To determine if subepithelial cells

To determine if subepithelial cells, such as SEMFs, are a required physiologic Wnt source, we used Myh11-Cre mice. Myh11-encoded smooth-muscle myosin heavy chain is expressed in much of the intestinal subepithelium (Wirth et al., 2008). To define the precise expression domain, we crossed Myh11-Cre to Rosa26-lox-STOP-lox-YFP (Rosa26R) reporter mice and treated with TAM. Yellow fluorescent protein (YFP) expression was evident in circular and longitudinal muscle layers as well as in SEMF 5 days post-Cre induction, colocalizing with α-smooth muscle nmda receptor (Figures 2A and S2). Most subepithelial cells expressed YFP, but the lamina propria was not totally marked, indicating that Myh11-Cre is expressed in most, but not all, subepithelial lineages. We then crossed Myh11-Cre and Porcn mice to ablate Wnt-ligand secretion specifically in SEMF and smooth muscle (Figure 2B), while preserving Porcn expression in the epithelium (Figure 2C). To estimate the mesenchymal cell fraction with recombined Porcn, we genotyped Porcn alleles in unfractionated Porcn;Myh11-Cre (Porcn) intestines, expecting no recombination in epithelial, serosal, endothelial, or blood cells, which lack Myh11 expression. In this light, a substantial contribution from recombined null Porcn DNA implied efficient recombination in most Myh11+ cells (Figure 2D), a point we demonstrate with greater confidence below (Figure 3B). Three weeks following Cre activation in Porcn males, we did not observe weight loss (Figure S1A), implying preserved intestinal function. Gross and microscopic intestinal anatomy were intact, and Porcn did not differ from control mice in the numbers of proliferating Ki67+ crypt cells; Paneth or other secretory cells (Figures 2E, S1B, and S1C); expression of nuclear β-catenin; or levels of Wnt target genes in crypt epithelium (Figure 2F). Thus, disruption of a subepithelial Myh11+ cell source of Wnts also did not perturb intestinal homeostasis. Intestinal organoid cultures from isolated Lgr5+ ISCs require supplemental WNT3A, which is thought to derive in vivo from Paneth cells (Sato et al., 2011). However, mouse intestines devoid of epithelial Wnt3 are normal and coculture of Lgr5+ ISCs with mesenchymal cells can substitute for WNT3A in organoid formation, suggesting redundant Wnt sources (Farin et al., 2012). To test this idea in vivo, we crossed Porcn mice onto a compound Villin-Cre;Myh11-Cre background and treated conditional null mice with TAM to force Porcn loss from both compartments (Porcn). Three weeks post-Cre activation, we observed nearly complete recombination of the floxed mutant allele in unfractionated intestines (Figure 3A), producing total absence of Porcn mRNA in the epithelium (Figure 3B). Unfractionated intestines showed 40% less Porcn mRNA in Porcn mice and 85% less in compound Porcn mice (Figure 3B). Together, these data reveal highly efficient Porcn depletion in both compartments, with the expected residual contribution from Villin;Myh11 cells. Compound mutant mice showed no weight loss (Figure S1A) or clinical compromise, decrease in proliferation as assessed by Ki67 expression or bromodeoxyuridine (BrdU) uptake (Figures 3C and 3D), or defects in intestine morphology (Figures 3D, S1B, and S1C). Strong immunostaining for nuclear β-catenin provided direct evidence for sustained Wnt-signaling activity, and qRT-PCR analysis revealed subtle, statistically insignificant deficits in Wnt target transcripts (Figure 3E). Thus, combined loss of Porcn in the gut epithelium and the dominant subepithelial compartment, including SEMFs, preserves all measurable Wnt-dependent functions. These findings conflict with previous results from forced expression of Wnt antagonists, which produced crypt atrophy, villus shortening, loss of Wnt target transcripts, and failure of secretory cell differentiation within 2–4 days (Kuhnert et al., 2004). There are several possible reasons for the absence of similar defects in PorcnDel intestines. First, Wnts necessary for intestinal homeostasis might circumvent the requirement for PORCN in secretion. This is unlikely because two independent studies reveal that PORCN seems necessary to palmitoylate all human Wnts; this in turn is required for Wnts to bind the carrier protein Wntless for secretory transport (Coombs et al., 2010; Liu et al., 2013) and to bind Frizzled receptors (Janda et al., 2012; Najdi et al., 2012). Second, the potent effect of Wnt antagonists on crypt functions might not reflect the native activity of Wnts per se but rather of R-spondin or another family of ligands. Available evidence, however, indicates that R-spondins act in conjunction with, and not separate from, Wnts (Niehrs, 2012). A third possibility is that Wnt reserves in the subepithelial basement membrane or elsewhere persisted for the duration of our experiments. Wnts can bind heparan sulfate proteoglycans present on the surface of Wnt-recipient cells, a proposed mechanism to prevent their diffusion and allow prolonged activity (Mikels and Nusse, 2006). However, even in the absence of information on intestinal Wnt concentrations, turnover, or reserves, secreted Wnts are unlikely to have persisted for the length of our studies. Porcn deletion was efficient, and we deliberately examined mice weeks after Cre activation, giving time for Wnt reserves to decay. Moreover, in a tissue that self-renews continually and responds quickly to injury or cell loss (Clevers, 2013), Wnts probably turn over rapidly to allow responsive homeostasis.