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The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration.

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. In the present study, we have utilized the ALM to identity the buttonhead-like zinc-finger transcription factor, Sp9, as being involved in the formation of the regeneration epithelium. Sp9 expression is induced in basal keratinocytes of the apical blastema epithelium in a pattern that is comparable to its expression in developing limb buds, and it thus is an important marker for dedifferentiation of the epidermis. Induction of Sp9 expression is nerve-dependent, and we have identified KGF as an endogenous nerve factor that induces expression of Sp9 in the regeneration epithelium.

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.

The amphibian limb is a model that has provided numerous insights into the principles and mechanisms of tissue and organ regeneration. While later stages of limb regeneration share mechanisms of growth control and patterning with limb development, the formation of a regeneration blastema is controlled by early events that are unique to regeneration. In this study, we present a stepwise experimental system based on induction of limb regeneration from skin wounds that will allow the identification and functional analysis of the molecules controlling this early, critical stage of regeneration. If a nerve is deviated to a skin wound on the side of a limb, an ectopic blastema is induced. If a piece of skin is grafted from the contralateral side of the limb to the wound site concomitantly with nerve deviation, the ectopic blastema continues to grow and forms an ectopic limb. Our analysis of dermal cell migration, contribution, and proliferation indicates that ectopic blastemas are equivalent to blastemas that form in response to limb amputation. Signals from nerves are required to induce formation of both ectopic and normal blastemas, and the diversity of positional information provided by blastema cells derived from opposite sides of the limb induces outgrowth and pattern formation. Hence, this novel and convenient stepwise model allows for the discovery of necessary and sufficient signals and conditions that control blastema formation, growth, and pattern formation during limb regeneration.

The expression of Hox complex genes in correct spatial and temporal order is critical to patterning of the body axis and limbs during embryonic development. In order to understand the role such genes play in appendage regeneration, we have compared the expression of two 5' Hox complex genes: Hoxb13 and Hoxc10 during development and regeneration of the body axis and the limbs of axolotls. In contrast to higher vertebrates, Hoxb13 is expressed not only in the tip of the developing tail, but also in the distal mesenchyme of developing hind limbs, and at low levels in developing forelimbs. Hoxc10 is expressed as two transcripts during both development and regeneration. The short transcript (Hoxc10S) is expressed in the tip of the developing tail, in developing hind limbs, and at low levels in developing forelimbs. The long transcript (Hoxc10L) is expressed in a similar pattern, with the exception that no expression in developing forelimbs could be detected. Hoxb13 and both transcripts of Hoxc10 are expressed at high levels in the regenerating spinal cord during tail regeneration, and in both regenerating hind limbs and forelimbs. The up-regulation of expression of these genes during forelimb regeneration, relative to the very low levels of expression during forelimb development, suggests that they play a critical and perhaps unique role in regeneration. This is particularly true for Hoxc10L, which is not expressed during forelimb development, but is expressed during forelimb regeneration; thus making it the first truly "regeneration-specific" gene transcript identified to date.

Axolotls, with their extensive abilities to regenerate as adults, provide a useful model in which to study the mechanisms of regeneration in a vertebrate, in hopes of understanding why other vertebrates cannot regenerate. Although the expression of many genes has been described in regeneration, techniques for functional analysis have so far been limited. In this paper we demonstrate a new method for efficient overexpression of foreign genes in axolotls. Using vaccinia virus expressing beta-galactosidase microinjected into regenerating limbs, we show that vaccinia can infect both dividing and nondividing limb cells. The site of infection remains discrete and there is no secondary spread of infection to nearby cells. beta-Gal is expressed at high levels in blastema cells for about a week and in differentiated cells for longer. Blastemas that have been injected with vaccinia at different stages regenerate normally. As a test of the utility of vaccinia for functional analysis in regeneration, we constructed a virus expressing Shh and injected it into the anterior of regenerating limbs. Ectopic Shh expression caused extra digits, carpals, and tarsals in the hands and feet of regenerating limbs, suggesting that despite differences in the timing of expression and the eventual pattern, the function of Shh appears to be similar to that in the developing limbs of other vertebrates. Our results demonstrate that vaccinia virus is an excellent vector for ectopically expressing genes for secreted proteins and is a useful tool to study the function of signaling molecules during the process of regeneration in urodeles.

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.

