Targeting of Wnt signaling represents a promising anti-cancer therapy. However, the consequences of systemically attenuating the Wnt pathway in an adult organism are unknown. Here, we globally prevent Wnt secretion by genetically ablating Wntless. We find that preventing Wnt signaling in the entire body causes mortality due to impaired intestinal homeostasis. This is caused by the loss of intestinal stem cells. Reconstitution of Wnt/β-catenin signaling via delivery of external Wnt ligands prolongs the survival of intestinal stem cells and reveals the essential role of extra-epithelial Wnt ligands for the renewal of the intestinal epithelium. Wnt2b is a key extra-epithelial Wnt ligand capable of promoting Wnt/β-catenin signaling and intestinal homeostasis. Wnt2b is secreted by subepithelial mesenchymal cells that co-express either Gli1 or Acta2. Subepithelial mesenchymal cells expressing high levels of Wnt2b are predominantly Gli1 positive.

In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/β-catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/β-catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/β-catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/β-catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the β-catenin-activated Wnt-reporter mouse and by pharmacological stimulation of Wnt/β-catenin by infusion of the specific glycogen synthase kinase 3β inhibitor, AR-A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/β-catenin, respectively, increased and decreased the differentiation of dSVZ-NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/β-catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/β-catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/β-catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs. Stem Cells 2014;32:1301–1312

β‐catenin‐mediated Wnt signaling is an ancient cell‐communication pathway in which β‐catenin drives the expression of certain genes as a consequence of the trigger given by extracellular WNT molecules. The events occurring from signal to transcription are evolutionarily conserved, and their final output orchestrates countless processes during embryonic development and tissue homeostasis. Importantly, a dysfunctional Wnt/β‐catenin pathway causes developmental malformations, and its aberrant activation is the root of several types of cancer. A rich literature describes the multitude of nuclear players that cooperate with β‐catenin to generate a transcriptional program. However, a unified theory of how β‐catenin drives target gene expression is still missing. We will discuss two types of β‐catenin interactors: transcription factors that allow β‐catenin to localize at target regions on the DNA, and transcriptional co‐factors that ultimately activate gene expression. In contrast to the presumed universality of β‐catenin's action, the ensemble of available evidence suggests a view in which β‐catenin drives a complex system of responses in different cells and tissues. A malleable armamentarium of players might interact with β‐catenin in order to activate the right “canonical” targets in each tissue, developmental stage, or disease context. Discovering the mechanism by which each tissue‐specific β‐catenin response is executed will be crucial to comprehend how a seemingly universal pathway fosters a wide spectrum of processes during development and homeostasis. Perhaps more importantly, this could ultimately inform us about which are the tumor‐specific components that need to be targeted to dampen the activity of oncogenic β‐catenin.This article is categorized under: Cancer > Molecular and Cellular Physiology Cancer > Genetics/Genomics/Epigenetics Cancer > Stem Cells and Development

Background: Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions. Results: We compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified. Conclusions: We propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

The fate of neural progenitor cells (NPCs) is determined by a complex interplay of intrinsic programs and extrinsic signals, very few of which are known. β-Catenin transduces extracellular Wnt signals, but also maintains adherens junctions integrity. Here, we identify for the first time the contribution of β-catenin transcriptional activity as opposed to its adhesion role in the development of the cerebral cortex by combining a novel β-catenin mutant allele with conditional inactivation approaches. Wnt/β-catenin signaling ablation leads to premature NPC differentiation, but, in addition, to a change in progenitor cell cycle kinetics and an increase in basally dividing progenitors. Interestingly, Wnt/β-catenin signaling affects the sequential fate switch of progenitors, leading to a shortened neurogenic period with decreased number of both deep and upper-layer neurons and later, to precocious astrogenesis. Indeed, a genome-wide analysis highlighted the premature activation of a corticogenesis differentiation program in the Wnt/β-catenin signaling-ablated cortex. Thus, β-catenin signaling controls the expression of a set of genes that appear to act downstream of canonical Wnt signaling to regulate the stage-specific production of appropriate progenitor numbers, neuronal subpopulations, and astroglia in the forebrain. Stem Cells 2015;33:170–182