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Before limbs or fins, can be patterned and grow they must be initiated. Initiation of the limb first involves designating a portion of lateral plate mesoderm along the flank as the site of the future limb. Following specification, a myriad of cellular and molecular events interact to generate a bud that will grow and form the limb. The past three decades has provided a wealth of understanding on how those events generate the limb bud and how variations in them result in different limb forms. Comparatively, much less attention has been given to the earliest steps of limb formation and what impacts altering the position and initiation of the limb have had on evolution. Here, we first review the processes and pathways involved in these two phases of limb initiation, as determined from amniote model systems. We then broaden our scope to examine how variation in the limb initiation module has contributed to biological diversity in amniotes. Finally, we review what is known about limb initiation in fish and amphibians, and consider what mechanisms are conserved across vertebrates.

We dissect genetically a gene regulatory network that involves the transcription factors Tbx4, Pitx1 and Isl1 acting cooperatively to establish the hindlimb bud, and identify key differences in the pathways that initiate formation of the hindlimb and forelimb. Using live image analysis of murine limb mesenchyme cells undergoing chondrogenesis in micromass culture, we distinguish a series of changes in cellular behaviours and cohesiveness that are required for chondrogenic precursors to undergo differentiation. Furthermore, we provide evidence that the proximal hindlimb defects observed in Tbx4 mutant mice result from a failure in the early differentiation step of chondroprogenitors into chondrocytes, providing an explanation for the origins of proximally biased limb defects., Summary: Examination of the effects of loss of Tbx4 reveals molecular and cellular mechanisms underlying proximally biased limb defects, which could explain the origins of the congenital limb abnormality, phocomelia.

Retinoic acid (RA) was one of the first molecules in the modern era of experimental embryology to be shown capable of generating profound effects on limb development. In this review, we focus on the earliest events of limb development and specifically on the role of RA in establishing the domain of cells that will go on to form the limb itself. Although there is some consensus on the role of RA during the earliest stages of limb formation, some controversy remains on the mechanism of RA action and the requirement for RA signaling in forming the hindlimb buds.

Anuran tadpoles can regenerate their tails after amputation. However, they occasionally form ectopic limbs instead of the lost tail part after vitamin A treatment. This is regarded as an example of a homeotic transformation. In this phenomenon, the developmental fate of the tail blastema is apparently altered from that of a tail to that of limbs, indicating a realignment of positional information in the blastema. Morphological observations and analyses of the development of skeletal elements during the process suggest that positional information in the blastema is rewritten from tail to trunk specification under the influence of vitamin A, resulting in limb formation. Despite the extensive information gained from morphological observations, a comprehensive understanding of this phenomenon also requires molecular data. We review previous studies related to anuran homeotic transformation. The findings of these studies provide a basis for evaluating major hypotheses and identifying molecular data that should be prioritized in future studies. Finally, we argue that positional information for the tail blastema changes to that for a part of the trunk, leading to homeotic transformations. To suggest this hypothesis, we present published data that favor the rewriting of positional information.

The single anastomosis duodeno-ileostomy (SADI) was introduced to eliminate the complications seen with Roux limb formation. It involves a loop anastomosis rather than traditional Roux-en-Y reconstruction. The procedure has been used as a primary, staged, or revision procedure to treat morbid obesity as well as obesity-related coexisting conditions.

International audience; Current knowledge on limbs development lacks a physical description of the forces leading to formation of the limbs precursors or "buds". Earlier stages of development are driven by large scale morphogenetic movements, such as dipolar vortical flows and mechanical buckling, pulled by rings of cells. It is a natural hypothesis that similar phenomena occur during limb formation. However it is difficult to experiment on the developmental forces, in such a complex dynamic system. Here, we report a physical study of hindlimb bud formation in the chicken embryo. We use electrical stimulation to enhance the physical forces present in the tissue, prior to limb bud formation. By triggering the physical forces in a rapid and amplified pattern, we reveal the mechanism of formation of the hindlimbs : the early presumptive embryonic territory is composed of a set of rings encased like russian-dolls. Each ring constricts in an excitable pattern of force, and the limb buds are generated by folding at a pre-existing boundary between two rings, forming the dorsal and ventral ectoderms. The amniotic sac buckles at another boundary. Physiologically, the actuator of the excitable force is the tail bud pushing posteriorly along the median axis. The developmental dynamics suggests how animals may evolve by modification of the magnitude of these forces, within a common broken symmetry. On a practical level, localized electrical stimulation of morphogenetic forces opens the way to in vivo electrical engineering of tissues.

