Multiple potential roles of thymosin β4 in the growth and development of hair follicles

The hair follicle (HF) is an important mini-organ of the skin, composed of many types of cells. Dermal papilla cells are important signalling components that guide the proliferation, upward migration and differentiation of HF stem cell progenitor cells to form other types of HF cells. Thymosin β4 (Tβ4), a major actin-sequestering protein, is involved in various cellular responses and has recently been shown to play key roles in HF growth and development. Endogenous Tβ4 can activate the mouse HF cycle transition and affect HF growth and development by promoting the migration and differentiation of HF stem cells and their progeny. In addition, exogenous Tβ4 increases the rate of hair growth in mice and promotes cashmere production by increasing the number of secondary HFs (hair follicles) in cashmere goats. However, the molecular mechanisms through which Tβ4 promotes HF growth and development have rarely been reported. Herein, we review the functions and mechanisms of Tβ4 in HF growth and development and describe the endogenous and exogenous actions of Tβ4 in HFs to provide insights into the roles of Tβ4 in HF growth and development.     

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.     

Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles

Hair follicles (HFs) are self-renewing structures that reconstitute themselves through the hair cycle. They maintain reservoirs of stem cells (SC) that are thought to reside in the bulge area, a region localized in the lowermost permanent portion of HFs. In mice and humans, HF bulge cells express nestin and present stem features as pluripotency. Nestin is a class VI intermediate filament protein; it was first described as a specific marker of CNS stem cells, but recent studies suggest that it may represent a more general stem cell marker (Wiese et al., 2004; Hoffman, 2006). Bulge cell characteristics have mainly been studied in mice and humans, but recently, a bulge-like region was identified also in dog HFs (Pascucci et al., 2006). In this work we investigate the presence and localization of nestin in dog HFs with the aim of evaluating its expression and to correlate it with the location of the bulge-like region. Immunostaining of skin samples collected from healthy dogs was performed by using a rabbit anti-nestin polyclonal antibody. The presence of a population of immunoreactive cells was revealed in the hair follicle middle region, at the arrector pili muscle insertion level. An immunohistochemical signal was detected only in primary hair follicles throughout the hair cycle. These observations led us to conclude that nestin positive cells are located in the bulge-like region of dog HFs and strengthen our hypothesis regarding the correlation between this region and the dog HF stem compartment.     

Inhibition of trophoblast stem cell potential in chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the mouse

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm - early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm - were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.     

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1

The directed movement of cells is critical for numerous developmental and disease processes. A developmentally reiterated form of migration is radial intercalation; the process by which cells move in a direction orthogonal to the plane of the tissue from an inner layer to an outer layer. We use the radial intercalation of cells into the skin of Xenopus laevis embryos as a model to study directed cell migration within an epithelial tissue. We identify a novel function for both the microtubule-binding protein CLAMP and members of the microtubule-regulating Par complex during intercalation. Specifically, we show that Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration. We propose a model in which the Par complex defines the orientation of apical migration during intercalation and in which subcellular localization of CLAMP promotes the establishment of an axis of microtubule stability required for the active migration of cells into the outer epithelium.     

Embryonic Stem Cells: Spontaneous and Directed Differentiation

The specific structural features of embryonic stem cells and embryoid bodies and mechanisms of their differentiation in different cell types are considered. The mouse embryonic stem cells (line R1) formed multilayer colonies which enlarged as a result of fast cell division. Embryoid bodies that derived from embryonic stem cells consisted of an outer layer, an inner layer, and an internal cavity. The structure of cells of the outer and inner layers markedly differed. Spontaneous and directed differentiation of embryoid bodies is determined by some unspecific and specific factors (growth and differentiation factors and extracellular matrix proteins). Retinoic acid, the most commonly used inducer of differentiation of the embryonic stem cells, induces different types of differentiation when applied at different concentrations. The sequence of expression of tissue specific genes and proteins during differentiation of the embryonic stem cells in vitrois similar to that in vivo.     

