The latent dedifferentiation capacity of newt limb muscles is unleashed by a combination of metamorphosis and body growth.

Newts can regenerate their limbs throughout their life-span. Focusing on muscle, certain species of newts such as Cynops pyrrhogaster dedifferentiate muscle fibers in the limb stump and mobilize them for muscle creation in the regenerating limb, as they grow beyond metamorphosis. However, which developmental process is essential for muscle dedifferentiation, metamorphosis or body growth, is unknown. To address this issue, we tracked muscle fibers during limb regeneration under conditions in which metamorphosis and body growth were experimentally shifted along the axis of development. Our results indicate that a combination of metamorphosis and body growth is necessary for muscle dedifferentiation. On the other hand, ex vivo tracking of larval muscle fibers revealed that newt muscle fibers have the ability to dedifferentiate independently of metamorphosis and body growth. These results suggest that newt muscle fibers have an intrinsic ability to dedifferentiate, but that metamorphosis and body growth are necessary for them to exhibit this hidden ability. Presumably, changes in the extracellular environment (niche) during developmental processes allow muscle fibers to contribute to limb regeneration through dedifferentiation. This study can stimulate research on niches as well as gene regulation for dedifferentiation, contributing to a further understanding of regeneration and future medical applications.

Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Antiosteoporotic effect of orally administered yolk-derived peptides on bone mass in women.

The main objective of this study was to verify the effect of oral intake of a yolk-derived peptide preparation (HYP) obtained by enzymatic hydrolysis of yolk water-soluble protein on bone markers and bone density in 65 perimenopausal women with an average age of 47.6 ± 5.2 years. Subjects were divided into three groups, and then enrolled in a 6-month, randomized, double-blind clinical trial. Bone formation and resorption markers were measured at 0, 3, and 6 months, while bone mineral density (BMD) in the lumbar spines was measured at 0 and 6 months. Although the bone formation marker levels showed the similarity changes among the groups, bone resorption markers in the test HYP group were significantly decreased after 3 and 6 months in comparison to other groups (P < 0.05). After 6 months, BMD in the test HYP group maintained at healthy numerical values whereas BMD values were decreased in other groups. Hence HYP would be an antiosteoporotic agent originated from natural food to maintain bone health, especially for women.

Subchronic toxicity evaluation of γ-aminobutyric acid (GABA) in rats.

γ-Aminobutyric acid (GABA) is an amino acid compound contained in vegetables such as tomatoes and also widely distributed in mammals. GABA acts as an inhibitory neurotransmitter and promotes parasympathetic activity to provide several beneficial effects, for instance, relaxation, anti-stress, and insomnia. GABA, produced via a fermentation process, has been available as a functional food ingredient. As part of a program to assess its safety, GABA was administered by oral gavage at doses of 500, 1250, and 2500mg/kg body weight to groups of 10 male and 10 female Sprague-Dawley rats for 13weeks. Treatment was not associated with the test substance-related mortality and appeared to be well tolerated. There were no toxicologically and statistically significant changes in urinalysis, hematology, clinical chemistry parameters, and in necropsy findings. A few statistically significant changes in food consumption and body weights were noted in the male groups while any significant changes were not noted in female groups. There was no effect of treatment on organ weights or on the results of the histopathological examinations. The results of toxicity evaluation support the safety use of GABA and the potential use as a functional food ingredient.

Conserved expression control and shared activity between cognate T-box genes Tbx2 and Tbx3 in connection with Sonic hedgehog signaling during Xenopus eye development.

Tbx2 and Tbx3 are considered to be cognate genes within a Tbx2/3/4/5 subfamily of T-box genes and are expressed in closely overlapping areas in a variety of tissues, including the eye. Herein, we show that misexpression of Tbx2 and Tbx3 in Xenopus embryos gave rise to defective eye morphogenesis, which was reminiscent of the defect caused by attenuated Sonic hedgehog (Shh) signaling. Indeed, Tbx2/3 misexpression suppressed Gli1, Gli2, Ptc2 and Pax2, mediators or targets of Hedgehog (Hh) signals. From these data, Tbx2/3 may have a shared function in inhibiting Gli-dependent Shh signaling during eye development. Conversely, the expression of Tbx2/3 was severely affected by both Shh and a putative dominant negative form of Hh, as well as by both transactivator and transrepressor forms of Gli-fusion proteins, suggesting that the expression of Tbx2/3 may be regulated by a Gli-dependent Hh signal transduction pathway. Because the Shh signal has been considered to play crucial roles in the formation of the proximal-distal and dorsal-ventral axes in the eyes, these findings about the mutual regulatory mechanism between Tbx2/3 and Gli-dependent Hh signaling provide valuable insight into the cause of the localized expression of Tbx2/3 and their role during the formation of these axes. In addition, our findings also imply the conserved regulation and shared activity between the cognate genes of Tbx2 and Tbx3.

Improved mRNA electroporation method for Xenopus neurula embryos.

