According to the current model of adult epidermal homeostasis, skin tissue is maintained by two discrete populations of progenitor cells: self-renewing stem cells; and their progeny, known as transit amplifying cells, which differentiate after several rounds of cell division. By making use of inducible genetic labelling, we have tracked the fate of a representative sample of progenitor cells in mouse tail epidermis at single-cell resolution in vivo at time intervals up to one year. Here we show that clone-size distributions are consistent with a new model of homeostasis involving only one type of progenitor cell. These cells are found to undergo both symmetric and asymmetric division at rates that ensure epidermal homeostasis. The results raise important questions about the potential role of stem cells on tissue maintenance in vivo.

Diseases of esophageal epithelium (EE) such as reflux esophagitis and cancer are rising in incidence. Despite this, the cellular behaviors underlying EE homeostasis and repair remain controversial. Here we show that in mice, EE is maintained by a single population of cells that divide stochastically to generate proliferating and differentiating daughters with equal probability. In response to challenge with all-trans Retinoic Acid (atRA) the balance of daughter cell fate is unaltered but the rate of cell division increases. However, following wounding, cells reversibly switch to producing an excess of proliferating daughters until the wound has closed. Such fate switching enables a single progenitor population to both maintain and repair tissue without the need for a "reserve" slow-cycling stem cell pool.Murine EE consists of layers of keratinocytes. These tissue lacks structures such as crypts or glands which form stem cell niches in other epithelia . Proliferation is confined to cells in the basal layer (6). On commitment to terminal differentiation, basal cells exit the cell cycle and subsequently migrate to the tissue surface from which they are shed. Early studies suggested all proliferating cells were functionally equivalent, but recent reports propose that a discrete population of slow-cycling stem cells is responsible for both maintenance and wound healing (7)(8)(9)(10)(11). This controversy and the importance of EE in disease motivated us to resolve the proliferative cell behavior in homeostatic EE and in tissue challenged by systemic treatment with the vitamin A metabolite all-trans Retinoic Acid (atRA) or acute local wounding (12-13).To investigate cell division rates in EE we used a transgenic label retaining cell (LRC) assay ( Fig. 1C) (1, 14-15). Doxycycline (DOX) induction of Histone-2B EGFP fusion protein (HGFP) expression in Rosa26 M2rtTA /TetO-HGFP mice resulted in nuclear fluorescent labeling throughout the EE (Fig. 1D and fig. S1A). When DOX is withdrawn, HGFP is diluted by cell division, leaving 0.4% basal layer cells (561 out of 140000) retaining label after a 4 week chase (Figs. 1E and S1B). 3D imaging showed these label retaining cells * To whom correspondence should be addressed. phj20@cam.ac.uk. 5 These authors contributed equally to this work Supplementary Materials: Materials and Methods Figures S1-S13 However, 99.9% (2457 out of 2459) of LRC were positive for the pan leukocyte marker CD45 (Fig. 1E inset), comprising of a mixture of Langerhan's cells and lymphocytes (Figs. S1E and F). These findings lead to the surprising conclusion that, unlike tissues such as the epidermis, there are no slow-cycling or quiescent epithelial stem cells in EE (1, 17). Indeed, HGFP dilution in basal cells was strikingly homogeneous, suggesting that all cells divide at a similar rate of approximately twice per week (Fig. S1G).Although epithelial cells have the same rate of division, they may still differ in their ability to generate cycling and differentiated progeny. We therefore used inducible ...

Typical murine epidermis has a patterned structure, seen clearly in ear skin, with regular columns of differentiated cells overlying the proliferative basal layer. It has been proposed that each column is a clonal epidermal proliferative unit maintained by a central stem cell and its transit amplifying cell progeny. An alternative hypothesis is that proliferating basal cells have random fate, the probability of generating cycling or differentiated cells being balanced so homeostasis is achieved. The stochastic model seems irreconcilable with an ordered tissue. Here we use lineage tracing to reveal that basal cells generate clones with highly irregular shapes that contribute progeny to multiple columns. Basal cell fate and cell cycle time is random. Cell columns form due to the properties of postmitotic cells. We conclude that the ordered architecture of the epidermis is maintained by a stochastic progenitor cell population, providing a simple and robust mechanism of homeostasis.