Patterns of Cell Division,Cell Differentiation and Cell Elongation in Epidermis and Cortex of Arabidopsis pedicels in the Wild Type and in erecta

Plant organ shape and size are established during growth by a predictable, controlled sequence of cell proliferation, differentiation, and elongation. To understand the regulation and coordination of these processes, we studied the temporal behavior of epidermal and cortex cells in Arabidopsis pedicels and used computational modeling to analyze cell behavior in tissues. Pedicels offer multiple advantages for such a study, as their growth is determinate, mostly one dimensional, and epidermis differentiation is uniform along the proximodistal axis. Three developmental stages were distinguished during pedicel growth: a proliferative stage, a stomata differentiation stage, and a cell elongation stage. Throughout the first two stages pedicel growth is exponential, while during the final stage growth becomes linear and depends on flower fertilization. During the first stage, the average cell cycle duration in the cortex and during symmetric divisions of epidermal cells was constant and cells divided at a fairly specific size. We also examined the mutant of ERECTA, a gene with strong influence on pedicel growth. We demonstrate that during the first two stages of pedicel development ERECTA is important for the rate of cell growth along the proximodistal axis and for cell cycle duration in epidermis and cortex. The second function of ERECTA is to prolong the proliferative phase and inhibit premature cell differentiation in the epidermis. Comparison of epidermis development in the wild type and erecta suggests that differentiation is a synchronized event in which the stomata differentiation and the transition of pavement cells from proliferation to expansion are intimately connected.     

Finite Element Modeling Predictions of Region-specific Cell-matrix Mechanics in the Meniscus

The knee meniscus exhibits significant spatial variations in biochemical composition and cell morphology that reflect distinct phenotypes of cells located in the radial inner and outer regions. Associated with these cell phenotypes is a spatially heterogeneous microstructure and mechanical environment with the innermost regions experiencing higher fluid pressures and lower tensile strains than the outer regions. It is presently unknown, however, how meniscus tissue mechanics correlate with the local micromechanical environment of cells. In this study, theoretical models were developed to study mechanics of inner and outer meniscus cells with varying geometries. The results for an applied biaxial strain predict significant regional differences in the cellular mechanical environment with evidence of tensile strains along the collagen fiber direction of ~0.07 for the rounded inner cells, as compared to levels of 0.02–0.04 for the elongated outer meniscus cells. The results demonstrate an important mechanical role of extracellular matrix anisotropy and cell morphology in regulating the region-specific micromechanics of meniscus cells, that may further play a role in modulating cellular responses to mechanical stimuli.     

Radial intercalation of ciliated cells during Xenopus skin development

Cells with motile cilia cover the skin of Xenopus tadpoles in a characteristic spacing pattern. This pattern arises during early development when cells within the inner layer of ectoderm are selected out by Notch to form ciliated cell precursors (CCPs) that then radially intercalate into the outer epithelial cell layer to form ciliated cells. When Notch is inhibited and CCPs are overproduced, radial intercalation becomes limiting and the spacing of ciliated cells is maintained. To determine why this is the case, we used confocal microscopy to image intercalating cells labeled using transplantation and a transgenic approach that labels CCPs with green fluorescent protein (GFP). Our results indicate that inner cells intercalate by first wedging between the basal surface of the outer epithelium but only insert apically at the vertices where multiple outer cells make contact. When overproduced, more CCPs are able to wedge basally, but apical insertion becomes limiting. We propose that limitations imposed by the outer layer, along with restrictions on the apical insertion of CCPs, determine their pattern of radial intercalation.     

Ultrastructural studies of the mantle and the periostracal lamina in the manila clam, Ruditapes philippinarum

The four folds of the mantle and the periostracal lamina of R. philippinarum were studied using light, transmission and scanning electron microscopy to determine the histochemical and ultrastructural relationship existing between the mantle and the shell edge. The different cells lining the four folds, and in particular those of the periostracal groove, are described in relation to their secretions. The initial pellicle of the periostracum arises in the intercellular space between the basal cell and the first intermediate cell. In front of the third cell of the inner surface of the outer fold, the periostracal lamina is composed of two major layers; an outer electron-dense layer or periostracum and an inner electron-lucent fibrous layer or fibrous matrix. The role and the fate of these two layers differ; the outer layer will recover the external surface of the shell and the inner layer will contribute to shell growth.     

Abnormal hair development and apparent follicular transformation to mammary gland in the absence of hedgehog signaling

We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF.     