Electroporation has led to new approaches to the analysis of gene regulation of the chick embryonic system. However, application of this method to Xenopus, another model organism of embryology, has left many difficulties to be overcome. The specially devised electrodes, the examination of luciferase activities expressed, and the direct visualization of green fluorescence protein allow us to optimize the conditions of electroporation for Xenopusembryos. The use of mRNA rather than DNA improved the expression efficiency 120 times more than for the case of plasmid DNA, and the effect emerged more immediately after electroporation. The noncontact electroporation adopted here caused less damage to cells and tissues than with the needle type electrode, making it practical for efficient application to early embryos. Furthermore, the mRNA electroporation technique is applicable for other systems in which the DNA electroporation has not had any significant effect because of its low expression efficiency.

Axes establishment during eye morphogenesis in Xenopus by coordinate and antagonistic actions of BMP4, Shh, and RA.

We have examined the roles of BMP4, Shh, and retinoic acid in establishing the proximal-distal and dorsal-ventral axes in the developing Xenopus eye. Misexpression of BMP4 caused the absence of an optic stalk and the expansion of dorsal and distal markers, tbx2/3/5, and pax6, at the expense of ventral and proximal markers vax2 and pax2. When Shh or Noggin, an antagonist of BMPs, was misexpressed, the reverse expression patterns of these marker genes were observed. These results suggest that BMP4 is involved in the specification of not only dorsal in the optic cup but also distal in the optic vesicle. Because Shh did not suppress bmp4 expression, unlike Noggin, Shh and BMP4 may antagonistically regulate common downstream genes in developing eye. We also found the difference between the effects of Shh and retinoic acid, another possible ventralizing factor, suggesting that Shh could promote ventralization independently of retinoic acid. These findings provide important clues to the coordinate and antagonistic actions of BMP4, Shh, and retinoic acid in axes specifications of Xenopus eyes.

Temporal alteration of dose-dependent response to activin in newt animal-cap explants.

In this study, we examined the dose-dependent responses of animal-pole cells of the Japanese newt, Cynops pyrrhogaster, to activin. Cynops has a slower developmental rate and a simpler animal cap structure than Xenopus. These features enable temporal differences in animal-cap competence to be identified more easily and relatively sharp dose-response profiles can be obtained without cell dissociation. When Cynops caps were excised at the mid-blastula stage and transcript levels of marker genes were examined at the early gastrula stage, the strongest induction of brachyury occurred at a low activin dose, suggesting that cells can recognize changing concentrations of an inducing signal in the embryo. Furthermore, the time course of brachyury expression revealed that caps from the mid-blastula stage exposed to a high dose of activin maintained a low expression level after induction. This suggests that Cynops animal-pole cells can assess activin concentration in a simple and direct manner. In addition, we found that animal-cap competence significantly changes during the blastula stage. The data presented here suggest that this change does not autonomously occur within animal-pole cells but requires signals that emanate from other germ layers.

Cloning of an Epidermis-specific Cynops cDNA from Neurula Library.

A cDNA encoding one of the epidermis-specific proteins designated as the spot 6 was isolated from the Cynops embryo. Cynops neurula cDNA library was constructed with the plasmid vector containing the promoter sequence for SP6 RNA polymerase. After transcription and translation in vitro the final protein products were screened for the presence of spot 6 by two-dimensional gel electrophoresis. The total library was digested by 11 restriction endonucleases selected not to destroy the sequence in both the vector and the insert encoding spot 6 protein. The ATP-dependent DNase digestion eliminated the cDNA population sensitive to these endonucleases. These steps effectively enriched the sequence for spot 6 protein. The resultant sublibrary was repeatedly divided into smaller pools and was screened. The tryptic peptide analysis showed that the isolated clone produced the protein identical to the spot 6 protein originally defined in vivo. Northern analyses showed that the cloned gene was expressed as expected from the developmental behavior of the spot 6 in vivo.

Protein Synthesis during Neural and Epidermal Differentiation in Cynops Embryo: (Cynops pyrrhogaster/protein synthesis/neural induction/concanavalin A/epidermal differentiation).

Two-dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with 35 S-methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.

Concanavalin A Acts as a Factor in Establishing the Dorso-Ventral Gradient in the Ventral Mesoderm of Newt Gastrula Embryos1 : (concanavalin A/organizer/dorsalization/dorso-ventral gradient/newt).

Con A induced dorsal differentiation in the ventral mesoderm of Cynops gastrula embryo. This process apparently requires a certain amount of Con A to be internalized as supported by the following evidence: 1) Oligomannose-type oligosaccharide, a potent inhibitor of Con A, considerably inhibited dorsalization of ventral mesoderm by Con A. The incorporation of 125 I-Con A into the ventral mesoderm was greatly inhibited by this sugar. 2) Sepharose-immobilized Con A did not dorsalize the ventral mesoderm. Con A-induced dorsalization was found to be concentration-dependent. Microautoradiograms of 125 I-Con A-treated ventral mesoderm suggest that the target site (some receptor molecules) of Con A exists inside the cell. Con A is the first pure substance reported to mimic the two properties of the organizer-neural induction of the competent ectoderm and dorsalization of the ventral mesoderm. In neural induction, Con A acts on the cell surface, while Con A apparently needs to be internalized to trigger dorsal differentiation. Interestingly, Con A-dorsalized ventral mesoderm acquired the neural inducing function of the organizer within the early phase of dorsalization.