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YAP/TAZ Transcriptional Activation Is Required for Cellular Reprogramming (A) Normal organoids (WT) and Apc-knockout (Apc KO ) spheres derived from the small intestine cultured in collagen type I with the indicated cytokine cocktail. Time point of analysis is indicated in the left bar. Scale bar, 100 mm. (legend continued on next page)
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Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate t...
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... signaling has previously been demonstrated to stimulate YAP/TAZ activation via the inhibition of Adenomatous Polyposis Coli (Apc)-mediated degradation ( Azzolin et al., 2012Azzolin et al., , 2014. As expected, in collagen cultures Wnt3a could be substituted by the loss of Apc (Figures 5A and 5B). Moreover, Apc KO organoid growth could be blocked by treatment with the YAP/TAZ inhibitor verteporfin ( Figure 5C). ...
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... expected, in collagen cultures Wnt3a could be substituted by the loss of Apc (Figures 5A and 5B). Moreover, Apc KO organoid growth could be blocked by treatment with the YAP/TAZ inhibitor verteporfin ( Figure 5C). This demonstrates that Wnt via inhibition of the Apc complex supports growth in collagen type I via YAP/TAZ activation. ...
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... is similar to the reported requirement for YAP/TAZ during tumor formation upon the loss of Apc ( Azzolin et al., 2014). Notably, blunting YAP/ TAZ mechano-transduction by the inhibition of Rho signaling also opposed the growth of Apc KO organoids, a result consistent with the requirement of cell mechanics for YAP/TAZ activation in the repairing epithelium in vivo ( Figure 5C). Importantly, even in the Apc KO model, cell mechanics were still causal for sustaining YAP/TAZ target genes (Cyr61 and Ctgf) and the associated appearance of markers associated with the fetal epithelium ( Tacstd2 and Ly6a). ...
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... directly assess the requirements for YAP/TAZ in the context of collagen-induced cellular reprogramming, we took advantage of both loss-and gain-of-function mutants. Using an inducible conditional double-knockout (cDKO) model for YAP/TAZ ( Azzo- lin et al., 2014), it was evident that YAP/TAZ are required for growth in collagen type I matrices ( Figure S5A). Intriguingly, overexpression of YAP in epithelial cells derived from the R26-rtTA; tetO-YAPS127A mouse model could compensate for the presence of Wnt in the culture medium ( Figures 5E and 5F). ...
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... an inducible conditional double-knockout (cDKO) model for YAP/TAZ ( Azzo- lin et al., 2014), it was evident that YAP/TAZ are required for growth in collagen type I matrices ( Figure S5A). Intriguingly, overexpression of YAP in epithelial cells derived from the R26-rtTA; tetO-YAPS127A mouse model could compensate for the presence of Wnt in the culture medium ( Figures 5E and 5F). Moreover, induction of exogenous YAPS127A expression in collagen rescued the detrimental effects of FAK and Src inhibition on (E) Detection of Sca1 (green, left) and Anxa1 (green, right) in collagen type I cultures from the small intestine. ...
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... bars, 50 mm. growth ( Figure 5F), demonstrating that YAP/TAZ activation lies downstream of the integrin-mediated mechano-transduction pathway. Importantly, the establishment of the repairing epithelium-like status in vitro via culturing in collagen type I, with either Wnt3a supplemented or by overexpression of YAP, led to a similar upregulation of markers of the fetal epithelium as well as a pronounced downregulation of markers associated with adult stem cells and differentiated lineages ( Figure 5G). ...
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... ( Figure 5F), demonstrating that YAP/TAZ activation lies downstream of the integrin-mediated mechano-transduction pathway. Importantly, the establishment of the repairing epithelium-like status in vitro via culturing in collagen type I, with either Wnt3a supplemented or by overexpression of YAP, led to a similar upregulation of markers of the fetal epithelium as well as a pronounced downregulation of markers associated with adult stem cells and differentiated lineages ( Figure 5G). Collectively, gain-and loss-of-function lines of evidence concur at demonstrating that YAP/TAZ-mediated activation is required and sufficient for establishing the repairing epithelium-like state in vitro. ...
