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• W4362651014 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The origin and differentiation mechanism of articular chondrocytes remain poorly understood. Broadly, the difference in developmental mechanisms of articular and growth-plate cartilage is still less elucidated. Here, we identified that the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) is a crucial regulator of articular, but not growth-plate, chondrocyte differentiation during development. At the early stage of mouse knee development (embryonic day 13.5), NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With development, NFATc1-expressing cells generated almost all articular chondrocytes but not chondrocytes in limb growth-plate primordium. NFATc1-expressing cells displayed prominent capacities for colony formation and multipotent differentiation. Transcriptome analyses revealed a set of characteristic genes in NFATc1-enriched articular cartilage progenitors. Strikingly, the expression of NFATc1 was diminished with articular chondrocyte differentiation, and suppressing NFATc1 expression in articular cartilage progenitors was sufficient to induce spontaneous chondrogenesis while overexpressing NFATc1 suppresses chondrogenesis. Mechanistically, NFATc1 negatively regulated the transcriptional activity of the Col2a1 gene. Thus, our results reveal that NFATc1 characterizes articular, but not growth-plate, cartilage progenitors during development and negatively determines articular chondrocyte differentiation at least partly through regulating COL2A1 gene transcription. Editor's evaluation NFATc1 was known as a crucial regulator in osteoclast differentiation. The current study presented surprising and novel findings, showing the specific expression of NFATc1 in articular cartilage and its function in cartilage biology. This is a significant discovery since it will help us understand the regulatory mechanism of articular chondrocyte differentiation and the development of osteoarthritis disease. https://doi.org/10.7554/eLife.81569.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Within the body are about 300 joints connecting bones together. Many factors – including trauma, inflammation, aging, and genetic changes – can affect the cushion tissue covering the end of the bones in these joints known as articular cartilage. This can lead to diseases such as osteoarthritis which cause chronic pain, and in some cases disability. To treat such conditions, it is essential to know how cells in the articular cartilage are formed during development. In the embryo, most cells come from groups of progenitor cells that are programmed to produce specific types of tissue. But which progenitor cells are responsible for producing the main cells in articular cartilage, chondrocytes, and the mechanisms that govern this transformation are poorly understood. In 2016, a group of researchers found that the gene for the protein NFATc1, which is important for building bone, is also expressed in a group of progenitor cells at the site where ligaments insert into bone in mice. Inactivation of NFATc1 in these progenitor cells has also been shown to cause abnormal cartilage to form, a condition termed osteochondromas. Building on this work, Zhang, Wang et al. – including some of the researchers involved in the 2016 study – set out to find whether NFATc1 is also involved in the normal development of articular chondrocytes. To investigate, the team used genetically modified mice in which any cells with NFATc1 also had a green fluorescent protein, and tracked these cells and their progeny over the course of joint development. This led them to discover a group of NFATc1-containing progenitor cells that gave rise to almost all articular chondrocytes in the knee joint. Further experiments revealed that when NFATc1 was removed, this made the progenitors become articular chondrocytes very quickly. In contrast, when the cells had excess amounts of the protein, the formation of articular chondrocytes was significantly reduced. This suggests that the level of NFATc1 governs when progenitors develop into articular chondrocytes. These findings have provided a way to track the progenitors of articular chondrocytes