Mature Limb Research Articles

Integration failure of regenerated limb tissue is associated with incongruencies in positional information in the Mexican axolotl.

Introduction: Little is known about how the newly regenerated limb tissues in the Mexican axolotl seamlessly integrate with the remaining stump tissues to form a functional structure, and why this doesn't occur in some regenerative scenarios. In this study, we evaluate the phenomenological and transcriptional characteristics associated with integration failure in ectopic limb structures generated by treating anterior-located ectopic blastemas with Retinoic Acid (RA) and focusing on the "bulbus mass" tissue that forms between the ectopic limb and the host site. We additionally test the hypothesis that the posterior portion of the limb base contains anterior positional identities. Methods: The positional identity of the bulbus mass was evaluated by assaying regenerative competency, the ability to induce new pattern in the Accessory Limb Model (ALM) assay, and by using qRTPCR to quantify the relative expression of patterning genes as the bulbus mass deintegrates from the host site. We additionally use the ALM and qRTPCR to analyze the distribution of anterior and posterior positional identities along the proximal/distal limb axis of uninjured and regenerating limbs. Results: The bulbus mass regenerates limb structures with decreased complexity when amputated and is able to induce complex ectopic limb structure only when grafted into posterior-located ALMs. Expressional analysis shows significant differences in FGF8, BMP2, TBX5, Chrdl1, HoxA9, and HoxA11 expression between the bulbus mass and the host site when deintegration is occuring. Grafts of posterior skin from the distal limb regions into posterior ALMs at the base of the limb induce ectopic limb structures. Proximally-located blastemas express significantly less HoxA13 and Ptch1, and significantly more Alx4 and Grem1 than distally located blastemas. Discussion: These findings show that the bulbus mass has an anterior-limb identity and that the expression of limb patterning genes is mismatched between the bulbus mass and the host limb. Our findings additionally show that anterior positional information is more abundant at the limb base, and that anterior patterning genes are more abundantly expressed in proximally located blastemas compared to blastemas in the more distal regions of the limb. These experiments provide valuable insight into the underlying causes of integration failure and further map the distribution of positional identities in the mature limb.

Study of histochemical characterizations of skeletal muscle fiber types in the rabbits ( Oryctolagus Cuniculus )

This project has been conducted on New Zealand White Rabbits (newborns and adults) . Biceps brachii, soleus and abdominal wall of muscles were used in this study. Fresh pieces of muscle tissues were frozen to be cut at (10 μm) thick sections by the cryostat.Skeletal musclefibre types were investigated histochemically to demonstrate myosin adenosin triphosphatase (ATPase) activity using acid and alkaline preincubation and succinate dehydrogenase ( SDHase ) activity . In this way it may be possible to recognize the fast and slow twitch muscle fibers, as well as to explore the difference in reaching maturity between different muscles of the body. The results showed that, the biceps brachii and abdominal wall muscles consist predominantly of fast fibers, whilst the soleus muscle is made up of a near homogenous population of slow fibers. The immature abdominal wall muscle showed some reversal of the mature limb muscles staining pattern of the same animal.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.

Molecular mechanisms in the control of limb regeneration: the role of homeobox genes.

Axolotls are unique among vertebrates in their ability to regenerate lost appendages as adults. They provide the opportunity to study the mechanism of regeneration in vertebrates and are an inspiration to pursue the goal of appendage regeneration in humans. In this article, we review data on the role of homeobox-containing genes in the regulation of limb regeneration. As a group, these genes are important in pattern formation in the primary body axis, developing limbs and regenerating limbs. To date, a total of 22 homeobox genes have been identified as being expressed in regenerating limbs. Nearly all of these are also expressed during limb regeneration, further supporting the view that limb development and regeneration involve similar regulatory mechanisms. Our recent results on the expression of HoxA genes demonstrate that once a blastema has formed, subsequent outgrowth and pattern formation are similar to those of limb development. In contrast to developing limbs, reexpression of the HoxA genes in regeneration occurs by a non-colinear mechanism that likely is related to the necessity of mature limb cells to undergo dedifferentiation in order to give rise to the blastema. These studies also indicate that the pattern is respecified by a distal-first mechanism during regeneration in contrast to the apparent proximal-to-distal sequence observed in developing limbs. Expression of the HoxA genes is altered coordinately in response to retinoic acid in a manner consistent with the transformation of a distal blastema to a proximal blastema. Given the recent increase in studies of the molecules involved in regeneration, it is likely that many of the functionally important regeneration genes will be identified and characterized in the near future.