Dynamic microtubules under the radial and outer tangential walls of microinjected pea epidermal cells observed by computer reconstruction

By microinjecting rhodamine-conjugated pig brain tubulin into living pea stem epidermal cells it has been possible to follow cortical microtubules beneath the outer tangential wall (OTW) as they re-orientate from a transverse to a longitudinal alignment. Earlier immunofluorescence studies on fixed material have shown that parallel cortical microtubules circumnavigate the cell forming apparently continuous arrays which are transverse, oblique or longitudinal to the cell's long axis. If the array re-orientates as a whole then microtubules along the radial walls would be expected to share the alignment of those on the tangential walls. There are, however, reports that microtubules beneath the outer tangential wall have a different orientation from microtubules at the radial cell walls, raising important questions about the construction and behaviour of the array. Using computer-rotated stacks of optical sections collected by confocal scanning laser microscopy it has been possible to display the microtubules along radial as well as tangential walls of the same microinjected cells. These observations demonstrate for living epidermal cells that when microtubules are aligned longitudinally at the outer epidermal wall they remain oblique or transverse at the radial walls. The array may not therefore re-orientate as a whole but seems to undergo re-organization on only one cell face. However, despite the differing angles between the OTW and radial walls microtubules still form patterns which at the level of the confocal microscope are continuous from one cell face to another, around the cell.
It is concluded that some organizing principle attempts to establish overall organization at the cellular level but that this can be perturbed by local re-organization of dynamic microtubules in subcellular domains. This study emphasizes the importance of the outer epidermal wall and its associated cytoskeleton in initiating changes in the direction of cell expansion.     

Radial glia regulate Cajal-Retzius cell positioning in the early embryonic cerebral cortex

The organization of neocortex, along its radial axis, into a six-layered structure is one of the most exquisite features of the brain. Because of their strategic localization in the marginal zone, and their expression of reelin, a signal that controls spatial ordering of cortical layers, Cajal–Retzius (C-R) cells play a crucial role in cortical patterning along this axis. Yet, it remains less well understood how C-R cell targeting itself is regulated. At the onset of corticogenesis when C-R cells first arrive in the cortex via tangential migration, radial glia (RG) are the main cell type present. This suggests that RG may play a role in C-R cell localization. To test this, we used genetic approaches to perturb RG scaffold during early corticogenesis. We found that disrupting RG endfoot adhesion to basal lamina consistently results in C-R cell displacement. These displacements do not appear to result from primary defects in neural progenitor cell proliferation, deficits in the meninges or basement membrane, or cell autonomous defects in C-R cells. Instead, they show close temporal and spatial correlation with RG endfoot retraction. Moreover, ablation of RG via cell cycle blockade similarly results in local displacement of C-R cells. These lines of evidence thus indicate that, during early corticogenesis, RG play a primary role in regulating spatial targeting of C-R cells. Since RG are also neural progenitors as well as neuronal migration scaffolds, these findings suggest that, during nervous system development, neuroepithelial stem cells may not only be responsible for generating a diverse array of neuronal cell types and facilitating their radial migration. They may also, through regulating the placement of guidepost cells, coordinate spatial patterning of the nervous system along its radial axis.     

Localization and Pattern of Graviresponse across the Pulvinus of Barley Hordeum vulgare

Pulvini of excised stem segments from barley (Hordeum vulgare cv `Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0° and the lowermost point 180°. A gravity-induced increase in pulvinus thickness was observable within 40° of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.     

The comparative characteristics of the effect of retinoic acid on the regeneration of the crystalline lens and the extremity in adult tritons]

Effect of retinoic acid (RA) on morphogenesis and proliferation of regenerating extremity and lens cells was studied using 3H-thymidine autoradiography and morphometry. The 3H-thymidine incorporation into the inner layer of dorsal and ventral iris was 1.5-3 times reduced by the 8th day following the RA administration. The applied RA dose (0.25 mg per animal) exerted no significant effect on the morphogenesis of regenerating lens with the exception of the case of forming an additional lens from dorsal iris. The RA effect on the regeneration of extremity corresponds to available data of literature and manifests itself in the decelerated regeneration and the appearance of additional structures along the proximodistal axis.     

Heparan Sulfate Regulates Hair Follicle and Sebaceous Gland Morphogenesis and Homeostasis

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1^{StEpiΔ/StEpiΔ} mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1^{StEpiΔ/StEpiΔ} mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling.