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.

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.

Smooth muscle-specific MMP17 (MT4-MMP) regulates the intestinal stem cell niche and regeneration after damage

Nature communications

2021 Nov 18

Martín-Alonso, M;Iqbal, S;Vornewald, PM;Lindholm, HT;Damen, MJ;Martínez, F;Hoel, S;Díez-Sánchez, A;Altelaar, M;Katajisto, P;Arroyo, AG;Oudhoff, MJ;
PMID: 34795242 | DOI: 10.1038/s41467-021-26904-6

Smooth muscle is an essential component of the intestine, both to maintain its structure and produce peristaltic and segmentation movements. However, very little is known about other putative roles that smooth muscle cells may have. Here, we show that smooth muscle cells may be the dominant suppliers of BMP antagonists, which are niche factors essential for intestinal stem cell maintenance. Furthermore, muscle-derived factors render epithelium reparative and fetal-like, which includes heightened YAP activity. Mechanistically, we find that the membrane-bound matrix metalloproteinase MMP17, which is exclusively expressed by smooth muscle cells, is required for intestinal epithelial repair after inflammation- or irradiation-induced injury. Furthermore, we propose that MMP17 affects intestinal epithelial reprogramming after damage indirectly by cleaving diffusible factor(s) such as the matricellular protein PERIOSTIN. Together, we identify an important signaling axis that establishes a role for smooth muscle cells as modulators of intestinal epithelial regeneration and the intestinal stem cell niche.

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.

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.

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.

Charting human development using a multi-endodermal organ atlas and organoid models

Cell

2021 May 17

Yu, Q;Kilik, U;Holloway, EM;Tsai, YH;Harmel, C;Wu, A;Wu, JH;Czerwinski, M;Childs, CJ;He, Z;Capeling, MM;Huang, S;Glass, IA;Higgins, PDR;Treutlein, B;Spence, JR;Camp, JG;
PMID: 34019796 | DOI: 10.1016/j.cell.2021.04.028

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

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

Glucagon-like peptide-2 stimulates S-phase entry of intestinal Lgr5+ stem cells

Cellular and molecular gastroenterology and hepatology

2022 Feb 23

Chen, ME;Naeini, SM;Srikrishnaraj, A;Drucker, DJ;Fesler, Z;Brubaker, PL;
PMID: 35218981 | DOI: 10.1016/j.jcmgh.2022.02.011

Leucine-rich repeat-containing G-protein coupled receptor-5 (Lgr5)+/olfactomedin-4 (Olfm4)+ intestinal stem cells (ISCs) in the crypt-base are crucial for homeostatic maintenance of the epithelium. The gut hormone, glucagon-like peptide-21-33 (GLP-2), stimulates intestinal proliferation and growth; however, the actions of GLP-2 on the Lgr5+ ISCs remain unclear. The aim of this study was to determine whether and how GLP-2 regulates Lgr5+ ISC cell cycle dynamics and number.Lgr5-eGFP-IRES-creERT2 mice were acutely administered human Gly2-GLP-2, or the GLP-2 receptor antagonist, GLP-23-33. Intestinal epithelial-insulin-like growth factor-1 receptor knockout and control mice were treated chronically with hGly2-GLP-2. Cell cycle parameters were determined by EdU, BrdU, Ki67 and phosphohistone-3 labeling and cell cycle gene expression.Acute hGly2-GLP-2 treatment increased the proportion of eGFP+EdU+/OLFM4+EdU+ cells by 11-22% (p<0.05), without affecting other cell cycle markers. hGly2-GLP-2 treatment also increased the ratio of eGFP+ cells in early-to-late S-phase by 97% (p<0.001), and increased the proportion of eGFP+ cells entering S-phase by 218% (p<0.001). hGly2-GLP-2 treatment induced jejunal expression of genes involved in cell cycle regulation (p<0.05), and increased expression of Mcm3 in the Lgr5-expressing cells by 122% (p<0.05). Conversely. GLP-23-33 reduced the proportion of eGFP+EdU+ cells by 27% (p<0.05), as well as the expression of jejunal cell cycle genes (p<0.05). Finally, chronic hGly2-GLP-2 treatment increased the number of OLFM4+ cells/crypt (p<0.05), in an intestinal epithelial insulin-like growth factor-1 receptor-dependent manner.These findings expand the actions of GLP-2 to encompass acute stimulation of Lgr5+ ISC S-phase entry through the GLP-2R, and chronic induction of Lgr5+ ISC expansion through downstream intestinal insulin-like growth factor-1 signaling.

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.

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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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