throughout development and study how articular cartilage is formed. In the future, this work could help researchers develop treatment strategies for osteoarthritis and other cartilage-based diseases. However, before this can happen, further work is needed to confirm that the effects observed in this study also relate to humans. Introduction The basic mechanism underlying articular cartilage development, particularly the origin and differentiation of articular chondrocytes, remains poorly understood. It is well appreciated that synovial joint tissues, including the articular cartilage, originate from a distinct group of progenitors from those that generate the limb primary cartilaginous anlagen Koyama et al., 2008; Chijimatsu and Saito, 2019. GDF5 is one of the genes widely used for tracking synovial joint and articular cartilage development Decker, 2017. Using reporter mice, multiple groups have displayed that Gdf5-expressing cell lineages form almost all articular chondrocytes Koyama et al., 2008; Rountree et al., 2004; Shwartz et al., 2016; Decker et al., 2017. As GDF5 is expressed in both interzone cells and its flanking cells at the early stage of joint morphogenesis, current results cannot discriminate which site is the origin of articular chondrocytes. Also, GDF5 expression is greatly diminished at the late stage of embryonic development and almost undetectable in articular cartilage in neonatal mice Decker, 2017. Thus, GDF5 cannot be used to track articular cartilage progenitors postnatally. PRG4 is an articular cartilage progenitor marker in the late stage of synovial joint development Chijimatsu and Saito, 2019; Chagin and Medvedeva, 2017. This gene encodes lubricin, a major component of synovial fluid and responsible for joint lubricity Coles et al., 2010. PRG4 is detected from the stage of joint cavitation and is predominantly expressed in the surficial layer of developed articular cartilage Rhee et al., 2005. Several studies exploited Prg4CreERT2 reporter mice to track postnatal articular cartilage development and identified this gene as a marker for postnatal and adult articular cartilage progenitors Decker et al., 2017; Kozhemyakina et al., 2015. Since PRG4 starts to predominantly express and function at the late stage of articular cartilage development, it does not label the primary progenitors of articular cartilage. Several other molecules, such as Sox9, Dkk3, and Tgfbr2, were also utilized to track articular cartilage and synovial joint development Shwartz et al., 2016; Decker et al., 2017; Li et al., 2013, but none of these molecules have been shown to specifically and constantly label articular cartilage progenitors and to be able to distinguish the origin of articular chondrocytes. In addition to the origin of articular chondrocytes, molecular mechanisms regulating articular chondrocyte differentiation remains largely unknown. In particular, the transcriptional regulation of articular chondrocyte differentiation is far from clear. SOX9 is essential in multiple steps of chondrogenesis, but it was originally and mainly studied in growth-plate chondrocytes Lefebvre and Dvir-Ginzberg, 2017. Although SOX9 is also expressed in articular cartilage and is essential for maintaining adult articular cartilage homeostasis Haseeb et al., 2021, its detailed functions and mechanisms in articular cartilage development remain to be elucidated. Also, SOX9 starts to express in mesenchymal cells from the very early stage of limb development before the cartilage template formation and it alone is not sufficient to induce chondrogenesis Lefebvre and Dvir-Ginzberg, 2017; Akiyama et al., 2005. Therefore, the identification of a core transcriptional regulator of articular chondrocyte differentiation is paramount for understanding the basic mechanism of articular cartilage development and exploring new strategies for treating disorders of articular cartilage. The nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) is one of the five members of the NFAT family, which share a similar DNA binding domain of approximately 300 amino acid residues Hogan et al., 2003; Vaeth and Feske, 2018. NFAT signaling plays a broad function in various physiological and pathological processes, including immune cell differentiation