Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin.

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.

The cell sorting process of Xenopus gastrula cells involves the acto-myosin system and TGF-ß signaling.

We have previously shown that the cell sorting process of animal pole cells (AC) and vegetal pole cells (VC) from Xenopus gastrulae is considered to involve two steps: concentrification and polarization. In this study, we addressed the question of what specified the spatial relationship of the AC and VC clusters during the process. First, we examined the inhibitory or facilitatory treatment for myosin 2 activity during each of the two steps. The aggregates treated with Y27632 or blebbistatin during the concentrification step showed a cluster random arrangement, suggesting the prevention of the cell sorting by inhibition of myosin 2. Meanwhile, the treatment with a Rac1 inhibitor, NSC23766, during the same step resulted in promotion of the fusion of the AC clusters and the progression of the cell sorting, presumably by an indirect activation of myosin 2. On the other hand, the treatments with any of the three drugs during the polarization step showed that the two clusters did not appose, and their array remained concentric. Thus, the modulation of cell contraction might be indispensable to each of the two steps. Next, the activin/nodal TGF-ß signaling was perturbed by using a specific activin receptor-like kinase inhibitor, SB431542. The results revealed a bimodal participation of the activin/nodal TGF-ß signaling, i.e., suppressive and promotive effects on the concentrification and the polarization, respectively. Thus, the present in vitro system, which permits not only the cell contraction-mediated cell sorting but also the TGF-ß-directed mesodermal induction such as cartilage formation, may fairly reflect the embryogenesis in vivo.

Possible roles of ENaC and Cl(-) channel in wound closure in Xenopus laevis embryos.

Our previous report showed that rapid wound closure in Xenopus laevis embryos was associated with a decrease in the extracellular concentration of either Na(+) or Cl(-) ions. In this study, we examined the wound closure in Xenopus embryos when epithelial Na(+) channel (ENaC), Na(+)/K(+) ATPase (Na(+) pump) or CICs (members of Cl(-) channel) were blocked by each specific inhibitor. Blockage of ENaC and CIC restricted the rate of wound closure during the first 30 min PW and during the subsequent period, respectively. In contrast, inhibition of Na(+) pump had no effect on the rate of wound closure. Furthermore, simultaneous administration of both ENaC and CIC inhibitors resulted in the cumulative reduction of wound closure. Thus, it is plausible that these ion channels play active roles in wound closure in Xenopus embryos. NPPB is known to inhibit both CIC-2 and CIC-3. Immunohistochemical experiments showed that CIC-3, but not CIC-2, was expressed in Xenopus embryos, suggesting that the reduced wound closure by NPPB was due to blockage of CIC-3. A local enhancement of CIC-3 expression at the leading edge of the wounded epidermis was found to be specific to closing wounds that were kept in 10% NAM. An in vitro wounding assay also showed a pattern of CIC-3 expression at the margin of the scratch wound comparable to the results in vivo. These findings suggest that intracellular translocation of CIC-3 is involved in wound closure. We propose that the ion channels, including CIC-3, play a crucial role in wound closure in Xenopus embryos.

Exposure to external environment of low ion concentrations is the trigger for rapid wound closure in Xenopus laevis embryos.

Wounds in Xenopus laevis embryos close rapidly, as previously described. In this study, we examined the dependence on extracellular Na(+) and/or Cl(-) ion concentrations of the closure of wounds in Xenopus embryos inflicted by thermal injury. Wound closure did not occur in normal amphibian medium (100% NAM), while wound areas remarkably decreased either in 10-50% NAM or in 100% NAM lacking Na(+) or Cl(-). Similarly, wound areas did not change in a set of Na(+) and Cl(-) ion concentrations equivalent to those of the humoral fluids of intact Xenopus embryos, but rapid wound closure was induced by decreasing the concentration of either of the two ions. A tangential accumulation of actin cytoskeleton along the wound edge was associated with wound closure. However, a similar actin alignment formed even under the 100% NAM condition, in which wounds did not close, as stated above. The epidermis around the wound edge exhibited ellipse-shaped hypertrophy, and the marginal cells centripetally elongated during wound closure. On the other hand, no distinct morphological changes occurred in 100% NAM, although the epidermis was somewhat thickened. Thus, the morphological changes in the epidermis specific to the low ionic environment most likely play active roles in the wound closure of Xenopus laevis embryos, whereas the tangential actin alignment alone may be insufficient. Taken together, we propose that the wound closure in Xenopus embryos is triggered by a decline in either the extracellular Na(+) or Cl(-) ion concentration, and that this process is required for the abovementioned changes in the shape of the marginal 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.