The axolotl limb blastema: cellular and molecular mechanisms driving blastema formation and limb regeneration in tetrapods.

The axolotl is one of the few tetrapods that are capable of regenerating complicated biological structures, such as complete limbs, throughout adulthood. Upon injury the axolotl generates a population of regeneration-competent limb progenitor cells known as the blastema, which will grow, establish pattern, and differentiate into the missing limb structures. In this review we focus on the crucial early events that occur during wound healing, the neural-epithelial interactions that drive the formation of the early blastema, and how these mechanisms differ from those of other species that have restricted regenerative potential, such as humans. We also discuss how the presence of cells from the different axes of the limb is required for the continued growth and establishment of pattern in the blastema as described in the polar coordinate model, and how this positional information is reprogrammed in blastema cells during regeneration. Multiple cell types from the mature limb stump contribute to the blastema at different stages of regeneration, and we discuss the contribution of these types to the regenerate with reference to whether they are "pattern-forming" or "pattern-following" cells. Lastly, we explain how an engineering approach will help resolve unanswered questions in limb regeneration, with the goal of translating these concepts to developing better human regenerative therapies.

Computational and mathematical models of chondrogenesis in vertebrate limbs

The production of cartilage (chondrogenic patterning) in the limb is one of the best-studied examples of the emergence of form in developmental biology. At the core of the theoretical study is an effort to understand the mechanism that establishes the characteristic distribution of cartilage in the embryonic limb, which defines the future sites and shapes of bones that will be present in the mature limb. This review article gives an overview of the history and current state of a rich literature of mathematical and computational models that seek to contribute to this problem. We describe models for the mechanisms of limb growth and shaping via interaction with various chemical fields, as well as models addressing the intrinsic self-organization capabilities of the embryonic mesenchymal tissue, such as reaction-diffusion and mechanochemical models. We discuss the contributions of these models to the current understanding of chondrogenesis in vertebrate limbs, as well as their relation to the varied conceptual models that have been proposed by experimentalists.

Comparison of the Paley Method Using Chronological Age with Use of Skeletal Maturity for Predicting Mature Limb Length in Children

Treating patients with congenital or acquired limb-length inequality requires accurate estimations of limb length at skeletal maturity. There is controversy over the best indicator of maturity to be used for limb-length calculations. Paley popularized the multiplier method, in which chronological age is used, which has the virtue of simplicity but does not account for the wide variance in timing of the adolescent growth spurt. The purpose of this study was to determine whether the use of chronological age or the level of skeletal maturity provides more accurate limb-length predictions. We identified patients with limb-length inequality, for whom scanograms had been obtained before and at maturity, and who had had no surgical procedures on their normal lower limb. Skeletal maturity was determined with use of the Greulich and Pyle atlas, Tanner-Whitehouse-3 method, and simplified stages described by Sanders et al. The length of the lower extremity was compared with the ultimate limb length and the actual multiplier (final limb length divided by current limb length) for each point in time. A linear model was used to determine the log-transformed multipliers for the level of skeletal maturity, and Paley's multipliers were used for chronological age. Residual standard errors were determined to compare the results of the methods. We also conducted piecewise linear regression on each of the methods and used the residual standard errors to rank their performance and cross-validated the results. We identified twenty-four patients (twelve girls and twelve boys) who met the study criteria. Most subjects had had multiple scanograms along with skeletal age radiographs (average, 4.5) at different ages. When all ages are considered, the Paley method had the best overall performance, with residual standard errors that were typically =5 cm. However, the Paley method did not perform best for subjects at stage-2 skeletal maturity or above; in those cases, skeletal-maturity-based predictions had residual standard errors of <2 cm. While the Paley method, which is based on chronological age, provides reasonable estimates of ultimate limb length for most patients, use of skeletal-maturity determinations appears to provide better predictions of mature limb length during adolescence.

