Elaine Fuchs

Socializing with the Neighbors: Stem Cells and Their Niche

The potential of stem cells in regenerative medicine relies upon removing them from their natural habitat, propagating them in culture, and placing them into a foreign tissue environment. To do so, it is essential to understand how stem cells interact with their microenvironment, the so-called stem cell niche, to establish and maintain their properties. In this review, we examine adult stem cell niches and their impact on stem cell biology.

Directed Actin Polymerization Is the Driving Force for Epithelial Cell–Cell Adhesion

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.

De Novo Hair Follicle Morphogenesis and Hair Tumors in Mice Expressing a Truncated β-Catenin in Skin

An effector of intercellular adhesion, beta-catenin also functions in Wnt signaling, associating with Lef-1/Tcf DNA-binding proteins to form a transcription factor. We report that this pathway operates in keratinocytes and that mice expressing a stabilized beta-catenin controlled by an epidermal promoter undergo a process resembling de novo hair morphogenesis. The new follicles formed sebaceous glands and dermal papilla, normally established only in embryogenesis. As in embryologically initiated hair germs, transgenic follicles induce Lef-1, but follicles are disoriented and defective in sonic hedgehog polarization. Additionally, proliferation continues unchecked, resulting in two types of tumors also found in humans. Our findings suggest that transient beta-catenin stabilization may be a key player in the long-sought epidermal signal leading to hair development and implicate aberrant beta-catenin activation in hair tumors.

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.

Epidermal homeostasis: a balancing act of stem cells in the skin

The skin epidermis and its array of appendages undergo ongoing renewal by a process called homeostasis. Stem cells in the epidermis have a crucial role in maintaining tissue homeostasis by providing new cells to replace those that are constantly lost during tissue turnover or following injury. Different resident skin stem cell pools contribute to the maintenance and repair of the various epidermal tissues of the skin, including interfollicular epidermis, hair follicles and sebaceous glands. Interestingly, the basic mechanisms and signalling pathways that orchestrate epithelial morphogenesis in the skin are reused during adult life to regulate skin homeostasis.

Stem Cells: A New Lease on Life

offspring, including the germ cell compartment. To During embryogenesis, a single fertilized oocyte gives maintain cultured ES cells in their relatively undifferentirise to a multicellular organism whose cells and tissues ated, pluripotent state, they must both express the inhave adopted differentiated characteristics or fates to trinsic transcription factor Oct4, and constitutively reperform the specified functions of each organ of the ceive the extrinsic signal from the cytokine leukemia body. As embryos develop, cells that have acquired their inhibitory factor (LIF) (Nichols et al., 1998; Niwa et al., particular fate proliferate, enabling tissues and organs 1998 and references therein). to grow. Even after an animal is fully grown, however, Upon LIF withdrawal, cultured ES cells spontaneously many tissues and organs maintain a process known as aggregate into embryo-like bodies, where they differenhomeostasis, where as cells die, either by natural death tiate and spawn many cell lineages, including beating or by injury, they are replenished. This remarkable feature heart muscle cells, blood islands, neurons, pigmented has ancient origins, dating back to the most primitive cells, macrophages, epithelia, and fat-producing adipoanimals, such as sponges and hydrozoans. Throughout cytes (Figure 1; for review, see Bradley, 1990). Similarly, evolution, nature has exerted considerable fun and fancy when ES cells are injected into nude mice, they differenin elaborating on this theme. Some amphibians, for intiate into multicellular masses, called teratocarcinomas. stance, can regenerate a limb or tail when severed, and Although the programs of gene expression in these the neurons of bird brains can readily regenerate. While structures often bear strong resemblance to the differmammals seem to have lost at least some of this wonentiation pathways typical of developing animals, the derful plasticity, their liver can partially regenerate protriggering of these programs is chaotic, yielding a jumviding that the injury is not too severe, and the epidermis bled grab bag of tissue types. These examples graphiand hair of their skin can readily repair when wounded cally illustrate the importance of intercellular interactions or cut. Additionally, the epidermis, hair, small intestine, and cellular organization in orchestrating development and hematopoietic system are all examples of adult tisand embryo shape. sues that are naturally in a state of dynamic flux: even During development, intercellular cross-talk results in in the absence of injury, these structures continually give the generation and transmission of specific signals from rise to new cells, able to transiently divide, terminally a cell to its neighbor, altering in some key way the subsedifferentiate and die. quent behavior of the neighbor. Of prime importance is The fabulous ability of an embryo to diversify and of sifting through the galaxy of environmental signals to certain adult tissues to regenerate throughout life is a determine which constellations of cues can selectively direct result of stem cells, nature’s gift to multicellular coax ES cells down a specific cell lineage pathway at organisms. Stem cells have both the capacity to selfthe expense of all others. To this end, Brüstle et al. renew, that is, to divide and create additional stem cells, (1999) were recently able to obtain pure populations of and also to differentiate along a specified molecular multipotent progenitor cells expressing glial precursor pathway. Embryonic stem cells are very nearly totipomarkers. They achieved this goal by taking aggregates tent, reserving the elite privileges of choosing among of cultured mouse ES cells and propagating them semost if not all of the differentiation pathways that specify quentially in medium containing first fibroblast growth the animal. In contrast, stem cells that reside within an factor (FGF) 2 alone, then a mixture of FGF2 and epideradult organ or tissue have more restricted options, often mal growth factor (EGF), and finally a mix of FGF2 and able to select a differentiation program from only a few platelet-derived growth factor (PDGF). Bathed in this possible pathways. Or so it seemed, until very recently. last broth of growth factors, these pluripotent cells could In the last year, some spectacular fireworks have exbe maintained for many generations in culture. Upon ploded many long-standing dogmas in the stem cell growth factor withdrawal, they subsequently differentiworld, giving adult stem cells a new lease on life, and enated into either of two specific lineages, oligodendroabling them to be what researchers previously thought cytes or astrocytes (Brüstle et al., 1999). they were not. Illustrating the enormous potential of this type of research for clinical application, McKay and coworkers Embryonic Stem Cells transplanted these cloned glial precursor cells into the Emanating from the pioneering mouse research of Marventricle of myelin-deficient rats. Myelin sheaths formed tin Evans in the 1970s and culminating with the recent around host axons in various brain regions, including cortex, hippocampus, and hypothalamus (Figure 2; Brüstle et al., 1999). No signs of nonneuronal tissue were * To whom correspondence should be addressed (e-mail: liptack@

