Protein arginine methyltransferase 1 regulates cell proliferation and differentiation in adult mouse adult intestine

Cell & bioscience

2021 Jun 22

Xue, L;Bao, L;Roediger, J;Su, Y;Shi, B;Shi, YB;
PMID: 34158114 | DOI: 10.1186/s13578-021-00627-z

Adult stem cells play an essential role in adult organ physiology and tissue repair and regeneration. While much has been learnt about the property and function of various adult stem cells, the mechanisms of their development remain poorly understood in mammals. Earlier studies suggest that the formation of adult mouse intestinal stem cells takes place during the first few weeks after birth, the postembryonic period when plasma thyroid hormone (T3) levels are high. Furthermore, deficiency in T3 signaling leads to defects in adult mouse intestine, including reduced cell proliferation in the intestinal crypts, where stem cells reside. Our earlier studies have shown that protein arginine methyltransferase 1 (PRMT1), a T3 receptor coactivator, is highly expressed during intestinal maturation in mouse.We have analyzed the expression of PRMT1 by immunohistochemistry and studied the effect of tissue-specific knockout of PRMT1 in the intestinal epithelium.We show that PRMT1 is expressed highly in the proliferating transit amplifying cells and crypt base stem cells. By using a conditional knockout mouse line, we have demonstrated that the expression of PRMT1 in the intestinal epithelium is critical for the development of the adult mouse intestine. Specific removal of PRMT1 in the intestinal epithelium results in, surprisingly, more elongated adult intestinal crypts with increased cell proliferation. In addition, epithelial cell migration along the crypt-villus axis and cell death on the villus are also increased. Furthermore, there are increased Goblet cells and reduced Paneth cells in the crypt while the number of crypt base stem cells remains unchanged.Our finding that PRMT1 knockout increases cell proliferation is surprising considering the role of PRMT1 in T3-signaling and the importance of T3 for intestinal development, and suggests that PRMT1 likely regulates pathways in addition to T3-signaling to affect intestinal development and/or homeostasis, thus affecting cell proliferating and epithelial turn over in the adult.

A Glo1-Methylglyoxal Pathway that Is Perturbed in Maternal Diabetes Regulates Embryonic and Adult Neural Stem Cell Pools in Murine Offspring.

Cell Rep.

2016 Oct 18

Yang G, Cancino GI, Zahr SK, Guskjolen A, Voronova A, Gallagher D, Frankland PW, Kaplan DR, Miller FD.
PMID: 27760310 | DOI: 10.1016/j.celrep.2016.09.067

Maternal diabetes is known to adversely influence brain development in offspring. Here, we provide evidence that this involves the circulating metabolite methylglyoxal, which is increased in diabetes, and its detoxifying enzyme, glyoxalase 1 (Glo1), which when mutated is associated with neurodevelopmental disorders. Specifically, when Glo1 levels were decreased in embryonic mouse cortical neural precursor cells (NPCs), this led to premature neurogenesis and NPC depletion embryonically and long-term alterations in cortical neurons postnatally. Increased circulating maternal methylglyoxal caused similar changes in embryonic cortical precursors and neurons and long-lasting changes in cortical neurons and NPCs in adult offspring. Depletion of embryonic and adult NPCs was also observed in murine offspring exposed to a maternal diabetic environment. Thus, the Glo1-methylglyoxal pathway integrates maternal and NPC metabolism to regulate neural development, and perturbations in this pathway lead to long-lasting alterations in adult neurons and NPC pools.

Deep crypt secretory cell differentiation in the colonic epithelium is regulated by Sprouty2 and interleukin-13

Cellular and molecular gastroenterology and hepatology

2022 Nov 19

Schumacher, MA;Liu, CY;Katada, K;Thai, MH;Hsieh, JJ;Hansten, BJ;Waddell, A;Rosen, MJ;Frey, MR;
PMID: 36414210 | DOI: 10.1016/j.jcmgh.2022.11.004

Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal IL-33/IL-13 signaling loop. Here we tested the hypothesis that IL-13, induced by epithelial Sprouty2 downregulation, promotes DCS cell differentiation and function.Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL-13 and IL-13 receptor; bulk RNA sequencing and qPCR were used to quantify changes in expression of identified HDPs. Cytokine treated colonoids were assessed for DCS cells. A requirement for an IL-33/IL-13 signaling loop in the regulation of DCS cells was assessed in vivo using IL-13 null mice.Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single cell transcriptomics showed the HDP Retnlb/RELMβ is highly enriched in DCS cells. Retnlb/RELMβ expression was increased in Spry2ΔIE colons. IL-13, but not IL-33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL-33-mediated expansion of the DCS cell population in vivo was dependent on IL-13, which was predominantly expressed by type II innate lymphoid cells (ILC2s) in the colonic mucosa.Sprouty2 limits colonic DCS cell differentiation through suppression of IL-13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMβ. Loss of epithelial Sprouty2 activates ILC2s to release IL-13, promoting expansion of the DCS cell population and increased colonic RELMβ levels.