Direct-developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct-developing frogs, but little attention has been given to direct-developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct-developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior-to-posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote-like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct-developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct-developing salamander species.

The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in TBX5, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor Tbx5 in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of Tbx5 produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In Tbx hypomorphic mutants maintained on an INV mutant background, with situs inversus, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of Tbx5 are required to overcome this asymmetry to ensure symmetric forelimb formation., Author Summary Externally, the human form appears bilaterally symmetric. For example, each of our pairs of arms and legs are the same length. This external symmetry masks many asymmetries found in internal organs. In most people the heart is found on the left side of the chest. The stomach, liver and spleen are also positioned asymmetrically. The authors of this study demonstrate, using a mouse model, that bilateral symmetry of the arms is not a default, passive state but that mechanisms are in place that ensure symmetrical formation of the left and right limbs. Bilateral symmetry of the arms is achieved by the action of a gene Tbx5 that masks the effects of signals that acted earlier during embryogenesis, many days before limb formation, and imposed asymmetries on the forming internal organs. Maintaining bilateral symmetry of the arms is important for them to carry out their normal functions but this process can go wrong. Holt-Oram syndrome patients have upper limb defects, including shortened arms. Consistently the defects are more severe in their left arm than right. This birth defect is caused by disruption of the TBX5 gene. By linking the action of Tbx5 to symmetrical limb formation, the authors provide an explanation for why Holt-Oram syndrome patients have more severe defects in the left arms than right.

SOST is a negative regulator of bone formation, and mutations in human SOST are responsible for sclerosteosis. In addition to high bone mass, sclerosteosis patients occasionally display hand defects, suggesting that SOST may function embryonically. Here we report that overexpression of SOST leads to loss of posterior structures of the zeugopod and autopod by perturbing anterior–posterior and proximal–distal signaling centers in the developing limb. Mutant mice that overexpress SOST in combination with Grem1 and Lrp6 mutations display more severe limb defects than single mutants alone, while Sost−/− significantly rescues the Lrp6−/− skeletal phenotype, signifying that SOST gain-of-function impairs limb patterning by inhibiting the WNT signaling through LRP5/6.

Embryogenesis of the upper limb occurs between 4 and 8 weeks after fertilization. Limb development is controlled by signaling centers that guide spatial axes formation and genes that encode limb arrangement. This article will discuss the signaling centers, genes, and molecular events that yield limb formation.

An increased understanding of embryogenesis has advanced our fundamental knowledge of limb anomalies. Animal models with similar limb patterning have been used to dissect and manipulate crucial signaling centers that affect limb development and orientation. Experimental embryologists can produce limb anomalies that are similar to the human phenotype encountered in clinical practice. The evaluating physician must possess a basic comprehension of embryogenesis and limb formation to comprehend congenital limb anomalies and to communicate relevant knowledge to the family. This Current Concepts article is intended to provide an update of limb development that is germane to the clinical scenario.

The paper considers conditions sufficient for the existence of classical C solutions to a new model of chondrogenesis during the vertebrate limb formation. We assume that the diffusion coefficient of the fibronectin is positive and that the function describing the interaction between the fibronectin and cells satisfies some additional properties. Copyright © 2008 John Wiley & Sons, Ltd.

Seventy trees from seven stands 50–650 years old were selected for this investigation of crown structural development in Pseudotsuga menziesii. All branches, limbs, and trunks were nondestructively measured for size, structure, and location while climbing the trees with ropes. These data were used to generate a computer model of each tree's crown that was error-checked trigonometrically. Leaves, bark, cambium, and wood were quantified by using limited destructive sampling to develop predictive equations that were applied to the complete inventory of structures in each tree's crown. Summations of these values yielded whole-tree estimates of several structural variables. A second set of equations was then developed to predict these whole-tree parameters from simple, ground-based measurements. Principal components analysis of 24 tree-level variables revealed two orthogonal dimensions of structure that accounted for 71.3% and 12.4% of total variation in the 70 trees. The first dimension represented a gradient...

Diffusing morphogens in cooperation can control gene expression in developing limbs. Additive cooperation corresponds to the Boolean operator OR and implies the equivalent action of the (suitably scaled) concentrations of two morphogens, either by their alternative binding to the same receptor or by another way of convergence of their effects during the signal transduction procedure. This cooperation can explain the spatial and temporal collinearities of the expression of hoxd genes in the vertebrate limb bud. A multiplicative cooperation of morphogens (corresponding to the Boolean operator AND), produced at the DPP and WG domains in the Drosophila leg imaginal disc, may account for the expression domains observed for Dll and dac . A molecular interpretation of the multiplicative morphogen cooperation is proposed. Some experiments are suggested for further testing of the model.

