Lgr5(+) gastric stem cells divide symmetrically to effect epithelial homeostasis in the pylorus

SummaryThe pyloric epithelium continuously self-renews throughout life, driven by limited reservoirs of resident Lgr5+ adult stem cells. Here, we characterize the population dynamics of these stem cells during epithelial homeostasis. Using a clonal fate-mapping strategy, we demonstrate that multiple Lgr5+ cells routinely contribute to epithelial renewal in the pyloric gland and, similar to what was previously observed in the intestine, a balanced homeostasis of the glandular epithelium and stem cell pools is predominantly achieved via neutral competition between symmetrically dividing Lgr5+ stem cells. Additionally, we document a lateral expansion of stem cell clones via gland fission under nondamage conditions. These findings represent a major advance in our basic understanding of tissue homeostasis in the stomach and form the foundation for identifying altered stem cell behavior during gastric disease

E-cadherin can limit the transforming properties of activating β-catenin mutations

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

Funktionelle Charakterisierung von Genen, die auf die intestinale Tumor Progression Einfluß nehmen

The inner surface of the intestine is covered by a single epithelial cell layer that is subdivided into crypt and villus domains. The mammalian intestinal epithelium constantly renews every 4 to 6 days via stem cells that reside at the bottom of intestinal crypts, while organ size and cellular composition remain steady throughout adulthood. Multiple signaling pathways interact to control intestinal stem cell renewal and tissue homeostasis, and their deregulation can cause tumor formation and progression. To identify novel genes and signaling pathways involved in intestinal tumor progression, I assessed the function of a shortlist of 364 metastasis candidate genes in intestinal cells. To this end, these genes were analyzed in a loss-of-function phenotypic screen in mesenchymal SW480 human colon cancer cells. I screened for genes whose inactivation promoted both an epithelial cell morphology and localization of the epithelial cell adhesion molecule E-cadherin to cell-cell contacts. Among 18 positively tested candidate genes, I identified FGF9 and subsequently established novel roles for FGF signals in transformed and untransformed intestinal cells. In colon cancer cells, silencing of FGF9 or inhibition of FGF receptors induced an epithelial cell morphology and blocked cell motility. These effects were transduced via the MAP kinase pathway, regardless of the existence of an activating KRAS mutation in these colon cancer cells. In line with a proposed function of FGF9 in intestinal tumor cells, I found generalized expression of FGF9 in the tumor epithelium of a subgroup of colon carcinomas. In addition, I identified Fgf9 specifically expressed in Paneth cells of the untransformed mouse small intestine. I therefore also investigated functions of FGF9 in the normal intestine. Using organotypic culture of primary intestine, I found FGF9 to promote intestinal stem cell proliferation leading to an increased number of intestinal stem cells and crypt domains. On the other hand, interference with Fgf9 or general inhibition of FGF receptors resulted in loss of stem cell proliferation, crypt degeneration, and biased specification of intestinal progenitors towards the absorptive cell lineage. These results implicate FGF9 as a growth factor that regulates intestinal stem cell maintenance. The finding of specific expression in Paneth cells that intermingle with stem cells suggests that FGF9 is among the growth factors that constitute the epithelial stem cell niche. Taken together, my studies revealed different functions for FGF signals in the normal and transformed intestinal cells. Interestingly, I found increased FGF9 expression in a subgroup of colon cancer patients, which negatively correlated with patient’s survival. FGF9 could thus have functional roles in colon cancer by regulating tumor cell morphology and/or by regulating the tumor stem cell pool.Die Innenseite des Darms ist mit einer einzelligen Epithelschicht ausgekleidet, die in Krypten und Villus-Domänen unterteilt ist. Das intestinale Epithel eines Säugetiers erneuert sich kontinuierlich innerhalb von 4 bis 6 Tagen. Die Erneuerung geht von Stammzellen aus, die sich im unteren Teil der Krypten-Domäne befinden. Die Organgröße, sowie die zelluläre Zusammensetzung, bleiben während des gesamten Erwachsendaseins konstant. Eine Vielzahl von Signalwegen interagiert miteinander, um Stammzellerhalt und Gewebehomöostase zu gewährleisten. Eine fehlerhafte Regulierung dieser Signalkaskaden hingegen kann Darmkrebsentstehung bzw. -progression verursachen. Zur Identifizierung neuartiger Gene und Signalwege, die an der Darmkrebsprogression beteiligt sind, habe ich die Funktion von 364 potentiellen Metastasierungsgenen untersucht. Hierzu habe ich eine Funktionsverlust-Phänotyp-Analyse dieser Gene in mesenchymalen SW480 humanen Kolonkarzinom-Zellen durchgeführt. Ich habe die Gene selektiert, deren Inaktivierung eine epitheliale Zellmorphologie bzw. eine membranständige Lokalisierung des Zelladhäsionmolekül E-cadherin in SW480 Zellen hervorgerufen hat. Eines von 18 positiv getesteten Genen war das FGF9-Gen. Im weiteren Verlauf meiner Arbeit habe ich bisher unbekannte Funktionen der FGF-Signale in transformierten und nicht-transformierten intestinalen Zellen aufgedeckt. In Kolonkarzinom-Zellen führte die Hemmung von FGF9 bzw. die Inhibition der FGF- Rezeptoren zu einer epithelialen Zellmorphologie und zur Behinderung von Zellmotilität. Diese Effekte werden durch den MAP-Kinase Signalweg übermittelt, unabhängig von der in dieser Kolonkarzinom Zelllinie vorhandenen aktivierenden KRAS Mutation. Übereinstimmend mit einer vermuteten Funktion von FGF9 in Darmtumoren konnte ich eine erhöhte FGF9-Expression in einigen Kolonkarzinomen nachweisen. Desweiteren habe ich eine spezifische FGF9-Expression in den Paneth-Zellen des Dünndarms von Mäusen detektiert. Da dieses Expressionsmuster auf eine FGF9 Signalaktivität im Normalgewebe schließen lässt, habe ich potentielle Funktionen von FGF9 im Normaldarmepithel untersucht. Mithilfe der organotypischen Primärkultivierung von intestinalem Gewebe konnte ich zeigen, dass FGF9 die intestinale Stammzell-Proliferation anregt, wodurch die Anzahl der Stammzellen bzw. Krypten-Domänen ansteigt. Andererseits führte die Hemmung von Fgf9 oder die Inhibition der FGF- Rezeptoren zur Abnahme der Stammzell-Proliferation bzw. zum Kryptenabbau sowie zu einer Verschiebung der intestinalen Zelltyp-Spezifizierung zu Gunsten der absorbierenden Darmzellen. Diese Ergebnisse identifizieren FGF9 als Wachstumsfaktor, der den Erhalt von intestinalen Stammzellen mitbestimmt. Aufgrund der spezifischen Expression in Paneth-Zellen ist anzunehmen, dass FGF9 zu den Faktoren gehört, die die epitheliale Stammzell-Nische bilden. Zusammengefasst haben meine Untersuchungen verschiedene Funktionen der FGF- Signale in normalen und transformierten Darmzellen aufgedeckt. Interessanterweise zeigte sich bei einer Darmkrebs-Patientengruppe mit erhöhter FGF9 Expression eine im Durchschnitt geringere Lebenserwartung. Dieses Ergebnis legt die Vermutung nahe, dass FGF9 Darmkrebs sowohl durch die Regulierung der Tumor-Zellmorphologie als auch durch Einflussnahme auf die Anzahl der Tumor-Stammzellen funktional beeinflussen kann

