“…R3 was also reported by Khateb et al. ( 66 ) to control Pax7 expression and downstream myogenic and neurogenic programs in differentiating ESCs. Nevertheless, leveraging our in house–developed in vivo genome editing system, we provided in vivo evidence to show that deletion of R2, R3, and R4 not only disturbed Pax7 RNA expression in QSCs but also resulted in distinct effects on 3D genome organization at Pax7 locus.…”
Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer–containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at
Pax7
locus and
Pax7
expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.
“…R3 was also reported by Khateb et al. ( 66 ) to control Pax7 expression and downstream myogenic and neurogenic programs in differentiating ESCs. Nevertheless, leveraging our in house–developed in vivo genome editing system, we provided in vivo evidence to show that deletion of R2, R3, and R4 not only disturbed Pax7 RNA expression in QSCs but also resulted in distinct effects on 3D genome organization at Pax7 locus.…”
Little is known about three-dimensional (3D) genome organization in skeletal muscle stem cells [also called satellite cells (SCs)]. Here, we comprehensively map the 3D genome topology reorganization during mouse SC lineage progression. Specifically, rewiring at the compartment level is most pronounced when SCs become activated. Marked loss in topologically associating domain (TAD) border insulation and chromatin looping also occurs during early activation process. Meanwhile, TADs can form TAD clusters and super-enhancer–containing TAD clusters orchestrate stage-specific gene expression. Furthermore, we uncover that transcription factor PAX7 is pivotal in enhancer-promoter (E-P) loop formation. We also identify cis-regulatory elements that are crucial for local chromatin organization at
Pax7
locus and
Pax7
expression. Lastly, we unveil that geriatric SC displays a prominent gain in long-range contacts and loss of TAD border insulation. Together, our results uncover that 3D chromatin extensively reorganizes at multiple architectural levels and underpins the transcriptome remodeling during SC lineage development and SC aging.
“…Various applications based on FACS have been developed in developmental biology, immunology, and oncology, such as immunophenotyping, cell cycle analysis, and single‐cell gene expression analyses (Bose et al, 2023; Jensen & Wnek, 2021; Kang & Sánchez Alvarado, 2009). Recently, the combination of transcriptome/epigenome (Keuls et al, 2023; Khateb et al, 2022) and transcriptome/spatial information (Cui et al, 2023; Mantri et al, 2021) has enabled the investigation of new aspects of developmental and cell biology. Single‐cell multi‐omics analyses have revealed cell type‐specific gene expression dynamics that can help to elucidate the heterogeneity of cell populations, gene regulatory networks, and the trajectories of cell state transitions.…”
Preparing viable single cells is critical for conducting single‐cell RNA sequencing (scRNA‐seq) because the presence of ambient RNA from dead or damaged cells can interfere with data analysis. Here, we developed a method for isolating viable single cells from adult planarian bodies using fluorescence‐activated cell sorting (FACS). This method was then applied to both adult pluripotent stem cells (aPSCs) and differentiating/differentiated cells. Initially, we employed a violet instead of ultraviolet (UV) laser to excite Hoechst 33342 to reduce cellular damage. After optimization of cell staining conditions and FACS compensation, we generated FACS profiles similar to those created using a previous method that employed a UV laser. Despite successfully obtaining high‐quality RNA sequencing data for aPSCs, non‐aPSCs produced low‐quality RNA reads (i.e., <60% of cells possessing barcoding mRNAs). Subsequently, we identified an effective FACS gating condition that excluded low‐quality cells and tissue debris without staining. This non‐staining isolation strategy not only reduced post‐dissociation time but also enabled high‐quality scRNA‐seq results for all cell types (i.e., >80%). Taken together, these findings imply that the non‐staining FACS strategy may be beneficial for isolating viable cells not only from planarians but also from other organisms and tissues for scRNA‐seq studies.
“…Then, the medium is switched to a HIFLR medium supplemented with hepatocyte growth factor (HGF), insulin growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2), LDN193189, and Rspo3 for additional 48 h and Pax3-GFP positive can be FACS sorted ( Figure 1 A and 1B). At this time, cells express Pax7 protein as shown 1 Figure 1 Induction and FACS-isolation of mESCs-derived Pax3-GFP + cells (A) Bright-field and fluorescence microscopy images of naïve mESCs and mESCs-derived Pax3-GFP + cells at different stages of induced differentiation. Bar, 100 μm (B) FACS plot of mESCs-derived Pax3-GFP + cells at day 6 (aPSM cells) and day 8 (HIFLR cells) of differentiation.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Using defined factors at specific time points, Chal and coworkers 2 have described the transition from mESCs towards myogenic progenitors and further commitment towards muscles formation. Employing the protocol described here, we have provided a comprehensive map of gene regulation and chromatin state change during mESCs differentiation 1 …”
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