The homeotic genes regulate segmental identity by repressing the formation of or influencing the morphology of segmental structures during development. The specific roles these genes have played in the generation of insect diversity are discussed relative to abdominal limb formation and to the evolution of wings as novel repeated structures. Other recent findings discussed suggest possible 'homeotic' origins for the non-homeotic genes found within the homeotic complexes of higher insects.

Among the objectives in writing the current chapter were the curiosity and the interest in allocating the sites and routes of migration of the reservoirs of the mesenchymal precartilaginous stem cells of the developing limbs in health and in disease. We chose to emphasize the events believed to initiate in these regions of stem cells, which may lead to growth retardation disorders. Thus, this narrow niche touches an enlarged scope of developmental biology angles and fields. The enclosed coverage sheds light on part of the musculoskeletal system, skeletogenesis, organogenesis of mobile structures and organs, the limbs, joints and digits (arthrology). It appears that the key role of the cartilage-bone regions is their responsibility to replenish the physis with committed chondrocytes, during the developmental, maturation and puberty periods. We shall start by outlining the framework of normal limb formation, the modalities, signals and the agents participating in this biological creation and regulation, illustrating potential sites that might deviate from normal development during the growth periods.

Harrison (1918: J. Exp. Zool. 25: 413–461) described a developmental field as an “equipotential self-differentiating system.” The present study was undertaken to address the question: To what extent can the pre-limb territory of a chick embryo be considered a developmental field? To what extent is the chick pre-limb territory an equipotential self-differentiating system? Two sets of experiments were undertaken to address these questions: (1) Whole and half limb territories were explanted to the celoma of host embryos, and (2) portions of the wing territories were extirpated. The wing exhibited the quality of self-differentiation after stage 12, in that the isolated wing territory, grafted to a host celom, could form limbs beginning at stage 12 (however, complete wings formed only from wing territories of stage 16 and older). On the other hand, the chick wing territory did not appear to exhibit equipotentiality. No posterior half limb graft formed normal limbs, and only in two exceptional cases did anterior half limb grafts form limbs. If part or all of the wing territory was removed from chick embryos, normal limbs formed in less than 15% of the cases after stage 15, in about 30% of the cases at stages 13 and 14, but in over half the cases at stages 10–12. Wound healing and reintiation of limb potential may be responsible for the higher incidence of limb formation at the younger ages. © 1993 Wiley-Liss, Inc.

Limb outgrowth is initiated and maintained by the secretion of FGFs in the apical ectodermal ridge (AER), and patterning regulated by SHH emanating from the posteriorly positioned zone of polarizing activity (ZPA). These factors are tightly linked during progressive limb formation by the SHH-FGF loop. Distal limb bud amputation (removing AER and ZPA) followed by application of FGF to the posterior (but not anterior) stump reactivates SHH expression and regeneration of the distal limb structures. We wondered whether SHH was sufficient to induce regenerative capacity and FGF-responsiveness in limb mesoderm. To determine the role of SHH in limb regeneration, we amputated developing wings (HH 23) and ectopically expressed SHH by confined microeclectroporation (CMEP) in the anterior and posterior stump margins in the presence or absence of FGF soaked beads. Also we ectopically expressed SHH in the anterior margin at HH 19 by CMEP, then applied FGF soaked beads to the anterior margin 24 h later (HH 23-24). Chic...

We have examined the distribution of transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), and the chicken EGF receptor (c-erbB), in embryonic chick limbs. Prior to limb budding, TGF-alpha is present in prospective limb-forming mesoderm and in prospective apical ectodermal ridge (AER)-forming ectoderm, but is not detected in non-limb-forming flank mesoderm or ectoderm, nor in presumptive non-AER-forming limb ectoderm, suggesting possible roles in initial limb formation and AER induction. Consistent with this possibility, TGF-alpha is present in the mesoderm of the wing buds of the amelic chick mutants limbless and wingless, which form and bud normally, but is absent from limbless and wingless ectoderm, which fails to form an AER. TGF-alpha and EGF are present in the AER of the developing limb, and TGF-alpha, EGF, and c-erbB are present in the underlying subridge mesoderm, suggesting possible roles in reciprocal AER/subridge mesoderm interactions required for limb outgrowth. We found that exogenous TGF-alpha and EGF can promote the outgrowth of limb mesoderm in the absence of the AER in vitro and can also promote the outgrowth of limbless and wingless wing bud explants. EGF is present in ventral but not dorsal limb ectoderm, suggesting a role for EGF in specification of ventral ectoderm. TGF-alpha and EGF are not detected in the differentiating cartilaginous elements or muscle primordia of the limb, suggesting that cessation of TGF-alpha and EGF expression may be required for cartilage and muscle formation. We have found that exogenous TGF-alpha and EGF inhibit chondrogenesis and myogenesis of limb mesenchyme in vitro. Together these results indicate that signaling through the EGF receptor via endogenous TGF-alpha and EGF may be important for initial limb formation, AER induction, outgrowth of limb mesoderm, and regulation of limb chondrogenic and myogenic differentiation.

