Disruption of mitochondrial complex III in cap mesenchyme but not in ureteric progenitors results in defective nephrogenesis associated with amino acid deficiency

Kidney international

2022 Mar 24

Guan, N;Kobayashi, H;Ishii, K;Davidoff, O;Sha, F;Ikizler, TA;Hao, CM;Chandel, NS;Haase, VH;
PMID: 35341793 | DOI: 10.1016/j.kint.2022.02.030

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development.

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

WNT signaling in pre-granulosa cells is required for ovarian folliculogenesis and female fertility

Development (Cambridge, England)

2021 May 01

Habara, O;Logan, CY;Kanai-Azuma, M;Nusse, R;Takase, HM;
PMID: 33914868 | DOI: 10.1242/dev.198846

In mammalian ovaries, immature oocytes are reserved in primordial follicles until their activation for potential ovulation. Precise control of primordial follicle activation (PFA) is essential for reproduction, but how this is achieved is unclear. Here, we show that canonical wingless-type MMTV integration site family (WNT) signaling is pivotal for pre-granulosa cell (pre-GC) activation during PFA. We identified several WNT ligands expressed in pre-GCs that act in an autocrine manner. Inhibition of WNT secretion from pre-GCs/GCs by conditional knockout (cKO) of the wntless (Wls) gene led to female infertility. In Wls cKO mice, GC layer thickness was greatly reduced in growing follicles, which resulted in impaired oocyte growth with both an abnormal, sustained nuclear localization of forkhead box O3 (FOXO3) and reduced phosphorylation of ribosomal protein S6 (RPS6). Constitutive stabilization of β-catenin (CTNNB1) in pre-GCs/GCs induced morphological changes of pre-GCs from a squamous into a cuboidal form, though it did not influence oocyte activation. Our results reveal that canonical WNT signaling plays a permissive role in the transition of pre-GCs to GCs, which is an essential step to support oocyte growth.

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.

Creb5 coordinates synovial joint formation with the genesis of articular cartilage

Nature communications

2022 Nov 26

Zhang, CH;Gao, Y;Hung, HH;Zhuo, Z;Grodzinsky, AJ;Lassar, AB;
PMID: 36435829 | DOI: 10.1038/s41467-022-35010-0

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

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.

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling

Nature communications

2022 Feb 07

Maimets, M;Pedersen, MT;Guiu, J;Dreier, J;Thodberg, M;Antoku, Y;Schweiger, PJ;Rib, L;Bressan, RB;Miao, Y;Garcia, KC;Sandelin, A;Serup, P;Jensen, KB;
PMID: 35132078 | DOI: 10.1038/s41467-022-28369-7

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Generation of the organotypic kidney structure by integrating pluripotent stem cell-derived renal stroma

Nature communications

2022 Feb 01

Tanigawa, S;Tanaka, E;Miike, K;Ohmori, T;Inoue, D;Cai, CL;Taguchi, A;Kobayashi, A;Nishinakamura, R;
PMID: 35105870 | DOI: 10.1038/s41467-022-28226-7

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.

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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