A unique Coxiella burnetii lipoprotein involved in metal binding (LimB)

Coxiella burnetii is the bacterial agent of Q fever in humans. Here, we describe a unique, ~7.2 kDa, surface-exposed lipoprotein involved in metal binding which we have termed LimB. LimB was initially identified as a potential metal-binding protein on far-Western (FW) blots containing whole-cell lysate proteins when probed with nickel-coated horseradish peroxidase (Ni-HRP) and developed with a chemiluminescent HRP substrate. The corresponding identity of LimB as CBU1224a was established by matrix-assisted laser desorption ionization-tandem time-of-flight mass spectrometry. blast analyses with CBU1224a showed no significant similarity to sequences outside strains of C. burnetii. Additional in silico analyses revealed a putative 20 residue signal sequence with the carboxyl end demarcated by a potential lipobox (LSGC) whose Cys residue is predicted to serve as the N-terminal, lipidated Cys of mature LimB. The second residue of mature LimB is predicted to be Ala, an uncharged envelope localization residue. These features suggest that CBU1224a is synthesized as a prolipoprotein which is subsequently lipidated, secreted and anchored in the outer membrane. Mature LimB is predicted to contain 45 aa, of which there are 10 His and 5 Cys; both amino acids are frequently involved in binding transition metal cations. Recombinant LimB (rLimB) was generated and its Ni-HRP-binding activity demonstrated on FW blots. Ni-HRP binding by rLimB was inhibited by >95 % on FW blots done in the presence of EDTA, imidazole, Ni(2+) or Zn(2+), and roughly halved in the presence of Co(2+) or Fe(3+). The limB gene was maximally expressed at 3-7 days post-infection in Coxiella-infected Vero cells, coinciding with exponential phase growth. Two isoforms of LimB were detected on FW and Western blots, including a smaller (~7.2 kDa) species that was the predominant form in small cell variants and a larger isoform (~8.7 kDa) in large cell variants. LimB is Sarkosyl-insoluble, like many omps. The predicted surface location of LimB was verified by immunoelectron and immunofluorescence microscopy using anti-rLimB antibodies. Overall, the results suggest that LimB is a unique Coxiella lipoprotein that serves as a surface receptor for divalent metal cations and may play a role in acquiring at least one of these metals during intracellular growth.

Rethinking the proximodistal axis of the vertebrate limb in the molecular era

The development of the vertebrate limb has long served as a paradigm for understanding the fundamental processes by which an undifferentiated field of cells gains spatial pattern and undergoes coordinated differentiation to produce the exquisitely complex structures that characterize our functional anatomy. The limb bud emerges from the flank as a mound of seemingly homogeneous mesenchymal cells within an ectodermal jacket. Yet within days this mass of cells gives rise to skeletal elements, muscles, and connective tissues organized in the form of a mature limb. Looking at one’s own arm or leg, this organization can immediately be appreciated along three different axes: The back of the hand (dorsal) is different from the palm (ventral), the thumb (anterior) is distinct from the little finger (posterior), and the upper arm (proximal) is different from the lower arm and the hand (distal). The process by which differences along the proximodistal axis are established has been particularly controversial, with competing models proposed to explain the outcome of both classical and genetic manipulations. However, if one examines the two major models that have been previously proposed, neither of them is tenable in the context of our current knowledge of gene activity in the developing limb. Proximodistal patterning therefore needs to be placed into a new framework based directly on the molecular data. The first key insight into the process of limb patterning came almost 60 yr ago when John Saunders discovered that the apical ectodermal ridge (AER), a thickened ridge of ectodermal cells that runs along the anterior– posterior axis of the distal limb bud (equivalent to a ridge running along the distal edge of the hand from which the finger tips will eventually form) is necessary for the successful outgrowth of the limb along the proximodistal axis (Saunders 1948). To explain how different structures arise at different proximodistal levels as the limb bud grows out, a model was subsequently proposed based on progressive specification of increasingly distal cell fates in a domain ∼300 µm deep directly below the AER (Summerbell et al. 1973). According to this view, under the influence of the AER, cells are maintained in a so-called progress zone. These cells continuously acquire ever more distal positional information through the influence of an internal clock that is kept active as long as the cells receive signaling from the overlying AER. This process gives the cells a distal fate proportional to the length of time the cells remain in the progress zone. When cells move out of the range of AER signaling— i.e., out of the progress zone—the clock stops and their proximodistal fate therefore becomes fixed. This happens continuously, as all the cells in the progress zone—indeed, all of the cells of the limb bud—are dividing. As more cells are produced, only the members of the population closest to

