Functional rice with tandemly repeated Cbl-b ubiquitin ligase inhibitory pentapeptide prevents denervation-induced muscle atrophy in vivo.

Ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) play a critical role in nonloading-mediated skeletal muscle atrophy: Cbl-b ubiquitinates insulin receptor substrate-1 (IRS-1), leading to its degradation and a resulting loss in muscle mass. We reported that intramuscular injection of a pentapeptide, DGpYMP, which acts as a mimic of the phosphorylation site in IRS-1, significantly inhibited denervation-induced skeletal muscle loss. In order to explore the possibility of the prevention of muscle atrophy by diet therapy, we examined the effects of oral administration of transgenic rice containing Cblin (Cbl-b inhibitor) peptide (DGYMP) on denervation-induced muscle mass loss in frogs. We generated transgenic rice seeds in which 15 repeats of Cblin peptides with a WQ spacer were inserted into the rice storage protein glutelin. A diet of the transgenic rice seeds had significant inhibitory effects on denervation-induced atrophy of the leg skeletal muscles in frogs, compared with those receiving a diet of wild-type rice.

Optimal stimulation toward the dermal papilla lineage can be promoted by combined use of osteogenic and adipogenic inducers.

Dermal papilla cells (DPCs) play crucial roles in hair regeneration, but they readily lose their hair-forming ability during in vitro culture. Although the formation of spheroids partially restores the ability, shrinkage of the spheroids makes it difficult to maintain cellular viability. To address this problem, we stimulated DPCs with factors known to induce adipogenic and/or osteogenic differentiation, because DPCs share unique gene expression profiles with adipocytes and osteocytes. We isolated DPCs from versican (vcan)-GFP mice, in which GFP is expressed under the control of a vcan promoter, which is strongly active in DPCs of anagen hair follicles. GFP fluorescence was most intense when the spheroids were made from DPCs cultured in a half-diluted combination of adipogenic and osteogenic media (CAO1/2), a Dulbecco's modified Eagle's medium-based medium that contains 10% FBS, 275 nm dexamethasone, 2.5 mm ß-glycerol phosphate, 12.5 µg·mL-1 ascorbic acid, 0.125 µm isobutylmethylxanthine and 2.5 ng·mL-1 insulin. The dose of each additive used was less than the optimal dose for adipogenic or osteogenic differentiation, and shrinkage of the spheroids was avoided through the addition of fibroblast growth factor 2 and platelet-derived growth factor-AA to  CAO1/2. In addition, the gene and protein expression of vcan, osteopontin, alkaline phosphatase and α-smooth muscle actin in the spheroids were augmented to levels similar to those of the intact dermal papillae, which exhibited restored hair-forming activity. In conclusion, a combination of certain adipogenic and osteogenic inducers, together with fibroblast growth factor 2 and platelet-derived growth factor-AA, can promote differentiation toward the DPC lineage.

Isolation and Primary Culture Methods of Adult and Larval Myogenic Cells from Xenopus laevis.

During amphibian metamorphosis, larval-to-adult conversion of the myogenic system occurs and there are two distinct types of muscle stem cells; larval myogenic cells have a death-fate by apoptosis in the presence of thyroid hormone T3, and adult myogenic cells have a life-fate under the same conditions. Here, we describe isolation and culture methods for adult and larval myogenic cells from the frog, Xenopus laevis. Both types of cultured myogenic cells undergo cell division and cell differentiation, i.e., formation of multinucleated myotubes in serum-containing medium. Insulin-like growth factor-1 promotes cell division and differentiation in both cells. Basic properties and some applications of primary culture are also described.

Insulin-like growth factor 1 regulation of proliferation and differentiation of Xenopus laevis myogenic cells in vitro.

To understand the mechanism of muscle remodeling during Xenopus laevis metamorphosis, we examined the in vitro effect of insulin-like growth factor 1 (IGF-1) on growth and differentiation of three different-fate myogenic cell populations: tadpole tail, tadpole dorsal, and young adult leg muscle. IGF-1 promoted growth and differentiation of both tail and leg myogenic cells only under conditions where these cells could proliferate. Inhibition of cell proliferation by DNA synthesis inhibitor cytosine arabinoside completely canceled the IGF-1's cell differentiation promotion, suggesting the possibility that IGF-1's differentiation-promotion effect is an indirect effect via IGF-1's cell proliferation promotion. IGF-1 promoted differentiation dose dependently with maximum effect at 100-500 ng/ml. RT-PCR analysis revealed the upregulation (11-fold) of ifg1 mRNA expression in developing limbs, suggesting that IGF-1 plays a role in promoting muscle differentiation during limb development. The combined effect of triiodo-L-thyronine (T3) and IGF-1 was also examined. In adult leg cells, IGF-1 promoted growth and differentiation irrespective of the presence of T3. In larval tail cells, cell count was 76% lower in the presence of T3, and IGF-1 did not promote proliferation and differentiation in T3-containing medium. In larval dorsal cells, cell count was also lower in the presence of T3, but IGF-1 enhanced proliferation and differentiation in T3-containing medium. This result is likely due to the presence among dorsal cells of both adult and larval types (1:1). Thus, IGF-1 affects only adult-type myogenic cells in the presence of T3 and helps accelerate dorsal muscle remodeling during metamorphosis.

