Intestinal Paneth cell differentiation relies on asymmetric regulation of Wnt 2 signaling by Daam1/2

The mammalian intestine is one of the most rapidly self-renewing tissues, driven by actively cycling stem cells residing at the crypt bottom . Together with stromal cells, Paneth cells form a major element of the niche microenvironment that provides various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. With 19 family members, different Wnt ligands can selectively activate βcatenin dependent (canonical) or independent (non-canonical) signaling. Here, we report that Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and non-canonical Wnt (Wnt/PCP) signaling, and their function is required for Paneth cell progenitor differentiation. We found that Daam1/2 interacts with the Wnt antagonist Rnf43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing Rnf43-dependent endo-lysosomal degradation of the ubiquitinated Wnt receptor, Frizzled (Fzd). Moreover, single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion, as a result of defective Wnt/PCP signaling. Taken together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and non-canonical Wnt signaling, the regulation of which is fundamental for specifying an adequate number of Paneth cells while maintaining intestinal stem cell homeostasis. Mouse Small intestine oragnoids from Vil-CreERT2-Rnf43fl/fl-Znrf3fl/fl mice (RZ sample) and Vil-CreERT2-Daam1fl/fl-Daam2fl/fl mice (D1/2 sample). For the samll intestinal organoid lines, 1 μg/ml 4-hydroxytamoxifen (4-OHT) was added overnight in WENR+Nic (Wnt, Egf, Noggin, R-spondin1 + nicotinamide) organoid culture medium. After recombination, D1/2 and RZ mutant organoids were cultured in WEN+Nic medium for selection purposes, as only successfully recombined mutant cells survive in the absence of Rspo1. WT, D1/2 cDKO and RZ cDKO organoids were maintained in regular WENR+Nic (WT) or WEN+Nic (D1/2 and RZ cDKO) culture medium for 10 days prior to single-cell analysis.