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The concept that tumors are maintained by dedicated stem cells, the so-called cancer stem cell hypothesis, has attracted great interest but remains controversial. Studying mouse models, we provide direct, functional evidence for the presence of stem cell activity within primary intestinal adenomas, a precursor to intestinal cancer. By \"lineage retracing\" using the multicolor Cre-reporter R26R-Confetti, we demonstrate that the crypt stem cell marker Lgr5 (leucine-rich repeat—containing heterotrimeric guanine nucleotide—binding protein—coupled receptor 5) also marks a subpopulation of adenoma cells that fuel the growth of established intestinal adenomas. These Lgr5 + cells, which represent about 5 to 10% of the cells in the adenomas, generate additional Lgr5 + cells as well as all other adenoma cell types. The Lgr5 + cells are intermingled with Paneth cells near the adenoma base, a pattern reminiscent of the architecture of the normal crypt niche.

Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5⁺) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5⁺ stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5⁺ stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4⁺ DCS cells promote organoid formation of single Lgr5⁺ colon stem cells. DCS cells can be massively produced from Lgr5⁺ colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4⁺ DCS cells serve as Paneth cell equivalents in the colon crypt niche.

Paneth cells carve out a niche Paneth cells, specialized cells found in the intestinal epithelium, are known to protect stem cells by producing bactericidal compounds. Now another crucial function is reported: they provide the essential niche signals (EGF/TGFα, Notch and Wnt) for Lgr5 -expressing stem cells in the small intestine. Multipotent stem cells expressing Lgr5 generate all intestinal epithelium cell types — Paneth cells included. Stem-cell niches are often seen as pre-existing sites to which stem cells migrate; this work shows that intestinal stem cells receive niche support from their own progeny. Multipotent stem cells expressing Lgr5 are known to generate all cell types of the intestinal epithelium (enterocytes, goblet cells, Paneth cells and enteroendocrine cells). A new study shows that Paneth cells have an essential role for intestinal crypt and stem cell maintenance by supplying essential niche signals to the Lgr5-expressing cells. Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms 1 , 2 . Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins 3 . Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt–villus organoids in the absence of non-epithelial niche cells 4 . Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro . CD24 + Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24 + cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.

Rnf43 (RING finger protein 43) and Znrf3 (zinc/RING finger protein 3) (RZ) are two closely related transmembrane E3 ligases, encoded by Wnt target genes, that remove surface Wnt (wingless-int) receptors. The two genes are mutated in various human cancers. Such tumors are predicted to be hypersensitive to, yet still depend on, secreted Wnts. We previously showed that mutation of RZ in the intestine yields rapidly growing adenomas containing LGR5 ⁺ (leucine-rich repeat-containing G-protein coupled receptor 5) stem cells and Wnt3-producing Paneth cells. We now show that removal of Paneth cells by Math1 mutation inhibits RZ ⁻/⁻ tumor formation. Similarly, deletion of Wnt3 inhibits tumorigenesis. Treatment of mice carrying RZ ⁻/⁻ intestinal neoplasia with a small molecule Wnt secretion inhibitor (porcupine inhibitor C59) strongly inhibited growth, whereas adjacent normal crypts remained intact. These results establish that paracrine Wnt secretion is an essential driver of RZ ⁻/⁻ tumor growth and imply that a therapeutic window exists for the use of porcupine inhibitors for RZ-mutant cancers. Significance Rnf43 (RING finger protein 43) and Znrf3 (zinc/RING finger protein 3), encoded by stem cell-specific Wnt (wingless-int) target genes, constitute a crucial negative feedback loop in the Wnt signaling pathway. Rnf43 is mutated in subsets of human cancers of the colon, pancreas, stomach, ovary, and liver, while Znrf3 is mutated in adrenocortical carcinoma and osteoblastoma. Indeed, when both genes are mutated simultaneously in small intestinal stem cells in mice, tumors arise within a few weeks. Treatment of mice carrying RZ ⁻/⁻ intestinal neoplasia with a small molecule Wnt secretion inhibitor strongly inhibited growth, while adjacent normal crypts remained intact. These results establish that paracrine Wnt secretion is an essential driver of RZ ⁻/⁻ tumor growth and imply that a therapeutic window exists for the use of porcupine inhibitors for RZ-mutant cancers.

Cycling intestinal Lgr5⁺ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5⁺ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5⁺ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

Notch signalling in the intestinal crypt is modulated to drive commitment to the secretory fate. Clevers and colleagues find that cells expressing the Notch ligand DLL1 are intermediate secretory cells that can revert to Lgr5+ stem cells upon damage. Lgr5 + intestinal stem cells generate enterocytes and secretory cells. Secretory lineage commitment requires Notch silencing. The Notch ligand Dll1 is expressed by a subset of immediate stem cell daughters. Lineage tracing in Dll1 GFP – ires – CreERT2 knock-in mice reveals that single Dll1 high cells generate small, short-lived clones containing all four secretory cell types. Lineage specification thus occurs in immediate stem cell daughters through Notch lateral inhibition. Cultured Dll1 high cells form long-lived organoids (mini-guts) on brief Wnt3A exposure. When Dll1 high cells are genetically marked before tissue damage, stem cell tracing events occur. Thus, secretory progenitors exhibit plasticity by regaining stemness on damage.

Intestinal cancer: stem-cell destinies Inappropriate activation of the Wnt signalling pathway in intestinal stem cells causes them to become cancerous. Two papers in this issue help identify the cell type at the root of this cancer, which should in turn aid therapeutic design. Zhu et al . report that prominin 1, a surface protein found on both normal stem cells and cancer stem cells, is a marker for stem cells that are prone to neoplastic transformation. Barker et al . show that in cells expressing Lgr5 , previously identified as a marker for intestinal stem cells, activation of Wnt signalling is sufficient to initiate tumour formation. Intestinal tumours can originate from Lgr5 + intestinal stem cells after genetic activation of the Wnt signalling pathway. Intestinal cancer is initiated by Wnt-pathway-activating mutations in genes such as adenomatous polyposis coli ( APC ). As in most cancers, the cell of origin has remained elusive. In a previously established Lgr5 (leucine-rich-repeat containing G-protein-coupled receptor 5) knockin mouse model, a tamoxifen-inducible Cre recombinase is expressed in long-lived intestinal stem cells 1 . Here we show that deletion of Apc in these stem cells leads to their transformation within days. Transformed stem cells remain located at crypt bottoms, while fuelling a growing microadenoma. These microadenomas show unimpeded growth and develop into macroscopic adenomas within 3-5weeks. The distribution of Lgr5 + cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. When Apc is deleted in short-lived transit-amplifying cells using a different cre mouse, the growth of the induced microadenomas rapidly stalls. Even after 30weeks, large adenomas are very rare in these mice. We conclude that stem-cell-specific loss of Apc results in progressively growing neoplasia.

The transcription factor NF-κB is indispensable for intestinal immune homeostasis, but contributes to chronic inflammation and inflammatory bowel disease (IBD). A20, an inhibitor of both NF-κB and apoptotic signalling, was identified as a susceptibility gene for multiple inflammatory diseases, including IBD. Despite absence of spontaneous intestinal inflammation in intestinal epithelial cell (IEC) specific A20 knockout mice, we found additional myeloid-specific A20 deletion to synergistically drive intestinal pathology through cell-specific mechanisms. A20 ensures intestinal barrier stability by preventing cytokine-induced IEC apoptosis, while A20 prevents excessive cytokine production in myeloid cells. Combining IEC and myeloid A20 deletion induces ileitis and severe colitis, characterized by IEC apoptosis, Paneth and goblet cell loss, epithelial hyperproliferation and intestinal microbiota dysbiosis. Continuous epithelial cell death and regeneration in an inflammatory environment sensitizes cells for neoplastic transformation and the development of colorectal tumours in aged mice. Aetiology of colitis is highly complex and incompletely understood. Here the authors show in mouse models that A20 deubiquitinase limits pro-inflammatory cytokine production in myeloid cells while inhibiting proapoptotic response to these cytokines in enterocytes, and that only upon losing both functions intestinal pathologies develop.

Little is known about the signaling mechanisms that determine the highly regular patterning of the intestinal epithelium into crypts and villi. With the use of mouse models, we show that bone morphogenetic protein (BMP)-4 expression occurs exclusively in the intravillus mesenchyme. Villus epithelial cells respond to the BMP signal. Inhibition of BMP signaling by transgenic expression of noggin results in the formation of numerous ectopic crypt units perpendicular to the crypt-villus axis. These changes phenocopy the intestinal histopathology of patients with the cancer predisposition syndrome juvenile polyposis (JP), including the frequent occurrence of intraepithelial neoplasia. Many JP cases are known to harbor mutations in BMP pathway genes. These data indicate that intestinal BMP signaling represses de novo crypt formation and polyp growth.

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development.