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Aligning single-cell developmental and reprogramming trajectories identifies molecular determinants of reprogramming outcome

ABSTRACT: Aligning single-cell developmental and reprogramming trajectories identifies molecular determinants of reprogramming outcome

PROVIDER: PRJNA415061 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6191750_1.fastq.gz	Fastqsanger.gz
	SRR6191750_2.fastq.gz	Fastqsanger.gz
	SRR6191760_1.fastq.gz	Fastqsanger.gz
	SRR6191760_2.fastq.gz	Fastqsanger.gz
	SRR6191770_1.fastq.gz	Fastqsanger.gz

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Aligning single-cell developmental and reprogramming trajectories identifies molecular determinants of reprogramming outcome

Project description:Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy, as well as for expanding our understanding of the general principles of gene regulation. MYOD-mediated myogenic reprogramming, which converts many cell types into contractile myotubes, remains one of the best characterized model system for direct conversion by defined factors. However, why MYOD can efficiently convert some cell types into myotubes but not others remains poorly understood. Here, we analyze MYOD-mediated reprogramming of human fibroblasts at pseudotemporal resolution using single-cell RNA-Seq. Successfully reprogrammed cells navigate a trajectory with two branches that correspond to two barriers to reprogramming, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Differential analysis of the major branch points alongside alignment of the successful reprogramming path to a primary myoblast trajectory revealed Insulin and BMP signaling as crucial molecular determinants of an individual cell’s reprogramming outcome, that when appropriately modulated, increased efficiency more than five-fold. Our single-cell analysis reveals that MYOD is sufficient to reprogram cells only when the extracellular milieu is favorable, supporting MYOD with upstream signaling pathways that drive normal myogenesis in development.

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Project description:Understanding the molecular programs that guide differentiation during development is a major challenge. Here, we introduce Waddington-OT, an approach for studying developmental time courses to infer ancestor-descendant fates and model the regulatory programs that underlie them. We apply the method to reconstruct the landscape of reprogramming from 315,000 single-cell RNA sequencing (scRNA-seq) profiles, collected at half-day intervals across 18 days. The results reveal a wider range of developmental programs than previously characterized. Cells gradually adopt either a terminal stromal state or a mesenchymal-to-epithelial transition state. The latter gives rise to populations related to pluripotent, extra-embryonic, and neural cells, with each harboring multiple finer subpopulations. The analysis predicts transcription factors and paracrine signals that affect fates and experiments validate that the TF Obox6 and the cytokine GDF9 enhance reprogramming efficiency. Our approach sheds light on the process and outcome of reprogramming and provides a framework applicable to diverse temporal processes in biology.

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Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis

Project description:During embryogenesis, cells acquire distinct fates by transitioning through transcriptional states. To uncover these transcriptional trajectories during zebrafish embryogenesis, we sequenced 38,731 cells and developed URD, a simulated diffusion-based computational reconstruction method. URD identified the trajectories of 25 cell types through early somitogenesis, gene expression along them, and their spatial origin in the blastula. Analysis of Nodal signaling mutants revealed that their transcriptomes were canalized into a subset of wild-type transcriptional trajectories. Some wild-type developmental branchpoints contained cells expressing genes characteristic of multiple fates. These cells appeared to trans-specify from one fate to another. These findings reconstruct the transcriptional trajectories of a vertebrate embryo, highlight the concurrent canalization and plasticity of embryonic specification, and provide a framework to reconstruct complex developmental trees from single-cell transcriptomes. This SuperSeries is composed of the SubSeries listed below.

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