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... reported previously, loss of YAP/TAZ does not affect normal tissue homeostasis (Figures S5B and S5C;Azzolin et al., 2014). Upon the administration of DSS, at concentrations with minor effects on controls animals, cDKO animals rapidly lost weight, and ulcerated lesions spread throughout the colon similarly to the animals treated with either FAK or Src inhibitors ( Figures 5H-5K). Importantly, the epithelium remaining in the YAP/TAZ cDKO could be divided into what appeared to be normal regenerating epithelium positive for YAP, demonstrating that these cells had escaped recombination, and thin-walled cysts found exclusively in the cDKO animals, where YAP and Sca1 were absent (Figures 5L, 5M, and S5E). ...
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Damage to intestinal epithelial cell proliferation or intestinal stem cell (ISC) maintenance may trigger inflammatory bowel disease (IBD), and protecting the ISCs is critical for IBD treatment. Here, we found that in the dextran sulfate sodium (DSS)-induced ulcerative colitis mice model, colon epithelium and Lgr5+ intestinal stem cells (ISCs) renew...
Citations
... [4][5][6][7][8][9][10] Work from us and many others demonstrated a critical requirement for epithelial Hippo signaling in intestinal stem cell maintenance and regeneration. 6,[11][12][13][14][15][16] Furthermore, our prior work also uncov-ered the previously unappreciated requirement of YAP/TAZ in the embryonic gut mesenchyme, linking the Hippo-YAP pathway to intestinal mesenchymal homeostasis. 17 We found that YAP and TAZ function as a molecular switch to coordinate mesenchymal growth and patterning in the developing gut. ...
... 40 Escalating studies have supported the existence of in vivo reprogramming in injured or tumor tissues. [41][42][43] Notably, as neural crest-derived cells, SCs or Schwann cell precursors have been proved to directly contribute to neurons, odontoblasts, and skeletal progenitors in both developing embryo and injured sites. 18,23,44,45 Repair SCs seem to manifest characteristics reminiscent of their parental neural crest population. ...
Plp1-lineage Schwann cells (SCs) of peripheral nerve play a critical role in vascular remodeling and osteogenic differentiation during the early stage of bone healing, and the abnormal plasticity of SCs would jeopardize the bone regeneration. However, how Plp1-lineage cells respond to injury and initiate the vascularized osteogenesis remains incompletely understood. Here, by employing single-cell transcriptional profiling combined with lineage-specific tracing models, we uncover that Plp1-lineage cells undergoing injury-induced glia-to-MSCs transition contributed to osteogenesis and revascularization in the initial stage of bone injury. Importantly, our data demonstrated that the Sonic hedgehog (Shh) signaling was responsible for the transition process initiation, which was strongly activated by c-Jun/SIRT6/BAF170 complex-driven Shh enhancers. Collectively, these findings depict an injury-specific niche signal-mediated Plp1-lineage cells transition towards Gli1 ⁺ MSCs and may be instructive for approaches to promote bone regeneration during aging or other bone diseases.
... Accumulating evidence indicates that inflammation in the intestine leads to uncontrolled ECM remodeling and alters the mechanical properties. Yui et al. demonstrated that DSS-induced colitis leads to significant ECM remodeling in a YAP/TAZ signalingdependent manner [118]. ECM remodeling is a key feature of IBD and is associated with disease progression and inflammation. ...
... This schematic shows the connections between mechanical stress and intestinal diseases. "−" means a negative correlation, and "+" means a positive correlation between stress and intestinal diseases[85,86,109,[115][116][117][118][119][120][121][122][123][124][125][126][127][129][130][131][132][133][134]. ...
The intestinal area is composed of diverse cell types that harmonize gut homeostasis, which is influenced by both endogenous and exogenous factors. Notably, the environment of the intestine is exposed to several types of mechanical forces, including shear stress generated by fluid flow, compression and stretch generated by luminal contents and peristaltic waves of the intestine, and stiffness attributed to the extracellular matrix. These forces play critical roles in the regulation of cell proliferation, differentiation, and migration. Many efforts have been made to simulate the actual intestinal environment in vitro. The three-dimensional organoid culture system has emerged as a powerful tool for studying the mechanism of the intestinal epithelial barrier, mimicking rapidly renewing epithelium from intestinal stem cells (ISCs) in vivo. However, many aspects of how mechanical forces, such as shear stress, stiffness, compression, and stretch forces, influence the intestinal area remain unresolved. Here, we review the recent studies elucidating the impact of mechanical forces on intestinal immunity, interaction with the gut microbiome, and intestinal diseases.