The rules governing cell division and differentiation are central to understanding the mechanisms of development, aging and cancer. By utilising inducible genetic labelling, recent studies have shown that the clonal population in transgenic mouse epidermis can be tracked in vivo. Drawing on these results, we explain how clonal fate data may be used to infer the rules of cell division and differentiation underlying the maintenance of adult murine tail-skin. We show that the rates of cell division and differentiation may be evaluated by considering the long-time and short-time clone fate data, and that the data is consistent with cells dividing independently rather than synchronously. Motivated by these findings, we consider a mechanism for cancer onset based closely on the model for normal adult skin. By analysing the expected changes to clonal fate in cancer emerging from a simple two-stage mutation, we propose that clonal fate data may provide a novel method for studying the earliest stages of the disease.

SignificanceMost epithelia are turned over throughout adult life as cells are lost from the surface and replaced by the proliferation of stem cells. Precise regulation of stem cells by signals from the local microenvironment or niche is important to maintain epithelial homeostasis. Here, using intestinal stem cells of the Drosophila midgut as a model system, we use transcriptome profiling to identify genes expressed specifically in stem and progenitor cells and not their differentiated daughters. We find that stem and progenitor cells express ligands of major developmental signaling pathways to both contribute to the niche and regulate the production of niche signals from other cell types.

The dynamics of a genetically-labelled cell population may be used to infer the laws of cell division in mammalian tissue. Recently, we showed that in mouse tail-skin, where proliferating cells are confined to a two-dimensional layer, cells proliferate and differentiate according to a simple stochastic model of cell division involving just one type of proliferating cell that may divide both symmetrically and asymmetrically. Curiously, these simple rules provide excellent predictions of the cell population dynamics without having to address their spatial distribution. Yet, if the spatial behaviour of cells is addressed by allowing cells to diffuse at random, one deduces that density fluctuations destroy tissue confluence, implying some hidden degree of spatial regulation in the physical system. To infer the mechanism of spatial regulation, we consider a two-dimensional model of cell fate that preserves the overall population dynamics. By identifying the resulting behaviour with a three-species variation of the "Voter" model, we predict that proliferating cells in the basal layer should cluster. Analysis of empirical correlations of cells stained for proliferation activity confirms that the expected clustering behaviour is indeed seen in nature. As well as explaining how cells maintain a uniform two-dimensional density, these findings present an interesting experimental example of voter-model statistics in biology.

In the 1970s, studies of tissue architecture and cell proliferation were used to formulate a new model of epidermal homeostasis. This asserted that the tissue was maintained by long-lived, slow-cycling, self-renewing stem cells that generate a short-lived population of transit amplifying (TA) cells, which undergo terminal differentiation after a set number of cell divisions. It was further hypothesized that in the epidermis, the tissue was organized into clonal epidermal proliferative units (EPUs) comprising a central stem cell with surrounding TA cells, which maintain the overlying differentiated cell layers. The stem ⁄ TA and EPU hypotheses have been widely influential.Here, we first revaluate older literature, finding numerous studies that conflict with the EPU model. We then review recent large-scale lineage tracing studies in transgenic mice which exclude the stem ⁄ TA and EPU hypotheses, and reveal that the epidermis is maintained by a single population of functionally equivalent cycling progenitor cells. The outcome of individual progenitor cell divisions is random, but the probabilities of generating differentiated and progenitor cell daughters are equal, so that homeostasis is maintained across the progenitor population. We reconcile this model with the older literature and place the epidermis in the context of other tissues that are also maintained by continually cycling cells with stochastic fate.

The use of genome-wide proteomic and RNA interference approaches has moved our understanding of signal transduction from linear pathways to highly integrated networks centered on core nodes. However, probing the dynamics of flow of information through such networks remains technically challenging. In particular, how the temporal dynamics of an individual pathway can elicit distinct outcomes in a single cell type and how multiple pathways may interact sequentially or synchronously to influence cell fate remain open questions in many contexts. The development of fluorescence-based reporters and optogenetic regulators of pathway activity enables the analysis of signaling in living cells and organisms with unprecedented spatiotemporal resolution and holds the promise of addressing these key questions. We present a brief overview of the evidence for the importance of temporal dynamics in cellular regulation, introduce these fluorescence-based tools, and highlight specific studies that leveraged these tools to probe the dynamics of information flow through signaling networks. In particular, we highlight two studies in Caenorhabditis elegans sensory neurons and cultured mammalian cells that demonstrate the importance of signal dynamics in determining cellular responses.