Localizations of γ-Actins in Skin, Hair, Vibrissa, Arrector Pili Muscle and Other Hair Appendages of Developing Rats.

Six isoforms of actins encoded by different genes have been identified in mammals including α-cardiac, α-skeletal, α-smooth muscle (α-SMA), ß-cytoplasmic, γ-smooth muscle (γ-SMA), and γ-cytoplasmic actins (γ-CYA). In a previous study we showed the localization of α-SMA and other cytoskeletal proteins in the hairs and their appendages of developing rats (Morioka K., et al. (2011) Acta Histochem. Cytochem. 44, 141-153), and herein we determined the localization of γ type actins in the same tissues and organs by immunohistochemical staining. Our results indicate that the expression of γ-SMA and γ-CYA is suggested to be poor in actively proliferating tissues such as the basal layer of the epidermis and the hair matrix in the hair bulb, and as well as in highly keratinized tissues such as the hair cortex and hair cuticle. In contrast, the expression of γ-actins were high in the spinous layer, granular layer, hair shaft, and inner root sheath, during their active differentiations. In particular, the localization of γ-SMA was very similar to that of α-SMA. It was located not only in the arrector pili muscles and muscles in the dermis, but also in the dermal sheath and in a limited area of the outer root sheath in both the hair and vibrissal follicles. The γ-CYA was suggested to be co-localized with γ-SMA in the dermal sheath, outer root sheath, and arrector pili muscles. Sparsely distributed dermal cells expressed both types of γ-actin. The expression of γ-actins is suggested to undergo dynamic changes according to the proliferation and differentiation of the skin and hair-related cells.

Steady and temporary expressions of smooth muscle actin in hair, vibrissa, arrector pili muscle, and other hair appendages of developing rats.

The hair erection muscle, arrector pili, is a kind of smooth muscle located in the mammalian dermis. The immunohistochemical study using an antibody against smooth muscle alpha actin (SMA) showed that the arrector pili muscle develops approximately 1-2 weeks after birth in dorsal and ventral skin, but thereafter they degenerate. The arrector pili muscle was not detected in the mystacial pad during any stage of development, even in the neighboring pelage-type hair follicle. A strong signal of SMA in the skin was located in the dermal sheath as well as in some outer root sheath cells in the hair and vibrissal follicles. Positive areas in the dermal and outer root sheaths were restricted to a lower moiety, particularly areas of similar height, where keratinization of the hair shaft occurs. This rule is valid for both pelage hair follicles and vibrissal follicles. At medium heights of the follicle, SMA staining in the dermal sheath was patchy and distant from the boundary between dermis and epidermis. In contrast to SMA, vimentin was expressed over the entire height of the dermal sheath. Unlike the arrector pili muscle, the expression of SMA in the dermal sheath was observed during fetal, neonatal, and adult stages. The presence of actin-myosin and vimentin fibers in supporting cells is thought to be beneficial for the hair follicle to cope with the movement of the hair shaft, which may be caused by physical contacts with outside materials or by the contraction of internal muscles.

Hair follicular expression and function of group X secreted phospholipase A2 in mouse skin.

Although perturbed lipid metabolism can often lead to skin abnormality, the role of phospholipase A(2) (PLA(2)) in skin homeostasis is poorly understood. In the present study we found that group X-secreted PLA(2) (sPLA(2)-X) was expressed in the outermost epithelium of hair follicles in synchrony with the anagen phase of hair cycling. Transgenic mice overexpressing sPLA(2)-X (PLA2G10-Tg) displayed alopecia, which was accompanied by hair follicle distortion with reduced expression of genes related to hair development, during a postnatal hair cycle. Additionally, the epidermis and sebaceous glands of PLA2G10-Tg skin were hyperplasic. Proteolytic activation of sPLA(2)-X in PLA2G10-Tg skin was accompanied by preferential hydrolysis of phosphatidylethanolamine species with polyunsaturated fatty acids as well as elevated production of some if not all eicosanoids. Importantly, the skin of Pla2g10-deficient mice had abnormal hair follicles with noticeable reduction in a subset of hair genes, a hypoplasic outer root sheath, a reduced number of melanin granules, and unexpected up-regulation of prostanoid synthesis. Collectively, our study highlights the spatiotemporal expression of sPLA(2)-X in hair follicles, the presence of skin-specific machinery leading to sPLA(2)-X activation, a functional link of sPLA(2)-X with hair follicle homeostasis, and compartmentalization of the prostanoid pathway in hair follicles and epidermis.

A guide to hair follicle analysis by transmission electron microscopy: technique and practice.