... Ly6a (Sca-1), an 18-kDa GPI-anchored surface protein of the Ly6 gene family, has been identified as a marker for RSCs with fetal characteristics. Ly6a expression is upregulated during intestinal damage, such as radiation exposure, injury, and inflammation, and is associated with tissue regeneration (9). Additionally, Ly6a expression has been reported to increase in response to Helminth infections and Enterococcus faecalis exposure (10,11). ...
Intestinal stem cells are crucial for maintaining intestinal homeostasis, yet their transformation into tumor stem cells in the context of microbial infection remains poorly understood. Fusobacterium nucleatum (F. nucleatum) is frequently associated with the onset and progression of colorectal cancer (CRC). In this study, we uncovered that F. nucleatum colonized the depths of gut crypts in both human CRC patients and mouse models. Through single-cell sequencing analysis, we demonstrated that F. nucleatum infection reprogrammed crypt cells and activated LY6A+ revival stem cells (RSCs), promoting their hyperproliferation and subsequent transformation into tumor stem cells, which accelerated intestinal carcinogenesis. Mechanistically, we identified LY6A as a GPI-anchored membrane receptor for F. nucleatum. Upon binding, F. nucleatum induced upregulation of RPS14 via the LY6A receptor, driving RSC hyperactivity and tumorigenic conversion. Functional studies showed that genetic ablation of Ly6a in intestinal epithelial cells or Rps14 in LY6A+ RSCs substantially reduced F. nucleatum colonization and tumorigenesis. Moreover, clinical CRC cohorts analysis revealed a strong correlation between F. nucleatum infection, RSC expansion, and elevated RPS14 expression in tumor tissues. These findings highlight an alternative F. nucleatum-LY6A-RPS14 signaling axis as a critical driver of CRC progression and propose potential therapeutic targets for effective CRC intervention.
... Additionally, a subpopulation of quiescent Lgr5 + stem cells, marked by Mex3a, can reestablish stem cell function (Barriga et al. 2017). Furthermore, "revival" stem cells, characterized by transient expression of clusterin (Clu) and Sca1, have also been identified as a player in intestinal regeneration (Nusse et al. 2018;Yui et al. 2018). ...
Intestinal epithelium regeneration is crucial for homeostatic maintenance of the intestinal functions. A recent study published in Nature uncovers tuft cells as an unexpected key player in the regenerative process. Human tuft cells, traditionally recognized for their involvement in immune defense and pathogen protection, were found to exhibit stem cell-like properties following radiation-induced injury. These cells not only resist damage but also have the ability to generate functional stem cells, promoting the repair of the intestinal epithelium. This finding suggests that tuft cells may function as a reserve pool of stem cells, essential for efficient intestinal regeneration after injury.
... epithelium is highly adaptable to environmental perturbations that include broad activation of some foetal signature genes across damage models 3,4 , as well as pathogen-specific shifts in cell proportions and variable activation of immune and stress response programmes 39 . Our previous work identified revSCs, a regenerative population that expresses aspects of foetal signatures and is induced by various forms of injury that include IR, Lgr5 + ISC ablation, dextran sodium sulfate (DSS) colitis and parasites 4,7,11 . Here, we provide deep single-nucleus multi-omics profiling of homeostatic and irradiated intestinal epithelia and identify important crypt-associated chromatin dynamics and a critical role for parallel TGFβ and Hippo signalling that provides a redundant system that robustly supports intestinal regeneration. ...
