RESUMO
Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.
Assuntos
Análise de Célula Única/métodos , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Condrócitos/citologia , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Fator 5 de Diferenciação de Crescimento/deficiência , Fator 5 de Diferenciação de Crescimento/genética , Hibridização In Situ , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Análise de Sequência de RNA , Células-Tronco/citologia , Membrana Sinovial/citologiaRESUMO
Single-cell methods offer a high-resolution approach for characterizing cell populations. Many studies rely on single-cell transcriptomics to draw conclusions regarding cell state and behavior, with the underlying assumption that transcriptomic readouts largely parallel their protein counterparts and subsequent activity. However, the relationship between transcriptomic and proteomic measurements is imprecise, and thus datasets that probe the extent of their concordance will be useful to refine such conclusions. Additionally, novel single-cell analysis tools often lack appropriate gold standard datasets for the purposes of assessment. Integrative (combining the two data modalities) and predictive (using one modality to improve results from the other) approaches in particular, would benefit from transcriptomic and proteomic data from the same sample of cells. For these reasons, we performed single-cell RNA sequencing, mass cytometry, and flow cytometry on a split-sample of human peripheral blood mononuclear cells. We directly compare the proportions of specific cell types resolved by each technique, and further describe the extent to which protein and mRNA measurements correlate within distinct cell types.
Assuntos
Citometria de Fluxo , Leucócitos Mononucleares , Análise de Sequência de RNA , Análise de Célula Única , Humanos , Leucócitos Mononucleares/metabolismo , Transcriptoma , ProteômicaRESUMO
Elucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.
Assuntos
Redes Reguladoras de Genes/genética , Camadas Germinativas/metabolismo , Animais , Diferenciação Celular , Endoderma/citologia , Endoderma/metabolismo , Camadas Germinativas/citologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/genética , Análise de Célula Única , Proteínas Wnt/metabolismoRESUMO
One snapshot of the peer review process for "Genomic rewiring of SOX2 chromatin interaction network during differentiation of ESCs to postmitotic neurons" (Bunina et al., 2020).
Assuntos
Cromatina , Neurônios , Diferenciação Celular , Cromatina/genética , GenômicaRESUMO
Directed differentiation of pluripotent stem cells provides an accessible system to model development. However, the distinct cell types that emerge, their dynamics, and their relationship to progenitors in the early embryo has been difficult to decipher because of the cellular heterogeneity inherent to differentiation. Here, we used a combination of bulk RNA-Seq, single cell RNA-Seq, and bioinformatics analyses to dissect the cell types that emerge during directed differentiation of mouse embryonic stem cells as embryoid bodies and we compared them to spatially and temporally resolved transcriptional profiles of early embryos. Our single cell analyses of the day 4 embryoid bodies revealed three populations which had retained related yet distinct pluripotent signatures that resemble the pre- or post-implantation epiblast, one population of presumptive neuroectoderm, one population of mesendoderm, and four populations of neural progenitors. By day 6, the neural progenitors predominated the embryoid bodies, but both a small population of pluripotent-like cells and an anterior mesoderm-like Brachyury-expressing population were present. By comparing the day 4 and day 6 populations, we identified candidate differentiation paths, transcription factors, and signaling pathways that mark the in vitro correlate of the transition from the mid-to-late primitive streak stage.