Retinoic acid-induced pattern duplication in regenerating urodele limbs

The effects of varying doses of retinoic acid on forelimb regeneration in larval Ambystoma mexicanum amputated through the wrist joint and in adult Notophthalmus viridescens amputated through the basal carpals were compared. In both species, the major effect of retinoic acid was to cause the proximodistal duplication, in the regenerate, of stump segments proximal to the amputation plane. Transverse axial duplications (anteroposterior and dorsoventral) occurred in a smaller percentage of cases; these consisted of cartilage spurs in axolotls, and extra digits in newts. The frequency and magnitude of the proximodistal and (in the newt) transverse duplications were dose dependent, and the regenerating limbs were maximally sensitive to the retinoid during the period of dedifferentiation and accumulation of blastema cells. The effect of retinoic acid is exerted on cells local to the amputation surface, as shown by the fact that retinoic acid caused the proximodistal duplication of stump segments in regenerates derived from amputated distal lower arm segments grafted to the eyesocket.     

Stage-dependent effects of retinoic acid on regenerating urodele limbs

Summary Following amputation through the distal zeugopodium, regenerating limbs of larvalAmbystoma mexicanum and pre and post-metamorphicPleurodeles waltlii were treated with 150 g of retinoic acid (RA) per gram of body weight, at the dedifferentiation, early bud, medium bud, late bud or early redifferentiation stages of regeneration. The effect of RA on regenerate morphogenesis differed as a function of the stage at which it was administered. When given during dedifferentiation or at early bud stages, RA evoked proximodistal duplications of stump segments in the regenerates. The maximum duplication index (DI) inAbystoma was achieved when RA was injected at 4 days post-amputation, which corresponds to the stage of dedifferentiation; and inPleurodeles at 10 days post-amputation, which corresponds to a stage midway between early bud and medium bud. When RA was administered at later stages, the DI declined progressively to zero or nearly zero by the stage of early redifferentiation in both species. The decline in DI was due to a decreased frequency of duplication, not to a decrease in the magnitude of duplication in individual regenerates. At the same time, there was an increase in hypomorphism and aberrant morphogenesis of both duplicating and non-duplicating regenerates. These results indicate that regenerative cells are differentially sensitive to RA in a stage-dependent way.     

Regeneration potency of mouse limbs

Mammalians have a low potency for limb regeneration compared to that of amphibians. One explanation for the low potency is the deficiency of cells for regenerating amputated limbs in mammals. Amphibians can form a blastema with dedifferentiated cells, but mammals have few such cells. In this paper, we report limb formation, especially bone/cartilage formation in amputated limbs, because bone/cartilage formation is a basic step in limb pattern regeneration. After the amputation of limbs of a neonatal mouse, hypertrophy of the stump bone was observed at the amputation site, which was preceded by cell proliferation and cartilage formation. However, no new elements of bone/cartilage were formed. Thus, we grafted limb buds of mouse embryo into amputated limbs of neonatal mice. When the intact limb bud of a transgenic green fluorescent protein (GFP) mouse was grafted to the limb stump after amputation at the digit joint level, the grafted limb bud grew and differentiated into bone, cartilage and soft tissues, and it formed a segmented pattern that was constituted by bone and cartilage. The skeletal pattern was more complicated when limb buds at advanced stages were used. To examine if the grafted limb bud autonomously develops a limb or interacts with stump tissue to form a limb, the limb bud was dissociated into single cells and reaggregated before grafting. The reaggregated limb bud cells formed similar digit-like bone/cartilage structures. The reaggregated grafts also formed segmented cartilage. When the reaggregates of bone marrow mesenchymal cells were grafted into the stump, these cells formed cartilage, as do limb bud cells. Finally, to examine the potency of new bone formation in the stump tissue without exogenously supplied cells, we grafted gelatin gel containing BMP-7. BMP induced formation of several new bone elements, which was preceded by cartilage formation. The results suggest that the environmental tissues of the stump allow the formation of cartilage and bone at least partially, and that limb formation will be possible by supplying competent cells endogenously or exogenously in the future.     

竹节虫的断足再生观察

竹节虫的断足再生现象,是生物学上很有趣的问题。蔡邦华先生(1956)指出:“腿节与转节间有缝,使足易于脱落。”[1]忻介六等(1985)说,断足在蜕皮时又能长出,但明显短小。[2]戚永和等(1992)发现金平巨树Ｔｉｒａｃｈｉｄｅａｗｅｓｔｗｏｏｄｉ(ＷｏｏｄＭａｓｏｎ)的雌虫或雄虫常会缺少一只或两只足,有些个体则在断足的基部长出了细弱弯曲的“肢芽”,并能够逐渐发育成一只完整的足,但是,看上去比原有那只弱小一些。[3]ＧＯＢｅｄｆｏｒｄ(1978)援引ＪＴＳａｌｍｏｎ(1955)的文章,竹节虫若虫的附肢,蜕皮时可能被损坏(或实验性损坏),在随后的…     