Myonuclear Addition to Uninjured Laryngeal Myofibers in Adult Rabbits

In normal mature limb skeletal muscle, satellite cells are quiescent and myonuclei do not divide after formation of their associated myofibers in the absence of injury. The possibility of myonuclear addition in uninjured laryngeal myofibers of adult rabbits was investigated in an immunohistochemical pilot study. Bromodeoxyuridine (brdU), a marker for cell division, was administered by intraperitoneal injection over a 12-hour period in rabbits. The number of brdU-positive myonuclei per myofiber was determined on cross sections through the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. In the TA muscle, 0.13% +/- 0.03% (mean +/- SEM) of the myofibers counted had a brdU-positive nucleus. In the PCA muscle, 0.13% +/- 0.01% of the myofibers counted had a brdU-positive nucleus. Approximately 0.2% and 0.3% of the myofibers of the TA and PCA muscles, respectively, had brdU-positive satellite cells associated with them. Tibialis anterior and pectoralis major muscle controls were negative for brdU-positive myonuclei. These data support the possibility of continuous remodeling in uninjured adult laryngeal myofibers and accentuate the distinct nature of laryngeal muscle relative to limb skeletal muscle in the rabbit model.

Continuous myofiber remodeling in uninjured extraocular myofibers: myonuclear turnover and evidence for apoptosis.

Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax-7, and the cell-cycle marker, Ki-67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU-positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross-section using a 12-hour brdU-labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL). Activated caspase-3 was present in localized cytoplasmic domains extending from 10 to 210 microm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease.

Development of epiphyseal structure and function in Didelphis virginiana (Marsupiala, Didelphidae)

This study addressed the question of how the epiphyses of growing mammals change their external shape and internal architecture during postnatal development. Ontogenetic transformations in the external form and internal structure of the fore- and hindlimb epiphyses were examined in a mixed cross-sectional sample of Didelphis virginiana using two methods: morphometric analysis of linear epiphyseal dimensions and histological staining of serially sectioned epiphyses. Metric data indicate that Virginia opossums are born with relatively short hindlimbs and long forelimbs, but by the time they are weaned their hindlimbs are longer than their forelimbs. Functional integration of the locomotor system in D. virginiana involves a decoupling of fore- and hindlimb growth rates so that between birth and weaning, femoral length, diaphyseal cross-sectional area, and articular surface area increase at a significantly faster rate than the corresponding humeral dimensions. Histological results demonstrate that these differences in growth rate are reflected in morphology of the humeral and femoral growth plate and epiphyseal cartilages. The humeral cartilages exhibit a level of cellular organization characteristic of more mature limb elements at earlier developmental stages compared to the femoral cartilages, which assume this anisotropic structure relatively later in postnatal development. Results presented here also reveal that the formation of articular cartilage and the initiation of epiphyseal ossification in D. virginiana are both correlated with the development of independent positional behaviors prior to weaning. These histological data, therefore, suggest that mechanical loading associated with the postnatal onset of locomotor and postural development may provide an important stimulus for the progression of ossification and the formation of articular cartilage in the epiphyses of growing mammals. J. Morphol. 239:283–296, 1999. © 1999 Wiley-Liss, Inc.

