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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 05, 2024 |
| Title |
Reconstructing regulative progenitor behaviors in synthetic embryos |
| Organisms |
Mus musculus; synthetic construct |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Advanced stem cell-based in vitro models of embryogenesis offer great promise to decipher developmental programs with high-throughput and resolution. However, the rapid changes in morphology and cell state that underly these models mask multiple higher order parameters, including the regulative balance between differentiation and self-renewal that govern transient progenitor cell populations. Here, we apply a continuous single-cell Cas9-based molecular recorder to mouse embryonic stem cells, which enables the recovery of can reproducibly recovere and high-content information regarding the historical relationship between individual cells. We apply our system to Trunk-Like Structures (TLS), a complex in vitro model of embryogenesis that captures differentiation and morphological dynamics from pluripotency exit to the specification of the somitic and neural lineages. By recording cell lineages alongside the transcriptional identities of extant cells, we are able to build high-resolution phylogenetic trees, identify clonal subpopulations, and reveal recover the step-wisestepwise series of events that govern germ layer specification. Moreover, we examine are able to functionally characterize the specification and maintenance of multipotent neuro-mesodermal progenitor (NMP) populations as they coordinate self-renewal and commitment into more specialized cell types, including how thehow highly disparateplastic individual nature of individual branches trajectories ultimately aggregateultimately aggregate to ensure to generate the generation of highly consistent embryonic structures. Finally, we demonstrate the potential of this technology for high-throughput perturbation screening by combining small molecule treatment with individual structure labeling, allowing us to we demonstrate that these parameters can be independently tuned to modulate TLS morphology, including in reversible ways that extend the duration through which NMPs can contribute to both lineages of the embryonic trunkconnect perturbation of early differentiation pathways to progenitor cell behavior and structure morphology. Our work represents the first continuous molecular recording approach applied to dynamic in vitro models, a toolkit that can be combined with inhibitor libraries or other screening modalities to uncover the genetic and epigenetic mechanisms that enable highly orchestrated and robust changes in cell state composition during critical windows of morphogenesis.
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| Overall design |
Single-cell RNAseq data were collected from Tracer TLS at 120h after aggregation (2 individual structures and one Multiseq experiment containing 24 structures, 12 Tracer TLS and 12 Tracer TLSCL). Libraries were prepared and sequenced with 10X Genomics single cell RNA sequencing system (RNAseq fraction). Individul Amplicon seq libraries for evolving, lenti and multiseq barcodes have been prepared separately from the 10x cDNA from each single-cell reaction. Bulk Amplicon seq data were collected from Tracer mESCs at various timepoints after Cas9 activation (Cas9-GFP+ sorted Tracer mESCs at day 0, 1, 2, 3, 4, 5 after aggregation) and from 8 individual Tracer TLS 120h after aggregation. Libraries were prepared from both gDNA and cDNA from the same cell suspensions.
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| Contributor(s) |
Bolondi A, Law BK, Kretzmer H, Gassalog SI, Riemenschneider C, Yang D, Buschow R, Walther M, McGinnis CS, Gartner ZJ, Veenvliet JV, Meissner A, Smith ZD, Chan MM |
| Citation missing |
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| Submission date |
Dec 14, 2022 |
| Last update date |
Jun 05, 2024 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
kretzmer@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platforms (3) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL26526 |
Illumina NovaSeq 6000 (synthetic construct) |
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| Samples (29)
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| Relations |
| BioProject |
PRJNA912143 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE220949_RAW.tar |
997.3 Mb |
(http)(custom) |
TAR (of TAR, TSV, TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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