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro.

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were "separately" co-cultured with Nc cells without direct cell-cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The "spinal cord-promotion" and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range "Nc suppression" will be determined by future studies. Interestingly, these effects, "Nc suppression" and "SC promotion" were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.

Accelerated proliferation and abnormal differentiation of epidermal keratinocytes in endo-beta-galactosidase C transgenic mice.

Transgenic (TG) mice that have systemically expressed endo-beta-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Galalpha1-3Gal disaccharide (alphaGal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the alphaGal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1-2 layers to 4-5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the alphaGal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice.

Hair cycle-dependent changes of alkaline phosphatase activity in the mesenchyme and epithelium in mouse vibrissal follicles.

Alkaline phosphatase (ALP) activity was detected in the restricted mesenchymal and epithelial regions in mouse vibrissal follicles. Its localization and strength dramatically changed during the hair cycle. Activity in the dermal papilla (DP) was moderate in very early anagen, reached a maximal level in early anagen, decreased at the proximal region of DP after mid anagen, and was kept at a low level during catagen. The bulbar dermal sheath showed intense ALP activity only in early anagen. Although most bulbar epithelium did not show ALP activity, germinative epidermal cells that were adjacent to the ALP-negative DP cells became ALP-positive in mid anagen and rearranged in a single layer so as to encapsulate the DP in mid catagen. During catagen, the outermost layer of bulbar epithelium became ALP-positive, which could be follicular epithelial precursors migrating from the bulge. Before the initiation of hair formation, ALP activity in the bulbar epithelium rapidly decreased and that in DP increased. These dynamic changes of ALP expression might be related to DP's functions in hair induction and also to reconstruction of the bulbar structure during the hair cycle.

Follicular epithelia and dermal papillae of mouse vibrissal follicles qualitatively change their hair-forming ability during anagen.

We studied the hair-forming ability of epithelium and the relevant activity of dermal papilla (DP) in mouse vibrissal follicles during the hair cycle. Follicles were transversely cut into four pieces and each of them was associated with an isolated DP and grafted beneath the kidney capsule to induce hair formation. Various hair-cycle combinations of the fragments and DPs were examined. Hairs were generated not only in the follicle fragment containing the bulge (fragment III) but also in the fragment between the bulge and hair bulb (fragment II). The hair-forming frequencies were affected by the hair cycle stages of both the follicle fragments and DPs. Fragment III at late anagen (LA) and fragment II at catagen frequently generated hairs when associated with early anagen (EA)-DPs, but infrequently with mid-anagen (MA)-DPs. Oppositely, anagen fragment II produced hairs at a high frequency with MA-DPs and at a low frequency with EA-DPs. Hair generation in anagen fragment II is an unexpected finding because previous studies suggested that, during anagen, this region does not contain clonogenic epithelial cells that have been believed to be crucial for hair formation. Therefore, non-clonogenic epithelial cells would be able to generate hairs as well as clonogenic ones, and they should have a latent hair-forming ability that could be more effectively awakened by MA-DP than by EA-DP stimuli. Non-clonogenic epithelial cells might be a dormant phase of hair precursor cells. Proliferating follicular epithelial cells were detected in the middle and lower outer root sheath throughout the hair cycle but scarcely at LA. These findings suggest that the hair inductivity of DPs should be altered between EA and MA, and follicular epithelial cells would change their DP stimuli-directed hair-forming ability around LA, probably linked to the proliferative activity.

Wound healing ability of Xenopus laevis embryos. I. Rapid wound closure achieved by bisectional half embryos.

We examined wound closure in 'half embryos' produced by the transverse bisection of Xenopus laevis embryos at the primary eye vesicle stage. Both the anterior- and posterior-half embryos survived for more than 6 days, and grew into 'half tadpoles'. Histology and videomicroscopy revealed that the open wound in the half embryo was rapidly closed by an epithelial sheet movement in the wound marginal zone. The time-course of wound closure showed a downward convex curve: the wound area decreased to one-fifth of the original area within 30 min, and the wound continued to contract slowly thereafter. The rapidity of closure of the epidermis as well as the absence of inflammatory cells are typical features of an embryonic type of wound healing. There was a dorso-ventral polarity in the motility of the epidermis: the wound was predominantly closed by the ventral and lateral epidermis. The change in the contour of the wound edge with time suggested a complex mechanism involved in the wound closure that could not be explained only by the purse-string theory. The present experimental system would be a unique and useful model for analyses of cellular movements in the embryonic epithelia.

Wound healing ability of Xenopus laevis embryos. II. Morphological analysis of wound marginal epidermis.

We previously showed that bisectional wounds made in Xenopus laevis embryos at the primary eye vesicle stage were rapidly closed. In this study, microscopic analyses, including scanning electron microscopy, on the morphology of the epidermis were conducted during wound closure in the half embryos. Bright fluorescence of Texas red-phalloidin showing actin filaments started to be visualized at the cut edge 10 min after wounding. It increased with time, forming a distinguished, though discontinuous, bundle along the wound margin. The wound closure was completely inhibited by 20 microm cytochalasin B, and almost completely by 50 mm 2,3-butanedione 2-monoxime, an inhibitor to myosin ATPase activity. Scanning electron microscopy revealed that the outer epidermal cells became extensively elongated in the radial direction, and the contour of the closing wound edge did not become smoother but remained ragged. Thus, a representative embryonic type of wound closure may be driven in Xenopus embryos by a complex mechanism, involving not only the actin 'purse-string' but also an inward movement of individual cells. Anyhow, the wound closure is a movement of the epidermal sheet maintaining cell-cell contact, and not involving locomotion of single cells separated from the wound edge.

Expression of P-cadherin distinct from that of E-cadherin in re-epithelialization in neonatal rat skin.

Our previous study showed that an open wound made in neonatal rat skin was covered by migration of certain undifferentiated populations of keratinocytes as a multilayered cell sheet. In this study, the expression of the components of adherens junctions (AJ), E- and P-cadherins, and beta-catenin, was examined to understand the underlying mechanisms. Both E- and P-cadherins were downregulated in the basal layer at 6 h post-wounding (PW), indicating a reduction in the intercellular adhesiveness. The expression of P-cadherin but not E-cadherin was expanded to the suprabasal layers at the wound margin at 12 h PW. Moreover, the expression pattern of P-cadherin at sites of cell-cell contact was punctate rather than linear. By 24 h PW, cells accumulated beta-catenin in the cytoplasm in a suprabasal layer contacting the basal layer at the wound margin. Both the E- and P-cadherins showed a punctate AJ pattern at the confined suprabasal layer. Such differential expression of the E- and P-cadherins strongly suggests that these two classic cadherins play distinct roles in re-epithelialization. The changing of the E- and/or P-cadherin expression may participate in a delay of terminal differentiation of keratinocytes for cell supply toward a wound.

The subsets of keratinocytes responsible for covering open wounds in neonatal rat skin.

Full-thickness excisional wounds were made on the dorsal skin of 1-day-old rats to elucidate from where the cells move into the defect and what kinds of cells they are. Immunohistochemical analyses of the wound sites revealed that the following two subsets of keratinocytes were the major contributors to reepithelialization: first, the cells at the forefront of the migrating epithelium, termed "leading edge cells," which expressed K14 keratin, known as basal cell-specific keratin, but not K6 or K10 keratins, so that they had probably moved from the basal cell layer; and, second, the cells tentatively termed "immature spinous cells," which expressed K14 and K6 but not K10, and formed an "ingrowth region" following the leading edge cells. These two kinds of cells moved to the open wound area, as a multilayered cell sheet. Fluorescent phalloidin staining experiments indicated that actin filaments became concentrated in the leading edge cells within 6 h postwounding (PW), whereas weak signals of actin filaments were detected in the immature spinous cells. Taken together, the present findings support the view that wound covering in neonatal rat skin is accomplished by a movement en masse of keratinocytes from the bottom half of the surrounding epidermis.