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The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

Huriez syndrome (HRZ, OMIM181600) is a rare genodermatosis characterized by scleroatrophic hands and feet, hypoplastic nails, palmoplantar keratoderma, and predisposition to cutaneous squamous cell carcinoma (cSCC). We report herein three HRZ families from Croatia, the Netherlands, and Germany. Deep sequencing followed by Sanger validation, confirmed the presence of germline causative SMARCAD1 heterozygous pathogenic variants. All seven HRZ patients displayed hypohidrosis, adermatoglyphia, and one patient developed cSCC at 32 years of age. Two novel monoallelic germline mutations were identified which are predicted to disrupt the first exon-intron boundary of the skin-specific SMARCAD1 isoform. On the basis of phenotypic and genotypic convergence with Adermatoglyphia (OMIM136000) and Basan syndrome (OMIM129200), our results lend credence to the notion that these three Mendelian disorders are allelic. We propose adding Huriez syndrome to the previously suggested SMARCAD syndrome designation, which was originally invoked to describe the spectrum of monogenic disorders between Adermatoglyphia and Basan syndrome. Agency for Science, Technology and Research (A*STAR) This study was supported by the Strategic Positioning Fund for Genetic Orphan Diseases, a SureKids grant by the Biomedical Research Council from the Agency for Science, Technology and Research in Singapore. Neil Rajan's research is supported by the New-castle NIHR Biomedical Research Centre (BRC).

Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1δ/ε, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of renal stem cells. Here we show that the two signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/β-catenin signalling and their genetic deletion leads to a rapid decline of renal progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss ofBmp7expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activateLef1, Fgf8andWnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and revealLgr-dependent and independent functions for these ligands during kidney formation.

// Amanda M. Goh 1,* , Yuezhen Xue 1,* , Marc Leushacke 2 , Ling Li 1 , Julin S. Wong 1 , Poh Cheang Chiam 1 , Siti Aishah Binte Rahmat 1 , Michael B. Mann 3,5 , Karen M. Mann 3,5 , Nick Barker 2 , Guillermina Lozano 4 , Tamara Terzian 4 and David P. Lane 1 1 p53 Laboratory, A*STAR, Singapore 2 Institute of Medical Biology, A*STAR, Singapore 3 Institute of Molecular and Cell Biology, A*STAR, Singapore 4 Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Cancer Research Program, Houston Methodist Research Institute, Houston, TX, USA * These authors have contributed equally to this work Correspondence to: David P. Lane, email: // Keywords : mutant p53, p53 R172H mouse model, small intestine, proliferation, Mdm2 inhibitor, Pathology Received : June 25, 2015 Accepted : July 05, 2015 Published : July 22, 2015 Abstract The tumour suppressor p53 is regulated primarily at the protein level. In normal tissues its levels are maintained at a very low level by the action of specific E3 ligases and the ubiquitin proteosome pathway. The mutant p53 protein contributes to transformation, metastasis and drug resistance. High levels of mutant p53 can be found in tumours and the accumulation of mutant p53 has previously been reported in pathologically normal cells in human skin. We show for the first time that similarly elevated levels of mutant p53 can be detected in apparently normal cells in a mutant p53 knock-in mouse model. In fact, in the small intestine, mutant p53 spontaneously accumulates in a manner dependent on gene dosage and cell type. Mutant p53 protein is regulated similarly to wild type p53, which can accumulate rapidly after induction by ionising radiation or Mdm2 inhibitors, however, the clearance of mutant p53 protein is much slower than wild type p53. The accumulation of the protein in the murine small intestine is limited to the cycling, crypt base columnar cells and proliferative zone and is lost as the cells differentiate and exit the cell cycle. Loss of Mdm2 results in even higher levels of p53 expression but p53 is still restricted to proliferating cells in the small intestine. Therefore, the small intestine of these p53 mutant mice is an experimental system in which we can dissect the molecular pathways leading to p53 accumulation, which has important implications for cancer prevention and therapy.

The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate Lgr5(+) stem cells in the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian-fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate Lgr5(+) populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult Lgr5(+) populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, Lgr5 marks stem/progenitor cells of the ovary and tubal epithelia.

The four R-spondin secreted ligands (RSPO1–RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1–3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers. Independently of the LGR4/5/6 receptors, RSPO2 acts as a direct antagonistic ligand to RNF43 and ZNRF3 during embryogenesis, and specifies the position and number of limbs that an embryo should form.

