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.

R-spondin2 signaling is required for oocyte-driven intercellular communication and follicular growth

Cell Death Differ

2020 Apr 27

De Cian MC, Gregoire EP, Le Rolle M, Lachambre S, Mondin M, Bell S, Guigon CJ, Chassot AA, Chaboissier MC
PMID: 32341451 | DOI: 10.1038/s41418-020-0547-7

R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/?-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility

Partial male-to-female reprogramming of mouse fetal testis by sertoli cell ablation

Development (Cambridge, England)

2023 Jun 28

Imaimatsu, K;Hiramatsu, R;Tomita, A;Itabashi, H;Kanai, Y;
PMID: 37376880 | DOI: 10.1242/dev.201660

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3βHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids

JCI insight

2023 Feb 23

Childs, CJ;Holloway, EM;Sweet, CW;Tsai, YH;Wu, A;Vallie, A;Eiken, MK;Capeling, MM;Zwick, RK;Palikuqi, B;Trentesaux, C;Wu, JH;Pellon-Cardenas, O;Zhang, CJ;Glass, IA;Loebel, C;Yu, Q;Camp, JG;Sexton, JZ;Klein, OD;Verzi, MP;Spence, JR;
PMID: 36821371 | DOI: 10.1172/jci.insight.165566

Epithelial organoids derived from intestinal tissue, called 'enteroids', recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Single-cell RNA sequencing of human nail unit defines RSPO4 onychofibroblasts and SPINK6 nail epithelium

Communications biology

2021 Jun 07

Kim, HJ;Shim, JH;Park, JH;Shin, HT;Shim, JS;Jang, KT;Park, WY;Lee, KH;Kwon, EJ;Jang, HS;Yang, H;Lee, JH;Yang, JM;Lee, D;
PMID: 34099859 | DOI: 10.1038/s42003-021-02223-w

Research on human nail tissue has been limited by the restricted access to fresh specimen. Here, we studied transcriptome profiles of human nail units using polydactyly specimens. Single-cell RNAseq with 11,541 cells from 4 extra digits revealed nail-specific mesenchymal and epithelial cell populations, characterized by RSPO4 (major gene in congenital anonychia) and SPINK6, respectively. In situ RNA hybridization demonstrated the localization of RSPO4, MSX1 and WIF1 in onychofibroblasts suggesting the activation of WNT signaling. BMP-5 was also expressed in onychofibroblasts implicating the contribution of BMP signaling. SPINK6 expression distinguished the nail-specific keratinocytes from epidermal keratinocytes. RSPO4+ onychofibroblasts were distributed at close proximity with LGR6+ nail matrix, leading to WNT/β-catenin activation. In addition, we demonstrated RSPO4 was overexpressed in the fibroblasts of onychomatricoma and LGR6 was highly expressed at the basal layer of the overlying epithelial component, suggesting that onychofibroblasts may play an important role in the pathogenesis of onychomatricoma.

Single-cell roadmap of human gonadal development

Nature

2022 Jul 01

Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Opposing effects of Wnt/β-catenin signaling on epithelial and mesenchymal cell fate in the developing cochlea

Development (Cambridge, England)

2021 Jun 01

Billings, SE;Myers, NM;Quiruz, L;Cheng, AG;
PMID: 34061174 | DOI: 10.1242/dev.199091

During embryonic development, the otic epithelium and surrounding periotic mesenchymal cells originate from distinct lineages and coordinate to form the mammalian cochlea. Epithelial sensory precursors within the cochlear duct first undergo terminal mitosis before differentiating into sensory and non-sensory cells. In parallel, periotic mesenchymal cells differentiate to shape the lateral wall, modiolus and pericochlear spaces. Previously, Wnt activation was shown to promote proliferation and differentiation of both otic epithelial and mesenchymal cells. Here, we fate-mapped Wnt-responsive epithelial and mesenchymal cells in mice and found that Wnt activation resulted in opposing cell fates. In the post-mitotic cochlear epithelium, Wnt activation via β-catenin stabilization induced clusters of proliferative cells that dedifferentiated and lost epithelial characteristics. In contrast, Wnt-activated periotic mesenchyme formed ectopic pericochlear spaces and cell clusters showing a loss of mesenchymal and gain of epithelial features. Finally, clonal analyses via multi-colored fate-mapping showed that Wnt-activated epithelial cells proliferated and formed clonal colonies, whereas Wnt-activated mesenchymal cells assembled as aggregates of mitotically quiescent cells. Together, we show that Wnt activation drives transition between epithelial and mesenchymal states in a cell type-dependent manner.

