The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon.

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Cold atmospheric plasma differentially affects cell renewal and differentiation of stem cells and APC-deficient-derived tumor cells in intestinal organoids.

Cold atmospheric plasma (CAP) treatment has been proposed as a potentially innovative therapeutic tool in the biomedical field, notably for cancer due to its proposed toxic selectivity on cancer cells versus healthy cells. In the present study, we addressed the relevance of three-dimensional organoid technology to investigate the biological effects of CAP on normal epithelial stem cells and tumor cells isolated from mouse small intestine. CAP treatment exerted dose-dependent cytotoxicity on normal organoids and induced major transcriptomic changes associated with the global response to oxidative stress, fetal-like regeneration reprogramming, and apoptosis-mediated cell death. Moreover, we explored the potential selectivity of CAP on tumor-like Apc-deficient versus normal organoids in the same genetic background. Unexpectedly, tumor organoids exhibited higher resistance to CAP treatment, correlating with higher antioxidant activity at baseline as compared to normal organoids. This pilot study suggests that the ex vivo culture system could be a relevant alternative model to further investigate translational medical applications of CAP technology.

Interleukin-6 is an activator of pituitary stem cells upon local damage, a competence quenched in the aging gland.

Stem cells in the adult pituitary are quiescent yet show acute activation upon tissue injury. The molecular mechanisms underlying this reaction are completely unknown. We applied single-cell transcriptomics to start unraveling the acute pituitary stem cell activation process as occurring upon targeted endocrine cell-ablation damage. This stem cell reaction was contrasted with the aging (middle-aged) pituitary, known to have lost damage-repair capacity. Stem cells in the aging pituitary show regressed proliferative activation upon injury and diminished in vitro organoid formation. Single-cell RNA sequencing uncovered interleukin-6 (IL-6) as being up-regulated upon damage, however only in young but not aging pituitary. Administering IL-6 to young mice promptly triggered pituitary stem cell proliferation, while blocking IL-6 or associated signaling pathways inhibited such reaction to damage. By contrast, IL-6 did not generate a pituitary stem cell activation response in aging mice, coinciding with elevated basal IL-6 levels and raised inflammatory state in the aging gland (inflammaging). Intriguingly, in vitro stem cell activation by IL-6 was discerned in organoid culture not only from young but also from aging pituitary, indicating that the aging gland's stem cells retain intrinsic activatability in vivo, likely impeded by the prevailing inflammatory tissue milieu. Importantly, IL-6 supplementation strongly enhanced the growth capability of pituitary stem cell organoids, thereby expanding their potential as an experimental model. Our study identifies IL-6 as a pituitary stem cell activator upon local damage, a competence quenched at aging, concomitant with raised IL-6/inflammatory levels in the older gland. These insights may open the way to interfering with pituitary aging.

LGR4 deficiency results in delayed puberty through impaired Wnt/ß-catenin signaling.

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/ß-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/ß-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/ß-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

LGR5 controls extracellular matrix production by stem cells in the developing intestine.

The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs.

Ex vivo Culture of Adult Mouse Antral Glands.

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker's protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016 ).

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors.

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016 ).

LGR4 and LGR5 Regulate Hair Cell Differentiation in the Sensory Epithelium of the Developing Mouse Cochlea.

In the developing cochlea, Wnt/ß-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/ß-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/ß-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/ß-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage.

Mouse fetal intestinal progenitors lining the epithelium prior to villogenesis grow as spheroids when cultured ex vivo and express the transmembrane glycoprotein Trop2 as a marker. Here, we report the characterization of Trop2-expressing cells from fetal pre-glandular stomach, growing as immortal undifferentiated spheroids, and their relationship with gastric development and regeneration. Trop2(+) cells generating gastric spheroids differed from adult glandular Lgr5(+) stem cells, but appeared highly related to fetal intestinal spheroids. Although they shared a common spheroid signature, intestinal and gastric fetal spheroid-generating cells expressed organ-specific transcription factors and were committed to intestinal and glandular gastric differentiation, respectively. Trop2 expression was transient during glandular stomach development, being lost at the onset of gland formation, whereas it persisted in the squamous forestomach. Undetectable under homeostasis, Trop2 was strongly re-expressed in glands after acute Lgr5(+) stem cell ablation or following indomethacin-induced injury. These highly proliferative reactive adult Trop2(+) cells exhibited a transcriptome displaying similarity with that of gastric embryonic Trop2(+) cells, suggesting that epithelium regeneration in adult stomach glands involves the partial re-expression of a fetal genetic program.

Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium.

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

LGR5 deficiency deregulates Wnt signaling and leads to precocious Paneth cell differentiation in the fetal intestine.

The orphan Leucine-rich repeat G protein-coupled receptor 5 (LGR5/GPR49), a target of Wnt signaling, is a marker of adult intestinal stem cells (SC). However, neither its function in the adults, nor during development of the intestine have been addressed yet. In this report, we investigated the role of LGR5 during ileal development by using LGR5 null/LacZ-NeoR knock-in mice. X-gal staining experiments showed that, after villus morphogenesis, Lgr5 expression becomes restricted to dividing cells clustered in the intervillus region and is more pronounced in the distal small intestine. At day E18.5, LGR5 deficiency leads to premature Paneth cell differentiation in the small intestine without detectable effects on differentiation of other cell lineages, nor on epithelial cell proliferation or migration. Quantitative RT-PCR experiments showed that expression from the LGR5 promoter was upregulated in LGR5-null mice, pointing to the existence of an autoregulatory negative feedback loop in intact animals. This deregulation was associated with overexpression of Wnt target genes in the intervillus epithelium. Transcriptional profiling of mutant mice ileums revealed that LGR5 function is associated with expression of SC and SC niche markers. Together, our data identify LGR5 as a negative regulator of the Wnt pathway in the developing intestine.

Mutation in the VP1-LDV motif of the murine polyomavirus affects viral infectivity and conditions virus tissue tropism in vivo.

The first contact of a virus with the host cell surface and further entry are important steps for a successful outcome of the infection process and for the virus-associated pathogenicity. We have previously shown that the entry of the murine Polyomavirus (Py) into fibroblasts is a multi-step process involving, at least, the attachment to primary sialic acids (SA)-containing cell receptors followed by post-binding interaction with secondary receptors, such as the alpha4beta1 integrin, likely through the VP1-LDV motif. Here we report on the functional role of the VP1-LDV motif in Py infectivity and in vivo virus tissue tropism. For this purpose, we have characterized a recombinant virus mutant, PyLNV, harboring a single aa substitution in this motif (D138N). Although not critical for virus viability, the D138N substitution abrogates the post-attachment Py-alpha4beta1 interaction, rendering the PyLNV mutant virus twofold less infectious than the Py wild-type (Wt) in alpha4beta1-positive fibroblasts. To study the putative role of the VP1-LDV motif in vivo, newborn C57BL/6 mice were inoculated with PyWt or PyLNV and, after six days, organs were analyzed for the presence of viral DNA. Intriguingly, PyLNV showed an altered spectrum of in vivo replication compared with PyWt, particularly in the skin and in the kidney. The implication of Py-alpha4beta1 integrin interaction in conditioning tissue-specificity of virus replication is discussed.

PARP-1 interaction with VP1 capsid protein regulates polyomavirus early gene expression.

Poly(ADP-ribose)polymerases are involved in fundamental cellular events as well as they seem to be associated to some viral infection process. In this work, the poly(ADP-ribose)polymerase-1 (PARP-1) role in the polyomavirus life cycle has been investigated. Early viral transcription was reduced by competitive inhibitors of PARPs in Swiss 3T3 cells and almost abolished in PARP-1 knockout fibroblasts and in wild-type fibroblasts when PARP-1 was silenced by RNA interference. In vivo chromatin immunoprecipitation assays showed that poly(ADP-ribosyl)ation (poly(ADP-ribose)) facilitates the release of the capsid protein viral protein 1 (VP1) from the chromatin of infecting virions. In vitro experiments demonstrated that VP1 stimulates the enzymatic activity of PARP-1 and binds non-covalently both protein-free and PARP-1-bound poly(ADP-ribose). Our studies suggest that PARP-1 promotes the complete VP1 displacement from viral DNA favouring the viral early transcription.