Cell state dynamics underlying successful tissue regeneration are undercharacterized. In the intestine, damage prompts epithelial reprogramming into revival stem cells (revSCs) that reconstitute Lgr5⁺ intestinal stem cells (ISCs). Here single-nuclear multi-omics of mouse crypts regenerating from irradiation shows revSC chromatin accessibility overlaps with ISCs and differentiated lineages. While revSC genes themselves are accessible throughout homeostatic epithelia, damage-induced remodelling of chromatin in the crypt converges on Hippo and the transforming growth factor-beta (TGFβ) signalling pathway, which we show is transiently activated and directly induces functional revSCs. Combinatorial gene expression analysis further suggests multiple sources of revSCs, and we demonstrate TGFβ can reprogramme enterocytes, goblet and paneth cells into revSCs and show individual revSCs form organoids. Despite this, loss of TGFβ signalling yields mild regenerative defects, whereas interference in both Hippo and TGFβ leads to profound defects and death. Intestinal regeneration is thus poised for activation by a compensatory system of crypt-localized, transient morphogen cues that support epithelial reprogramming and robust intestinal repair.
... 152 The cytoskeleton assembly is involved in adjusting Hippo-YAP pathway and further the inhibitor of Rho kinase protein can intercept YAP activation in response to cytoskeleton-mediated mechanosensitivity. 153,154 The ECM protein of Agrin can improve the division of mice cardiomyocytes and human iPSC-derived cardiomyocytes. The underlying mechanism is revealed by the disassembly of dystrophin glycoprotein structures and YAP-ERK-dependent signaling pathway. ...
Force-sensing biophysical cues in microenvironment, including extracellular matrix performances, stretch-mediated mechanics, shear stress and flow-induced hemodynamics, have a significant influence in regulating vascular morphogenesis and cardiac remodeling by mechanotransduction. Once cells perceive these extracellular mechanical stimuli, Piezo activation promotes calcium influx by forming integrin-adhesion-coupling receptors. This induces robust contractility of cytoskeleton structures to further transmit biomechanical alternations into nuclei by regulating Hippo-Yes associated protein (YAP) signaling pathway between cytoplasmic and nuclear translocation. Although biomechanical stimuli are widely studied in cardiovascular diseases, the expression of force-sensing proteins in response to cardiovascular mechanotransduction has not been systematically concluded. Therefore, this review will summarize the force-sensing Piezo, cytoskeleton and YAP proteins to mediate extracellular mechanics, and also give the prominent emphasis on intrinsic connection of these mechanical proteins and cardiovascular mechanotransduction. Extensive insights into cardiovascular mechanics may provide some new strategies for cardiovascular clinical therapy.
... When intestinal epithelial cells are damaged, the Yes-associated protein (YAP) signaling pathway induces intestinal cell reprogramming, enabling rapid repair. [15][16][17][18][19] Small intestinal organoids have emerged in recent years as powerful tools for studying intestinal development and disease. Intestinal organoids are three-dimensional (3D) mini-intestines with intact crypts and villus structural domains. ...
... 25 But when intestinal epithelial cells sustain injury, adult intestinal organoids undergo transient reprogramming to generate fetal-like organoids that proliferate rapidly to promote damage repair. 16,25,26 We have effectively restored the damaged intestine by transplanting and colonizing intestinal organoids into the injured intestinal mucosa. 3 ...
... During the repair process, the damaged epithelial cells undergo transient reprogramming, resulting in the upregulation of fetal and regeneration markers, while the markers of adult stem cell and differentiation are suppressed. 16,26 When cultured in vitro, parasite-infected crypts can develop a spheroid with a fetal-like transcriptome, known as fetal-like organoid. 57 The WNT, Notch, and YAP/transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways are currently recognized as key contributors to intestinal damage repair. ...
Metabolism serves not only as the organism's energy source but also yields metabolites crucial for maintaining tissue homeostasis and overall health. Intestinal stem cells (ISCs) maintain intestinal homeostasis through continuous self‐renewal and differentiation divisions. The intricate relationship between metabolic pathways and intestinal homeostasis underscores their crucial interplay. Metabolic pathways have been shown to directly regulate ISC self‐renewal and influence ISC fate decisions under homeostatic conditions, but the cellular and molecular mechanisms remain incompletely understood. Understanding the intricate involvement of various pathways in maintaining intestinal homeostasis holds promise for devising innovative strategies to address intestinal diseases. Here, we provide a comprehensive review of recent advances in the regulation of intestinal homeostasis. We describe the regulation of intestinal homeostasis from multiple perspectives, including the regulation of intestinal epithelial cells, the regulation of the tissue microenvironment, and the key role of nutrient metabolism. We highlight the regulation of intestinal homeostasis and ISC by nutrient metabolism. This review provides a multifaceted perspective on how intestinal homeostasis is regulated and provides ideas for intestinal diseases and repair of intestinal damage.
