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.     

A growth-promoting influence from the mesonephros during limb outgrowth.

It has been suggested that the mesonephros has a role in normal limb development. This hypothesis was directly tested by removing the mesonephros adjacent to the presumptive limb region of stage 12-18 chick embryos using microsurgery or laser ablation. The experimental manipulation resulted in reduced limb outgrowth on the operated side. The poor limb outgrowth was correlated with either the lack of or the presence of a rudimentary mesonephros on the operated side. Furthermore, the presence of nephric tissue in limb bud organ culture enhanced growth and morphological differentiation of cartilage formed in culture. In vivo, the influence of the mesonephros resulted in significantly higher cell proliferation in the adjoining medial half of the limb mesoderm compared with the lateral half. The removal of the mesonephros adjoining the prospective limb region reduced the number of dividing cells in the medial mesoderm. The higher proliferation in the medial limb mesoderm is significant to limb outgrowth since grafting experiments showed that most of the cells that form the limb are derived from the medial mesoderm. The results suggest that the influence from the mesonephros may provide some signal for limb outgrowth.     

Characteristics of human knee muscle coordination during isometric contractions in a standing posture: The effect of limb task

Different functional roles for the hands have been demonstrated, however leg control is not as well understood. The purpose of the present study was to evaluate bilateral knee neuromuscular control to determine if the limb receiving greater attention would have more well-tuned control compared to an unattended limb. Surface electrodes were placed on seven muscles of each limb, before standing on two force platforms. Visual feedback was given of the forces and moments of the “focus limb,” but not the “unattended limb.” Static isometric forces were matched with their focus limb, requiring their unattended limb to push in the opposite direction, using a combination of forward–backward–medial–lateral shear forces while muscle activity was collected bilaterally. There was a significant main effect for limb task (p = 0.02), with the medial hamstrings being more specific (p = 0.001) while performing the unattended limb and the lateral hamstring being more well-tuned (p = 0.007) while performing the focus limb task. The focus limb’s medial and lateral gastrocnemius were principally active in the forwards direction, but only the unattended limb’s lateral gastrocnemius was active in the backwards direction. Findings suggest unique neuromuscular control strategies are used for the legs depending on limb task.     

Limb Initiation and Development Is Normal in the Absence of the Mesonephros

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression ofcSim1andLmx1,molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked.fgf-8expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically itsfgf-8expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.     

Fibroblast-growth-factor-induced additional limbs in the study of initiation of limb formation, limb identity, myogenesis, and innervation

In this review, we focus on the additional limb induced by members of the fibroblast growth factor (FGF) family in the flank of chick embryos. The "additional limb" was first reported 73 years ago by Balinsky in 1925. He grafted otic vesicle to the flank of newt embryos and observed the formation of the "additional limb." In 1995, formation of an additional limb was found to be induced by FGF in the chick embryo. This finding subsequently led to the recent understanding of how the limb bud is initially formed, how the limb position is determined, and how the limb identity is determined. Thus, the additional limb has been recognized as a useful experimental system for the study of limb development and its relation to the regionalization of the body. Furthermore, since limb muscles are formed from cells which have migrated from somites and innervation to them takes place from the spinal cord, the additional limb would also be a powerful tool with which to study the relation of limb morphogenesis to developmental processes of the spinal cord and somites. This review consists of five sections: (1) "Introduction," (2) "How to make additional limbs," (3) "Characteristics of the additional limb," (4) "Studies with the additional limb," and (5) "Concluding remarks." In the second section, techniques to make additional limbs are reviewed, showing that additional limbs can be made by fairly easy manipulation of the chick embryo. In the third section, the characteristics analyzed so far of the additional limb are summarized, focusing on its morphology. In the fourth section, recent studies on the use of the additional limb are reviewed: experiments on the additional limb have been performed to elucidate the mechanisms governing determination of limb identity by Hox codes and the Tbx family and initiation of limb formation by FGF10. In addition, the roles of SF/HGF in the formation of limb muscles have also been investigated using the additional limb. In the near future, the additional limb will be also used in the study of innervation from the spinal cord, and probably migration of neural crest cells.     

Effects of limb mass distribution on the ontogeny of quadrupedalism in infant baboons (Papio cynocephalus) and implications for the evolution of primate quadrupedalism

Primate quadrupedal kinematics differ from those of other mammals. Several researchers have suggested that primate kinematics are adaptive for safe travel in an arboreal, small-branch niche. This study tests a compatible hypothesis that primate kinematics are related to their limb mass distribution patterns. Primates have more distally concentrated limb mass than most other mammals due to their grasping hands and feet. Experimental studies have shown that increasing distal limb mass by adding weights to the limbs of humans and dogs influences kinematics. Adding weights to distal limb elements increases the natural period of a limb's oscillation, leading to relatively long swing and stride durations. It is therefore possible that primates' distal limb mass is responsible for some of their unique kinematics. This hypothesis was tested using a longitudinal ontogenetic sample of infant baboons (Papio cynocephalus). Because limb mass distribution changes with age in infant primates, this project examined how these changes influence locomotor kinematics within individuals. The baboons in this sample showed a shift in their kinematics as their limb mass distributions changed during ontogeny. When their limb mass was most distally concentrated (at young ages), stride frequencies were relatively low, stride lengths were relatively long, and stance durations were relatively long compared to older ages when limb mass was more proximally concentrated. These results suggest that the evolution of primate quadrupedal kinematics was tied to the evolution of grasping hands and feet.     

