cells provide redundant supportive factors, with certain BMPi and the most potent Wnt co-factor, RSPO2, restricted to single cell types. Niche functions refine during a critical period of postnatal crypt morphogenesis, in part to oppose dense aggregation of BMP-expressing sub-epithelial myofibroblasts that promote epithelial differentiation. A specialized muscle layer, the muscularis mucosae, first appears during this period and supplements neighboring RSPO and BMPi sources. In vivo ablation of smooth muscle raises BMP activity and potently limits a pre-weaning burst of crypt fission. Thus, distinct and progressively specialized mesenchymal components together create the milieu required to propagate crypts during rapid organ growth and to sustain adult ISCs.