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

Cell2location maps fine-grained cell types in spatial transcriptomics

Nature biotechnology

2022 Jan 13

Kleshchevnikov, V;Shmatko, A;Dann, E;Aivazidis, A;King, HW;Li, T;Elmentaite, R;Lomakin, A;Kedlian, V;Gayoso, A;Jain, MS;Park, JS;Ramona, L;Tuck, E;Arutyunyan, A;Vento-Tormo, R;Gerstung, M;James, L;Stegle, O;Bayraktar, OA;
PMID: 35027729 | DOI: 10.1038/s41587-021-01139-4

Spatial transcriptomic technologies promise to resolve cellular wiring diagrams of tissues in health and disease, but comprehensive mapping of cell types in situ remains a challenge. Here we present сell2location, a Bayesian model that can resolve fine-grained cell types in spatial transcriptomic data and create comprehensive cellular maps of diverse tissues. Cell2location accounts for technical sources of variation and borrows statistical strength across locations, thereby enabling the integration of single-cell and spatial transcriptomics with higher sensitivity and resolution than existing tools. We assessed cell2location in three different tissues and show improved mapping of fine-grained cell types. In the mouse brain, we discovered fine regional astrocyte subtypes across the thalamus and hypothalamus. In the human lymph node, we spatially mapped a rare pre-germinal center B cell population. In the human gut, we resolved fine immune cell populations in lymphoid follicles. Collectively, our results present сell2location as a versatile analysis tool for mapping tissue architectures in a comprehensive manner.

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.

Odontoblast death drives cell-rich zone-derived dental tissue regeneration

Bone

2021 May 18

Zhao, L;Ito, S;Arai, A;Udagawa, N;Horibe, K;Hara, M;Nishida, D;Hosoya, A;Masuko, R;Okabe, K;Shin, M;Li, X;Matsuo, K;Abe, S;Matsunaga, S;Kobayashi, Y;Kagami, H;Mizoguchi, T;
PMID: 34020080 | DOI: 10.1016/j.bone.2021.116010

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.

Arid1a-Plagl1-Hh signaling is indispensable for differentiation-associated cell cycle arrest of tooth root progenitors

Cell reports

2021 Apr 06

Du, J;Jing, J;Yuan, Y;Feng, J;Han, X;Chen, S;Li, X;Peng, W;Xu, J;Ho, TV;Jiang, X;Chai, Y;
PMID: 33826897 | DOI: 10.1016/j.celrep.2021.108964

Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.

R-SPONDIN2+ mesenchymal cells form the bud tip progenitor niche during human lung development

Developmental cell

2022 Jun 07

Hein, RFC;Wu, JH;Holloway, EM;Frum, T;Conchola, AS;Tsai, YH;Wu, A;Fine, AS;Miller, AJ;Szenker-Ravi, E;Yan, KS;Kuo, CJ;Glass, I;Reversade, B;Spence, JR;
PMID: 35679862 | DOI: 10.1016/j.devcel.2022.05.010

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Mouse Dspp frameshift model of human dentinogenesis imperfecta

Scientific reports

2021 Oct 19

Liang, T;Hu, Y;Zhang, H;Xu, Q;Smith, CE;Zhang, C;Kim, JW;Wang, SK;Saunders, TL;Lu, Y;Hu, JC;Simmer, JP;
PMID: 34667213 | DOI: 10.1038/s41598-021-00219-4

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Arid1a regulates cell cycle exit of transit-amplifying cells by inhibiting the Aurka-Cdk1 axis in mouse incisor

Development (Cambridge, England)

2021 Apr 15

Du, J;Jing, J;Chen, S;Yuan, Y;Feng, J;Ho, TV;Sehgal, P;Xu, J;Jiang, X;Chai, Y;
PMID: 33766930 | DOI: 10.1242/dev.198838

Stem cells self-renew or give rise to transit-amplifying cells (TACs) that differentiate into specific functional cell types. The fate determination of stem cells to TACs and their transition to fully differentiated progeny is precisely regulated to maintain tissue homeostasis. Arid1a, a core component of the switch/sucrose nonfermentable complex, performs epigenetic regulation of stage- and tissue-specific genes that is indispensable for stem cell homeostasis and differentiation. However, the functional mechanism of Arid1a in the fate commitment of mesenchymal stem cells (MSCs) and their progeny is not clear. Using the continuously growing adult mouse incisor model, we show that Arid1a maintains tissue homeostasis through limiting proliferation, promoting cell cycle exit and differentiation of TACs by inhibiting the Aurka-Cdk1 axis. Loss of Arid1a overactivates the Aurka-Cdk1 axis, leading to expansion of the mitotic TAC population but compromising their differentiation ability. Furthermore, the defective homeostasis after loss of Arid1a ultimately leads to reduction of the MSC population. These findings reveal the functional significance of Arid1a in regulating the fate of TACs and their interaction with MSCs to maintain tissue homeostasis.

	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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