Marc Leushacke

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 Henles 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.

Lgr5 and Lgr6 as markers to study adult stem cell roles in self-renewal and cancer

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.

Ex vivo culture of the intestinal epithelium: strategies and applications

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.

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

The 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.

Lgr5-expressing chief cells drive epithelial regeneration and cancer in the oxyntic stomach

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.

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

Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of the 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 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 higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.

Wnt and BMP signals control intestinal adenoma cell fates

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 (Ctnnb1mut) 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 Ctnnb1mut 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.

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 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.

Quantifying Lgr5-positive stem cell behaviour in the pyloric epithelium.

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.

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

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.