Long-term heightened larval production in nursery-bred coral transplants

The ‘gardening coral reefs’ method is part of the approaches proposed for counteracting the substantial impacts of global climate change on the survival of coral reefs. It incorporates ecosystem engineering strategies for coral nursery farming and coral colonies out-planting. This study explores the reproductive output of three sets of nursery-grown Stylophora pistillata colonies along eight reproductive seasons following transplantation, as compared to that of native corals. When native and transplanted corals grew side by side in a disturbed environment, the nursery-grown transplants showed enhanced larval release (2.6–22.5 times more planulae/colony; multiyear average: 11.6±1.8 planulae/transplant vs. 1.5±0.3 planulae/native colony) with higher percentages of gravid colonies (91±2.1% transplants vs. 34±7.6% native colonies). The inherently enhanced larval production of transplants, maintained for such a long period of time post-transplantation, reveals a possible enduring impact of the nursery conditions on future fitness and ecological traits of transplants. This is further supported by the emerging documentation regarding the enhanced growth of corals under nursery conditions, which continues to be detected even years after transplantation was conducted on the natural reef. The above enhancement of coral reproduction can be harnessed as a human intervention tool for countering global climate change impacts.     

Regeneration of mirror symmetrical limbs in the axolotl

Measurements of tubulin exchange into and from bovine brain microtubules at steady state in vitro were made with 3H-GTP as a marker for tubulin addition to or loss from microtubules. Tubulin has an exchangeable GTP binding site that becomes nonexchangeable in the microtubule. We found that tubulin addition to and loss from microtubules under steady state conditions occurred at equivalent rates, that loss and gain were linear, and that exchange rates (percentage of total tubulin in microtubules lost or gained per hour) were dependent upon microtubule length. Furthermore, we found that podophyllotoxin blocked steady state assembly, but did not alter the rate of steady state tubulin loss. When the assembling microtubule end was pulsed with 3H-GTP at steady state, the label was almost completely retained during a subsequent chase. We conclude that the microtubule assembly-disassembly "equilibrium" is a steady state summation of two different reactions which occur at opposite ends of the microtubule, and that assembly and disassembly occur predominantly and perhaps exclusively at the opposite ends under steady state conditions in vitro.     

Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs

Hyaluronate-mediated expansion of the extracellular matrix has been suggested as an important element of growth and morphogenesis in several developing systems. In vitro, various growth factors have been shown to stimulate hyaluronate synthesis as well as cell proliferation. A similar link between proliferation and hyaluronate production during in vivo growth is difficult to demonstrate, because in most systems the source of growth-promoting factors is either not known or not amenable to experimental manipulation. During amphibian limb regeneration, cell proliferation depends upon paracrine release of factors from axons in the limb stump, and the nerve supply can be eliminated or augmented experimentally for study of growth in this system. Denervated and amputated limbs of larval salamanders do not begin to regenerate until distal areas of the limb stumps are reinnervated. We have used such limbs to examine the effect exerted by the reappearance of nerves on the amount of hyaluronate in the tissue undergoing the growth response. Hyaluronate was demonstrated by the metachromatic dye Ethyl Stains-all, which stains hyaluronate blue while sulfated glycosaminoglycans (GAGs) and proteins in the extracellular matrix stain various shades of violet, and by microspectrophotometry of alcian-blue-stained GAGs in serial sections pretreated with buffer or with Streptomyces hyaluronidase (SH) to remove hyaluronate specifically. Both methods showed little hyaluronate in the distal region of limb stumps prior to reinnervation, while reinnervated stumps had amounts of hyaluronate similar to those of control blastemas. Autoradiography of ³H-glucosamine-labeled limbs indicated that hyaluronate in the blastemas of reinnervated limb stumps included material newly synthesized by cells throughout the growing tissue. The microspectrophotometric study revealed that the relative concentration of hyaluronate increased during the time distal limb areas were undergoing reinnervation, which was monitored by staining of nerve fibers. The increase in hyaluronate concentration was followed immediately by an increase in mitotic activity and a decrease in mesenchymal cell density, two changes leading to blastema formation that others have shown to be associated with reinnervation in this system. These observations indicate that the growth-promoting influence of nerves includes stimulation of hyaluronate production, an effect similar to that of serum or purified mitogens on many cultured cells. Hyaluronate synthesis appears to promote expansion of the limb stump, which occurs when denervated-amputated larval limbs are reinnervated.     

Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground-born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.     

Preparation of cultured myofibers from larval salamander limbs for cellular plasticity studies

Identification of the mechanisms underlying cellular plasticity in salamander cells is important because these may give pointers to the restricted regenerative ability of mammals. The myofibers from salamanders are remarkable for their ability to undergo cellularization and cell-cycle re-entry during regeneration. Here, we describe a detailed method for the isolation and culture of larval salamander myofibers in numbers suitable for cellular plasticity studies. The basic protocol for isolation and purification of cells can be completed in 4 h.