ERK basal and pulsatile activity are differentially regulated in mammalian epidermis to control proliferation and exit from the stem cell compartment

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to a range of differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse downregulation promotes initiation of differentiation whereas DUSP10-mediated downregulation of mean ERK activity promotes and stabilizes post-commitment differentiation. Quantification of MAPK1/3, DUSP6 and DUSP10 transcripts in individual cells demonstrated that ERK activity is controlled both transcriptionally and post-transcriptionally. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity and downregulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics.SignificanceUnderstanding how intracellular signaling cascades control cell fate is a key issue in stem cell biology. Here we show that exit from the stem cell compartment in mammalian epidermis is characterised by pulsatile ERK MAPK activity. Basal activity and pulses are differentially regulated by DUSP10 and DUSP6, two phosphatases that have been shown previously to regulate differentiation commitment in the epidermis. ERK activity is controlled both transcriptionally and post-transcriptionally. Spatial segregation of mean ERK activity and pulses is observed both in reconstituted human epidermis and in mouse epidermis. Our findings demonstrate the tight spatial and temporal regulation of ERK MAPK expression and activity in mammalian epidermis.

SingleCellSignalR: inference of intercellular networks from single-cell transcriptomics

Single-cell transcriptomics offers unprecedented opportunities to infer the ligand–receptor (LR) interactions underlying cellular networks. We introduce a new, curated LR database and a novel regularized score to perform such inferences. For the first time, we try to assess the confidence in predicted LR interactions and show that our regularized score outperforms other scoring schemes while controlling false positives. SingleCellSignalR is implemented as an open-access R package accessible to entry-level users and available from https://github.com/SCA-IRCM. Analysis results come in a variety of tabular and graphical formats. For instance, we provide a unique network view integrating all the intercellular interactions, and a function relating receptors to expressed intracellular pathways. A detailed comparison of related tools is conducted. Among various examples, we demonstrate SingleCellSignalR on mouse epidermis data and discover an oriented communication structure from external to basal layers.

A G1 sizer mechanism coordinates growth and division in the mouse epidermis

SummaryCell size homeostasis is often achieved by coupling cell cycle progression to cell growth. Studies of cell size homeostasis in single-celled bacteria and yeast have observed several distinct phenomena. Growth can be coupled to division through a range of mechanisms, including a ‘sizer’, wherein cells of varying birth size divide at similar final sizes [1–3], and an ‘adder’, wherein cells increase in size a fixed amount per cell cycle [4–6]. Importantly, intermediate control mechanisms are observed, and even the same organism can exhibit distinct control phenomena depending on growth conditions [2,7,8]. While studying unicellular organisms in laboratory conditions may give insight into their growth control in the wild, this is less apparent for studies of mammalian cells growing outside the organism. Sizer, adder, and intermediate mechanisms have been observed in vitro [9–12], but it is unclear how these diverse size homeostasis phenomena relate to mammalian cell proliferation in vivo. To address this gap, we analyzed time-lapse images of the mouse epidermis taken over one week during normal tissue turnover [13]. We quantified the 3D volume growth and cell cycle progression of single cells within the mouse skin. In dividing epidermal stem cells, we found that cell growth is coupled to division through a sizer mechanism operating largely in the G1 phase. Thus, while the majority of tissue culture studies to-date identified adder mechanisms, our analysis demonstrates that sizer mechanisms are important in vivo and highlights the need to determine their underlying molecular origin.