Hox genes play a critical role in the development of the vertebrate axis and limbs, and previous studies have implicated them in the specification of positional identity, the control of growth, and the timing of differentiation. Axolotl limbs offer an opportunity to distinguish these alternatives because the sequence of skeletal differentiation is reversed along the anterior-posterior axis relative to that of other tetrapods. We report that during early limb development, expression patterns of HoxD genes in axolotls resemble those in amniotes and anuran amphibians. At later stages, the anterior boundary of Hoxd-11 expression is conserved with respect to morphological landmarks, but there is no anterior-distal expansion of the posterior domain of Hoxd-11 expression similar to that observed in mice and chicks. Since axolotls do not form an expanded paddle-like handplate prior to digit differentiation, we suggest that anterior expansion of expression in higher vertebrates is linked to the formation of the handplate, but is clearly not necessary for digit differentiation. We also show that the 5' HoxD genes are reexpressed during limb regeneration. The change in the expression pattern of Hoxd-11 during the course of regeneration is consistent with the hypothesis that the distal tip of the regenerate is specified first, followed by intercalation of intermediate levels of the pattern. Both Hoxd-8 and Hoxd-10 are expressed in non-regenerating wounds, but Hoxd-11 is specific for regeneration. It is also expressed in the posterior half of nerve-induced supernumerary outgrowths.

The relationship between growth and pattern specification during development remains elusive. Some molecules known to function as growth factors are also potent agents of pattern formation. This raises the possibility that growth factors could act in pattern formation via an effect on the cell cycle. We have tested the significance of the length of the cell cycle for gene expression and pattern formation in developing chick limb buds by locally slowing the cell cycle. When anterior cell cycles are lengthened by reversible inhibition of DNA replication or by other means, some genes characteristic of the posterior polarizing region are expressed, and digit duplication is observed. Conversely, when posterior cell cycles are slowed, expression of some posterior-specific genes is inhibited, but the pattern is normal. These results indicate that control of the length of the cell cycle could play a primary role in pattern formation by influencing the complement of genes expressed in a particular region of the embryo.

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-beta 2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression of shh.

It is well established that Hensen's nodes can induce the formation of supernumerary digits after grafting into the anterior margin of the developing limb bud. The recent finding that distinct mesodermal cell populations are segregated within the node has made it possible to isolate different prospective cell types in an attempt to correlate digit-inducing ability with cell fate. We find that the prospective notochord cells contained within Hensen's node are able to induce supernumerary digits, whereas presumptive somite cells cannot. This early difference in inducing ability persists into later stages of development: epithelial somites are unable to induce while notochord from all lengths of the neuraxis continues to induce. Using probes to retinoic acid receptor-beta 2 and sonic hedgehog (shh) we find no evidence to support the idea that inducing tissues generate extra digits by releasing retinoic acid into adjacent limb tissue but find that the inducing ability of a tissue correlates with its expression of shh.

Removal of specific extracellular matrix (ECM) components has been implicated in the initiation of salamander limb regeneration. Remodeling of the ECM at the distal stump is necessary for the release of cells that eventually contribute to the blastema. Several matrix metalloproteinases (MMPs) have been well characterized as important to various physiological and pathological processes, such as bone remodeling and tumor invasion. The goal of this study is to identify and characterize MMPs that modulate the ECM during appendage regeneration in the axolotl Ambystoma mexicanum. By analyzing axolotl tissue extracts using gelatin-substrate gels, we have identified a 90-kDa gelatinase/collagenase that is upregulated within hours after limb amputation. This gelatinase shows a dramatic elevation in activity during the dedifferentiation and blastema stages. Its activity declines by the palette stage and returns to its basal level by the digit stages. The increase in activity of the 90-kDa gelatinase in response to amputation is independent of the nerve supply and the wound epithelium but these factors affect its subsequent downregulation. In addition to the blastema, the 90-kDa gelatinase can be detected in the stump at least 4 mm proximal to the regenerate. Similar regulation of the 90-kDa gelatinolytic activity is observed during tail regeneration and flank would healing. We suggest that this 90-kDa gelatinase/collagenase may play a role in the initiation and rapid growth phase of axolotl regeneration and wound healing.