Formation of a new limb bud at the boundary between a transplanted limb bud and the tail surface of Xenopus tadpoles.

Through transplantation experiments with Xenopus laevis tadpoles, we found a new morphogenetic phenomenon consisting of limb bud formation at the boundary between transplanted whole limb buds and the tail surface. This phenomenon occurs without limb-limb stump interaction and has a number of unique features: (1) Only one extra limb bud was formed per transplant and the new limb and the transplanted limb were bilaterally symmetrical, forming a pair of limb girdles. (2) Extra new limb bud formation occurred not only at the tail but also at other non-limb regions, including abdominal and head surfaces. (3) Successful limb formation required the presence of basal 1/4 region (presumptive limb girdle) of a limb bud explant. (4) New limb formation was host-stage-dependent: before metamorphosis, limb bud formation ratio was high (> 90%), but as the host tadpole entered metamorphosis, this potential declined and morphological abnormalities of new limbs increased. (5) Cell lineage analysis showed that epidermis of the new limb bud always contained many (about 60%) host-derived cells, while new limb cartilage cells were completely graft-derived. These results suggest that heterotopic new limb formation occurs through interaction between graft mesenchyme and host epidermis. Thus, the present study has clarified the two important aspects of limb ontogeny: the importance of presumptive limb girdle for the limb bud initiation and the relationship between limb bud formation potential and metamorphic tissue remodeling. The present experimental system may help to improve our understanding of epithelial-mesenchymal interaction during limb bud initiation and subsequent limb cell differentiation during metamorphosis.

Differential muscle regulatory factor gene expression between larval and adult myogenesis in the frog Xenopus laevis: adult myogenic cell-specific myf5 upregulation and its relation to the notochord suppression of adult muscle differentiation.

During Xenopus laevis metamorphosis, larval-to-adult muscle conversion depends on the differential responses of adult and larval myogenic cells to thyroid hormone. Essential differences in cell growth, differentiation, and hormone-dependent life-or-death fate have been reported between cultured larval (tail) and adult (hindlimb) myogenic cells. A previous study revealed that tail notochord cells suppress terminal differentiation in adult (but not larval) myogenic cells. However, little is known about the differences in expression patterns of myogenic regulatory factors (MRF) and the satellite cell marker Pax7 between adult and larval myogenic cells. In the present study, we compared mRNA expression of these factors between the two types. At first, reverse transcription polymerase chain reaction analysis of hindlimb buds showed sequential upregulation of myf5, myogenin, myod, and mrf4 during stages 50-54, when limb buds elongate and muscles begin to form. By contrast, in the tail, there was no such increase during the same period. Secondary, these results were duplicated in vitro: adult myogenic cells upregulated myf5, myod, and pax7 in the early culture period, followed by myogenin upregulation and myotube differentiation, while larval myogenic cells did not upregulate these genes and precociously started myotube differentiation. Thirdly, myf5 upregulation and early-phase proliferation in adult myogenic cells were potently inhibited by the presence of notochord cells, suggesting that notochord cells suppress adult myogenesis through inhibiting the transition from Myf5(-) stem cells to Myf5(+) committed myoblasts. All of the data presented here suggest that myf5 upregulation can be a good criterion for the activation of adult myogenesis during X. laevis metamorphosis.

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.

Strategies to detect interdigital cell death in the frog, Xenopus laevis: T3 accerelation, BMP application, and mesenchymal cell cultivation.

Fates of digits in amniotes, i.e., free or webbed digits, are determined by the size of programmed interdigital cell death (ICD) area. However, no (or very few) cell death has thus far been observed in developing limb buds of non-amniotic terrestrial vertebrates including other anuran or urodela amphibians. We speculate that the undetectable situation of amphibian ICD is the result of their less frequency due to slow developmental speed characteristic to most amphibian species. Here, we present three strategies for detecting difficult-to-find ICD in the frog, Xenopus laevis. (1) Addition of triiodo-L-thyronine (T(3)) accelerated two to three times the limb development and increased two to four times the appearance frequency of vital dye-stainable cells in limb buds of the accelerated tadpoles (stage 54 to 55). (2) Application of human bone morphogenetic protein-4 to the autopods of tadpoles at stage 53 to 54 enhanced digital cartilage formation and induced vital dye-stainable cells around the enhanced digital cartilages within 2 d. (3) In cell culture, T(3) increased the chondrogenic and cell death activities of limb mesenchymal cells. The augmentation of both activities by T(3) was stronger in the forelimb cells than in the hindlimb cells. This situation is well coincided with the limb fates of non-webbed forelimbs and webbed hindlimbs in X. laevis adulthood. Collectively, all three approaches showed that it become possible to detect X. laevis ICD with appropriate strategies.

