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Reconstructing regulative progenitor behaviors in synthetic embryos

ABSTRACT: 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.

ORGANISM(S): Mus musculus synthetic construct

PROVIDER: GSE220949 | GEO | 2024/03/05

REPOSITORIES: GEO

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Project description:deBack2012 - Lineage Specification in Pancreas Development This model of two neighbouring pancreas precursor cells, describes the exocrine versus endocrine lineage specification process. To account for the tissue scale patterns, this couplet model has been extended to hundreds of coupled cells. This model is described in the article: On the role of lateral stabilization during early patterning in the pancreas de Back W., Zhou JX, Brusch L J. R. Soc. Interface 6 February 2013 vol. 10 no. 79 20120766 Abstract: The cell fate decision of multi-potent pancreatic progenitor cells between the exocrine and endocrine lineages is regulated by Notch signalling, mediated by cell–cell interactions. However, canonical models of Notch-mediated lateral inhibition cannot explain the scattered spatial distribution of endocrine cells and the cell-type ratio in the developing pancreas. Based on evidence from acinar-to-islet cell transdifferentiation in vitro, we propose that lateral stabilization, i.e. positive feedback between adjacent progenitor cells, acts in parallel with lateral inhibition to regulate pattern formation in the pancreas. A simple mathematical model of transcriptional regulation and cell–cell interaction reveals the existence of multi-stability of spatial patterns whose simultaneous occurrence causes scattering of endocrine cells in the presence of noise. The scattering pattern allows for control of the endocrine-to-exocrine cell-type ratio by modulation of lateral stabilization strength. These theoretical results suggest a previously unrecognized role for lateral stabilization in lineage specification, spatial patterning and cell-type ratio control in organ development. This model is hosted on BioModels Database and identified by: MODEL1211010000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:The bHLH transcription factor stem cell leukemia gene (Scl) is a master regulator for hematopoiesis essential for hematopoietic specification and proper differentiation of the erythroid and megakaryocyte lineages. However, the critical downstream targets of Scl remain undefined. Here, we identified a novel Scl target gene, transcription factor myocyte enhancer factor 2 C (Mef2C) from Sclfl/fl fetal liver progenitor cell lines. Analysis of Mef2C-/- embryos showed that Mef2C, in contrast to Scl, is not essential for specification into primitive or definitive hematopoietic lineages. However, adult VavCre+Mef2Cfl/fl mice exhibited platelet defects similar to those observed in Scl deficient mice. The platelet counts were reduced, while platelet size was increased and the platelet shape and granularity was altered. Furthermore, megakaryopoiesis was severely impaired in vitro. ChIP-on-chip analysis revealed that Mef2C is directly regulated by Scl in megakaryocytic cells, but not in erythroid cells. In addition, an Scl independent requirement for Mef2C in B-lymphoid homeostasis was observed in Mef2C-deficient mice, characterized as severe age-dependent reduction of specific B cell progenitor populations reminiscent of premature aging. In summary, this work identifies Mef2C as an integral member of hematopoietic transcription factors with distinct upstream regulatory mechanisms and functional requirements in megakaryocyte and B-lymphoid lineages. Experiment Overall Design: Sclfl/fl hematopoietic progenitor lines were generated from fetal liver progenitors from E12.5 Sclfl/fl embryos9 by immortalization with Hox11 retrovirus.17 Sclfl/fl progenitor cells were cultured with IL3, and a clonal line containing cells with megakaryocyte morphology and acetylcholinesterase (AchE) activity was selected. The Sclfl/fl cell line was transduced with Cre- GFP retrovirus to generate the Scl Experiment Overall Design: Î?/Î? cell line, and the Scl Î?/Î? cell line was transduced with Scl retrovirus to re-introduce Scl expression (Scl Î?/Î? +Scl cell line). Megakaryocyte differentiation was enhanced by adding Tpo for 5 days before harvesting the cells. RNA was extracted with Trizol (Gibco BRL) and RNEasy (Qiagen) kits. Differential gene expression between Sclfl/fl, Scl Î?/Î? and Scl Î?/Î? +Scl cell lines was analyzed by Affymetrix MOE430_2 microarray in the microarray core facility at the Dana-Farber Cancer Institute.

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Reconstructing regulative progenitor behaviors in synthetic embryos

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