Asymmetric cell divisions promote stratification and differentiation of mammalian skin

The epidermis is a stratified squamous epithelium forming the barrier that excludes harmful microbes and retains body fluids. To perform these functions, proliferative basal cells in the innermost layer periodically detach from an underlying basement membrane of extracellular matrix, move outward and eventually die. Once suprabasal, cells stop dividing and enter a differentiation programme to form the barrier. The mechanism of stratification is poorly understood. Although studies in vitro have led to the view that stratification occurs through the delamination and subsequent movement of epidermal cells, most culture conditions favour keratinocytes that lack the polarity and cuboidal morphology of basal keratinocytes in tissue. These features could be important in considering an alternative mechanism, that stratification occurs through asymmetric cell divisions in which the mitotic spindle orients perpendicularly to the basement membrane. Here we show that basal epidermal cells use their polarity to divide asymmetrically, generating a committed suprabasal cell and a proliferative basal cell. We further demonstrate that integrins and cadherins are essential for the apical localization of atypical protein kinase C, the Par3–LGN–Inscuteable complex and NuMA–dynactin to align the spindle.

Sticky Business: Orchestrating Cellular Signals at Adherens Junctions

Cohesive sheets of epithelial cells are a fundamental feature of multicellular organisms and are largely a product of the varied functions of adherens junctions. These junctions and their cytoskeletal associations contribute heavily to the distinct shapes, polarity, spatially oriented mitotic spindle planes, and cellular movements of developing tissues. Deciphering the underlying mechanisms that govern these conserved cellular rearrangements is a prerequisite to understanding vertebrate morphogenesis.

Regulation of terminal differentiation of cultured human keratinocytes by vitamin A

Vitamin A is known to exert an important influence on epithelial differentiation. The fetal calf serum supplement of cell-culture medium contains enough of the vitamin to affect the differentiation of cultured keratinocytes derived from epidermis and from other stratified squamous epithelia. The cellular and molecular properties of the cultures are altered when the medium is supplemented with serum from which the vitamin A has been removed by solvent extraction (delipidized serum). Cell motility is reduced, the adhesiveness of cells increases and pattern formation is prevented. In both epidermal and conjunctival keratinocytes, removal of vitamin A leads to the synthesis of a 67 kd keratin characteristic of terminally differentiating epidermis and to much reduced synthesis of the 52 kd and 40 kd keratins typical of conjunctiva. These changes, both cellular and molecular, are reversed by the addition of retinyl acetate to the medium containing delipidized serum. Cell motility and pattern formation are restored, and detachment of the most mature cells from the surface of the stratified epithelium is promoted. Synthesis of the 67 kd keratin is prevented and the synthesis of the 40 and 52 kd keratins is stimulated. The nature of the keratins synthesized is regulated by the concentration of vitamin A, and each cell type adjusts its synthesis differently at a given vitamin concentration.

A skin microRNA promotes differentiation by repressing ‘stemness’

In stratified epithelial tissues, homeostasis relies on the self-renewing capacity of stem cells located within the innermost basal layer. As basal cells become suprabasal, they lose proliferative potential and embark on a terminal differentiation programme. Here, we show that microRNA-203 is induced in the skin concomitantly with stratification and differentiation. By altering miR-203’s spatiotemporal expression in vivo, we show that miR-203 promotes epidermal differentiation by restricting proliferative potential and inducing cell-cycle exit. We identify p63 as one of the conserved targets of miR-203 across vertebrates. Notably, p63 is an essential regulator of stem-cell maintenance in stratified epithelial tissues. We show that miR-203 directly represses the expression of p63: it fails to switch off suprabasally when either Dicer1 or miR-203 is absent and it becomes repressed basally when miR-203 is prematurely expressed. Our findings suggest that miR-203 defines a molecular boundary between proliferative basal progenitors and terminally differentiating suprabasal cells, ensuring proper identity of neighbouring layers.