Hair follicles contain several tissues and cell types that differentiate down distinct pathways to provide for growth, keratinization and the maintenance of the hair shaft. Electron microscopy is useful for examining the morphological characteristics of developing hair follicles, including special types of keratinization, the timing of keratinization, programmed cell death, cell adhesion and separation, cell movement and changes in organelles. Hair follicles are one of the more challenging targets for electron microscopic analysis, and the use of neonatal animals combined with careful treatment of the samples can yield informative photomicrographs. Detailed protocols and examples of a number of techniques are presented here.

Age- and morph-dependent activation of the lysosomal system and Buchnera degradation in aphid endosymbiosis.

Endosymbiosis in aphids is maintained through a mutualistic association between the host and a symbiotic bacterium, Buchnera, which is harbored in specialized host cells called bacteriocytes. Here, we examined the changes in the Buchnera density in bacteriocytes in relation to the development and polyphenism of the host aphid. Buchnera density in the winged morph aphids, alatae, decreased drastically around the final ecdysis, whereas in the wingless morph aphids, apterae, Buchnera density decreased after the final ecdysis. Thereafter, in both apterae and alatae, Buchnera density was maintained at a constant level until 10 days and then again decreased gradually until 18 days after the final ecdysis. Cytochemical analysis with LysoTracker reagent and quantitative RT-PCR analysis revealed that the number of lysosome-like acidic organelles and the amount of lysosome-related gene (lysozyme and cathepsin L) transcripts increased drastically in the bacteriocytes of alatae around the final ecdysis. Electron microscopy of alatae bacteriocytes around the final ecdysis revealed many Buchnera with irregular electron-dense areas in their cytoplasm that were enclosed by a distended symbiosome membrane. These findings indicated that age- and morph-dependent decreases in Buchnera density coincided with activation of the host lysosomal system and the increased degradation of Buchnera.

Localization of myosin and actin in the pelage and whisker hair follicles of rat.

The combined effects of myosin II and actin enable muscle and nonmuscle cells to generate forces required for muscle contraction, cell division, cell migration, cellular morphological changes, the maintenance of cellular tension and polarity, and so on. However, except for the case of muscle contraction, the details are poorly understood. We focus on nonmuscle myosin and actin in the formation and maintenance of hair and skin, which include highly active processes in mammalian life with respect to the cellular proliferation, differentiation, and movement. The localization of nonmuscle myosin II and actin in neonatal rat dorsal skin, mystacial pad, hair follicles, and vibrissal follicles was studied by immunohistochemical technique to provide the basis for the elucidation of the roles of these proteins. Specificities of the antibodies were verified by using samples from the relevant tissues and subjecting them to immunoblotting test prior to morphological analyses. The myosin and actin were abundant and colocalized in the spinous and granular layers but scarce in the basal layer of the dorsal and mystacial epidermis. In hair and vibrissal follicles, nonmuscle myosin and actin were colocalized in the outer root sheath and some hair matrix cells adjoining dermal papillae. In contrast, most areas of the inner root sheath and hair matrix appeared to comprise very small amounts of myosin and actin. Hair shaft may comprise significant myosin during the course of its keratinization. These results suggest that the actin-myosin system plays a part in cell movement, differentiation, protection and other key functions of skin and hair cells.

Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster: (erythroid differentiation/embryonic erythroid cells/morphological change/image-processing analysis/syrian hamster).

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image-processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk-sac-derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10-10.5 µm on day 12, fell gradually to 8-9 µm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult-type erythrocytes were detected after birth with diameters of 5.5-6 µm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of "primitive" yolk-sac-derived erythroid cells is more closely correlated with that of the "definitive" fetal liver cells than has been considered to be the case to date.

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro.

The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.

Novel Inducers and Inhibitors of Differentiation of Friend Erythroleukemia Cells: Application of an Opal Glass Transmission Method to Study of Erythroid Differentiation: (erythroid differentiation/poly(ADP-ribose)/bile acids/opal glass method/Friend leukemia cells).

The potencies of poly(ADP-ribosylation)-inhibitors in inducing erythroid differentiation of Friend erythroleukemia cells were surveyed. Picolinamide and m-aminobenzamide were newly found to be inducers, whereas compounds related caffeine did not induce differentiation. In other series of experiments some bile acids suspected of being tumor promoters were found to inhibit the differentiation like typical tumor promoters such as phorbol esters. These modifications of erythroid differentiation were detected by an opal glass transmission method. This method is simpler than any previously reported methods, and is sufficiently reliable to use in determining hemoglobin in living cells as a quantitative marker of erythroid differentiation.