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries

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

2021 May 18

Meinsohn, MC;Saatcioglu, HD;Wei, L;Li, Y;Horn, H;Chauvin, M;Kano, M;Nguyen, NMP;Nagykery, N;Kashiwagi, A;Samore, WR;Wang, D;Oliva, E;Gao, G;Morris, ME;Donahoe, PK;Pépin, D;
PMID: 33980714 | DOI: 10.1073/pnas.2100920118

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.

Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

Cell death & disease

2022 Feb 21

Walter, RJ;Sonnentag, SJ;Munoz-Sagredo, L;Merkel, M;Richert, L;Bunert, F;Heneka, YM;Loustau, T;Hodder, M;Ridgway, RA;Sansom, OJ;Mely, Y;Rothbauer, U;Schmitt, M;Orian-Rousseau, V;
PMID: 35190527 | DOI: 10.1038/s41419-022-04607-0

Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44fl/fl;VillinCreERT2 mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration.

New Stable Cell Lines Derived from the Proximal and Distal Intestine of Rainbow Trout (Oncorhynchus mykiss) Retain Several Properties Observed In Vivo

Cells

2021 Jun 19

Pasquariello, R;Verdile, N;Pavlovic, R;Panseri, S;Schirmer, K;Brevini, TAL;Gandolfi, F;
PMID: 34205481 | DOI: 10.3390/cells10061555

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

PDGFRα-induced stromal maturation is required to restrain postnatal intestinal epithelial stemness and promote defense mechanisms

Cell stem cell

2022 May 05

Jacob, JM;Di Carlo, SE;Stzepourginski, I;Lepelletier, A;Ndiaye, PD;Varet, H;Legendre, R;Kornobis, E;Benabid, A;Nigro, G;Peduto, L;
PMID: 35523143 | DOI: 10.1016/j.stem.2022.04.005

After birth, the intestine undergoes major changes to shift from an immature proliferative state to a functional intestinal barrier. By combining inducible lineage tracing and transcriptomics in mouse models, we identify a prodifferentiation PDGFRαHigh intestinal stromal lineage originating from postnatal LTβR+ perivascular stromal progenitors. The genetic blockage of this lineage increased the intestinal stem cell pool while decreasing epithelial and immune maturation at weaning age, leading to reduced postnatal growth and dysregulated repair responses. Ablating PDGFRα in the LTBR stromal lineage demonstrates that PDGFRα has a major impact on the lineage fate and function, inducing a transcriptomic switch from prostemness genes, such as Rspo3 and Grem1, to prodifferentiation factors, including BMPs, retinoic acid, and laminins, and on spatial organization within the crypt-villus and repair responses. Our results show that the PDGFRα-induced transcriptomic switch in intestinal stromal cells is required in the first weeks after birth to coordinate postnatal intestinal maturation and function.

Immunohistochemical Study of a Correlation between Pemphigus Vulgaris Activity Score and Stem Cell Control

The Egyptian Journal of Hospital Medicine

2021 Apr 01

Bazid, H;Seleit, I;Abo Hegazy, S;Samaka, R;
| DOI: 10.21608/ejhm.2021.165168

BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease. PV autoantibodies disrupt desmosomal adhesion and cause acantholysis. Previous researches have shown that stem cells are indirectly involved as a result of desmoglein deficiency. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) as a follicular stem cell marker was evaluated in aim to correlate its intensity of expression with disease severity. OBJECTIVE: To correlate LGR5 intensity of expression with disease severity. PATIENT AND METHODS: This prospective cross sectional study was carried out on 20 PV patients. Patients were subjected to complete history taking, general, dermatological examination and assessment of disease severity by the Pemphigus Vulgaris Activity Score (PVAS), histopathological and immunohistochemical expression of LGR5 were done. RESULTS: All studied cases showed positive cytoplasmic basal LGR5 expression in patchy manner. 75% of cases had mild intensity of expression, 15% had moderate intensity and their Histo (H) score ranged from 50-130 with Mean ±SD 110±18.92. There were no significant correlation between PVAS scores ''skin, mucosa and total involvement'' and H score of LGR5 expression. CONCLUSION: The current study could shed a new light on the disease and its correlation with stem cells, LGR5 as a stem cell marker could be related to the healing process in PV. However, it didn't correlate PVAS scores either in skin, mucosa or total involvement.

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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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