NFATc1 marks articular cartilage progenitors and negatively determines articular chondrocyte differentiation
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Evaluation Summary:
In this study, the authors identified that NFATc1 acts as a key regulator of articular chondrocyte differentiation during early mouse development. Using multiple pulse-chase experiments the authors found that NFATc1 expressing cells generated most of the articular chondrocytes, but not growth plate chondrocytes. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1 after articular cartilage development. This is an important study since it provides valuable evidence to reveal the regulatory mechanism of articular chondrocyte differentiation. The data presented in this manuscript, in general, support their conclusion.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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.
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Evaluation Summary:
In this study, the authors identified that NFATc1 acts as a key regulator of articular chondrocyte differentiation during early mouse development. Using multiple pulse-chase experiments the authors found that NFATc1 expressing cells generated most of the articular chondrocytes, but not growth plate chondrocytes. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1 after articular cartilage development. This is an important study since it provides valuable evidence to reveal the regulatory mechanism of articular chondrocyte differentiation. The data presented in this manuscript, in general, support their conclusion.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the …
Evaluation Summary:
In this study, the authors identified that NFATc1 acts as a key regulator of articular chondrocyte differentiation during early mouse development. Using multiple pulse-chase experiments the authors found that NFATc1 expressing cells generated most of the articular chondrocytes, but not growth plate chondrocytes. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1 after articular cartilage development. This is an important study since it provides valuable evidence to reveal the regulatory mechanism of articular chondrocyte differentiation. The data presented in this manuscript, in general, support their conclusion.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The molecular mechanism of articular chondrocyte differentiation has not been fully revealed. In this study, the authors identified NFATc1 as a key regulator of articular chondrocyte differentiation during early mouse development. At E13.5, NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With cartilage development, NFATc1-expressing cells generated most of articular chondrocytes. Through transcriptome analyses the authors showed a set of genes expressed in NFATc1-enriched articular cartilage progenitor cells. The expression of NFATc1 was diminished during articular chondrocyte differentiation. The authors found that suppression of NFATc1 expression in articular cartilage progenitor cells is sufficient to induce spontaneous chondrogenesis; while overexpressing NFATc1 …
Reviewer #1 (Public Review):
The molecular mechanism of articular chondrocyte differentiation has not been fully revealed. In this study, the authors identified NFATc1 as a key regulator of articular chondrocyte differentiation during early mouse development. At E13.5, NFATc1-expressing cells were mainly located in the flanking region of the joint interzone. With cartilage development, NFATc1-expressing cells generated most of articular chondrocytes. Through transcriptome analyses the authors showed a set of genes expressed in NFATc1-enriched articular cartilage progenitor cells. The expression of NFATc1 was diminished during articular chondrocyte differentiation. The authors found that suppression of NFATc1 expression in articular cartilage progenitor cells is sufficient to induce spontaneous chondrogenesis; while overexpressing NFATc1 suppresses chondrogenesis. They further demonstrated that NFATc1 negatively regulated Col2a1 gene transcription. This is an important study since it will help us understand the regulatory mechanism of articular chondrocyte differentiation and pathological mechanisms of joint diseases, such as osteoarthritis.
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Reviewer #2 (Public Review):
An interesting work by Zhang and colleagues: through utilizing Nfatc1-Cre and Nfatc1-CreERT2 mice as the tracing tools to try to understand the origin and differentiation of articular chondrocytes based on their prior studies. It is an unexpected observation and important direction for the identification of stem-cell like subset generating articular cartilage that NFATc1 constantly and specifically labels articular cartilage progenitors throughout embryonic development and postnatal growth. The authors further revealed that NFATc1 negatively regulates articular chondrocyte differentiation partly through controlling Col2a1 expression. Albeit some approaches applied here could not entirely resolve the questions posed, topic addressed here is both novel and important for further studies related to articular …
Reviewer #2 (Public Review):
An interesting work by Zhang and colleagues: through utilizing Nfatc1-Cre and Nfatc1-CreERT2 mice as the tracing tools to try to understand the origin and differentiation of articular chondrocytes based on their prior studies. It is an unexpected observation and important direction for the identification of stem-cell like subset generating articular cartilage that NFATc1 constantly and specifically labels articular cartilage progenitors throughout embryonic development and postnatal growth. The authors further revealed that NFATc1 negatively regulates articular chondrocyte differentiation partly through controlling Col2a1 expression. Albeit some approaches applied here could not entirely resolve the questions posed, topic addressed here is both novel and important for further studies related to articular progenitors.
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Reviewer #3 (Public Review):
Zhang et al. examined a novel articular cartilage progenitors NFATc1 expressing cells. Through multiple pulse-chase experiments, they found that NFATc1 expressing cells generated most of the articular chondrocytes, but not chondrocytes in the growth plate primordium. In vitro and in vivo transplantation of NFATc1 expressing progenitors demonstrated that these cells exhibit pluripotency to chondrocytes, osteoblasts, and adipocytes. RNA-seq analysis of NFATc1 expressing progenitors demonstrated that these cells are enriched with articular cartilage stem cell markers such as Prg4. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1. Through CRISPR-mediated knockdown and conditional deletion in Prrx1-cre cells, …
Reviewer #3 (Public Review):
Zhang et al. examined a novel articular cartilage progenitors NFATc1 expressing cells. Through multiple pulse-chase experiments, they found that NFATc1 expressing cells generated most of the articular chondrocytes, but not chondrocytes in the growth plate primordium. In vitro and in vivo transplantation of NFATc1 expressing progenitors demonstrated that these cells exhibit pluripotency to chondrocytes, osteoblasts, and adipocytes. RNA-seq analysis of NFATc1 expressing progenitors demonstrated that these cells are enriched with articular cartilage stem cell markers such as Prg4. Interestingly, NFATc1 expression in chondrocytes diminished as mice aged, suggesting that NFATc1 expressing progenitors are no longer expressing NFATc1. Through CRISPR-mediated knockdown and conditional deletion in Prrx1-cre cells, authors found that NFATc1 negatively regulated chondrocyte differentiation with its putative binding sites on Col2a1 promoter and intron 1. These data support authors' conclusion that NFATc1 negatively regulates chondrocyte differentiation but it also marks chondrocyte progenitors.
The major strengths of the manuscript are the rigorous approach to examine NFATc1 expressing progenitors, including in vivo pulse-chase experiments, in vitro differentiation and in vivo transplantation studies, and transcriptomic profiling. Authors also use multiple approaches to demonstrate functional role of NFATc1, which is negatively regulating chondrocyte differentiation. All these findings generally support authors conclusions. There are some minor weaknesses, such as discordance between NFATc1 expression and NFATc1 expressing cells on articular cartilage, comparison of NFATc1 to the well-known articular chondrocyte progenitors such as GDF5 expressing progenitors, and lack of analyses of in vivo multipotency of NFATc1 expressing progenitors. Nevertheless, authors' findings will substantially advance the field that have long sought to examine the mechanism of joint development by revealing novel population of progenitors and chondrocyte differentiation mechanism. This could ultimately lead to novel treatment strategies for articular cartilage diseases.
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