Development of epiphyseal structure and function inDidelphis virginiana (Marsupiala, Didelphidae)

This study addressed the question of how the epiphyses of growing mammals change their external shape and internal architecture during postnatal development. Ontogenetic transformations in the external form and internal structure of the fore- and hindlimb epiphyses were examined in a mixed cross-sectional sample of Didelphis virginiana using two methods: morphometric analysis of linear epiphyseal dimensions and histological staining of serially sectioned epiphyses. Metric data indicate that Virginia opossums are born with relatively short hindlimbs and long forelimbs, but by the time they are weaned their hindlimbs are longer than their forelimbs. Functional integration of the locomotor system in D. virginiana involves a decoupling of fore- and hindlimb growth rates so that between birth and weaning, femoral length, diaphyseal cross-sectional area, and articular surface area increase at a significantly faster rate than the corresponding humeral dimensions. Histological results demonstrate that these differences in growth rate are reflected in morphology of the humeral and femoral growth plate and epiphyseal cartilages. The humeral cartilages exhibit a level of cellular organization characteristic of more mature limb elements at earlier developmental stages compared to the femoral cartilages, which assume this anisotropic structure relatively later in postnatal development. Results presented here also reveal that the formation of articular cartilage and the initiation of epiphyseal ossification in D. virginiana are both correlated with the development of independent positional behaviors prior to weaning. These histological data, therefore, suggest that mechanical loading associated with the postnatal onset of locomotor and postural development may provide an important stimulus for the progression of ossification and the formation of articular cartilage in the epiphyses of growing mammals. J. Morphol. 239:283-296, 1999. © 1999 Wiley-Liss, Inc.

A mechanistic model for morphogenesis and regeneration of limbs and imaginal discs

When an amphibian limb, cockroach leg or Drosophila imaginal disc is subjected to a surgical operation, it is capable of regenerating or duplicating certain parts. Although the structure of the regenerated tissue varies depending on the location and mass of the amputated or transplanted part, it can be predicted from a set of formal rules, called the polar coordinate model [French et al., (1976) Science 193–983; Bryant et al., (1981) Science 212, 993–1002]. In the polar coordinate model, it is assumed (and experimentally proven) that the juxtaposition of normally non-adjacent cells stimulates cell proliferation locally, which implies that the underlying mechanism which gives positional values to each cell, is also responsible for the control of cell growth. Because locally activated proliferation alters the shape and size of the developmental field, the question of how to control the cell growth is the central problem in the regeneration of the limbs and imaginal discs. In this paper, I propose a possible underlying mechanism for the ‘polar coordinate rules’, and show how this mechanistic model explains the experimental results using computer simulation. The proposed mechanism is an extension of Turing's model (1952). In addition to the reaction-diffusion of the molecules, cell proliferation is taken into consideration. With appropriate initial conditions, the computer simulation shows that a small mass of cells grows up to form a mature limb, and that the mature limb is able to respond to surgical operations as predicted by the polar coordinate model.

Nonsegmental organization of positional information in regenerating Ambystoma limbs

Positional information in cockroach appendages has previously been shown to be organized in homologous, segmentally repeating sequences ( H. Bohn, 1970, Wilhelm Roux' Arch. Entwicklungsmech. Organismen 165, 303–341; 1970, Develop. Biol. 23, 355–379 ). We have conducted experiments to determine whether similar sequences exist in Ambystoma limbs. The same or different segment levels of these limbs were combined by grafting together mature limb regions and by grafting regeneration blastemas to mature limb regions. The repeating segmental sequence hypothesis predicts that no intercalary regeneration should occur when the same segment levels are apposed, and that only the positional values necessary to restore a single segment sequence should be intercalated when different segment levels are apposed. When regeneration blastemas are grafted to mature limb regions, all of the missing limb regions are restored by intercalary regeneration, regardless of the segment levels that are combined. When mature limb regions are grafted together, only the host stump segment is intercalated, and this intercalation also takes place without regard to which segment levels are combined. These results are not compatible with the segmental sequence hypothesis, and suggest instead that positional information is organized in a single limb sequence. Histological data indicate that the reason more complete intercalation does not take place after grafting mature limb regions is that dedifferentiated cells are unable to display positional information and divide long enough to restore more than the host segment.