and functions, cardiac valve development, and cancer progression and metastasis Hogan et al., 2003; Mancini and Toker, 2009. In the skeletal system, NFATc1 is critical for osteoclast differentiation and functions Aliprantis et al., 2008; Takayanagi, 2007 and is also involved in osteoblast differentiation by cooperating with the Osterix gene Koga et al., 2005. Intriguingly, NFATc1 expression was found in the superficial layers of articular cartilage as well and decreased in human osteoarthritic cartilage Greenblatt et al., 2013. Following these studies, we recently identified a function of NFATc1 in restricting osteochondroma formation from entheseal progenitors Ge et al., 2016, revealing that NFATc1 is a suppressor of chondrogenesis in these cells. In this study, we unexpectedly found that NFATc1 constantly labels articular cartilage progenitors throughout embryonic development and postnatal growth. The expression of NFATc1 is diminished with articular chondrocyte differentiation, and suppression of NFATc1 in articular cartilage progenitors is sufficient to induce spontaneous chondrocyte differentiation through regulating the transcriptional activity of the Col2a1 gene. These findings provide novel insights into the identity and origin of articular cartilage progenitors and identify a fundamental function of NFATc1 in determining physiological articular chondrocyte differentiation. Results Articular cartilage is derived from NFATc1-expressing progenitors Following our previous discovery that NFATc1 identifies entheseal progenitors at the site of ligaments inserted onto the bone Ge et al., 2016, we unexpectedly found that in Nfatc1Cre;Rosa26mTmG/+ dual-fluorescence reporter mice, the majority of articular chondrocytes expressed green fluorescence protein (GFP) at 8 weeks of age [Figure 1(A and B), 90.55 ± 6.38%, n=5 mice]. As in this genetic reporter mouse line, both Nfatc1-expressing cells and their progenies express GFP, this finding suggests that articular chondrocytes were either expressing Nfatc1 or derived from Nfatc1-expressing progenitors. To clarify the expression pattern of NFATc1 during articular cartilage development, we mapped GFP+ cells in Nfatc1Cre;Rosa26mTmG/+ mice at the early stage of knee joint morphogenesis (E13.5), postnatal day 0 (P0), and 2 weeks of age [Figure 1(A)]. At E13.5, GFP+ cells were mainly localized to the flanking region of the joint interzone with only sporadic distribution in the interzone site. We further examined the expression of NFATc1 at this stage by crossing the tamoxifen-induced Nfatc1CreERT2 mouse line with Rosa26mTmG/+ mice to generate Nfatc1CreERT2;Rosa26mTmG/+ reporter mice, in which the real-time expression of NFATc1 could be reflected by GFP shortly after tamoxifen pulse. The localization of Nfatc1-expressing cells surrounding the joint interzone was verified after administering tamoxifen to Nfatc1CreERT2;Rosa26mTmG/+ mice at E11.5 and sampling at E13.5 [Figure 1—figure supplement 1A]. Figure 1 with 1 supplement see all Download asset Open asset Articular cartilage is derived from NFATc1-expressing progenitors. (A) Confocal microscopy images showing the distribution of GFP+ cells during articular cartilage development at the knee of Nfatc1Cre;Rosa26mTmG/+ mice (n=5 animals for each age, two knee joints per animal). Arrow indicating the main location of GFP+ cells at the knee at embryonic day 13.5 (E13.5). P0, postnatal day 0. (B) Quantification of GFP+ cells in the articular cartilage of Nfatc1Cre;Rosa26mTmG/+ mouse knee at 8 weeks of age (n=5 animals, one knee joint per animal). AC, articular cartilage. (C) Representative confocal images demonstrating the distribution of RFP+ cells in the articular cartilage at 2 weeks and 8 weeks of age in Nfatc1CreERT2;Rosa26RFP/+ mice 48 hrs after tamoxifen pulse for 5 consecutive days (n=3 mice for each age, two knee joints per animal). (D) Quantification of RFP+ cells in the articular cartilage of Nfatc1CreERT2;Rosa26RFP/+ mouse knee at 2 weeks and 8 weeks of age (n=3 mice for each age, one knee joint per animal). (E) Immunohistochemistry detecting the expression of NFATc1 during mouse articular cartilage development (n=3 mice for each age, two knee joints per animal). Data are mean ± SD of results from five