Lineage-specific differences and regulatory networks governing human chondrocyte development
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Curated by eLife
Evaluation Summary:
The study presented in this manuscript is of interest to cartilage biologists studying the mechanisms of chondrocyte differentiation. The authors investigated transcriptomic profiles of hESC-derived articular and growth plate chondrocytes. To characterize the regulatory landscapes with respective transcriptomes, they mapped chromatin accessibility in hESC derived chondrocyte lineages and mouse embryonic chondrocytes using ATAC-sequencing and revealed lineage-specific gene regulatory networks. They further validated functional interactions of two transcription factors, Runx2 and RELA, with their predicted genomic targets. This study could help us understand chondrocyte differentiation.
(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
To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology.
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Evaluation Summary:
The study presented in this manuscript is of interest to cartilage biologists studying the mechanisms of chondrocyte differentiation. The authors investigated transcriptomic profiles of hESC-derived articular and growth plate chondrocytes. To characterize the regulatory landscapes with respective transcriptomes, they mapped chromatin accessibility in hESC derived chondrocyte lineages and mouse embryonic chondrocytes using ATAC-sequencing and revealed lineage-specific gene regulatory networks. They further validated functional interactions of two transcription factors, Runx2 and RELA, with their predicted genomic targets. This study could help us understand chondrocyte differentiation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
The study presented in this manuscript is of interest to cartilage biologists studying the mechanisms of chondrocyte differentiation. The authors investigated transcriptomic profiles of hESC-derived articular and growth plate chondrocytes. To characterize the regulatory landscapes with respective transcriptomes, they mapped chromatin accessibility in hESC derived chondrocyte lineages and mouse embryonic chondrocytes using ATAC-sequencing and revealed lineage-specific gene regulatory networks. They further validated functional interactions of two transcription factors, Runx2 and RELA, with their predicted genomic targets. This study could help us understand chondrocyte differentiation.
(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):
In this manuscript, the authors relied on their well-established directed differentiation approach to differentiate pluripotent stem cells (hESCs) towards growth plate (BMP4-treated) or articular (TGFb3-treated) chondrocytes. Integrating RNA-seq data from hESCs-derived growth plate or articular chondrocytes with data from in vivo (fetal) counterparts, the authors showed similarities (and some divergences) in the transcriptional networks of in the in vitro-differentiated cells, uncovering genes with potential novel roles in cartilage biology. Integrating ATAC-seq (to assess chromatin accessibility) and transcriptomics data, the authors both characterized the regulatory landscapes in these cells, and also uncovered lineage-specific gene-regulatory networks. Using targeted ChIP-qPCR, and leveraging available …
Reviewer #1 (Public Review):
In this manuscript, the authors relied on their well-established directed differentiation approach to differentiate pluripotent stem cells (hESCs) towards growth plate (BMP4-treated) or articular (TGFb3-treated) chondrocytes. Integrating RNA-seq data from hESCs-derived growth plate or articular chondrocytes with data from in vivo (fetal) counterparts, the authors showed similarities (and some divergences) in the transcriptional networks of in the in vitro-differentiated cells, uncovering genes with potential novel roles in cartilage biology. Integrating ATAC-seq (to assess chromatin accessibility) and transcriptomics data, the authors both characterized the regulatory landscapes in these cells, and also uncovered lineage-specific gene-regulatory networks. Using targeted ChIP-qPCR, and leveraging available ChIP-seq datasets, the authors validated the functional interactions of two well-described DNA-binding trans-acting factors (RUNX2 and RELA) with putative genomic targets (both previously involved and with non-explored/novel roles in cartilage biology). Taken together, these analyses provide novel insight into the molecular mechanisms contributing to growth plate and articular cartilage specification.
Strengths:
This is a very well-written manuscript. The findings are of relevance to understanding cartilage development and maintenance and are of potential impact to understand (and correct) cartilage damage and pathology. The experiments are well conducted, and the conclusions and claims are supported by the data. The authors performed a superb job characterizing and defining gene regulatory networks, elegantly integrating in vitro systems with in vivo datasets, and combining transcriptomics and epigenomics tools. These approaches uncovered regulatory networks and novel genes with unexplored roles and contributions to growth plate and articular cartilage development.
Weaknesses:
The functional implication of the findings is somewhat limited: while the authors did evaluate and confirm interactions of selected transcription factors with putative target genes, the mechanistic contribution of these findings to chondrocyte specification is not fully explored.
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Reviewer #2 (Public Review):
The study is aiming to provide new information for understanding the development of human cartilage. The major weaknesses include its experimental design and technical approaches. Only terminal differentiated hESC-derived cartilage and E67 fetal femur samples were used for analysis, which is not enough for a developmental study. Also, bulk RNA sequencing, instead of single-cell RNA sequencing was used, which made the study much less valuable as a resource for other researchers.
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