Cellular contribution to supernumerary limbs resulting from the interaction between developing and regenerating tissues in the axolotl

The relationship between limb development and limb regeneration is considered with regard to the mechanisms by which pattern is established during limb outgrowth. In a previous paper (Muneoka, K. and Bryant, S. V. 1982 Nature (London) 298, 369-371) the interaction between cells from the developing limb bud and the regenerating limb blastema was found to result in the production of organized supernumerary limb structures. In this paper the relative cellular contribution from developing and regenerating cells to supernumerary limbs resulting from contralateral grafts between limb buds and blastemas has been analyzed using the triploid cell marker in the axolotl. Results show that there is substantial participation from both developing and regenerating limb cells to all supernumerary limbs analyzed. These data lend further support to the hypothesis that developing and regenerating limbs utilize the same patterning mechanisms during limb outgrowth. This conclusion is discussed in terms of patterning models for developing and regenerating limbs and it is proposed that the rules of the polar coordinate model can best explain the behavior of cells during limb development as well as limb regeneration.     

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.     

Effect of denervation on hindlimb regeneration in Xenopus laevis larvae

Xenopus laevis larvae at stages 51-57, according to Nieuwkoop and Faber, were subjected to amputation of the right hindlimb or of both limbs at the thigh or the tarsal level, as well as to somatic denervation of the right limb. Larvae at the same stage having undergone amputation of the right limb or of both limbs and sham denervation of the right limb were used as controls. In experimental series I a single denervation of the right limb was performed at the time of amputation. In experimental series II repeated denervations were performed (before, during and after amputation). Results show that in larvae at stages 51-53 subjected to limb amputation at the proximal level (thigh) even repeated denervation of the right limb did not prevent regeneration, although giving rise to various degrees of hypotrophy. In stage-55 larvae partial inhibition of the regenerative process in the right limb was clearly visible only after repeated denervations and amputation at the proximal level. After amputation at the distal level (tarsalia) the regenerative process in the right limb underwent no significant delay with respect to the controls, although the regenerated right limb was hypotrophic. In stage-57 larvae even a single denervation at the time of amputation was enough to inhibit regeneration of the right limb after either proximal or distal amputation. Therefore, in Xenopus laevis larvae, nerve-dependence for hindlimb regeneration takes place proximodistally as the nerve fibers grow in the limb and it gradually undergoes a process of proximodistal differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.     

Effect of apomorphine injection into the nucleus accumbens on the limb preference in manipulation movements in rats

The concentration of dophamine and its derivates is known to correlate with the degree of handedness in manipulative movements in rodents. In this work we studied a possibility to changing handedness in rats by injection of a dopamine agonist into the nucleus accumbens. Retrieving food from a horizontal tube was used to determine the limb preference (10 food retrievals by the preferred limb). Then apomorphine was injected into the n. accumbens ipsilateral to the preferred limb in the course of 7 days. The same volume of buffer solution was injected into the contralateral n. accumbens. Just after the last injection the limb preference was tested. It was shown that the chronic injection of the non-specific agonist of dophamine receptors significantly changed the limb preference.     

Lower limb skeletal muscle mass: development of dual-energy X-ray absorptiometry prediction model.

Although magnetic resonance imaging (MRI) can accurately measure lower limb skeletal muscle (SM) mass, this method is complex and costly. A potential practical alternative is to estimate lower limb SM with dual-energy X-ray absorptiometry (DXA). The aim of the present study was to develop and validate DXA-SM prediction equations. Identical landmarks (i.e., inferior border of the ischial tuberosity) were selected for separating lower limb from trunk. Lower limb SM was measured by MRI, and lower limb fat-free soft tissue was measured by DXA. A total of 207 adults (104 men and 103 women) were evaluated [age 43 +/- 16 (SD) yr, body mass index (BMI) 24.6 +/- 3.7 kg/m(2)]. Strong correlations were observed between lower limb SM and lower limb fat-free soft tissue (R(2) = 0.89, P < 0.001); age and BMI were small but significant SM predictor variables. In the cross-validation sample, the differences between MRI-measured and DXA-predicted SM mass were small (-0.006 +/- 1.07 and -0.016 +/- 1.05 kg) for two different proposed prediction equations, one with fat-free soft tissue and the other with added age and BMI as predictor variables. DXA-measured lower limb fat-free soft tissue, along with other easily acquired measures, can be used to reliably predict lower limb skeletal muscle mass.     