In both Drosophila wings and vertebrate limbs, signaling between dorsal and ventral cells establishes an organizer that promotes limb formation. Significant progress has been made recently towards characterizing the signaling interactions that occur at the dorsal—ventral limb border. Studies of chicks have indicated that, as in Drosophila, this signaling process requires the participation of Fringe. Studies of Drosophila have indicated that Fringe functions by inhibiting the ability of Notch to be activated by one ligand, Serrate, while potentiating the ability of Notch to be activated by another ligand, Delta. Recent studies of both Drosophila and vertebrates have also shed new light on the signaling activity of the dorsal—ventral boundary limb organizer, and have highlighted how this organizer is maintained by feedback mechanisms with neighboring cells.

An examination has been made of the supernumerary limbs produced as a result of tissue interaction following ipsilateral rotation of blastemas at the stage of early digits. The frequency of supernumerary limb formation peaks sharply at angles of rotation near 180°, where tissues from widely separated circumferential locations are opposed. This behavior is compatible with predictions of the polar coordinate model with positional values clustered in the anteriorventral quadrant of the circumference (French et al., '76; Bryant and Iten, '76).

The role of the polarizing zone mesoderm in development of supernumerary distal wing parts after 180° rotation of the wing tip was investigated. Postaxial mesoderm with and without polarizing tissue was repositioned preaxially in the wing bud and duplications occurred only when polarizing zone tissue was included. When the polarizing zone was removed and the distal tip of the wing reoriented, no duplication resulted. Similarly when the polarizing zone was removed, the distal tip reoriented and postaxial, nonpolarizing mesoderm introduced to restore the tissue mass of the stump, no twinning occurred. However, with the distal aspect reoriented on a stump from which postaxial, nonpolarizing mesoderm was removed, twinning occurred in 92.9% of the cases. Further, when the polarizing zone was removed, the distal aspect reoriented and a small piece of polarizing tissue returned, twinning resulted in 93.5% of the cases. These results indicate that polarizing zone tissue is required for the twinning that results after 180° rotation of the wing tip.

The interactions between irradiated and unirradiated blastemas and stumps in the newt forelimb were studied. Irradiated right blastemas at the stage of early digits were grafted to unirradiated left stumps and unirradiated left blastemas were grafted to irradiated right stumps. Grafts were oriented with their anterior-posterior axes opposed to that of the stumps. Supernumerary limbs ranging in completeness from one to four digits were found to arise predominantly on the anterior or posterior sides of the host limb. The graft developed well when the blastema was unirradiated and had reversed handedness with respect to the stump. Irradiated grafts developed poorly. On occasions, limbs with two supernumerary structures were found. The results are discussed in terms of the origin of the cells which comprise the supernumerary limbs and their bearing on a recently presented model concerned with pattern specification and regulation in epimorphic fields.

Normally, urodele limb regeneration is nerve-dependent. Reduction in nerve-dependency has been reported for regenerating, transplanted newt limbs (Singer and Mutterperl, '63). Aneurogenic limbs can regenerate without nerves (Yntema, '59). Induction of supernumerary limbs may be obtained from aneurogenic limbs of larval Ambystoma after transplantation orthotopically to innervated larvae and with normal nerve ingrowth to the limb transplant prevented by repeated section of brachial nerves. Of the 13 (of 43) grafts with supernumeraries, nerve counts showed 11 with 0–5; 1 with 5–10; and 1 with 20+ fibers. Orthotopically grafted aneurogenic limbs allowed to become innervated showed 14 supernumeraries in 49 grafts. This supernumerary limb induction is thus not nerve-dependent. Normally, innervated larval Ambystoma limbs grafted orthotopically and heteroplastically regenerated in 17 of 37 cases after repeated section of brachial nerves. Of the 17 regenerates nerve counts showed 4 with 0–5; 5 with 5–10; 7 with 10–19; and 1 with 20+ fibers. Larval limbs heteroplastically transplanted may require very few or no nerves for regeneration.