... In addition, T140D tumoroids suppressed a fetal intestinal stem cell (ISC) signature originally defined as genes enriched in murine fetal intestinal spheroids relative to adult intestinal organoids (Figs 4F and S4B) 41 . This fetal gene expression program is associated with worse prognosis in CRC patients and regenerative programs in the adult mouse intestine post-injury [42][43][44][45][46] . The transcriptional effector of Hippo signaling, YAP, is a central regulator of the fetal ISC gene expression program 42 . ...
Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths with increasing incidence globally. Mutations in the tumor suppressor APC initiate CRC at least in part by preventing the GSK3 kinase from phosphorylating β-CATENIN, leading to its constitutive stabilization and transactivation of mitogenic target genes. While the importance of β-CATENIN phosphorylation by GSK3 is well-established, APC regulation of GSK3 activity upon other targets with potential oncogenic relevance are not understood. Here, we identify the H4K20 methyltransferase SETD8 as target of APC-coordinated GSK3 phosphorylation in the intestinal epithelium. We found that phosphorylation by GSK3 restrains the oncogenic activity of SETD8, with loss of phosphorylation sensitizing mice to oncogenic insults. Mechanistically, phosphorylation alters the role of SETD8 in transcriptional regulation, most notably by preventing it from activating oncogenic YAP signaling and a fetal-like transcriptional program. These results underscore the importance of SETD8 in CRC and represent a novel β-CATENIN -independent oncogenic consequence of APC loss.
Significance
GSK3 is thought to restrain colorectal cancer primarily by phosphorylation of β-CATENIN. We show that GSK3 also phosphorylates SETD8, preventing SETD8 activation of oncogenic programs including YAP-driven fetal-like gene expression.
... In addition, the transcript levels of genes encoding the collagens implicated in wound healing (44) such as types I, II, IV, and VI (29) were higher in the Smad4 KO colon epithelium ( Fig 4C). Because collagen in the ECM is a regulator of the various processes involved in epithelial regeneration and homeostasis (45,46,47), we probed for Col1a1, a type I collagen, by immunostaining ( Fig 4D). Type I collagen increased significantly in the Smad4 IEC−KO pericryptal epithelium after 3 d of DSS (Fig 4E), suggesting increased collagen in the epithelial ECM. ...
... The decreased Cdkn1a transcripts encoding the cell cycle inhibitor p21 (Fig S2B) further indicated the lack of proliferative arrest in the Smad4 IEC−KO colon epithelium. The DSS-induced expansion of the proliferative zone in the Smad4 KO epithelium is consistent with the epithelial injury responses that support wound healing (47). In addition, Lgr5 transcript levels were significantly higher in the DSS-treated Smad4 IEC−KO colon epithelium (Fig S2H) of Lgr5+ stem cells in the intestinal epithelial regeneration (65,66). ...
Mucosal healing is associated with better clinical outcomes in patients with inflammatory bowel disease. But the epithelial-specific contribution to mucosal healing in vivo is poorly understood. We evaluated mucosal healing in an acute dextran sulfate sodium mouse model that shows an alleviated colitis response after epithelial-specific loss of Smad4. We find that enhanced epithelial wound healing alleviates the fibrotic response. Dextran sulfate sodium caused increased mesenchymal collagen deposition—indicative of fibrosis—within a week in the WT but not in the Smad4 KO colon. The fibrotic response correlated with decreased epithelial proliferation in the WT, whereas uninterrupted proliferation and an expanded zone of proliferation were observed in the Smad4 KO colon epithelium. Furthermore, the Smad4 KO colon showed epithelial extracellular matrix alterations that promote epithelial regeneration. Our data suggest that epithelium is a key determinant of the mucosal healing response in vivo, implicating mucosal healing as a strategy against fibrosis in inflammatory bowel disease patients.