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.

Robust and photocontrollable DNA capsules using azobenzenes.

Various three-dimensional structures have been created on a nanometer scale using the self-assembly of DNA molecules. However, ordinary DNA structures breakdown readily because of their flexibility. In addition, it is difficult to control them by inputs from environments. Here, we construct robust and photocontrollable DNA capsules using azobenzenes. This provides a method to construct DNA structures that can survive higher temperatures and can be controlled with ultraviolet irradiation.

Regulation of desmin expression in adult-type myogenesis and muscle maturation during Xenopus laevis metamorphosis.

Isoforms of myosin heavy chain and tropomyosin convert during metamorphosis of Xenopus laevis with larval-to-adult remodeling of dorsal muscle (Nishikawa and Hayashi, 1994 , Dev. Biol. 165: 86-94). In the present study, other markers for muscle remodeling during metamorphosis were determined by SDS-PAGE analysis. The amounts of twelve muscle proteins changed remarkably during metamorphosis. Among these, a 54-kDa molecule was found to be desmin, and the relative content/total proteins decreased dramatically through metamorphosis. In hindlimb muscle, desmin content increased fourfold during prometamorphosis, when myoblast proliferation and fusion occurred. With further myotube maturation, this content decreased by 1/2 while that of muscle actin continued to increase. Thus, desmin up- and down-regulation in hindlimbs mark early and late phases of myogenesis, respectively. In tall muscle, the desmin content decreased continuously to (1/8) before and during metamorphosis, due to tall muscle growth and maturation. In dorsal muscle, three desmin changes occurred: a pre-metamorphic decrease, a transient increase at prometamorphosis, and a rapid decrease at the climax stage. Immunohistochemical analysis showed desmin+ cells to be present between young (adult-type) myotubes and replicating (PCNA+) cells in dorsal muscles, correlating the transient desmin upregulation in dorsal muscle with the initiation of adult-type myogenesis. After the upregulation, dorsal muscle desmin decreased to (1/8). This rapid down-regulation was replicated by administration of triiodothyronine (T3) to tadpoles, suggesting a significant role for T3 in dorsal muscle remodeling during metamorphosis. Collectively, these results show that analysis of desmin expression and PCNA-immunohistochemistry are good tools for determining the sites and timing of larval-to-adult muscle remodeling during Xenopus laevis metamorphosis.

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis.

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130-160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.

Intercalary and supernumerary regeneration in the limbs of the frog, Xenopus laevis.

Anuran amphibians, such as Xenopus laevis, can regenerate their limbs only when they are young tadpoles, whereas urodele amphibians have a regenerative ability throughout their lives. It is still unclear whether anuran and urodele use the same mechanism during regeneration. In the present study, we analyzed intercalary and supernumerary regeneration in Xenopus. In contrast to urodele blastema that induces intercalary regeneration along the proximodistal (PD) axis, intercalation did not occur in the Xenopus limb bud when the presumptive zeugopodium (fibula and tibia) was removed. However, when the limb bud tip (presumptive autopodium) was transplanted to the presumptive stylopodium (femur) with a 180-degree rotation at stage 52, the complete zeugopodium was regenerated. These results were similar to the results of urodele mature limbs, suggesting that Xenopus limb buds are equivalent to the urodele mature limbs but not to the urodele blastemas. We hypothesized that the ability for intercalation depends on the expression pattern of fibroblast growth factor (fgf)-8, because the expression of fgf-8 in the urodele spreads over the whole blastema and is close enough to activate the growth of the stump. To test this hypothesis, an FGF-8-soaked bead was implanted at the boundary between the stump and tip of a Xenopus limb bud. Intercalary regeneration was induced at stages 52 and 53. These results suggest that the Xenopus limb bud possesses the potential for intercalation, but endogenous FGF-8 in the apical ectodermal ridge (AER) does not induce intercalation to the stump because of the long distance between the AER and stump.

Regulation of specific developmental fates of larval- and adult-type muscles during metamorphosis of the frog Xenopus.

During anuran metamorphosis, larval-type myotubes in both trunk and tail are removed by apoptosis, and only trunk muscles are replaced by newly formed adult-type myotubes. In the present study, we clarified the regulatory mechanisms for specific developmental fates of adult and larval muscles. Two distinct (adult and larval) types of myoblasts were found to exist in the trunk, but no or very few adult myoblasts were found in the tail. Each type of myoblast responded differently to metamorphic trigger, 3,3',5-triiodo-L-thyronine (T(3)) in vitro. T(3)-induced cell death was observed in larval myoblasts but not in adult myoblasts. These results suggest that the fates (life or death) of trunk and tail muscles are determined primarily by the differential distribution of adult myoblasts within the muscles. However, a transplantation study clarified that each larval and adult myoblast was not committed to fuse into particular myotube types, and they could form heterokaryon myotubes in vivo. Cell culture experiments suggested that the following two mechanisms are involved in the specification of myotube fate: (1) Heterokaryon myotubes could escape T(3)-induced death only when the proportion of adult nuclei number was higher than 70% in the myotubes. Apoptosis was not observed in any larval nuclei within the surviving heterokaryon myotubes, suggesting the conversion of larval nuclei fate. (2) Differentiation of adult myoblasts was promoted by the factor(s) released from larval myoblasts in a cell type-specific manner. Taken together, the developmental fate of myotubes is determined by the ratio of nuclei types, and the formation of adult nuclei-rich myotubes was specifically enhanced by larval myoblast factor(s).

Isolation and Characterization of Mesenchymal Cells from the Tail of Bullfrog Tadpoles*: (mesenchymal cells/macrophage/tadpole/metamorphosis/thyroid hormone).

Mesenchymal cells were separated with the aid of EDTA, dispase, collagenase and hyaluronidase from tail fins of Rana catesbeiana tadpoles and were cultured to examine their morphology and response to thyroid hormone, which controls metamorphosis. The mensenchymal tissue consisted of two main types of cells, macrophage-like cells (M cells) and fibroblastic cells (F cells), distinguishable by their morphological and biochemical characteristics. M cells were preferentially located near the boundary between the mesenchyme and the epidermis. They were more adhesive to the plastic culture dishes than F cells. The M cells developed a bizarre rod-like branching process during one week of culture, which appeared to be identical to the process formed by mammalian macrophages undergoing clasmatosis. M cells cultured for more than one week tended to fuse together forming multinuclear giant cells. F cells were found uniformly in the tissue and were active in collagen biosynthesis. Thyroid hormone at a physiological concentration greatly reduced the life span of F cells, but did not reduce that of M cells.

Progression throughout All Stages of Meiosis from the Early Prophase of Newt Primary Spermatocytes in vitro: (newt/meiosis/primary spermatocyte/prophase/collagen matrix).

Stages of prophase of living primary spermatocytes were determined by use of Rose culture chambers (1). Dissociated primary spermatocytes were cultured at low cell-density in a collagen matrix at 22°C or 27°C and the percentages of cells which had progressed from various stages in prophase through meiosis to various advanced stages were measured. In a standard medium (Leibovitz-15 + 10% fetal bovine serum), more than 70% of the primary spermatocytes at stages beyond the pachytene stage could advance to round spermatids with flagella within a few days at 22°C. The percentages of cells that progressed from stages before the late zygotene stage were less, but at least 13 % of leptotene cells reached metaphase I within a week at 22°C. The percentage of cells that progressed was slightly lower at 27°C than at 22°C: 6.3 and 4.3 days were required for progress from leptotene to metaphase I at 22°C and 27C, respectively. Fetal bovine serum was not indispensable for progression through meiosis. Moreover, 0.5-5.0 µg/ml ovine follicle stimulating hormone (NIAMDD-o-FSH-13), 0.01-1.0 µg/ml 5α-dihydrotestosterone and 1.0 µg/ml testosterone propionate had no significant effect in increasing the percentage of cell progression at 22°C.

Periodic Rotation of Chromosomes During the Mitotic Divisions in Secondary Spermatogonia of Newt, Cynops Pyrrhogaster.

Mitosis was frequently observed in the secondary spermatogonia of newt in in vitro cultures. From prometaphase to mid-anaphase, the whole set of the chromosomes rotated alternately clockwise and counterclockwise generally in the same plane as the bottom of a plastic dish. The axis of rotation was almost always perpendicular to the bottom of a dish, passing through the central part of the cell. This rotation of chromosomes was so fast that we could discern it directly by a phase contrast microscope. It was a rhythmic and regular motion with almost the constant frequency and magnitude. The average period of each cycle during metaphase varied from cell to cell and between 70 to 20 seconds (0.9-3.0 rotations/min) and the average angle traversed during each motion also varied and between 10 to 90 degrees at 25°C. By marking the cell surface with iron particles, it was demonstrated that the inner part of the cell actively rotated and not the cell as a whole. Colcemid at the concentration of 1.0 µg/ml reversibly arrested the chromosomal rotation and karyokinesis. Cytochalasin B (4.0 µg/ml) also reversibly disturbed the rotation though the karyokinesis continued. These results suggest that the rotation of chromosomes as a set may be mediated by filamentous organelles such as microtubules in the mitotic spindle and cytoplasmic microfilaments.