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding.

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.

Chromosome-protein interactions in polyomavirus virions.

In this work, we sought to determine whether the components of the murine polyomavirus capsid establish specific interactions with the minichromosome encapsidated into the mature viral particles by using the cis-diamminedichloroplatinum(II) cross-linking reagent. Our data indicated that VP1, but not minor capsid proteins, interacts with the viral genome in vivo. In addition, semiquantitative PCR assays performed on cross-linked DNA complexes revealed that VP1 binds to all regions of the viral genome but significantly more to the regulatory region. The implications of such an interaction for viral infectivity are discussed.

Role of sialic acid-containing molecules and the alpha4beta1 integrin receptor in the early steps of polyomavirus infection.

Murine polyomavirus (MPyV) infection occurs through recognition of sialic acid (SA) residues present on the host cell membrane, but the nature of the molecules involved and the exact role of this interaction in virus cell entry still need to be clarified. In this work, mutations at residues R(77) or H(298) of the MPyV VP1 protein were shown to lead to a complete loss of virus infectivity, which, however, could be restored by lipofection of virus particles into the cytoplasm of the host cells. Using virus-like particles (VLPs), it was demonstrated that the non-infectivity of these mutants was due to impaired cell entry caused by total abrogation of SA-dependent cell binding. This indicates that SA residues are essential primary cell receptors for MPyV. As the alpha4beta1 integrin has been identified recently as a cell receptor for MPyV, the relationship, if any, was investigated between SA-containing and alpha4beta1 integrin receptors. The ability of mutants R(77)Q and H(298)Q and wt VLPs to bind to cells overexpressing the alpha4beta1 integrin was studied in SA-positive (BALB/c 3T3 cells and Pro-5 cells) and SA-deficient (Pro5-derived Lec-2 cells) backgrounds. Overexpression of alpha4beta1 integrin did not restore binding of mutant VLPs in any of these cell lines or, indeed, that of wt VLPs in a SA-deficient background. Moreover, evidence is provided that overexpression of the sialylated alpha4beta1 integrin enhances wt VLP cell binding, suggesting that, in addition to its function at a post-attachment level, alpha4beta1 integrin acts also as one of the SA-containing receptors for initial cell binding.

Alpha4beta1 integrin acts as a cell receptor for murine polyomavirus at the postattachment level.

The initial interaction of murine polyomavirus (Py) with host cells occurs through direct binding of the major capsid protein VP1 with cell membrane molecules containing terminal sialic acids; however, these Py receptor molecules have not yet been identified. Analysis of the capsid protein primary sequences of all murine strains revealed the presence of integrin ligand motifs in the DE and EF loops of VP1 (LDV and DLXXL, respectively) and at the N terminus of VP2 (DGE). We show that infectivity of the Py A2 strain in mouse Swiss 3T3 fibroblasts is significantly reduced only in the presence of natural integrin ligands carrying an LDV motif or antibodies directed against the alpha4 and beta1 integrin subunits. Furthermore, we demonstrate that expression of the alpha4 subunit in the alpha4-deficient BALB/c 3T3 cells increases viral infectivity. Addition of alpha4 function-blocking antibodies, prior to or after virus adsorption, blocks this increased infectivity without affecting virus binding to cells. Taken together, these data indicate that expression of alpha4 integrin enhances permissivity to Py, probably by acting as one of the postattachment receptors.

Immunization of T-cell deficient mice against polyomavirus infection using viral pseudocapsids or temperature sensitive mutants.

A murine experimental model system aimed at developing potential vaccines to papovavirus infection in immunosuppressed individuals was explored. A VP1-pseudocapsid based on the major capsid protein of the murine polyomavirus A2 strain and a mutant, M17-pseudocapsid as well as four temperature sensitive (ts)-mutants were used as immunogens. T-cells deficient CD4-/-8-/- mice were immunized four times with each immunogen and then together with non-immunized control mice challenged with polyomavirus. In contrast to all control mice, only half of the immunized mice exhibited presence of polyoma DNA when assayed by PCR. The results indicate that pseudocapsids and ts-mutant immunization may potentially protect mice with an impaired T-cell function from polyomavirus infection.