Cellular hierarchies and signals that govern stemness and differentiation of intestinal adenoma cells are not well defined. In this study, we used organotypic culture to investigate the impact of β-catenin and BMP signals in cells that form intestinal adenoma in the mouse. We found that activation of β-catenin signaling by loss of APC or transgenic induction of oncogenic mutant β-catenin (Ctnnb1(mut) ) initiates the conversion of untransformed intestinal cells to tumor cells. These tumor cells display cancer stem cell (CSC) traits such as increased expression of the CSC markers Cd133 and Cd44, a high capacity for self-renewal and unlimited proliferative potential. Subsequent inactivation of transgenic Ctnnb1(mut) results in the reversion of tumor cells to normal intestinal stem cells, which immediately reinstall the cellular hierarchy of the normal intestinal epithelium. Our data demonstrate that oncogenic activation of β-catenin signaling initiates the early steps of intestinal cellular transformation in the absence of irreversible genetic or epigenetic changes. Interestingly, we found that tumor cells in culture and in adenoma produce BMP4, which counteracts CSC-like traits by initiating irreversible cellular differentiation and loss of self-renewal capacity. We conclude that the opposition of stemness-maintaining oncogenic β-catenin signals and autocrine differentiating BMP signals within the adenoma cell provides a rationale for the formation of cellular hierarchies in intestinal adenoma and may serve to limit adenoma growth.

The extended longevity of many mammals imposes the need for an effective tissue renewal capacity within the vital organs to maintain optimal function. Resident adult stem cells are instrumental in delivering this renewal capacity by virtue of their characteristic ability to maintain themselves long-term as a population (self-renewal), whilst also supplying all functional cell-lineages of the respective tissue (multipotency). The homeostatic activity of these adult stem cell reservoirs is tailored to meet the specific renewal requirements of individual tissues through a combination of intrinsic genetic programming and local cues delivered from the surrounding environment (the niche). Considerable phenotypic diversity therefore exists between adult stem cell populations in different organs, making it a considerable challenge to identify broadly applicable markers that facilitate their identification and characterization. However, the 7-transmembrane receptor, Lgr5 has recently gained prominence as a marker of Wnt-regulated adult stem cell populations in the hair-follicle, intestine and stomach. A closely-related protein, Lgr6 marks adult stem cells responsible for fueling the renewal of the sebaceous gland and skin. The discovery of these markers has already greatly improved our understanding of stem cell biology in these rapidly renewing tissues and has major implications for the identification and isolation of human adult stem cell populations for exploitation of their regenerative medicine potential in the clinic.

In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.

Using in-vivo lineage tracing data we quantified clonal expansion as well as proliferation and differentiation of the Lgr5-positive stem cell population in pyloric gastric glands. Fitting clone expansion models, we estimated that there are five effective Lgr5-positive cells able to give rise to monoclonal glands by replacing each other following a pattern of neutral drift dynamics. This analysis is instrumental to assess stem cell performance; however, stem cell proliferation is not quantified by clone expansion analysis. We identified a suitable mathematical model to quantify proliferation and differentiation of the Lgr5-positive population. As expected for populations in steady-state, the proliferation rate of the Lgr5-positive population was equal to its rate of differentiation. This rate was significantly faster than the rate at which effective cells are replaced, estimated by modelling clone expansion/contraction. This suggests that the majority of Lgr5-positive cell divisions serve to renew epithelial cells and only few result in the effective replacement of a neighbour to effect expansion to the entire gland. The application of the model under altered situations with uncoupled differentiation and proliferation was demonstrated. This methodology represents a valuable tool for quantifying stem cell performance in homeostasis and importantly for deciphering altered stem cell behaviour in disease.

Wnt pathway deregulation is a common characteristic of many cancers. But only Colorectal Cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve a Wnt deregulation and acquire a crypt-progenitor-cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with significantly higher expression of the β-catenin binding partner E-cadherin. This increased expression is associated with a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction of E-cadherin synergised with an activating mutation of β-catenin so there was now a rapid CPC phenotype within the colon and SI. Thus there is a threshold of β-catenin that is required to drive transformation and E-cadherin can act as a buffer to prevent β-catenin accumulation.

Limited pools of resident adult stem cells are critical effectors of epithelial renewal in the intestine throughout life. Recently, significant progress has been made regarding the isolation and in vitro propagation of fetal and adult intestinal stem cells in mammals. It is now possible to generate ever-expanding, three-dimensional epithelial structures in culture that closely parallel the in vivo epithelium of the intestine. Growing such organotypic epithelium ex vivo facilitates a detailed description of endogenous niche factors or stem-cell characteristics, as they can be monitored in real time. Accordingly, this technology has already greatly contributed to our understanding of intestinal adult stem-cell renewal and differentiation. Transplanted organoids have also been proven to readily integrate into, and effect the long-term repair of, mouse colonic epithelia in vivo, establishing the organoid culture as a promising tool for adult stem cell/gene therapy. In another exciting development, novel genome-editing techniques have been successfully employed to functionally repair disease loci in cultured intestinal stem cells from human patients with a hereditary defect. It is anticipated that this technology will be instrumental in exploiting the regenerative medicine potential of human intestinal stem cells for treating human disorders in the intestinal tract and for creating near-physiological ex vivo models of human gastrointestinal disease.

SummaryMultipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5+ve cells via in vivo lineage tracing. The appearance and localization of Lgr5+ve cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle’s loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.