Wnt secretion from epithelial cells and subepithelial myofibroblasts is not required in the mouse intestinal stem cell niche in vivo

Abstract

Wnt signaling is a crucial aspect of the intestinal stem cell niche required for crypt cell proliferation and differentiation. Paneth cells or subepithelial myofibroblasts are leading candidate sources of the required Wnt ligands, but this has not been tested in vivo. To abolish Wnt-ligand secretion, we used Porcupine (Porcn) conditional-null mice crossed to strains expressing inducible Cre recombinase in the epithelium, including Paneth cells (Villin-Cre (ERT2) ); in smooth muscle, including subepithelial myofibroblasts (Myh11-Cre (ERT2) ); and simultaneously in both compartments. Elimination of Wnt secretion from any of these compartments did not disrupt tissue morphology, cell proliferation, differentiation, or Wnt pathway activity. Thus, Wnt-ligand secretion from these cell populations is dispensable for intestinal homeostasis, revealing that a minor cell type or significant and unexpected redundancy is responsible for physiologic Wnt signaling in vivo.

Graphical abstract

Figure 1

Porcn Deletion in Epithelial Cells Spares Intestinal Epithelial Morphology and Function (A) Strategy for Porcn deletion by TAM-inducible Cre recombinase driven by epithelium-specific Villin promoter. ER, estrogen receptor. (B) Genomic PCR of epithelium isolated from four Porcn^E-Del intestines indicates complete recombination of the floxed allele (485 bp band) compared to two Porcn^+/Y control mice (685 bp band from the wild-type allele). For reference, DNA is also shown from single Porcn^Del/X (Δ/+; 485 bp and 685 bp products) and Porcn^Fl/Y;Cre− (762 bp band from the unrecombined floxed allele) mice. WT, wild-type. (C) qRT-PCR analysis of Porcn mRNA in isolated Porcn^E-Del intestinal crypts (N = 4) reveals reduction by orders of magnitude compared to controls (N = 2). (D) Histology and immunohistochemistry (IHC) of Porcn^E-Del (N = 5) and control (Porcn^+/Y;Villin-Cre^ERT2; N = 3) mice reveals no abnormalities. Left to right: hematoxylin and eosin (H&E) staining, Ki67 IHC, lysozyme IHC, and Alcian blue staining. High-magnification images are shown to the right of low-magnification views. (E) Left, β-catenin IHC in control and Porcn^E-Del mice shows nuclear-staining crypt-base cells (arrows). Right, qRT-PCR analysis of Wnt target mRNAs in isolated control (N = 2) and Porcn^E-Del (N = 4) crypt epithelium demonstrates unperturbed Wnt-pathway activity. Bars represent mean ± SEM of biological replicates; all scale bars, 50 μm.

Figure 2

Porcn Loss in Intestinal Smooth Muscle Cells and SEMFs Does Not Adversely Affect Epithelial Morphology and Function (A) Myh11-Cre^ERT2;Rosa26R mice activate Cre recombinase in all muscle layers, including subepithelial myofibroblasts. The dotted line in the high-magnification image (right) outlines a crypt, and arrows point to the thin SEMF layer of YFP+ cells enveloping the crypt. The scale bars represent 30 μm. See also Figure S2. (B) Strategy to induce muscle-cell-specific recombination in Porcn^Fl/Y;Myh11-Cre^ERT2 mice. (C) qRT-PCR analysis of isolated crypt epithelium shows no significant difference in Porcn mRNA expression in Porcn^M-Del (N = 4) mice compared to control (Porcn^+/Y;Myh11-Cre^ERT2, N = 4) mice, p = 0.35. (D) PCR from whole-intestine genomic DNA, revealing the expected proportion of recombined Porcn in smooth muscle, with residual unrecombined Porcn DNA contributed by epithelial and other cell types that lack Myh11-Cre^ERT2 expression. (E) Histology and immunostains on Porcn^M-Del (N = 7) and control (Porcn^+/Y;Myh11-Cre^ERT2, N = 6) intestines reveal no abnormalities. (F) Left, β-catenin IHC in control and Porcn^M-Del mice shows nuclear staining in crypt-base cells (arrows). Right, qRT-PCR reveals intact Wnt target expression in crypts isolated from Porcn^M-Del intestines, compared to controls (N = 4; statistics in Table S1). Bars represent mean ± SEM of biological replicates; scale bars (E and F), 50 μm.

Figure 3

Consequences of Simultaneous Loss of Porcn from Epithelial and Muscle Cells (A) DNA from whole intestines in TAM-treated Porcn^Fl/Y;Villin-Cre^ERT2;Myh11-Cre^ERT2 mice shows significant recombination at the floxed Porcn allele, with a minimal contribution of unrecombined DNA from nonepithelial, nonmuscle cells. (B) Left, qRT-PCR analysis of RNA from crypts of Porcn^EM-Del mutants (N = 3) compared to controls (Porcn^+/Y;Villin-Cre^ERT2;Myh11-Cre^ERT2; N = 3) shows total Porcn deficiency, p = 0.0013. Right, qRT-PCR of unfractionated Porcn^M-Del (N = 2) intestines shows lower Porcn expression than controls (Porcn^+/Y;Villin-Cre^ERT2;Myh11-Cre^ERT2 and Porcn^+/Y;Myh11-Cre^ERT2; N = 4), which is further reduced in Porcn^EM-Del (N = 2) mice. (C) Equivalent BrdU uptake in control and Porcn^EM-Del intestinal crypts. (D) Histology and immunostains from Porcn^EM-Del and control intestines reveal intact morphology, cell proliferation, and differentiation (N = 3). (E) Left, β-catenin IHC in control and Porcn^EM-Del mice shows nuclear staining in crypt-base cells (arrows). Right, qRT-PCR analysis of control (N = 3) and Porcn^EM-Del (N = 3) intestines indicates a statistically insignificant (Table S1) reduction in levels of Wnt target transcripts. Bars in graphs represent mean ± SEM of biological replicates; all scale bars, 50 μm.