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The involvement of epidermal stem cells in spatiotemporal ERK activity propagation

Periodic Reporting for period 1 - SKINFRET (The involvement of epidermal stem cells in spatiotemporal ERK activity propagation)

Reporting period: 2016-06-27 to 2018-06-26

In the project, we studied the spatial and temporal dynamics of ERK MAPK signal for the regulation of stem cell commitment to differentiation.
As proposed in the Annex1 of Grant Agreement, ERK MAPK signal was observed at single-cell resolution by EKAREV, a FRET probe for ERK MAPK. It was applied to multiple experimental platforms from cultured human keratinocytes, 3D patterned substrates, and mouse in vivo skin.
Together with ERK MAPK signal activity, we simultaneously monitored keratinocyte differentiation level by developing a reporter that expresses mCherry fluorescence under the regulation of Involucrin promoter. This revealed that stem cells and differentiated cells are characterized by stable high and low ERK MAPK activity, pattern, respectively. In contrast, cells that are committed to exit the stem cell compartment are characterized by pulsatile ERK MAPK activity. Differentiation was preceded by significant decrease in ERK MAPK pulses.

Milestones of the projects were achieved as described below.
(MS 1) in vivo imaging of stem cells and SPREAD : Achieved by the 6th month of Year 1
Pulse activation of ERK MAPK activity was successfully observed in both living mouse skin and cultured human epidermal stem cells.
(MS 2) ERK activation pattern on engineered substrate: Achieved by the 3rd month of Year 2
On the 3D patterned substrate, cells with different ERK MAPK patterns were segregated.
(MS 3) ERK activation pattern on 3D reconstituted skin: Still ongoing.
live imaging of 3D reconstituted skin requires optimization of microscopy due to its fragile structure.
(MS 4) Light-induced generation of SPREAD: Achieved by the 6th month of Year 2
Generation of ERK activation wave directed cells to migrate toward the origin of the waves.
1.1 Objectives
In the project, we aimed to answer the following questions.
Objective 1: Do epidermal stem cells operate as initiating cells of SPREAD?
Objective 2: If so, what is the influence of stem cell microenvironment on SPREAD?
Objective 3: Does SPREAD appear in human keratinocytes?
We monitored ERK activity in individual primary human neonatal keratinocytes (NHKs) via lentiviral expression of an ERK reporter, EKAREV-nls. NHKs were cultured on a 3T3 feeder layer under conditions that support clonal growth of stem cells and differentiation of their progeny, as occurs in skin (Fig. 1a). Within colonies, mean ERK activity was lower in large than small keratinocytes (Fig. 1b) and there was a wider range of mean ERK activity in smaller cells (tails and outliers in Fig.1b). This suggests that the patterns of ERK activity differ in stem cells and differentiated cells, since keratinocytes are known to enlarge as they undergo terminal differentiation.
The temporal patterns of ERK activity varied considerably between cells. Some maintained stable-high or stable-low activity, while others exhibited pulsating ERK activity (Fig. 1c, d). By plotting ERK activity as a function of time, we observed cells with stable-low or stable-high ERK activity (characterized by a low variance) (Fig. 1c, d). We also observed cells with pulsating ERK activity (characterized by a high variance). The pulses were not uniform between cells but differed in amplitude, frequency and duration (Fig. 1c, d). We ruled out the possibility that the pulses were an imaging artifact by using a kinase-dead control FRET biosensor, EKAREV-TA-nls (Extended Data Fig. 1d).
We generated a fluorescent reporter of the differentiation marker, Involucrin, which is expressed by all keratinocytes undergoing terminal differentiation in culture. We used the previously characterised Involucrin promoter to drive expression of mCherry (Fig. 1e). Cells were cultured in low Ca2+ keratinocyte serum-free medium (KSFM), which does not support full terminal differentiation, and then transferred to medium containing high Ca2+ (1.6 mM) or serum. Under those conditions, involucrin-positive cells are known to accumulate and there was a significant increase in mCherry fluorescence (Fig. 1f, g).
Simultaneous monitoring of ERK activity and Involucrin expression in individual cells revealed that ERK pulses are gradually lost, coinciding with the onset of involucrin expression (Fig. 1h). To gain an integrative view of the temporal relationship between ERK activity pulses and Involucrin expression, we computed the variance over a moving time window of ERK activity and a moving mean on the Involucrin time series. This allowed us to build trajectories in the ERK variance/mean Involucrin phase diagram, in order to discover how these qualities relate to each other as a function of time. For this, we incorporated the observations from 780 cells (Fig. 1i, j). The vectors point forward in time, showing how ERK variance and mean Involucrin expression coevolve as cells differentiate. This reveals that ERK activity pulses (measured by the variance) are drastically decreased prior to Involucrin expression, reaching a stable status of low variance-high Involucrin (red square in Fig. 1i). Conversely, the mean ERK activity gradually converges towards a low mean value, as the level of Involucrin increases (see arrows in Fig. 1j). This differential behavior indicates that ERK pulses and mean level are subject to distinct regulation, and that a dramatic decrease in pulsatile behaviour precedes differentiation (Fig. 1k).
The achievements in the project have a great impact not only on stem cell biology but also on cellular signaling and developmental biology. The dynamic switching of ERK activity profiles occurs at single cell level and has long been elusive in population-based approaches. This was achieved by the state-of-the-art approaches of this project and great supports from this Marie Skłodowska-Curie IF fellowship.
The research achievements are being submitted to a highly-ranked journal. Furthermore, the results are already presented in multiple domestic and international conferences including Cold Spring Harbor Meeting in the United States and Lindau Nobel Laureate Meeting in Germany.
The information on section 2.1 of the DoA (how your project will contribute to the expected impacts) is still relevant. Dr. Hiratsuka acquired skills in human epidermal stem cell culture systems, single cell live imaging, sophisticated image processing methods, and mathematical interpretations of quantitative results. These skills will be highly beneficial to pursue his interest in spatiotemporal ERK MAPK dynamics.
The work was supervised by Prof. Fiona Watt, a leading expert in stem cell biology and Dr. Hiratsuka had regular discussions with lab colleagues and other researchers in the Centre for Stem Cells and Regenerative Medicine. He has regularly attend KIN (King’s Imaging Network) meetings and created networks with other researchers working with live imaging in King’s College London.
During this project, Dr. Hiratsuka significantly extended his research networks, made full use of it for the completion of this project, and connected it to further future research projects.