In experiments designed to test the effect of physical manipulations upon the ability of frog kidney implants to induce supernumerary limbs in Triturus viridescens, kidney tissue from adult Rana pipiens was treated by boiling, lyophilization and homogenization. Normal kidney induces supernumerary growth in over 90% of cases. Boiling at 100°C for ten minutes destroyed the ability of the kidney implant to stimulate supernumerary growth. Lyophilization did not affect the inductive capacity of the implant. Normal percentages of accessory growth were observed after multiple injections of frog kidney homogenates. It was concluded that some chemical agent(s) present in frog kidney is responsible for the inductive effect.

1. In an experimental system of implant-induced supernumerary limb formation in the newt, two inhibitors of limb regeneration, X-rays and beryllium nitrate, were individually applied to either the fro

The possible relationship of homograft and heterograft reactions to stimulation of accessory urodele limb formation was discussed and a model, based upon the concept that implants taken from different organs may differ in their efficacy in initiating a host foreign body response and as such could theoretically deliver quantitatively varied amounts of qualitatively varied cytolytic products to neighboring host limb tissues, was proposed. Experiments, whose results seem to warrant consideration of the factors which play a role in the homograft and heterograft phenomena, were presented. It was suggested that though homograft and heterograft reactions play a role in this kind of post-embryonic "induction," by virtue of their relationship to implant-caused trauma, it is not yet known whether the varied "inductive" capabilities of different organs are directly related to them.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling.

Multiple limbs were obtained from either limb buds or mature limbs. By counting the fibers of the main nerves at homologous levels of both multiple limbs and contralateral single limbs, it was shown that the innervation of each duplicated distal part of the limb was quantitatively similar to the innervation of an equal single limb. Only in the late induced multiple limbs from regenerating mature limbs, the excess of nerve fibers distal to the branching level of the multiple limbs, modified a little if any the fiber amount of the proximal common base. In the early induced multiple limbs from limb buds, a large percentage of the distal fiber excess was found in the nerves of the common base of the limb. These results suggest a possible additional recruitment of neurons only at an early stage of limb formation. They are discussed in connection with results obtained in other vertebrates.

1. The eye extirpation experiment ofDuRKEN (1913) has been repeated over a period of years with larvae ofRana sylvatica, and conflicting results have been obtained. 2. In the experiments herein reported, larvae have been reared under varied conditions of illumination and of temperature. The few defective cases obtained (0.7% of the whole) were distributed equally between the operated individuals and the controls, and were independent of the influences of darkness or of light, and of varying temperatures. 3. The results of these experiments strongly indicate that there is no causal relationship between the early removal of an eye and the later development of the limbs. 4. The occurrence of cases with defective limbs among museum specimens and among material collected in the field suggests that abnormal limb formation may be due to an instability of the individual.

FROG kidney adenocarcinoma implants and normal adult frog kidney implants made subcutaneously on the dorsal radioulnar surface of non-regenerating larval salamander forelimbs cause the host limbs to respond to their presence by forming supernumerary hand-wrist structures. Larval host limbs may on occasion fail to elicit any morphological response1. On the other hand, adult salamander host limbs may form: (a) an accessory structure comparable with the larval response; (b) nothing; (c) single or clustered subcutaneous cartilaginous nodules as in Fig. 1 2,3. Although these nodules are commonly found in adult host limbs, they had never been produced by larvae in hundreds of cases assayed.

This book, originally published in 1957, is now in its fourth edition. It was then the first textbook of pediatric orthopedic surgery in English, and for some years it remained unchallenged, although now two other texts, those of Sharrard and Tachdjian, are worthy competitors. The present edition represents major changes over the last in that ten contributing authors have been enlisted, and substantial additions have been made in several portions, eg, developmental anatomy, prosthetics, treatment of scoliosis, and pictorialization of operative techniques. The original organization of the book is maintained: it is primarily regional, with chapters on the foot, the knee, the spine, etc. There are six such anatomical divisions. Six other chapters treat muscular affections, neurological disease, congenital diseases, tumors, infections, and metabolic diseases. The three remaining chapters—one on orthopedic treatment, including consideration of developmental anatomy, disturbances in limb formation, etc; one on the epiphyseal, metaphyseal and diaphyseal affections