G2 stem cells orchestrate time-directed, long-range coordination of calcium signaling during skin epidermal regeneration

Abstract

Regenerative processes in the mammalian skin require coordinated cell-cell communication. Ca2+ signaling can coordinate tissue-level responses in developing and wounded epithelia in tissue explants and invertebrates. However, its role in the homeostatic, regenerative basal layer of the skin epithelium is unknown due to significant challenges in studying signaling dynamics in a spatially complex tissue context in live mice. Here we combine in vivo imaging of dynamic Ca2+ signaling at the single cell level across thousands of cells with a novel computational approach, Geometric Scattering Trajectory Homology (GSTH). GSTH models Ca2+ as signals over a cell adjacency graph and uses a multi-level wavelet-like transform (called a scattering transform) to extract signaling patterns from our high dimensional in vivo datasets. We discover local Ca2+ signaling patterns are orchestrated so that signals flow in a coordinated and directed manner across the tissue, distinct from topographically uncoordinated Ca2+ signaling in excitatory tissues. Directed Ca2+ signaling is regulated by the major gap junction protein in the epidermal stem cell layer, Connexin 43 (Cx43). Cx43 gap junctions are dissociated as cells progress through the cell cycle out of G1 and play an essential role in the progression of stem cells from G2 towards mitosis. Finally, G2 cells display related signaling patterns and are essential for tissue-level signaling coordination. Together, our results provide insight into how such a ubiquitous signaling pathway regulates highly specific behaviors and outcomes at a tissue-wide level to maintain proper homeostasis.

Stress vesicles are induced by acute mechanical force and precede the commitment of epidermal stem cells to terminal differentiation

“…In untreated skin, live imaging of the mouse ear skin captured complex patters of intracellular calcium fluxes, exhibited specifically by basal layer keratinocytes, consistent with previous observations (Fig. 4B) 64 .…”

“…In untreated skin, live imaging of the mouse ear skin captured complex patters of intracellular calcium fluxes, exhibited specifically by basal layer keratinocytes, consistent with previous observations (Fig. 4B) 64 . To detect how epidermal stem cells modulate intracellular calcium in response to an acute mechanical stimulus, we applied a compressive force to the mouse ear skin and directly visualized the calcium dynamics in the affected epidermal cells.…”

“…Calcium is a key mediator of cellular mechanotransduction in various systems 35,38,39,44 . Moreover, calcium is a major regulator of keratinocyte differentiation in vivo and in vitro 37,64,73 . Our observation of stress vesicles in epidermal stem cells and their link to cell differentiation indicated that calcium may be implicated in this pathway.…”

“…3H). Previous studies have shown that cells in the basal layer of the epidermis experience major changes in cell shape, cell adhesiveness and cytoskeletal architecture during the different phases of cell the cycle, and that these cellular changes are also associated with the responses of cells to external mechanical stimuli [62][63][64][65] .…”

“…3H). Previous studies have shown that cells in the basal layer of the epidermis experience major changes in cell shape, cell adhesiveness and cytoskeletal architecture during the different phases of cell the cycle, and that these cellular changes are also associated with the responses of cells to external mechanical stimuli [62][63][64][65] . Interestingly, the pro-differentiation phenotype of epidermal stem cells as result of mechanical stress, appears to be linked to the degree of the nuclear deformation rather than to changes in nuclear volume (Fig.…”