or three animals; scale bars, 200 μm except for the right three images in (E), 50 μm. Figure 1—source data 1 Quantification data for GFP+ or RFP+ cells in articular cartilage. For Figure 1B Quantification of GFP+ cells in articular cartilage of Nfatc1Cre;Rosa26mTmG/+ mouse knee at 8 weeks of age (%); Figure 1D Quantification of RFP+ cells in articular cartilage of Nfatc1CreERT2;Rosa26RFP/+ mouse knee at 2 weeks and 8 weeks of age after tamoxifen pulse for 5 consecutive days (%). https://cdn.elifesciences.org/articles/81569/elife-81569-fig1-data1-v2.xlsx Download elife-81569-fig1-data1-v2.xlsx In neonatal Nfatc1Cre;Rosa26mTmG/+ mice (P0), GFP+ cells consisted of a portion of cells in the presumptive articular cartilage site [Figure 1A]. Strikingly, at 2 weeks of age, most articular chondrocytes turned out to be GFP+, similar to that at 8 weeks of age [Figure 1(A)]. To clarify the real-time expression of NFATc1 in articular cartilage at 2 weeks and 8 weeks of age, we used Nfatc1CreERT2;Rosa26RFP/+ reporter mice, in which the expression of NFATc1 is reflected by red fluorescence protein (RFP) shortly after tamoxifen administration. Of interest, with tamoxifen pulse, RFP+ cells were found scattered in the articular cartilage at 2 weeks of age accounting for about 22.75 ± 2.18% (n=3 mice) of all cells of articular cartilage, while most RFP+ cells were confined to the superficial layers of articular cartilage at 8 weeks of age (10.94 ± 1.26%, n=3) [Figure 1(C and D)]. Furthermore, the expression pattern of NFATc1 in mouse articular cartilage was verified by immunohistochemistry at E13.5, P0, 2 weeks, and 8 weeks of age [Figure 1(E) and Figure 1—figure supplement 1B]. Therefore, by mapping GFP and RFP expression in articular cartilage of Nfatc1Cre;Rosa26mTmG/+ and Nfatc1CreERT2;Rosa26RFP/+ mice respectively [Figure 1—figure supplement 1C], many GFP+ articular chondrocytes in Nfatc1Cre;Rosa26mTmG/+ mice at 2 weeks and 8 weeks of age should be derived from Nfatc1-expressing progenitors and had lost the expression of NFATc1 with development. Notably, there were no GFP+ chondrocytes in the primordium of growth-plate cartilage at E13.5 in both Nfatc1Cre;Rosa26mTmG/+ and tamoxifen-induced Nfatc1CreERT2;Rosa26mTmG/+ mice [Figure 1(A) and Figure 1—figure supplement 1A], suggesting that NFATc1-expressing cells do not generate the cartilaginous primordium of growth-plate. We did not detect GFP+ cells in articular cartilage in Rosa26mTmG/+ control mice and Nfatc1CreERT2;Rosa26mTmG/+ mice without tamoxifen induction [Figure 1—figure supplement 1D], suggesting that there was no Cre leakage in the articular cartilage in these two reporter mouse lines. Together, these results reveal that articular chondrocytes are derived from NFATc1-expressing progenitors and NFATc1 expression is diminished with articular cartilage development. Colony formation and multipotent differentiation of NFATc1-expressing progenitors The lineage tracing data in Nfatc1Cre and Nfatc1CreERT2 reporter mice suggest that NFATc1 characterizes articular cartilage progenitors. In this context, the fluorescence-labeled cells after tamoxifen-induced recombination in Nfatc1CreERT2 reporter mice should be able to form in vivo cell clones in or next to the articular cartilage with development. To verify this assumption, we exploited the Nfatc1CreERT2;Rosa26mTmG/+ double-fluorescence reporter mouse line and administered two dosages of tamoxifen to dams at P0 and P1, respectively. One week following the tamoxifen pulse, GFP+ cells were detected at the presumptive articular cartilage site at the mouse knee [Figure 2(A)]. Local GFP+ cell clusters with 3–6 cells each could be observed in articular cartilage by 2 weeks and 8 weeks of age [Figure 2(A and B)]. Notably, GFP+ cell clusters were also found in the meniscus, synovial lining, and ligament [Figure 2(A and B)], suggesting that NFATc1 also marks progenitor cells for joint tissues other than articular cartilage. Indeed, in Nfatc1Cre;Rosa26mTmG/+ mice, GFP+ cells also formed the meniscus, synovial lining, ligament, and primordium of the patella at the knee [Figure 1(A) and Figure 2(C)]. Figure 2 Download asset Open asset NFATc1-expressing progenitors form cell clusters with articular cartilage development and