The finding that retinoic acid (RA) has major effects on pattern formation in developing chick limbs has led to the conclusion that RA plays a central role in the normal development of the limb. In addition, it has been demonstrated that treatment with exogenous RA leads to changes in expression of homeobox-containing Hox complex genes within the developing limb. This has been used to argue that, since RA activates 5' Hox D complex genes in the same temporal and spatial sequence as in normal development, Hox genes might be regulated by RA during normal limb development. In this study, we further examine the temporal and spatial changes in expression of Hox D13 and RAR-beta 2 transcripts in wing buds in response to local application of RA in vivo. We confirm reports that RAR-beta 2 expression is induced early and locally by RA. However, we find that the effect of RA on Hox D13 gene expression at the distal end of wing buds at stage 25/26 is downregulation of transcript expression. Furthermore, we find that activation of ectopic Hox D13 expression in response to implantation of RA beads along the anterior margin at stage 20/21 is indirect. Finally, the effects of RA exposure on Hox D13 expression appear to directly correlate with effects on the pattern of distal skeletal elements.

The finding that retinoic acid (RA) has major effects on pattern formation in developing chick limbs has led to the conclusion that RA plays a central role in the normal development of the limb. In addition, it has been demonstrated that treatment with exogenous RA leads to changes in expression of homeobox-containing Hox complex genes within the developing limb. This has been used to argue that, since RA activates 5' Hox D complex genes in the same temporal and spatial sequence as in normal development, Hox genes might be regulated by RA during normal limb development. In this study, we further examine the temporal and spatial changes in expression of Hox D13 and RAR-beta 2 transcripts in wing buds in response to local application of RA in vivo. We confirm reports that RAR-beta 2 expression is induced early and locally by RA. However, we find that the effect of RA on Hox D13 gene expression at the distal end of wing buds at stage 25/26 is downregulation of transcript expression. Furthermore, we find that activation of ectopic Hox D13 expression in response to implantation of RA beads along the anterior margin at stage 20/21 is indirect. Finally, the effects of RA exposure on Hox D13 expression appear to directly correlate with effects on the pattern of distal skeletal elements.

The expression domains of genes located at the 5' end of the HoxD (formerly Hox-4) complex appear to correlate with pattern along both the proximal-distal (PrDi) and the anterior-posterior (AP) axes of the developing limb bud, and it has been suggested that the HoxD gene products are involved in the specification of positional information during limb development. The apical ectodermal ridge is required for limb outgrowth and is thought to influence mesodermal cells at the distal end of the limb bud in a region within which patterning events occur. In this paper, we examine the expression of 5' HoxD genes during PrDi pattern regulation in chick wing buds. In limbs undergoing pattern regulation, we demonstrate that the domains of HoxD11 and HoxD13 gene expression are "regenerated" within 24 hr of removal of the distal mesenchyme. In contrast, in limbs which will not form distal structures, HoxD13 expression becomes reduced.

Retinoic acid (RA), a derivative of vitamin A, has remarkable effects on developing and regenerating limbs. These effects include teratogenesis, arising from RA's ability to inhibit growth and pattern formation. They also include pattern duplication, arising as a result of the stimulation of additional growth and pattern formation. In this review we present evidence that the diverse effects of RA are consistent with a singular, underlying explanation. We propose that in all cases exogenously applied RA causes the positional information of pattern formation-competent cells to be reset to a value that is posterior-ventral-proximal with respect to the limb. The diversity of outcomes can be seen as a product of the mode of application of exogenous RA (global versus local) coupled with the unifying concept that growth and pattern formation in both limb development and limb regeneration are controlled by local cell-cell interactions, as formulated in the polar coordinate model. We explore the possibility that the major role of endogenous RA in limb development is in the establishment of the limb field rather than as a diffusible morphogen that specifies graded positional information across the limb as previously proposed. Finally, we interpret the results of the recent finding that RA can turn tail regenerates into limbs, as evidence that intercalary interactions may also be involved in the formation of the primary body axis.

This study describes the temporal pattern of posterior positional identity in mouse limb bud cells. To do this wedges of tissue from the posterior edge of mouse limb buds at various stages (limb stages: Wanek et al., 1989b. J. Exp. Zool. 249, 41-49) were grafted to the anterior edge of a host chick embryo wing bud. Grafts of mouse posterior cells are able to induce the formation of supernumerary digits every time when they are taken from buds from stage 3 through stage 6. At stage 7, the frequency declines and by stage 8 the chick cells no longer respond. The results indicate a change in tissue properties at stage 7, which progresses by stage 8 to the point at which posterior positional identity is no longer detectable by this assay. These temporal changes in this aspect of limb pattern formation can be used as an additional criterion to guide the identification of genes involved in the specification of posterior positional identity.