Supernumerary limgs in amphibians: experimental production in Notophthalmus viridescens and a new interpretation of their formation.

The formation of supernumerary limbs was studied in the adult newt, Notophthalmus viridescens. Forelimb blastemas at the stages of medium bud and early digits were either transplanted to the contralateral forelimb with their dorsal-ventral axis opposed to that of the limb stump, or removed, rotated through 180°, and replaced on the same limb stump with both dorsal-ventral and anterior-posterior axes opposed to those of the stump, or as a control, removed, and replaced in normal orientation. Supernumerary limbs were produced in both experimental series, but not in the controls.Following contralateral transplantation, supernumerary limbs arose close to the graft junction at the two positions where dorsal limb tissue was in contact with ventral limb tissue. Both dorsal and ventral supernumerary limbs were of the same handedness as the limb stump and they were mirror-images of the regenerate developing directly from the transplanted blastema. Following 180° rotation, supernumerary limbs arose close to the graft junction at those positions where anterior-ventral and posterior-dorsal limb tissues were in contact. The supernumerary limb which arose in the posterior-dorsal position with respect to the limb stump was a mirror-image of the transplant, and was therefore of opposite handedness to both transplant and stump. The supernumerary limb which arose in the anterior-ventral position was of the same handedness as both transplant and stump. A new model of pattern regulation in epimorphic fields which can account for these results and which has retrospective value in the interpretation of earlier experiments on developing limbs is discussed.     

Treatment of brachial nerves with colchicine inhibits limb regeneration in the newt Notophthalmus viridescens

In urodele amphibians, limb regeneration is dependent on innervation and is blocked by the administration of colchicine. The objective of this experiment was to determine if colchicine blocks limb regeneration by a direct action on the blastema cells or by an indirect action on the nerves, specifically, if colchicine treatment of the brachial nerves would inhibit limb regeneration in the newt Notophthalmus viridescens. Colchicine was applied to the nerves by implanting a colchicine-loaded silastin block adjacent to the brachial nerves of an amputated newt limb. With appropriate dose levels of colchicine, limb regeneration was completely inhibited. Contralateral control limbs, carrying unloaded silastin blocks, and control limbs with colchicine-loaded blocks implanted equidistant from the blastema, but not adjacent to the brachial nerves, regenerated normally. Thus, the results indicate that the colchicine inhibition of limb regeneration is mediated by colchicine effects on the nerves. The possible mechanism of colchicine action on nerves may involve either wallerian degeneration, or inhibition of axoplasmic transport, or both.     

Impaired Hyperemic Response to Exercise Post Stroke

Individuals with chronic stroke have reduced perfusion of the paretic lower limb at rest; however, the hyperemic response to graded muscle contractions in this patient population has not been examined. This study quantified blood flow to the paretic and non-paretic lower limbs of subjects with chronic stroke after submaximal contractions of the knee extensor muscles and correlated those measures with limb function and activity. Ten subjects with chronic stroke and ten controls had blood flow through the superficial femoral artery quantified with ultrasonography before and immediately after 10 second contractions of the knee extensor muscles at 20, 40, 60, and 80% of the maximal voluntary contraction (MVC) of the test limb. Blood flow to the paretic and non-paretic limb of stroke subjects was significantly reduced at all load levels compared to control subjects even after normalization to lean muscle mass. Of variables measured, increased blood flow after an 80% MVC was the single best predictor of paretic limb strength, the symmetry of strength between the paretic and non-paretic limbs, coordination of the paretic limb, and physical activity. The impaired hemodynamic response to high intensity contractions was a better predictor of lower limb function than resting perfusion measures. Stroke-dependent weakness and atrophy of the paretic limb do not explain the reduced hyperemic response to muscle contraction alone as the response is similarly reduced in the non-paretic limb when compared to controls. These data may suggest a role for perfusion therapies to optimize rehabilitation post stroke.     

Regulation and Function of SF/HGF during Migration of Limb Muscle Precursor Cells in Chicken

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.     

Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth

Outgrowth and patterning of the vertebrate limb requires a functional apical ectodermal ridge (AER). The AER is a thickening of ectodermal tissue located at the distal end of the limb bud. Loss of this structure, either through genetic or physical manipulations results in truncation of the limb. A number of genes, including Bmps, are expressed in the AER. Previously, it was shown that removal of the BMP receptor Bmpr1a specifically from the AER resulted in complete loss of hindlimbs suggesting that Bmp signaling in the AER is required for limb outgrowth. In this report, we genetically removed the three known AER-expressed Bmp ligands, Bmp2, Bmp4 and Bmp7 from the AER of the limb bud using floxed conditional alleles and the Msx2-cre allele. Surprisingly, only defects in digit patterning and not limb outgrowth were observed. In triple mutants, the anterior and posterior AER was present but loss of the central region of the AER was observed. These data suggest that Bmp ligands expressed in the AER are not required for limb outgrowth but instead play an essential role in maintaining the AER and patterning vertebrate digits.