Neuron stem cell NLRP6 sustains hippocampal neurogenesis to resist stress-induced depression

Acta Pharmaceutica Sinica B

2023 Mar 01

Tang, C;Wang, Q;Shen, J;Wang, C;Ding, H;Wen, S;Yang, F;Jiao, R;Wu, X;Li, J;Kong, L;
| DOI: 10.1016/j.apsb.2023.03.010

Neurogenesis decline in hippocampal dentate gyrus (DG) participates in stress-induced depressive-like behaviors, but the underlying mechanism remains poorly understood. Here, we observed low-expression of NOD-like receptor family pyrin domain containing 6 (NLRP6) in hippocampus of stress-stimulated mice, being consistent with high corticosterone level. NLRP6 was found to be abundantly expressed in neural stem cells (NSCs) of DG. Both Nlrp6 knockout (Nlrp6−/−) and NSC-conditional Nlrp6 knockout (Nlrp6CKO) mice were susceptible to stress, being more likely to develop depressive-like behaviors. Interestingly, NLRP6 was required for NSC proliferation in sustaining hippocampal neurogenesis and reinforcing stress resilience during growing up. Nlrp6 deficiency promoted esophageal cancer-related gene 4 (ECRG4) expression and caused mitochondrial dysfunction. Corticosterone as a stress factor significantly down-regulated NLRP6 expression, damaged mitochondrial function and suppressed cell proliferation in NSCs, which were blocked by Nlrp6 overexpression. ECRG4 knockdown reversed corticosterone-induced NSC mitochondrial function and cell proliferation disorders. Pioglitazone, a well-known clinical drug, up-regulated NLRP6 expression to inhibit ECRG4 expression in its protection against corticosterone-induced NSC mitochondrial dysfunction and proliferation restriction. In conclusion, this study demonstrates that NLRP6 is essential to maintain mitochondrial homeostasis and proliferation in NSCs, and identifies NLRP6 as a promising therapeutic target for hippocampal neurogenesis decline linked to depression.

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation

Cell stem cell

2021 Jun 11

Sun, T;Annunziato, S;Bergling, S;Sheng, C;Orsini, V;Forcella, P;Pikiolek, M;Kancherla, V;Holwerda, S;Imanci, D;Wu, F;Meylan, LC;Puehringer, LF;Waldt, A;Oertli, M;Schuierer, S;Terracciano, LM;Reinker, S;Ruffner, H;Bouwmeester, T;Sailer, AW;George, E;Roma, G;de Weck, A;Piscuoglio, S;Lohmann, F;Naumann, U;Liberali, P;Cong, F;Tchorz, JS;
PMID: 34129813 | DOI: 10.1016/j.stem.2021.05.013

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/β-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/β-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of β-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/β-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/β-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Single mRNA detection of Wnt signaling pathway in the human limbus

Experimental eye research

2023 Jan 23

Bonnet, C;Ruiz, M;Gonzalez, S;Tseng, CH;Bourges, JL;Behar-Cohen, F;Deng, SX;
PMID: 36702232 | DOI: 10.1016/j.exer.2022.109337

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the cornea (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules involved in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis

Research square

2023 Jan 10

Imada, S;Shin, H;Khawaled, S;Meckelmann, S;Whittaker, C;Correa, R;Pradhan, D;Calibasi, G;Nascentes, LN;Allies, G;Wittenhofer, P;Schmitz, O;Roper, J;Vinolo, M;Cheng, CW;Tasdogan, A;Yilmaz, ÃM;
PMID: 36711807 | DOI: 10.21203/rs.3.rs-2320717/v1

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.

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

Proceedings of the National Academy of Sciences of the United States of America

2021 Jun 22

Vennekens, A;Laporte, E;Hermans, F;Cox, B;Modave, E;Janiszewski, A;Nys, C;Kobayashi, H;Malengier-Devlies, B;Chappell, J;Matthys, P;Garcia, MI;Pasque, V;Lambrechts, D;Vankelecom, H;
PMID: 34161279 | DOI: 10.1073/pnas.2100052118

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.

