Intravital imaging reveals cell cycle-dependent satellite cell migration during muscle regeneration

Background:

A major hurdle to studying endogenous stem cell repair is the limitations of standard experimental methods. In vivo regeneration studies enable a snapshot assessment of cell behavior, whereas in vitro cultures allow for time-lapse analysis but cannot fully recapitulate the complex multicellular, biochemical and biophysical interactions that occur in the native environment. Consequently, recent efforts in skeletal muscle research have focused on developing novel ways to visualize endogenous muscle stem cell (satellite cell)-mediated repair. Intravital two-photon microscopy (IVM) enables the real-time visualisation of cells within living tissues, by tracking fluorescently labelled cells and/or proteins within an ‘imaging window’ of anaesthetised animals. In skeletal muscle, IVM has captured physiological events that could not be observed by other methods, including independent myofibre contractions (Mercier et al., 2016) and microvasculature permeability rates (Hotta et al., 2018). Notably, an IVM study of satellite cell migration and division orientation (which has been linked to cell fate) during regeneration contrasted with behaviors observed in ex vivo isolated myofibers (Webster et al., 2016), highlighting the importance of biochemical and biophysical cues from the native environment. However, questions remain about the spatiotemporal control of satellite cell behavior during regeneration, and to what extent different signaling pathways contribute to each step. This is in part due to a lack of real-time reporter analysis in vivo. In this preprint, the authors used transgenic mice containing satellite cell-specific Förster/fluorescence resonance energy transfer (FRET) biosensors and cell-cycle reporters to explore how extracellular signal-regulated kinase (ERK) signaling contributes to satellite cell proliferation and migration during endogenous repair.

Key findings:

To monitor physiological ERK signaling in satellite cells, the authors crossed R26R-EKAREV mice containing a floxed FRET biosensor for monitoring ERK activity (EKAREV) to the satellite cell-specific Pax7-CreERT2 strain. EKAREV emits a cyan (CFP) donor signal, but upon phosphorylation by ERK a conformational change occurs, causing the FRET intensity to change as yellow fluorescence (YFP) is emitted. Therefore, ERK activity can be measured as a ratio of FRET efficiency (FRET/CFP).

Figure 1: Schematic of the Cre-lox recombination strategy for satellite cell expression of the ERK FRET biosensor, the experimental set-up used and representative images of ERK activity. Adapted from Konagaya et al. and made available under a CC-BY-NC-ND 4.0 International license.

To study satellite cell-mediated regeneration, the authors administered a muscle injury via cardiotoxin and monitored ERK activity over a 5-day timeline via IVM. They found that ERK activity peaked 2 days post-injury, followed by a rapid increase in proliferation and migration speed at day 3. Whilst ERK signaling has been shown to regulate both these processes during muscle regeneration, the relationship between cell cycle modulation and migration isn’t well characterised. Further, evidence from other cell types suggests that migration rates are cell cycle stage specific. Therefore, the authors tested the relationship between ERK activity on proliferation and migration separately.

To examine the relationship between ERK signaling and migration, the authors measured relative ERK activity vs. migration speed in individual cells at day 3, but found no obvious correlation. Further, inhibition of ERK signaling with a MEK inhibitor (PD0325901) reduced the migration speed of some, but not all satellite cells, indicating that ERK signaling is only required for the migration of a subpopulation of cells.

Next they tested the requirement of ERK activity on cell cycle progression in vivo. For this, they used R26Fucci2aR/Pax7-CreERT2 mice, which express mCherry during G0/G1 and mVenus during S/G2/M stages of the cell cycle in satellite cells. PD0325901 administration reduced the number of mVenus+ cells in S/G2/M, indicating that ERK activity is required for G1/S progression in vivo. To further explore whether cell cycle stage and migration are linked, they tracked migration speed in mCherry+ and mVenus+ satellite cells. Again, day 3 post-injury was chosen for assessment, as cells were actively cycling. Interestingly, by dividing cells into cell cycle stage, it became clear that migration speed increased when cells were in the S/G2 phase. To understand how cell cycle relates to migration speed, R26R-EKAREV/ Pax7-CreERT2 mice were injected with cyclin dependant kinase (CDK) inhibitors 3 days post-injury, and the relative migration speed per cell was measured pre- and post-administration. This identified the CDK1/2 inhibitor roscovitine as specifically reducing migration speed.

Overall, the presented work shows that ERK activity modulates G1/S transition during in vivo satellite cell regeneration, and that cell cycle progression is important for the migration of cells to the area of injury. Further studies are required to understand how ERK signaling regulates specific cell cycle stages, and why ERK-mediated migration is required only in a subpopulation of satellite cells.

Take home messages:

• ERK activity peaks in satellite cells prior to division and migration
• ERK signaling regulates G1/S cell cycle progression
• Satellite cells migrate in a cell cycle-specific manner
• The relationship between ERK signaling and migration might be subpopulation specific

 

Why I chose this pre-print:

Unpinning the relationships between cell behaviour and molecular mechanisms, especially in vivo, is challenging. This preprint quantifies for the first time intracellular signaling in satellite cells in real-time during in vivo regeneration and compares signaling activity to key stages of the repair process. FRET is a notoriously challenging technique, so overcoming the challenges of quantification in an in vivo time-lapse experiment is a feat. It also presents an exciting step forward in our ability to visualize cell behavior in the native context.

 

Remaining questions

1. How deep into the muscle were you able to image, and did you observe any regional differences in behavior?
2. Your data shows that some cells have ERK activity in uninjured muscle, when the majority of satellite cells are expected to be in a quiescent state– why do you think this is the case? Is it background FRET activity, or could it be due to proportion of satellite cells being active during muscle homeostasis?
3. You observed no correlation between ERK activity and migration speed at day 3 post-injury, with only some cells responding to ERK inhibition. However, if ERK signaling is upstream of migration (it peaks at day 2 post-injury), would you expect ERK inhibition at day 2 to have an effect?
4. Would it be possible to combine your FRET biosensor reporter (CFP to YFP with TdKeima [RFP] in non-recombined cells) with reporters of potential downstream targets of ERK signaling or cell fate (for example MYOD) if they used far red fluorescence or bioluminescence?

References:

Hotta, K, Behnke, BJ, Masamoto, K, Shimotsu, R, Onodera, N, Yamaguchi, A, Poole, DC, and Kano, Y (2018). Microvascular permeability of skeletal muscle after eccentric contraction-induced muscle injury: in vivo imaging using two-photon laser scanning microscopy. J Appl Physiol 125, 369–380.

Mercier, L, Böhm, J, Fekonja, N, Allio, G, Lutz, Y, Koch, M, Goetz, JG, and Laporte, J (2016).  In vivo imaging of skeletal muscle in mice highlights muscle defects in a model of myotubular myopathy . IntraVital 5, e1168553.

Webster, MT, Manor, U, Lippincott-Schwartz, J, and Fan, C-M (2016). Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration. Cell Stem Cell 18, 243–252.

Tags: biosensor, cell cycle, muscle, repair, stem cell

doi: https://doi.org/10.1242/prelights.24064

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