Organization of the Morphogenetic Field in Regenerating Amphibian Limbs

The amphibian limb is an example of a secondary embryonic field that can be reactivated during larval or adult life so that amputated parts are regenerated. Two major questions are: (1) what is the origin of the morphogenetic field of the regeneration blastema, and (2) what is the nature of this field and how does it specify the spatial pattern of blastemal redifferentiation? Evidence is analyzed here which leads to the following propositions: (1) the field is represented in latent form by properties of the mature limb cells, and these properties are activated and inherited by the blastemal cells after amputation and dedifferentiation. At the same time, the inherited field is sensitive to the mature stump tissues and its spatial organization can be altered by a stump pattern alien to the one from which it was derived. (2) The properties of the mesodermal limb tissues represent positional values that are arranged in gradients along the proximal-distal, anterior-posterior and dorsal-ventral axes. These properties allow dedifferentiated mesodermal cells to change their positional value to any value between their original one in the limb and the value of any neighboring cell after creation of a discontinuity. The direction of change is always from proximal to distal in the PD axis; it is uncertain as to whether change can take place only centripetally or both centripetally and centrifugally along the AP and DV axes. (3) Epidermal cells have the same positional value everywhere in the limb and act as the distal and circumferential boundaries up to which the mesodermal cells may change their positional values. The proximal boundary is represented by the level-specific properties of the mesodermal cells at the maximum extent of distal to proximal dedifferentiation. Normal regeneration can then be visualized as occurring in the following way. When deletions are made in the limb pattern, cells with widely different positional values are confronted. During regeneration, blastema cells increase in number and continually interact with their neighbors to adjust their positional values within the boundaries until discontinuities are eliminated. The multiple limbs resulting from rearrangement of stump tissue patterns can also be accounted for by using these propositions. It is suggested that positional information is encoded on the cell surface and/or in the extracellular matrix.

Intercalary and supernumerary regeneration in regenerating the mature limbs of Notophthalmus viridescens.

AbstractWe describe the ability of advanced limb regenerates and mature limbs of Notophthalmus viridescens to undergo intercalary and supernumerary regeneration following grafting operations to confront normally nonadjacent cells. Although both of these types of regeneration are known to be exhibited by regenerating limbs up to the stage of early digits, this is the first investigation of these abilities in mature limbs of Notophthalmus viridescens using similar grafting procedures. The autopodia of mature limbs, or regenerates at the stage of late digits or older from a distal level of amputation (distal third of forearm), were transplanted to other limb sites so as to oppose either anterior and posterior, or dorsal and ventral positions of stump and transplant. Following transplantation of either the regenerate or the mature autopodium, supernumerary limbs were sometimes formed, and their handedness, position and orientation were, in those cases in which they could be accurately determined, similar to what has been found previously in experiments on younger regenerates. Unlike the results of the experiments on regenerating limbs, when mature autopodia were grafted ipsilaterally with 180° rotation, the handedness of the supernumerary limbs could seldom be determined, and their location appeared to be random. An enhancement of the incidence of supernumerary limb formation in mature limb grafts was noted when the grafts were made so as to oppose normally nonadjacent proximal‐distal as well as circumferential positions.When advanced regenerates were grafted ipsilaterally and without rotation to a more proximal limb level (mid upper arm), good intercalary regeneration of the distal portion of the humerus as well as the radius and/or ulna occurred in most, but not all cases. When similarly grafted mature autopodia were examined, it was found that none of the limbs formed a complete intercalary regenerate, but the majority formed partial intercalary regenerates by completing the humerus. When mature autopodia were grafted so as to oppose normally non‐adjacent circumferential as well as proximal‐distal positions, then the intercalary regenerate was more complete and frequently included a regenerated radius and/or ulna, normally in conjunction with one or more supernumerary limbs. The similarities between these findings and our earlier ones on intercalary and supernumerary regeneration in regenerating newt limbs are discussed in terms of a recently developed model for pattern regulation in epimorphic fields.