contribute to the meniscus and articular synovium formation. (A) Confocal microscopy images showing the distribution of GFP+ cells in the articular cartilage of Nfatc1CreERT2;Rosa26mTmG/+ mouse knee at 1 week and 8 weeks after administering tamoxifen to dams at P0 and P1. The most left image showing GFP+ cells in articular tissues after 1 week of tamoxifen administration. The right three images demonstrating GFP+ cell clusters in the articular cartilage (arrows), meniscus (arrowheads), and ligament. (B) Representative confocal images displaying GFP+ cells or cell clusters (arrows) in the meniscus and articular synovium at 1 week or 2 weeks after tamoxifen administration to dams at P0 and P1. (C) Confocal microscopy images demonstrating that GFP+ cells contribute to the formation of the ligament, synovial lining (left image, arrowheads, 2 weeks of age), and the patella of the knee (right image, arrow, P0) in Nfatc1Cre;Rosa26mTmG/+ mice. All images are representative of five mice at each time point or age, two knee joints per animal. Scale bars, 200 μm. To further characterize the colony formation capacity of NFATc1-expressing articular cartilage progenitors, we cultured and sorted GFP+ cells and their counterparts (GFP- cells) from the knee of neonatal Nfatc1Cre;Rosa26mTmG/+ mice [Figure 3(A)]. The ex vivo colony formation assay showed that GFP+ cells formed remarkably more numerous and larger cell clones in comparison with GFP- cells when plated at the same cell densities and cultured for the same time period [Figure 3(B)]. A similar outcome could be observed even after five consecutive cell passages [Figure 3—figure supplement 1A]. Thus, NFATc1-expressing articular cartilage progenitors display a rigorous capacity for colony formation both in vivo and ex vivo. Figure 3 with 1 supplement see all Download asset Open asset Colony formation and multipotent differentiation of Nfatc1-expressing progenitors. (A) Schematic diagram showing culturing and sorting GFP+ and GFP- cells from the knee of neonatal Nfatc1Cre;Rosa26mTmG/+ mice. (B) Colony formation assay of GFP+ and GFP- cells with 50 or 100 cells plated in 6-well-plates and cultured for 2 weeks. n=6 with cells from three animals, two replicates for each, nonparametric Mann-Whitney test, experiment repeated twice. (C) Alcian blue staining and immunohistochemistry of COL2A1 showing the chondrogenic potential of GFP+ and GFP- cell pellets after being cultured in the chondrogenic differentiation medium for 3 weeks. Isotype as a negative control for COL2A1 antibody. The maximum diameter of cell pellets reflecting the proliferative capacity of GFP+ and GFP- cells. n=9 with cells from three animals, three replicates for each. (D) Alizarin red staining and gene expression analysis of Ibsp and Sp7 demonstrating the osteogenic potential of GFP+ and GFP- cells after being cultured in the osteogenic differentiation medium for 4 weeks. n=6 with cells from three animals, two replicates for each, nonparametric Mann-Whitney test for colony counting data, two-way ANOVA followed by Sidak’s tests for gene expression data, experiments repeated twice. (E) Oil red O staining and gene expression analysis of Fabp4 and Lpl displaying adipogenesis in GFP+ and GFP- cells after being cultured in the adipogenic differentiation medium for 10 days. n=6 with cells from three animals, two replicates for each, nonparametric Mann-Whitney test for colony counting data, two-way ANOVA followed by Sidak’s tests for gene expression data, experiments repeated twice. (F) Schematic illustration and histology respectively showing transplantation of GFP+ cells along with Matrigel matrix underneath the dorsal skin of severe combined immune-deficient mice and the formation of chondrocytes, chondrocyte clusters, and hypertrophic cartilage-like structure (arrows) 4 weeks later. Images are representative of six animals, with GFP- cells as the control (results shown in Figure 3—figure supplement 1B). All data are mean ± SD. Scale bars, 400 μm (C), 500 μm (D), 200 μm (E, F). Figure 3—source data 1 Data of colony numbers, cell pellet diameters, and qPCR. For Figure 3B Colony formation assay of GFP+ and GFP- cells with 50 or 100 cells plated in 6-well-plates and cultured for 2 weeks (colony numbers); Figure 