Valentina Greco

Argosomes: A Potential Vehicle for the Spread of Morphogens through Epithelia

The formation of morphogen gradients is essential for tissue patterning. Morphogens are released from producing cells and spread through adjacent tissue; paradoxically, however, many morphogens, including Wingless, associate tightly with the cell membrane. Here, we describe a novel cell biological mechanism that disperses membrane fragments over large distances through the Drosophila imaginal disc epithelium. We call these membrane exovesicles argosomes. Argosomes are derived from basolateral membranes and are produced by many different regions of the disc. They travel through adjacent tissue where they are found predominantly in endosomes. Wingless protein colocalizes with argosomes derived from Wingless-producing cells. The properties of argosomes are consistent with their being a vehicle for the spread of Wingless protein.

Spatial organization within a niche as a determinant of stem-cell fate.

Stem-cell niches in mammalian tissues are often heterogeneous and compartmentalized; however, whether distinct niche locations determine different stem-cell fates remains unclear. To test this hypothesis, here we use the mouse hair follicle niche and combine intravital microscopy with genetic lineage tracing to re-visit the same stem-cell lineages, from their exact place of origin, throughout regeneration in live mice. Using this method, we show directly that the position of a stem cell within the hair follicle niche can predict whether it is likely to remain uncommitted, generate precursors or commit to a differentiated fate. Furthermore, using laser ablation we demonstrate that hair follicle stem cells are dispensable for regeneration, and that epithelial cells, which do not normally participate in hair growth, re-populate the lost stem-cell compartment and sustain hair regeneration. This study provides a general model for niche-induced fate determination in adult tissues.

Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration

Tissue development and regeneration depend on cell–cell interactions and signals that target stem cells and their immediate progeny. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, intravital two-photon imaging approach we study physiological hair-follicle regeneration over time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny during physiological hair regeneration and addressed how the mesenchyme influences their behaviour. Consistent with earlier studies, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny are more actively dividing. Moreover, stem cell progeny divisions are spatially organized within follicles. In addition to cell divisions, coordinated cell movements of the progeny allow the rapid expansion of the hair follicle. Finally, we show the requirement of the mesenchyme for hair regeneration through targeted cell ablation and long-term tracking of live hair follicles. Thus, we have established an in vivo approach that has led to the direct observation of cellular mechanisms of growth regulation within the hair follicle and that has enabled us to precisely investigate functional requirements of hair-follicle components during the process of physiological regeneration.

Stem cell dynamics in the hair follicle niche.

Hair follicles are appendages of the mammalian skin that have the ability to periodically and stereotypically regenerate in order to continuously produce new hair over our lifetime. The ability of the hair follicle to regenerate is due to the presence of stem cells that along with other cell populations and non-cellular components, including molecular signals and extracellular material, make up a niche microenvironment. Mounting evidence suggests that the niche is critical for regulating stem cell behavior and thus the process of regeneration. Here, we review the literature concerning past and current studies that have utilized mouse genetic models, combined with other approaches to dissect the molecular and cellular composition of the hair follicle niche. We also discuss our current understanding of how stem cells operate within the niche during the process of tissue regeneration and the factors that regulate their behavior.

β-Catenin Activation Regulates Tissue Growth Non–Cell Autonomously in the Hair Stem Cell Niche

Coordinated Hair Growth Wnt/β-catenin signaling is a key pathway that plays a conserved role in regulating stem cell function during adult tissue regeneration. Using time-lapse imaging of live mice, Deschene et al. (p. 1353) show that genetic activation of β-catenin within hair follicle stem cells generates axes of hair growth by coordinated cell divisions and cell movements, even when the normal niches—the dermal papillae—are laser-ablated. Activated β-catenin enhances Wnt ligand secretion, and these ligands can then activate Wnt signaling in adjacent cells that do not have activated β-catenin, indicating how activated stem cells could influence neighboring cells during normal growth and in cancer. Signals generated by mouse hair follicle stem cells generate new hair growth. Wnt/β-catenin signaling is critical for tissue regeneration. However, it is unclear how β-catenin controls stem cell behaviors to coordinate organized growth. Using live imaging, we show that activation of β-catenin specifically within mouse hair follicle stem cells generates new hair growth through oriented cell divisions and cellular displacement. β-Catenin activation is sufficient to induce hair growth independently of mesenchymal dermal papilla niche signals normally required for hair regeneration. Wild-type cells are co-opted into new hair growths by β-catenin mutant cells, which non–cell autonomously activate Wnt signaling within the neighboring wild-type cells via Wnt ligands. This study demonstrates a mechanism by which Wnt/β-catenin signaling controls stem cell–dependent tissue growth non–cell autonomously and advances our understanding of the mechanisms that drive coordinated regeneration.

Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.

Tracking stem cell fate in time and space After injury and during homeostasis, tissues rely on the balance of cell loss and renewal. Rompolas et al. visualized individual stem cells over their lifetime in the epidermis of live mice. Tracking stem cells over multiple generations revealed that tissue homeostasis in the mouse epidermis is not maintained by asymmetric cell division as previously thought, but through the coordination of sibling cell fate and lifetimes. Furthermore, differentiating stem cells reused the existing spatial organization of the epidermis. Science, this issue p. 1471 In mice a single stem cell population maintains the adult epidermis by coordinating cell fate and using existing tissue organization. Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors.

Mice cloned from skin cells

Adult stem cells represent unique populations of undifferentiated cells with self-renewal capacity. In many tissues, stem cells divide less often than their progeny. It has been widely speculated, but largely untested, that their undifferentiated and quiescent state may make stem cells more efficient as donors for cloning by nuclear transfer (NT). Here, we report the use of nuclei from hair follicle stem cells and other skin keratinocytes as NT donors. When keratinocyte stem cells (KSCs) were used as NT donors, 19 liveborn mice were obtained, 9 of which survived to adulthood. Embryonic keratinocytes and cumulus cells also gave rise to cloned mice. Although cloning efficiencies were similar (<6% per transferred blastocyst), success rates were consistently higher for males than for females. Adult keratinocyte stem cells were better NT donors than so-called transit amplifying (TA) keratinocytes in both sexes (1.6% vs. 0% in females and 5.4% vs. 2.8% in males). Our findings reveal skin as a source of readily accessible stem cells, the nuclei of which can be reprogrammed to the pluripotent state by exposure to the cytoplasm of unfertilized oocytes.

Compartmentalized organization: a common and required feature of stem cell niches?

A key question in the stem cell field is how to balance the slow cycling of stem cells with active organ growth. Recent studies of the hair follicle stem cell niche have shown that this can be achieved by organizing the stem cell niche into two compartments: one that engages in immediate, rapid new growth and one that contributes later to long-term growth that fuels hair regeneration. Based on these and other recent findings, we propose that several other adult stem cell niches, including those in the blood, intestine and brain, have a similar bi-compartmental organization and that stem cells might work cooperatively with their progeny to sustain tissue regeneration.

Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool

Tissue homeostasis is achieved through a balance of cell production (growth) and elimination (regression). In contrast to tissue growth, the cells and molecular signals required for tissue regression remain unknown. To investigate physiological tissue regression, we use the mouse hair follicle, which cycles stereotypically between phases of growth and regression while maintaining a pool of stem cells to perpetuate tissue regeneration. Here we show by intravital microscopy in live mice that the regression phase eliminates the majority of the epithelial cells by two distinct mechanisms: terminal differentiation of suprabasal cells and a spatial gradient of apoptosis of basal cells. Furthermore, we demonstrate that basal epithelial cells collectively act as phagocytes to clear dying epithelial neighbours. Through cellular and genetic ablation we show that epithelial cell death is extrinsically induced through transforming growth factor (TGF)-β activation and mesenchymal crosstalk. Strikingly, our data show that regression acts to reduce the stem cell pool, as inhibition of regression results in excess basal epithelial cells with regenerative abilities. This study identifies the cellular behaviours and molecular mechanisms of regression that counterbalance growth to maintain tissue homeostasis.

Spontaneous tumour regression in keratoacanthomas is driven by Wnt/retinoic acid signalling cross-talk

A fundamental goal in cancer biology is to identify the cells and signalling pathways that are keys to induce tumour regression. Here we use a spontaneously self-regressing tumour, cutaneous keratoacanthoma (KAs), to identify physiological mechanisms that drive tumour regression. By using a mouse model system that recapitulates the behaviour of human KAs, we show that self-regressing tumours shift their balance to a differentiation programme during regression. Furthermore, we demonstrate that developmental programs utilized for skin hair follicle regeneration, such as Wnt, are hijacked to sustain tumour growth and that the retinoic acid (RA) signalling pathway promotes tumour regression by inhibiting Wnt signalling. Finally, we find that RA signalling can induce regression of malignant tumours that do not normally spontaneously regress, such as squamous cell carcinomas. These findings provide new insights into the physiological mechanisms of tumour regression and suggest therapeutic strategies to induce tumour regression.