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Single-cell atlas of mouse limb development reveals that the skeleton forms nonconsecutively by progressive differentiation of Msx1+ naïve progenitors

ABSTRACT: Single-cell atlas of mouse limb development reveals that the skeleton forms nonconsecutively by progressive differentiation of Msx1+ naïve progenitors

PROVIDER: PRJNA771406 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16356700_1.fastq.gz	Fastqsanger.gz
	SRR16356700_2.fastq.gz	Fastqsanger.gz
	SRR16356701_1.fastq.gz	Fastqsanger.gz
	SRR16356701_2.fastq.gz	Fastqsanger.gz
	SRR16356702_1.fastq.gz	Fastqsanger.gz

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Project description:Limb development has long served as a model system for studying how a pool of progenitor cells undergoes coordinated spatial patterning and differentiation to produce complex and well-organized structures. Here, we identify a population of naïve-state limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern. First, by performing single-cell RNA sequencing of the developing mouse forelimb we created an atlas of cellular compositions and transcriptional states. This atlas revealed three progenitor states: naïve, proximal and autopodial, as well as Msx1 as a marker for the naïve progenitors. In vivo lineage tracing analysis firmly established this role of Msx1 and localized the naïve progenitors to the outer margin of the developing limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that Msx1 naïve progenitors undergo progressive and simultaneous transition into both proximal and autopodial progenitors and then to Sox9+ chondroprogenitors. Interestingly, the progressive differentiation to chondroprogenitors occurred along all the skeletal segments concurrently, suggesting that skeleton form in a nonconsecutive way. Indeed, by tracking the spatiotemporal sequence of naïve progenitor differentiation, we found that the skeleton forms in a complex nonconsecutive three-dimensional pattern. Overall, these findings propose a new model for limb skeleton development. Limb development has long served as a model system for studying how a pool of progenitor cells undergoes coordinated spatial patterning and differentiation to produce complex and well-organized structures. Here, we identify a population of naïve-state limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern. First, by performing single-cell RNA sequencing of the developing mouse forelimb we created an atlas of cellular compositions and transcriptional states. This atlas revealed three progenitor states: naïve, proximal and autopodial, as well as Msx1 as a marker for the naïve progenitors. In vivo lineage tracing analysis firmly established this role of Msx1 and localized the naïve progenitors to the outer margin of the developing limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that Msx1 naïve progenitors undergo progressive and simultaneous transition into both proximal and autopodial progenitors and then to Sox9+ chondroprogenitors. Interestingly, the progressive differentiation to chondroprogenitors occurred along all the skeletal segments concurrently, suggesting that skeleton form in a nonconsecutive way. Indeed, by tracking the spatiotemporal sequence of naïve progenitor differentiation, we found that the skeleton forms in a complex nonconsecutive three-dimensional pattern. Overall, these findings propose a new model for limb skeleton development. Limb development has long served as a model system for studying how a pool of progenitor cells undergoes coordinated spatial patterning and differentiation to produce complex and well-organized structures. Here, we identify a population of naïve-state limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern. First, by performing single-cell RNA sequencing of the developing mouse forelimb we created an atlas of cellular compositions and transcriptional states. This atlas revealed three progenitor states: naïve, proximal and autopodial, as well as Msx1 as a marker for the naïve progenitors. In vivo lineage tracing analysis firmly established this role of Msx1 and localized the naïve progenitors to the outer margin of the developing limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that Msx1 naïve progenitors undergo progressive and simultaneous transition into both proximal and autopodial progenitors and then to Sox9+ chondroprogenitors. Interestingly, the progressive differentiation to chondroprogenitors occurred along all the skeletal segments concurrently, suggesting that skeleton form in a nonconsecutive way. Indeed, by tracking the spatiotemporal sequence of naïve progenitor differentiation, we found that the skeleton forms in a complex nonconsecutive three-dimensional pattern. Overall, these findings propose a new model for limb skeleton development.

Project description:The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape. Disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2'704 putative chondrogenic enhancer regions. Most of these enhancers (74%) showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Moreover, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci each exhibited a significant reduction of cognate gene expression, therefore demonstrating their functional importance. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.

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Single-cell atlas of mouse limb development reveals that the skeleton forms nonconsecutively by progressive differentiation of Msx1+ naïve progenitors

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