3C Maximum diameter of cell pellets (mm); Figure 3D Colony numbers of mineralization, Relative gene expression of Ibsp (2-△Ct) and Relative gene expression of Sp7 (2-△Ct); Figure 3E Colony numbers of adipogenesis, Relative gene expression of Fabp4 (2-△Ct), and Relative gene expression of Lpl (2-△Ct). https://cdn.elifesciences.org/articles/81569/elife-81569-fig3-data1-v2.xlsx Download elife-81569-fig3-data1-v2.xlsx To study the differentiation potentials of NFATc1-expressing articular cartilage progenitors, we put GFP+ and GFP- cells under chondrogenic, osteogenic, and adipogenic differentiation conditions, respectively. Notably, GFP+ cells displayed a much higher potential to differentiate toward chondrocytes, osteoblasts, and adipocytes compared to GFP- cells [Figure 3(C–E)]. A more striking difference was noticed under the context of chondrocyte differentiation: in the 3D cell-pellet culture model, GFP+ cells always grew into larger pellets as shown by the diameter of cell pellets and displayed a robust capacity of chondrocyte differentiation as shown by alcian blue staining and COL2A1 protein expression, while GFP- cells formed relatively small pellets and only displayed faint cartilage formation at the margin of cell pellets [Figure 3(C)], indicating that GFP+ cells have a more prominent capacity for proliferation and chondrogenesis compared to GFP- cells. Furthermore, when transplanted alongside with Matrigel matrix underneath the dorsal skin of SCID mice, these GFP+ cells differentiated and formed typical chondrocytes as well as chondrocyte clusters within 4 weeks, while the formation of chondrocytes was rarely observed when transplanting GFP- cells [Figure 3(F) and Figure 3—figure supplement 1B]. Notably, many chondrocyte clusters from GFP+ cells had formed hypertrophic cartilage-like tissue with a certain hardness, similar to the physiological process of articular cartilage development [Figure 3(F)]. Taken together, these results demonstrate the intrinsic capacities of colony formation and multipotent differentiation of NFATc1-expressing articular cartilage progenitors. Transcriptional profile of NFATc1-enriched articular cartilage progenitors Next, we sought to dissect the molecular signature of NFATc1-enriched articular cartilage progenitors. In order to minimize the influence of differentiated cells in GFP+ and GFP- cell populations, single-clone cells were sorted at the first passage (P1), amplified for one more passage, and subjected to transcriptome analysis at P2 [Figure 4(A)]. Bioinformatics analysis identified 117 high- and 168 low-expressing genes in GFP+ vs. GFP- cells [Figure 4—figure supplement 1A]. High-expressing genes in GFP+ cells were mainly related to skeletal system development, cartilage development, or extracellular matrix component and organization [Figure 4(B) and Figure 4—figure supplement 1B]. Of note, these high-expressing genes in GFP+ cells included several previously reported articular cartilage progenitor cell markers Decker, 2017; Bian et al., 2020, such as Osr2, Prg4, Postn, Col3a1, Gdf6, and Tgfbr2 [Figure 4(C)]. In contrast, enriched genes in GFP- cells were mainly relevant to muscle cell development and differentiation [Figure 4—figure supplement 1C], suggesting that GFP- single-clone cells could be skeletal muscle progenitors. Importantly, the characteristic molecular signature and enriched biological pathways in GFP+ cells were verified by the second transcriptome analysis using bulk primary GFP+ cells [Figure 4—figure supplement 1D-F]. Figure 4 with 2 supplements see all Download asset Open asset Transcriptional profile of Nfatc1-expressing articular cartilage progenitors. (A) Schematic diagram showing the process of sorting single-clone cells for RNA-sequencing. (B) Cluster heatmap displaying 20 high-expressing genes associated with articular cartilage development in GFP+ vs. GFP- cells. Color descending from red to blue indicates log10(FPKM +1) from large to small. n=3 with cells from three animals in each group. (C) Transcriptome analysis revealing the enrichment of previously reported articular cartilage progenitor marker genes Osr2, Prg4, Postn, Col3a1, and Tgfbr2 in GFP+ relative to GFP- cells. (D) Transcriptome analysis identifying high expression