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Highly regenerative tissues continuously produce terminally differentiated cells to replace those that are lost. How they orchestrate the complex transition from undifferentiated stem cells towards post-mitotic, molecularly distinct and often spatially segregated differentiated populations is not well understood. In the adult skin epidermis, the stem cell compartment contains molecularly heterogeneous subpopulations1–4 whose relationship to the complete trajectory of differentiation remains unknown. Here we show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes with upregulation of differentiation genes preceding downregulation of typical stemness genes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit-amplifying programme but help to buffer the differentiating cell pool during heightened demand. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover.

Stem cell differentiation requires dramatic changes in gene expression and global remodeling of chromatin architecture. How and when chromatin remodels relative to the transcriptional, behavioral, and morphological changes during differentiation remain unclear, particularly in an intact tissue context. Here, we develop a quantitative pipeline which leverages fluorescently-tagged histones and longitudinal imaging to track large-scale chromatin compaction changes within individual cells in a live mouse. Applying this pipeline to epidermal stem cells, we reveal that cell-to-cell chromatin compaction heterogeneity within the stem cell compartment emerges independent of cell cycle status, and instead is reflective of differentiation status. Chromatin compaction state gradually transitions over days as differentiating cells exit the stem cell compartment. Moreover, establishing live imaging of Keratin-10 (K10) nascent RNA, which marks the onset of stem cell differentiation, we find that Keratin-10 transcription is highly dynamic and largely precedes the global chromatin compaction changes associated with differentiation. Together, these analyses reveal that stem cell differentiation involves dynamic transcriptional states and gradual chromatin rearrangement.

Skin epithelial stem cells detect and correct aberrancies induced by oncogenic mutations. Different oncogenes invoke different mechanisms of epithelial tolerance: while wild-type cells outcompete β-catenin-Gain-of-Function (βcatGOF) mutant cells, HrasG12Vmutant cells outcompete wild-type cells1,2. Here we ask how metabolic states change as wild-type stem cells interface with mutant cells, and how this ultimately drives different cell competition outcomes. By adapting our live-imaging platform to track endogenous redox ratio (NAD(P)H/FAD) with single cell resolution in the same mice over time, we show that wild-type epidermal stem cells maintain robust redox ratio despite their heterogeneous cell cycle states. We discover that both βcatGOF and HrasG12Vmodels lead to a rapid drop in redox ratios. However, the “winner” cells in each model (wild-type in βcatGOF and mutant in HrasG12V), rapidly recover their redox ratios, irrespective of the mutation induced. Using mass spectrometry (13C-LC-MS/MS)3, we find that both mutants increase flux through the oxidative tricarboxylic acid cycle, but the “winner” HrasG12Vcells and the “loser” βcatGOF cells modulate glycolytic flux differently. Hence, we reveal the metabolic adaptations that define the hallmarks of winners and losers during cell competitionin vivoand uncover the nodes of regulation unique to each cell fate.

A functional network of blood vessels is essential for organ growth and homeostasis. Yet, how the vasculature matures and maintains adult homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by network-wide vessel regression and transient angiogenesis. A fixed number of neonatal ECs rearrange their positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon injury, while neonatal ECs are predisposed to die, adult ECs survive through a plasmalemmal self-repair response. Furthermore, adult neighboring ECs reactivate migration to assist vessel repair. Lastly, neonatal vessel regression and adult vascular maintenance are orchestrated by temporally restricted VEGFR2 dependent signaling. Our work sheds light on fundamental cellular mechanisms that underlie both vascular maturation and adult homeostasisin vivo.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning results in the distension of the alveolar structures by the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GcAMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6 and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis. This is the result of increased TGFβ signaling and inhibition of cell cycle progression. Finally, we demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3, which also appears to be mediated by TGFβ signaling. In summary, these data suggest that intracellular calcium serves as an important biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.TeaserIncrease intracellular calcium leads to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

Healthy skin is a tapestry of wild-type and mutant clones. Although injury can cooperate with Ras mutations to promote tumorigenesis, the consequences in genetically mosaic skin are unknown. Here, we show that wild-type cells prevent oncogenic Ras-induced aberrant growth after injury. Although HrasG12V/+ and KrasG12D/+ cells outcompete wild-type cells in uninjured, mosaic tissue, their competitive advantage is suppressed after injury due to a selective increase in wild-type cell proliferation. EGFR inhibition abolishes the competitive advantage of wild-type cells after injury of HrasG12V/+-mosaic skin. Global loss of the cell cycle inhibitor p21 increases wild-type cell proliferation even without injury, suppressing the competitive advantage of HrasG12V/+ cells. Thus, injury plays an unanticipated role in switching the competitive balance between oncogenic and wild-type cells in genetically mosaic skin.One sentence SummaryInjury-repair selectively induces wild-type cell proliferation to suppress oncogenic growth in Ras-mosaic skin epithelium.

Mutations associated with tumor development in certain tissues can be nontumorigenic in others, yet the mechanisms underlying these different outcomes remains poorly understood. To address this, we targeted an activating Hras mutation to hair follicle stem cells and discovered that Hras mutant cells outcompete wild-type neighbors yet are integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium leads to benign outgrowths. Follicular Hras mutant cells autonomously and nonautonomously enhance regeneration, which directs mutant cells into continuous tissue cycling to promote integration rather than aberrancy. This follicular tolerance is maintained under additional challenges that promote tumorigenesis in the epidermis, including aging, injury, and a secondary mutation. Thus, the hair follicle possesses a unique, enhanced capacity to integrate and contain Hras mutant cells within both homeostatic and perturbed tissue, demonstrating that in the skin, multiple, distinct mechanisms exist to suppress oncogenic growth.

