A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

Abstract

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h to induce differentiation. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases -including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA -promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Their activity is antagonised by concomitant upregulation of DUSP10. Boolean network modelling of phosphatase interactions identifies commitment as an inherently unstable biological switch between the stem and differentiated cell states. Furthermore, phosphatase expression is spatially regulated both in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

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

“…This suggests the intriguing possibility that DUSP6-mediated ERK pulse downregulation promotes the initiation of differentiation whereas DUSP10-mediated downregulation of mean ERK activity promotes and stabilizes post-commitment differentiation. This is consistent with the finding that DUSP6 is transiently upregulated on commitment, while DUSP10 upregulation is more sustained(23).…”

“…Previous studies have demonstrated interactions between DUSP6, DUSP10 and ERK at the transcriptional level in addition to the post-translation level(23, 35-37). We therefore compared transcripts of MAPK3 and MAPK1, which code ERK1 and ERK2 proteins, together with DUSP6 and DUSP10, in individual cells using RNA fluorescence in situ hybridization (Fig.…”

“…We have previously reported that human epidermal stem cells have a patterned distribution in skin(38), leading us to predict that ERK dynamics would be spatially regulated. To examine this, we plated NHKs co-expressing a cytoplasmic ERK sensor (EKAR-EVnes) and Involucrin-mCherry on collagen-coated undulating polydimethylsiloxane (PDMS) substrates that mimic the topography of the human epidermal-dermal junction(23, 39). As in human epidermis, stem cells that express high β1-integrin and DUSP6 levels cluster at the tips of the features, whereas DUSP10 expression is more uniformly distributed(23).…”

“…Here we show, by live imaging of thousands of human epidermal cells, that there are dynamic transitions in ERK activity during stem cell colony expansion and differentiation. ERK pulse activity and basal levels are independently regulated by DUSP6 and DUSP10, components of the autoregulatory protein phosphatase network that acts as a switch between the stem cell state and the differentiated cell state(23). We also observe spatial segregation of cells with different ERK temporal patterns on substrates mimicking human dermis and in live mouse skin, establishing the physiological significance of our observations.…”

Generative adversarial networks simulate gene expression and predict perturbations in single cells

“…To further investigate our gene expression predictions, we focused on four clusters of dynamic gene expression profile, two corresponding to increasing and decreasing expression ( Figure 4B, Groups I and II) and two subsets of genes only transiently up-or down-regulated during commitment to differentiation ( Figure 4B, Group III and IV genes). To validate these findings we used a recently published dataset investigating human epidermal differentiation 28 . Mishra and colleagues utilised methylcellulose suspension-induced differentiation to obtain temporal differentiation gene expression data from human keratinocytes at 0, 4, 8 and 12 hours.…”