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.

The effects of exogenous transforming growth factor beta (TGF-beta) on chick limb development in vivo were studied by implanting carriers of TGF-beta 1 into developing wing buds. Agarose beads were soaked in solutions containing TGF-beta 1 and implanted into wing buds at stages 18 to 27. Localized application of TGF-beta 1 to distal regions of the wing bud caused specific skeletal elements in the limb to be reduced or absent. The particular proximal-distal limb element affected depended on the stage at which the bead was implanted. Position of the bead in the anterior-posterior axis also influenced the pattern of affected structures. Experiments in which TGF-beta 1 beads were implanted and then removed at 24- and 48-hr intervals indicate that there are specific periods during which a skeletal element appears to be sensitive to the effects of exogenous TGF-beta 1. In a few cases, beads placed in proximal positions in later staged limbs resulted in formation of ectopic cartilage near the bead. These results suggest that exposure to exogenous TGF-beta 1 in vivo influences the development of skeletal elements in the chick limb in a stage- and position-dependent manner.

We have experimentally tested the similarity of limb pattern-forming mechanisms in urodeles and anurans. To determine whether the mechanisms of limb outgrowth are equivalent, we compared the results of two kinds of reciprocal limb bud grafts between Xenopus and axolotls: contralateral grafts to confront anterior and posterior positions of graft and host, and ipsilateral grafts to align equivalent circumferential positions. Axolotl limb buds grafted to Xenopus hosts are immunologically rejected at a relatively early stage. Prior to rejection, however, experimental (but not control) grafts form supernumerary digits. Xenopus limb buds grafted to axolotl hosts are not rejected within the time frame of the experiment and therefore can be used to test the ability of frog cells to elicit responses from axolotl tissue that are similar to those that are elicited by axolotl tissue itself. When Xenopus buds were grafted to axolotl limb stumps so as to align circumferential positions, the majority of limbs did not form any supernumerary digits. However, in experimental grafts, where anterior and posterior of host and graft were misaligned, supernumerary digits formed at positional discontinuities. These results suggest that Xenopus/axolotl cell interactions result in responses that are similar to axolotl/axolotl cell interactions. Furthermore, axolotl and Xenopus cells can cooperate to build recognizable skeletal elements, despite large differences in cell size and growth rate between the two species. We infer from these results that urodeles and anurans share the same limb pattern-forming mechanisms, including compatible positional signals that allow appropriate localized cellular interactions between the two species. Our results suggest an approach for understanding homology of the tetrapod limb based on experimental cellular interactions.

Using the diploid/triploid cell marker in the axolotl (Ambystoma mexicanum) we have examined the movement of cells from the dermis into the early limb blastema. Cells of dermal origin begin to migrate beneath the wound epithelium at about 5 days postamputation, and by 10 days they are widely distributed across the amputation surface. By 15 days, a dense accumulation of blastema cells is present beneath the apical cap, and these cells are preferentially oriented in a circumferential direction. These results are discussed in relation to previous studies showing that the progeny of dermal cells become widely distributed during regeneration, and that cells of dermal origin are a major source of blastema cells. The results are also discussed in relation to ideas about how growth and patterning of the new appendage occur.

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. © 1976.

Surgically constructed symmetrical double-anterior and double-posterior upper forelimbs of the axolotl were amputated immediately after surgery. Double-anterior limbs either failed to regenerate or formed single digits or spikes. Double-posterior limbs formed symmetrical double-posterior regenerates in 60% of the cases, thus extending the previous finding that the amount of distal transformation in surgically constructed double-half limbs is inversely proportional to the time between grafting and amputation (Tank and Holder, 1978). When these symmetrical regenerates were amputated through the forearm region, all but one formed a symmetrical secondary regenerate. The majority of the secondary regenerates had a larger number of digits than did their corresponding primary regenerates. Reamputation of the secondary regenerates resulted in symmetrical tertiary regenerates, and the majority of these also had a larger number of digits than did their corresponding primary regenerates. The results are compared to those of Slack and Savage (1978a, b) on embryonically derived double-posterior limbs and they are discussed in terms of a formal model for distal transformation (Bryant and Baca, 1978). © 1980.