of Cd105, Cd10, and Cd13 and low expression of Cd146, Cd29, and Cd151 in GFP+ vs. GFP- cells. (E) Flow cytometry verifying the expression of CD105 in GFP+ relative to GFP- cells. (F) Flow cytometry showing the expression of cell surface molecules CD9, SCA1, CD166, and CD200 in GFP+ and GFP- cells. Representative results of cells from three mice, experiment repeated twice. Cell surface markers are important in identifying and sorting progenitor or stem cells. Transcriptome analyses showed that both GFP+ and GFP- progenitors expressed several surface markers of cells of mesenchymal origin, including Cd9, Sca1, Thy1, Cd73, Cd166, Cd200, and Cd51, but not hematopoietic or endothelial markers Cd11b, Cd45, or Cd31 (Supplementary file 1). When compared with GFP- cells, GFP+ cells displayed higher expression of Cd105, Cd10, and Cd13 and lower expression of Cd146, Cd29, and Cd151 [Figure 4(D)]. The expression of CD105, CD9, SCA1, CD166, CD200, CD11B, CD45, and CD31 were further verified by flow cytometry in GFP+ and GFP- cells [Figure 4(E and F) and Figure 4—figure supplement 2]. Combined, these data identify a set of genes preferentially expressed in NFATc1-expressing articular cartilage progenitors and provide a perspective to understand the transcriptional signature of these progenitors. NFATc1 negatively regulates articular chondrocyte differentiation The function of NFATc1 in articular cartilage progenitors remains unclear. As aforementioned, the lineage tracing of Nfatc1-expressing cells showed that most articular chondrocytes were GFP+ in Nfatc1Cre;Rosa26mTmG/+ mice at 8 weeks of age, but the real-time expression of NFATc1 was confined to the superficial layers of articular cartilage as shown by RFP expression after tamoxifen pulse in Nfatc1CreERT2;Rosa26RFP/+ mice [Figure 1—figure supplement 1C]. These results indicate that NFATc1 expression was diminished with articular chondrocyte differentiation. Consistently, the diminishment of Nfatc1 expression was also detected in ex vivo chondrogenesis in Nfatc1-expressing articular cartilage progenitors [Figure 5(A)]. Figure 5 with 1 supplement see all Download asset Open asset NFATc1 negatively determines articular chondrocyte differentiation through regulating Col2a1 gene transcription. (A) Gene expression analysis displaying the change of Nfatc1 expression in GFP+ cell-micromass cultured in the chondrogenic medium for 10 days. n=3, experiment repeated three times with cells from three mice. (B) Alcian blue staining and gene expression analysis of Acan, Col2a1, and Col10a1 after deleting Nfatc1 by CRISPR/CAS9 technique in ex vivo micromass culture of GFP+ cells from neonatal Nfatc1Cre;Rosa26mTmG/+ mice (day 7, without chondrogenic induction, n=3). Experiment repeated three times with cells from three animals. (C) Quantification of alcian blue staining and gene expression analysis of Col2a1 and Col10a1 showing decreased chondrogenesis after overexpressing NFATc1 in GFP+ cells by infecting a caNFATc1 retrovirus structure. For alcian blue staining, n=6 with cells from three animals, two replicates for each; for gene expression analysis, n=3, experiment repeated twice with cells from two animals. (D) Safranin O staining demonstrating enhanced articular cartilage staining in the hip of Prrx1Cre;Nfatc1fl/fl vs. Prrx1Cre;Nfatc1fl/+ mice at 12 weeks of age. Representative images from five animals in each group were displayed. (E) Representative images of alcian blue staining and polarized light on H&E staining manifesting increased staining (arrows) and thickness of articular cartilage (double arrows) in the knee of Prrx1Cre;Nfatc1fl/fl relative to Prrx1Cre;Nfatc1fl/+ mice at 16 weeks of age. n=5 animals for each group. (F) Quantitative PCR determining the expression of Acan, Col2a1, and Col10a1 genes in articular cartilage of Prrx1Cre;Nfatc1fl/fl relative to Prrx1Cre;Nfatc1fl/+ mice at 8 weeks of age." @default.
• W4362651014 created "2023-04-07" @default.
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• W4362651014 date "2022-08-29" @default.
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• W4362651014 title "Editor's evaluation: NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation" @default.
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