We examined the unique contributions of the cytokines IL-21 and IL-4 on germinal center (GC) B cell initiation and subsequent maturation in a murine model system. Similar to other reports, we found T follicular helper cell expression of IL-21 begins prior to T follicular helper cell migration into the B cell follicle and precedes that of IL-4. Consistent with this timing, IL-21 signaling has a greater influence on the perifollicular pre-GC B cell transition to the intrafollicular stage. Notably, Bcl6hi B cells can form in the combined absence of IL-21R– and STAT6-derived signals; however, these nascent GC B cells cease to proliferate and are more prone to apoptosis. When B cells lack either IL-21R or STAT6, aberrant GCs form atypical centroblasts and centrocytes that differ in their phenotypic maturation and costimulatory molecule expression. Thus, IL-4 and IL-21 play nonredundant roles in the phased progression of GC B cell development that can initiate in the combined absence of these cytokine signals.

Thermoelectrically coupled nanoantennas (TECNAs) for mid- to far-infrared radiation are developed to study the Sun. TECNAs are resonantly absorbing the IR radiation using a nanoantenna that provides wavelength and polarization selectivity, as well as providing uncooled, and fast detection. This technology will enable transient solar measurements in the mid- to far- and wavelength ranges.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplantation (alloSCT). By static microscopy, cutaneous GVHD lesions contain a mix of T cells and myeloid cells. We used 2-photon intravital microscopy to investigate the dynamics of CD4+ and CD8+ T cells and donor dendritic cells (DCs) in cutaneous GVHD lesions in an MHC-matched, multiple minor histocompatibility antigen-mismatched (miHA) model. The majority of CD4 and CD8 cells were stationary, and few cells entered and stopped or were stopped and left the imaged volumes. CD8 cells made TCR:MHCI-dependent interactions with CD11c+ cells, as measured by the durations that CD8 cells contacted MHCI+ vs MHCI− DCs. The acute deletion of Langerin+CD103+ DCs, which were relatively rare, did not affect CD8 cell motility and DC contact times, indicating that Langerin−CD103− DCs provide stop signals to CD8 cells. CD4 cells, in contrast, had similar contact durations with MHCII+ and MHCII− DCs. However, CD4 motility rapidly increased after the infusion of an MHCII-blocking antibody, indicating that TCR signaling actively suppressed CD4 movements. Many CD4 cells still were stationary after anti-MHCII antibody infusion, suggesting CD4 cell heterogeneity within the lesion. These data support a model of local GVHD maintenance within target tissues.

Fibroblasts are an essential cellular and structural component of our organs. Despite several advances, the critical behaviors that fibroblasts utilize to maintain their homeostasis in vivo have remained unclear. Here, by tracking the same skin fibroblasts in live mice, we show that fibroblast position is stable over time and that this stability is maintained despite the loss of neighboring fibroblasts. In contrast, fibroblast membranes are dynamic during homeostasis and extend to fill the space of lost neighboring fibroblasts in a Rac1-dependent manner. Positional stability is sustained during aging despite a progressive accumulation of gaps in fibroblast nuclei organization, while membrane occupancy continues to be maintained. This work defines positional stability and cell occupancy as key principles of skin fibroblast homeostasis in vivo, throughout the lifespan of mice, and identifies membrane extension in the absence of migration as the core cellular mechanism to carry out these principles.

Cells in healthy tissues acquire mutations with surprising frequency. Many of these mutations are associated with abnormal cellular behaviours such as differentiation defects and hyperproliferation, yet fail to produce macroscopically detectable phenotypes1–3. It is currently unclear how the tissue remains phenotypically normal, despite the presence of these mutant cells. Here we use intravital imaging to track the fate of mouse skin epithelium burdened with varying numbers of activated Wnt/β-catenin stem cells. We show that all resulting growths that deform the skin tissue architecture regress, irrespective of their size. Wild-type cells are required for the active elimination of mutant cells from the tissue, while utilizing both endogenous and ectopic cellular behaviours to dismantle the aberrant structures. After regression, the remaining structures are either completely eliminated or converted into functional skin appendages in a niche-dependent manner. Furthermore, tissue aberrancies generated from oncogenic Hras, and even mutation-independent deformations to the tissue, can also be corrected, indicating that this tolerance phenomenon reflects a conserved principle in the skin. This study reveals an unanticipated plasticity of the adult skin epithelium when faced with mutational and non-mutational insult, and elucidates the dynamic cellular behaviours used for its return to a homeostatic state.

Tissue repair is fundamental to our survival as tissues are challenged by recurrent damage. During mammalian skin repair, cells respond by migrating and proliferating to close the wound. However, the coordination of cellular repair behaviours and their effects on homeostatic functions in a live mammal remains unclear. Here we capture the spatiotemporal dynamics of individual epithelial behaviours by imaging wound re-epithelialization in live mice. Differentiated cells migrate while the rate of differentiation changes depending on local rate of migration and tissue architecture. Cells depart from a highly proliferative zone by directionally dividing towards the wound while collectively migrating. This regional coexistence of proliferation and migration leads to local expansion and elongation of the repairing epithelium. Finally, proliferation functions to pattern and restrict the recruitment of undamaged cells. This study elucidates the interplay of cellular repair behaviours and consequent changes in homeostatic behaviours that support tissue-scale organization of wound re-epithelialization.