The regulative ability of the limb regeneration blastema ofNotophlhalmus viridescens: Experimentsin situ.

1. The regulative ability of the regeneration blastema of the newt limb (Notophthalmus viridescens) was tested by operationsin situ. Either the anterior, posterior, dorsal, or ventral half of the blastema was removed at various stages during regeneration. 2. All blastemas operated on prior to the stage of four early digits showed a delay in reaching the subsequent stages of regeneration. 3. The blastema is capable of extensive regulation in the anterior-posterior and dorsoventral axes even after many of its cells have begun to differentiate. 4. Early digital stages of regeneration were found to be defective in regulative ability. Additional skeletal elements were present in limbs which had been operated on at the stage of three early digits. Supernumerary digits as well as additional skeletal elements were present in limbs which had been operated on at the stage of four early digits. Removal of the posterior half of the regenerate at one of these late stages resulted in more severe abnormalities than did removal of the anterior half. 5. Either the anterior or the posterior half of a mature limb was removed back to the level of the wrist. In several cases, an almost complete autopodium developed alongside the remaining half autopodium. 6. Removal of half of a regenerate at digital stages gave results similar to those obtained following removal of half of a mature limb. 7. The results are discussed in the context of other experiments on regenerating limbs, and of experiments on other developing systems. It is concluded that amphibian blastemas in common with a number of other systems can develop according to the presumptive fates of their cells, or they can regulate when they are given the opportunity for growth and cell division.

Asymmetry of the node of ranvier

ABSTRACT The nodal constrictions of Ranvier in normal limb nerves of mammals are bounded by asymmetric paranodal bulbs, the proximal bulb (nearer the cell body) being larger in all dimensions. This investigation was undertaken to determine whether the asymmetry results from partial damming of proximo-distally flowing axoplasm at the nodal constriction, or whether other features of local growth patterns are more relevant. The degree of asymmetry was estimated on teased, osmicated fibres from normal immature and mature limb nerves to skin and muscle and on ventral nerve roots. Estimates were also made on the central processes of dorsal root ganglionic cells with their contrasting direction of flow, after alteration of growth patterns in regenerates of crushed immature and mature nerves, and in the recurrent laryngeal nerve which pursues an exceptional course in relation to surrounding tissues. A polarization of asymmetry with larger proximal bulbs was found in uncomplicated limb nerves and after simultaneous regeneration and limb growth following crushing of immature nerves. Mixed populations (that is, with no preferred direction of asymmetry and often symmetrical bulbs) were found in dorsal and ventral nerve roots and the apical recurved segment of the recurrent laryngeal, whilst the mature regenerates closely approached the symmetrical condition. The descending and ascending limbs of the recurrent laryngeal nerve showed a reversal of polarization with respect to the cell body, but similar with respect to the cephalo-caudal body axis. It was concluded that damming of directionally flowing axoplasm was not causally related to the formation of asymmetric bulbs. The overall interstitial growth pattern of limb nerve elongation as revealed by intemodal distance studies contrasts with the differential growth and maturation gradients shown by the various limb segments through which the nerve passes. These differences result in a relative movement between the myelinating and elongating Schwann cell and the surrounding limb tissues. The movements are considerable and unidirectional throughout most of the limb, the Schwann cells becoming relatively further removed from the limb apex, whilst the movements minimize at the limb root and apex. It is suggested that the altered mechanical conditions operating at the ‘advancing’ and ‘trailing’ ends of the cell are related to the dimensional differences of the paranodal apparatus comprising the bulb at each end of the cell, and secondarily result in asymmetric nodes of Ranvier.