Panteleimon Rompolas

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.

Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor

Intraflagellar transport (IFT) is the bi-directional movement of particles along the length of axonemal outer doublet microtubules and is needed for the assembly and maintenance of eukaryotic cilia and flagella. Retrograde IFT requires cytoplasmic dynein 1b, a motor complex whose organization, structural composition and regulation is poorly understood. We have characterized the product of the Chlamydomonas FAP133 gene that encodes a new WD-repeat protein similar to dynein intermediate chains and homologous to the uncharacterized vertebrate protein WD34. FAP133 is located at the peri-basal body region as well as in punctate structures along the flagella. This protein is associated with the IFT machinery because it is specifically depleted from the flagella of cells with defects in anterograde IFT. Fractionation of flagellar matrix proteins indicates that FAP133 associates with both the LC8 dynein light chain and the IFT dynein heavy chain and light intermediate chain (DHC1b-D1bLIC) motor complex. In the absence of DHC1b or D1bLIC, FAP133 fails to localize at the peri-basal body region but, rather, is concentrated in a region of the cytoplasm near the cell center. Furthermore, we found that FAP133, LC8, DHC1b, D1bLIC, the FLA10 kinesin-2 necessary for anterograde IFT and other IFT scaffold components associate to form a large macromolecular assembly.

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.

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

Here we use the motile ventral cilia of the planarian S. mediterranea to examine the role of outer arm dynein in the generation and maintenance of metachronal synchrony. We demonstrate that a single dynein light chain plays a mechanosensory role necessary to entrain and maintain the metachronal synchrony of motile cilia.

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.

The lissencephaly protein Lis1 is present in motile mammalian cilia and requires outer arm dynein for targeting to Chlamydomonas flagella

Lissencephaly is a developmental brain disorder characterized by a smooth cerebral surface, thickened cortex and misplaced neurons. Classical lissencephaly is caused by mutations in LIS1, which encodes a WD-repeat protein involved in cytoplasmic dynein regulation, mitosis and nuclear migration. Several proteins required for nuclear migration in Aspergillus bind directly to Lis1, including NudC. Mammalian NudC is highly expressed in ciliated epithelia, and localizes to motile cilia in various tissues. Moreover, a NudC ortholog is upregulated upon deflagellation in Chlamydomonas. We found that mammalian Lis1 localizes to motile cilia in trachea and oviduct, but is absent from non-motile primary cilia. Furthermore, we cloned a gene encoding a Lis1-like protein (CrLis1) from Chlamydomonas. CrLis1 is a ∼37 kDa protein that contains seven WD-repeat domains, similar to Lis1 proteins from other organisms. Immunoblotting using an anti-CrLis1 antibody revealed that this protein is present in the flagellum and is depleted from flagella of mutants with defective outer dynein arm assembly, including one strain that lacks only the α heavy chain/light chain 5 thioredoxin complex. Biochemical experiments confirmed that CrLis1 associates with outer dynein arm components and revealed that CrLis1 binds directly to rat NudC. Our results suggest that Lis1 and NudC are present in cilia and flagella and may regulate outer dynein arm activity.

Regulation of Kalirin by Cdk5

Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.