Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations

Brink, Susanne C. van den; Sage, Fanny; Vértesy, Ábel; Spanjaard, Bastiaan; Peterson-Maduro, Josi; Baron, Chloé S.; Robin, Catherine; Oudenaarden, Alexander van

doi:10.1038/nmeth.4437

Cited by 842 publications

(667 citation statements)

References 8 publications

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“…On FACS plots, we found that the freshly isolated p110a-null MuSCs were even smaller in size than the control MuSCs based on the mean forward scatter (FSC) values ( Fig EV4C). This was most likely due to the fact that the control "MuSCs" was already partially activated during the isolation process (van den Brink et al, 2017;Machado et al, 2017;van Velthoven et al, 2017). Consistently, on freshly isolated myofibers, we found that the size of the mutant MuSCs was indeed smaller than that of the control MuSCs ( Fig EV4D and E).…”

Section: Deletion Of P110a In Adult Muscs In Uninjured Muscles Reducesupporting

confidence: 80%

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells

et al. 2018

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Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon injury, MuSCs exit quiescence to become activated, re-enter the cell cycle to proliferate, and differentiate to repair the damaged muscles. It remains unclear which extrinsic cues and intrinsic signaling pathways regulate quiescence exit during MuSC activation. Here, we demonstrated that inducible MuSC-specific deletion of, a catalytic subunit of phosphatidylinositol 3-kinase (PI3K), rendered MuSCs unable to exit quiescence, resulting in severely impaired MuSC proliferation and muscle regeneration. Genetic reactivation of mTORC1, or knockdown of s, in-null MuSCs partially rescued the above defects, making them key effectors downstream of PI3K in regulating quiescence exit. was found to be a key transcriptional target of the PI3K/mTORC1 signaling axis essential for MuSC quiescence exit. Moreover, induction of a constitutively active PI3K in quiescent MuSCs resulted in spontaneous MuSC activation in uninjured muscles and subsequent depletion of the MuSC pool. Thus, PI3K-p110α is both necessary and sufficient for MuSCs to exit quiescence in response to activating signals.

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Section: Deletion Of P110a In Adult Muscs In Uninjured Muscles Reducesupporting

confidence: 80%

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells

et al. 2018

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“…Interestingly, members of the AP‐1 complex, such as Jun , Fos and Fosb , were decreased in p110α‐null MuSCs. These findings are consistent with the recent demonstration that mRNA levels of MyoD and of the members of the AP‐1 family are specifically induced during the early activation of MuSCs (van den Brink et al , ; Machado et al , ; van Velthoven et al , ). Strikingly, combining rapamycin mTORC1 inhibition with MuSC‐specific ablation of p110α and Tsc1 confirmed Jun as downstream target of mTORC1, thereby defining a PI3K‐mTORC1‐Jun axis involved in controlling quiescence exit.…”

Section: Pi3k Signalling Controls Skeletal Muscle Stem Cell (Musc) Exsupporting

confidence: 91%

“…They hypothesized that fibre harvesting was essentially a massive injury and likely resulted in satellite cell activation. Recent studies using complementary strategies have shown that MuSC activation following dissociation is indeed a rapid process, with thousands of genes changing in a few hours, and that this early activation is associated with stress response transcriptional programmes (van den Brink et al , ; Machado et al , ; van Velthoven et al , ). This is in sharp contrast to the time required for the first division (around 24–36 h in response to muscle injury), probably reflecting the metabolic, epigenetic and cellular changes necessary to undergo a first cell division from a quiescent state.…”

Section: Pi3k Signalling Controls Skeletal Muscle Stem Cell (Musc) Exmentioning

confidence: 99%

“…The finding that PI3K signalling leads to a transcriptional response via AP‐1 factors is novel and of particular interest. AP‐1 expression has been identified as a major signature for early activation of MuSCs (van den Brink et al , ; Machado et al , ; van Velthoven et al , ). The precise role of this transcriptional complex with respect to the various aspects of MuSC activation needs to be investigated.…”

Section: Pi3k Signalling Controls Skeletal Muscle Stem Cell (Musc) Exmentioning

confidence: 99%

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Waking up muscle stem cells: PI 3K signalling is ringing

Relaix

Machado

2018

The EMBO Journal

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“…However, this method requires a suitably specific promoter that allows targeting UPRT expression only to the cell type of interest, and experience to date with cre lines suggests that such a promoter may be elusive in the skeletal system. Recent work on muscle comparing post‐isolation to “in vivo” transcriptional profiles provides insight into the likely scope and degree of the impact of tissue digestion and isolation; these procedures induce an immediate early stress response and loss of quiescence that may occur predominantly in subpopulations of cells . Particular care is warranted in assessing whether such isolation‐associated activating transcriptional changes may either drive cell clustering or confound efforts to assess the transcriptional response to environmental or genetic perturbations.…”

Section: Planning a Scrna‐seq Study Of Bonementioning

confidence: 99%

The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

Greenblatt

Ono

Ayturk

et al. 2019

Journal of Bone and Mineral Research

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Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well‐defined populations suitable for further studies. Single‐cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single‐cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single‐cell RNA sequencing (scRNA‐seq) experiments, bone biologists will be ideal leaders in the application of scRNA‐seq to the skeleton. Here we provide an overview of the steps involved with a single‐cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.

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Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations

Cited by 842 publications

References 8 publications

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells

p110α of PI3K is necessary and sufficient for quiescence exit in adult muscle satellite cells

Waking up muscle stem cells: PI 3K signalling is ringing

The Unmixing Problem: A Guide to Applying Single-Cell RNA Sequencing to Bone

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