An RNA interference phenotypic screen identifies a role for FGF signals in colon cancer progression

In tumor cells, stepwise oncogenic deregulation of signaling cascades induces alterations of cellular morphology and promotes the acquisition of malignant traits. Here, we identified a set of 21 genes, including FGF9, as determinants of tumor cell morphology by an RNA interference phenotypic screen in SW480 colon cancer cells. Using a panel of small molecular inhibitors, we subsequently established phenotypic effects, downstream signaling cascades, and associated gene expression signatures of FGF receptor signals. We found that inhibition of FGF signals induces epithelial cell adhesion and loss of motility in colon cancer cells. These effects are mediated via the mitogen-activated protein kinase (MAPK) and Rho GTPase cascades. In agreement with these findings, inhibition of the MEK1/2 or JNK cascades, but not of the PI3K-AKT signaling axis also induced epithelial cell morphology. Finally, we found that expression of FGF9 was strong in a subset of advanced colon cancers, and overexpression negatively correlated with patients' survival. Our functional and expression analyses suggest that FGF receptor signals can contribute to colon cancer progression

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors

During kidney development, WNT/β-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here we show in mice that the 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 nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, 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 reveal Lgr-dependent and independent functions for these ligands during kidney formation

Lgr5+ve Stem/Progenitor Cells Contribute to Nephron Formation during Kidney Development

Multipotent 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

MCC is a centrosomal protein that relocalizes to non-centrosomal apical sites during intestinal cell differentiation

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.Agency for Science, Technology and Research (A*STAR)Published versionThis work was funded by the Institute of Medical Biology [Agency for Science, Technology and Research (A*STAR), Singapore] as well as start-up funding provided by the Lee Kong Chian School of Medicine, Nanyang Technological University and the Ministry of Education (MOE), Singapore [Continuation Grant – Endodermal Development and Differentiation (EDD) Lab] to N.R.D. L.B.T. was initially funded by the Singapore International Graduate Award (SINGA), A*STAR Graduate Academy (A*GA). B.A.L. was funded by a Canadian Institute of Health Research postdoctoral fellowship

Huriez syndrome: additional pathogenic variants supporting allelism to SMARCAD syndrome

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)

Erratum to: RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6 (Nature, (2018), 557, 7706, (564-569), 10.1038/s41586-018-0118-y)

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

RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6

The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling(1-3). 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