Tissue of Origin, but Not XCI State, Influences Germ Cell Differentiation from Human Pluripotent Stem Cells

Cells

2021 Sep 13

Chang, YW;Overeem, AW;Roelse, CM;Fan, X;Freund, C;Chuva de Sousa Lopes, SM;
PMID: 34572048 | DOI: 10.3390/cells10092400

Human pluripotent stem cells (hPSCs) are not only a promising tool to investigate differentiation to many cell types, including the germline, but are also a potential source of cells to use for regenerative medicine purposes in the future. However, current in vitro models to generate human primordial germ cell-like cells (hPGCLCs) have revealed high variability regarding differentiation efficiency depending on the hPSC lines used. Here, we investigated whether differences in X chromosome inactivation (XCI) in female hPSCs could contribute to the variability of hPGCLC differentiation efficiency during embryoid body (EB) formation. For this, we first characterized the XCI state in different hPSC lines by investigating the expression of XIST and H3K27me3, followed by differentiation and quantification of hPGCLCs. We observed that the XCI state did not influence the efficiency to differentiate to hPGCLCs; rather, hPSCs derived from cells isolated from urine showed an increased trend towards hPGCLCs differentiation compared to skin-derived hPSCs. In addition, we also characterized the XCI state in the generated hPGCLCs. Interestingly, we observed that independent of the XCI state of the hPSCs used, both hPGCLCs and soma cells in the EBs acquired XIST expression, indicative of an inactive X chromosome. In fact, culture conditions for EB formation seemed to promote XIST expression. Together, our results contribute to understanding how epigenetic properties of hPSCs influence differentiation and to optimize differentiation methods to obtain higher numbers of hPGCLCs, the first step to achieve human in vitro gametogenesis.

Capybara: A computational tool to measure cell identity and fate transitions

Cell stem cell

2022 Mar 23

Kong, W;Fu, YC;Holloway, EM;Garipler, G;Yang, X;Mazzoni, EO;Morris, SA;
PMID: 35354062 | DOI: 10.1016/j.stem.2022.03.001

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.

Vasculature atrophy causes a stiffened microenvironment that augments epidermal stem cell differentiation in aged skin

Nature Aging

2022 Jul 01

Ichijo, R;Maki, K;Kabata, M;Murata, T;Nagasaka, A;Ishihara, S;Haga, H;Honda, T;Adachi, T;Yamamoto, T;Toyoshima, F;
| DOI: 10.1038/s43587-022-00244-6

Stem cell loss causes tissue deterioration associated with aging. The accumulation of genomic and oxidative stress-induced DNA damage is an intrinsic cue for stem cell loss1 [/articles/s43587-022-00244-6#ref-CR1],2 [/articles/s43587-022-00244-6#ref-CR2]; however, whether there is an external microenvironmental cue that triggers stem cell loss remains unclear. Here we report that the involution of skin vasculature causes dermal stiffening that augments the differentiation and hemidesmosome fragility of interfollicular epidermal stem cells (IFESCs) in aged mouse skin. Aging-related IFESC dysregulation occurs in plantar and tail skin, and is correlated with prolonged calcium influx, which is contributed by the mechanoresponsive ion channel Piezo1 (ref. 3 [/articles/s43587-022-00244-6#ref-CR3]). Epidermal deletion of Piezo1 ameliorated IFESC dysregulation in aged skin, whereas Piezo1 activation augmented IFESC differentiation and hemidesmosome fragility in young mice. The dermis stiffened with age, which was accompanied by dermal vasculature atrophy. Conversely, induction of the dermal vasculature softened the dermis and ameliorated IFESC dysregulation in aged skin. Single-cell RNA sequencing of dermal fibroblasts identified an aging-associated anti-angiogenetic secretory molecule, pentraxin 3 (ref. 4 [/articles/s43587-022-00244-6#ref-CR4]), which caused dermal sclerotization and IFESC dysregulation in aged skin. Our findings show that the vasculature softens the microenvironment for stem cell maintenance and provide a potential mechanobiology-based therapeutic strategy against skin disorders in aging.

NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model

International journal of molecular sciences

2022 Mar 17

Torz, L;Niss, K;Lundh, S;Rekling, JC;Quintana, CD;Frazier, SED;Mercer, AJ;Cornea, A;Bertelsen, CV;Gerstenberg, MK;Hansen, AMK;Guldbrandt, M;Lykkesfeldt, J;John, LM;Villaescusa, JC;Petersen, N;
PMID: 35328681 | DOI: 10.3390/ijms23063260

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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