The capacity for distal transformation of surgically produced symmetrical forearms of the axolotl, Ambystoma mexicanum, was tested. The polar coordinate model for pattern regulation in epimorphic fields states that positional information is a property of cells which reflects their position in the field, specifically, by their location on a radius and a circumference of the field. The model proposes that for complete distal transformation to occur, a complete circumference of positional values must be present at the plane of amputation. Previous experiments have shown that surgically constructed, symmetrical upper arms of axolotls and newts are not capable of regenerating a distally complete pattern. In the present experiment, double-posterior and double-anterior symmetrical forearms were produced by exchanging the anterior half of the right forearm with the posterior portion of the left forearm. The grafts were allowed to heal for 5-35 days, at which time the limb was amputated through the center of the grafted region. Ninety-five percent of the double-anterior (left) forearms and 77% of the double-posterior (right) forearms either did not regenerate or regenerated symmetrical patterns. The regenerated patterns always converged, i.e., lost structures along the line of symmetry. The length of the healing time did not affect the number of elements regenerated. The remaining limbs regenerated asymmetrical structures, either normal, or parts of normal, hands. These results are compared with those of similar experiments previously performed on the upper arms of axolots and newts and the hindlimbs of salamanders and are discussed in light of a model for distal transformation. © 1980.

Numerous experiments have demonstrated that skin has a profound influence on the pattern of limb regeneration in urodeles. In this investigation, the fate during regeneration of marked cells derived from narrow strips of skin inserted into different positions around the limb circumference has been followed. Skin strips were taken from triploid axolotls and transplanted into diploid sibling animals. The distribution of trinucleolate cells was determined at the site of amputation and in the regenerated limb. The results indicate that at the time of amputation marked cells appear to be localized to the graft, whereas in the regenerated marked cells may be found at all proximal-distal levels and at any position around the circumference of the limb. These results are discussed in terms of a possible mechanism for distal outgrowth.

Interactions between the limb stump and the developing regenerate were studied in the limbs of adult newts, Notophthalmus viridescens. Forelimb blastemas at various stages were transplanted to the contralateral forelimb such that the anterior-posterior axes of stump and blastema were opposed. The blastemas were transplanted either from a proximal to distal, distal to proximal, proximal to proximal, or distal to distal level limb stump. The results indicate that at the earliest stage studied the anterior-posterior axis of the blastema is established but is not stable. An interection between the stump and blastema at this early stage results in the production of a variety of limbs intermediate in polarity between the graft and the stump. At all later stages, the original anterior-posterior axis of the blastema can be retained, although under certain grafting conditions the stump can still exert considerable influence over the anterior-posterior organization of the final regenerate. In those circumstances in which the blastema retains its original handedness, the interaction between stump and blastema results in the production of separate anterior and posterior supernumerary regenerates. The results of transplanting proximal blastemas to a distal limb level indicate that the proximal boundary of the blastema has been established by the earliest stage studied, leading to the production of limbs with serially duplicated segments. However, irrespective of the stage of a blastema transplanted from a distal to proximal level, there are no deleted structures in the proximal-distal axis of the resulting limb. From both histological examination of transplanted regenerates and the arrangement of skeletal elements of the resulting limbs, it is postulated that the stump plays an important role in the production of the intercalary regenerate. © 1975.

Using the triploid cell marker, the cellular contribution from graft and stump to the supernumerary limbs which result from controlateral grafts of limb buds and regeneration blastemas in the axolotl has been analyzed. Grafts were made so as to appose anterior and posterior limb positions. Overall, the contribution from graft and stump tissue was found to be approximately equal although the position of the boundary between the two was variable from limb to limb. This result is consistent with models which suggest that intercalary regeneration is the driving force for patterning of the vertebrate limb. In addition, the pattern of cellular contribution to supernumerary limbs was consistently found to be asymmetrical in the dorsal-ventral axis. Hence, posterior limb tissue predominantly contributed cells to the posterior and dorsal part of the supernumerary limb whereas anterior limb tissue predominantly contributed cells to the anterior and ventral part of the supernumerary limb. The reason for this asymmetrical pattern remains unknown, but we suggest that it might result from a directional bias in intercalary regeneration, similar to that observed during intercalation in the proximal-distal axis of the urodele limb. Using the triploid cell marker in conjunction with a black/white pigmentation marker, the relationship between the cellular contribution boundary and the pigmentation boundary in supernumerary limbs has also been analyzed. It has been found that the positions of the two boundaries do not coincide, a result which suggests that the eventual location of pigment cells is not a good indicator of the location of nonpigment cells derived from graft and stump.

