In this study, we demonstrated the biological links between the shape of TJ-forming cells and a mechanism for the maintenance of TJ barrier homeostasis in the epidermis, as a representative example of how tissues adopt form to follow function. Our proposed f-TKD cell turnover model suggests that the local spatiotemporal orchestration of cell differentiation in the SG cell layer enables the constituent f-TKD cells to be renewed while maintaining TJ barrier homeostasis in cornified epidermis.

The regular columnar stack of flattened corneocytes in murine ear epidermis demonstrates a regular zig-zag interdigitation pattern between two adjacent cell columns (Figure 6—figure supplement 1 reproduced from Figure 1 of Mackenzie [1975]) (Mackenzie, 1969; Christophers, 1972; Menton, 1976; Ball, 2001). This regular interdigitation pattern was spontaneously reproduced by our f-TKD model (Figure 6A), in which cell differentiation in the SG2 cell layer occurs in turn in pairs of adjacent cell columns. The f-TKD model thus provides a coherent mechanistic explanation of how the regular stacks of corneocytes are constructed.

Figure 6 with 1 supplement with 1 supplement see all Download asset Open asset Spatiotemporal orchestration of cell differentiation in the f-TKD model generates interdigitated stacks of corneocytes. (A) The f-TKD model provides a coherent explanation of how the regular interdigitation of corneocytes is produced in the SG2 layer. Arrows show SG3 cells that are newly aligned to the columnar stack. One cycle of cell turnover (orange arrow) corresponds to one cycle indicated in Figure 5. The yellow-colored cell is differentiated from SG3 to SG1 in the time course. (B) Previously proposed columnar unit concept of epidermal structure and turnover. (C) Our proposed f-TKD model for epidermal homeostasis. https://doi.org/10.7554/eLife.19593.031

The f-TKD model further suggests that homeostasis of the SC–SG layers is maintained by the spatiotemporal orchestration of cell differentiation in the SG2 cell layer, rather than by the kinetics of stem cell proliferation and differentiation in the basal layer. Our model is consistent with a pioneering computational simulation suggesting that the regular 3D stacking structure of the SC can be spontaneously formed by randomly supplied cells (Honda et al., 1996). Our model also accords with in vivo cell-tracing studies demonstrating a random supply of cells from the spinous layer to the SG layer (Doupé et al., 2010), and in vivo live observations of the upward movement of spinous layer cells funneling into preexisting cell columns of the SG (Rompolas et al., 2016).

In the 1980s, the epidermal proliferative unit (EPU) concept postulated that each column of flattened corneocytes corresponds to a set of basal layer stem cells that proliferate directly under the column (Potten and Allen, 1975; Potten, 2004). In the EPU model, the regular interdigitation pattern of the SC is explained by the regular kinetics of basal layer stem cells in each EPU. However, recent in vivo cell-fate-tracing studies demonstrated more random cell fate decisions in the basal cell layer, with EPU model-like upward cell movement funneling into the cell columns of the SG, leading to a new concept: the epidermal differentiation unit (EDU) (Rompolas et al., 2016). However, the underlying mechanism dictating the regular interdigitation pattern of cell columns remains enigmatic (Figure 6B).

In our f-TKD model, the cell columns exist only in the SG and SC layers, rather than extending from the basal layer through to the SC. The cells originate from stem cells in the basal layer and are randomly supplied to a spinous layer. Once the cells enter the SG layer, cell turnover in adjacent columns is tightly coordinated in a spatiotemporal manner, leading to the regular interdigitation pattern (Figure 6C). Epidermal homeostasis is maintained by balancing cell proliferation in the basal layer, cell translocation (differentiation) from the basal to the spinous layer, cell integration to the SC/SG layer, and cell shedding from the top of the SC as squames. Future studies are needed to explore how this balance among critical processes in the epidermal layers is regulated to maintain a constant thickness of the epidermis.

The mammalian epidermis is a representative stratified epithelium. Nutrients for stratified cells are mostly supplied from the basal connective tissue via diffusion through paracellular pathways. If all the keratinocytes in the epidermis formed TJs, cells located in the upper epidermis would likely starve due to their segregation from the nutrient supply by multi-layered TJ barriers. Therefore, it is biologically reasonable that the TJ barrier is single-layered in stratified epithelia (Kubo et al., 2012; Yoshida et al., 2013). Our observations revealed that TJ formation is restricted only between SG2 cells (Figure 4—figure supplement 1E). The molecular mechanisms that coordinate the sequential differentiation steps from SG3 to SG1 cells and restrict TJ-forming activity to SG2 cells are currently unknown. The f-TKD model may help to reveal these mechanisms in future studies.

Various mammalian epidermis shows a regular interdigitation pattern in the SC (Christophers, 1972; Mackenzie et al., 1981), suggesting that the f-TKD cell turnover mechanism governs cell differentiation in mammals. Further investigation on whether this characteristic interdigitation pattern is observed in the SC of other vertebrates, such as amphibians, reptiles and birds, may reveal the general applicability of the f-TKD model to cornified stratified epithelia. Other mechanisms could be involved in maintaining TJ barrier homeostasis in simple epithelia and non-cornified stratified epithelia, where apoptotic cells are extruded to the outside TJ barrier by adjacent cells that migrate into the basal side of the apoptotic cells and form multi-junctional TJs (Pentecost et al., 2006; Eisenhoffer and Rosenblatt, 2011).

The actual structure of the optimal space-filling shape with minimal surface area could be more complex than Kelvin’s model (Lewis, 1943; Williams, 1968; Weaire and Phelan, 1994), and the shape and alignment of corneocytes are much more variegated in human skin compared to mouse ear skin (Mackenzie et al., 1981). Nonetheless, the basic concept of the spatiotemporal orchestration of SG cell differentiation in the f-TKD model would be sufficient to explain the regular interdigitation pattern of corneocytes observed in various types of cornified skin (Mackenzie et al., 1981). The f-TKD cell turnover model of the SG can be applied to stratified stacks of variously shaped polyhedral cells and provides a fundamental basis for the maintenance of barrier homeostasis during cell turnover in cornified stratified epithelia.