Techniques for using the triploid cell marker for studying cell lineage during the development and regeneration of the axolotl limb are described. Triploid animals possess cells with three nucleoli while diploid animals possess cells with two nucleoli. We have developed a technique for isolating the limb dermis as a sheet of cells for whole-mount analysis of cellular ploidy. Whole-mount tissue preparations as well as paraffin-embedded sectioned tissues were stained specifically for nucleoli with bismuth. Cell counts from a number of triploid and diploid dermal preparations show that (1) diploid dermal cells never possess three nucleoli, (2) the frequency of trinucleolate cells in whole-mount triploid dermal preparations is not 100% but varies between animals from 30 to 76%, (3) within a single triploid animal, the frequency of trinucleolate cells in different dermal preparations is constant. These data establish the usefulness of this technique and emphasize the need for appropriate control cell counts when using the triploid cell marker 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.

Using the triploid/diploid cell marker in the axolotl, Ambystoma mexicanum, we have analyzed the extent to which cells derived from the dermis and the skeleton contribute to the regenerating limb blastema. We found that dermal cells contribute 43% of the blastemal cell population whereas cells derived from skeletal tissue contribute only 2%. When compared to the availability of cells at the plane of amputation, dermal cells overcontribute by greater than twofold whereas skeletal cells undercontribute by several-fold. These data correlate with the effects that these two tissues have on the formation of the limb pattern during regeneration; dermis has a dramatic influence on pattern and skeletal tissue has virtually no effect. It is suggested that the fibroblasts present in the dermis and in other parts of the limb form virtually all of the mesodermal tissues in the regenerate with the exception of the muscle.

We investigated the expression of segment polarity genes during the development of overgrowing and duplicating imaginal discs in the lethal overgrowth mutant lethal (2) giant discs (l (2) gd) of Drosophila in order to explore the molecular basis of hyperplasia and axis establishment in imaginal discs. The expression of wingless (wg), detected using an enhancer trap, is initially restricted to a ventral sector of the leg disc, as in wild type, but expands toward the opposite end of the disc during overgrowth. In the third leg disc, the expanding wg expression stripe evolves to a new center of wg expression at the site where a duplicate leg is subsequently formed. Expression of decapentaplegic (dpp) also begins normally in l (2) gd discs, but the dpp expression domain expands into the posterior region of the disc where it enlarges to eventually become the center of dpp expression in the duplicate. In l (2) gd homozygotes that are simultaneously homozygous for wg or dpp mutations the leg discs overgrow but do not duplicate. Thus ectopic wg and dpp expression is associated with and appears to be required for disc duplication. The wing discs of l (2) gd homozygotes also show expansion of the wg and dpp expression domains, but in this disc wg and dpp mutations inhibit overgrowth as well as pattern duplication. Our results raise the possibility that hyperplasia in other mutants and in other systems may be caused by the misexpression of genes involved in the generation of positional information.

Antibodies generated against bombyxin and prothoracicotropic hormone (PTTH) of Bombyx mori and allatotropin, allatostatin, and diuretic hormone (DH) of Manduca sexta react with distinct sets of cells in the central nervous system of Drosophila larvae, pupae, and adults. Brain neurons immunoreactive with antibodies to bombyxin, PTTH, and DH are in strikingly similar positions to their lepidopteran counterparts, indicating that at least some Drosophila neuroendocrine cells are homologous to those of lepidopterans. Allatotropin and allatostatin-immunopositive neurons of Drosophila differ from those of lepidopterans, but many of them are identical with neurons that express the FMRFamide gene. Antibodies to bombyxin, PTTH, allatostatin, and DH also stain axons and axon terminals in the neurohemal part of the ring gland, and all tested antibodies except that against bombyxin show positive reaction in the neurohemal area of the ventral ganglion. Although immunoreactivity with all antibodies is variable during development, the highest levels of staining are found at developmental stages when the neuropeptides would be expected to be functioning. A genetic analysis of neuropeptide expression and function has been initiated by analyzing immunoreactivity in mutants that have prolonged larval life associated with imaginal disc overgrowth. Two of these mutants, dlg and dco, show abnormally strong immunoreactivity for allatotropin during the extended larval period and the former also show increased staining with the PTTH antibody. The reduced ecdysteroid titer and delayed or blocked metamorphosis in the mutants may be a result of altered neuropeptide production, which is probably secondary to the imaginal disc overgrowth.

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.