Leaping lopsided: a review of the current hypotheses regarding etiologies of limb malformations in frogs.

Recent progress in the investigation of limb malformations in free-living frogs has underlined the wide range in the types of limb malformations and the apparent spatiotemporal clustering of their occurrence. Here, we review the current understanding of normal and abnormal vertebrate limb development and regeneration and discuss some of the molecular events that may bring about limb malformation. Consideration of the differences between limb development and regeneration in amphibians has led us to the hypothesis that some of the observed limb malformations come about through misdirected regeneration. We report the results of a pilot study that supports this hypothesis. In this study, the distal aspect of the right hindlimb buds of X. laevis tadpoles was amputated at the pre-foot paddle stage. The tadpoles were raised in water from a pond in Minnesota at which 7% of surveyed newly metamorphosed feral frogs had malformations. Six percent (6 of 100) of the right limbs of the tadpoles raised in pond water developed abnormally. One truncated right limb was the only malformation in the control group, which was raised in dechlorinated municipal water. All unamputated limbs developed normally in both groups. Three major factors under consideration for effecting the limb malformations are discussed. These factors include environmental chemicals (primarily agrichemicals), encysted larvae (metacercariae) of trematode parasites, and increased levels of ultraviolet light. Emphasis is placed on the necessary intersection of environmental stressors and developmental events to bring about the specific malformations that are observed in free-living frog populations.

Development of an evolutionarily novel structure: fibroblast growth factor expression in the carapacial ridge of turtle embryos.

The turtle shell, an evolutionarily novel structure, contains a bony exoskeleton that includes a dorsal carapace and a ventral plastron. The development of the carapace is dependent on the carapacial ridge (CR), a bulge in the dorsal flank that contains an ectodermal structure analogous to the apical ectodermal ridge (AER) of the developing limb (Burke. 1989a. J Morphol 199:363-378; Burke. 1989b. Fortschr Zool 35:206-209). Although the CR is thought to mediate the initiation and outgrowth of the carapace, the mechanisms of shell development have not been studied on the molecular level. Here, we present data suggesting that carapace formation is initiated by co-opting genes that had other functions in the ancestral embryo, specifically those of limb outgrowth. However, there is divergence in the signaling repertoire from that involved in limb initiation and outgrowth. In situ hybridizations with antisense riboprobes derived from Trionyx spiniferous fibroblast growth factor-10 (tfgf10) and Trachemys scripta (T. scripta) fibroblast-growth factor 8 (tfgf8) cDNAs were performed on sections of early T. scripta embryos (< 30 days). Expression of tfgf10 was localized to the mesenchyme subjacent to the ectoderm of the CR. In the chick limb bud, FGF10 is known to be expressed in the early limb-forming mesenchyme and is capable of inducing FGF8 in the AER to initiate the outgrowth of the limb bud. Although the expression of tfgf8 was found in the AER of the developing turtle limb, it was not seen in the CR. Thus, the initiation of the carapace is in agreement with FGF10 expression in the CR, but FGF8 does not appear to have a role in mediating early carapace outgrowth.

Manifestation of the limb prepattern: limb development in the absence of sonic hedgehog function.

The secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior-posterior limb polarity. In the Shh(-/-) mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh(-/-) mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior-posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior-posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11-13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh(-/-) mutant buds with relatively normal morphology. We report that the architecture of the Shh(-/-) AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh(-/-) limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh(-/-) forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh(-/-) mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 during craniofacial morphogenesis.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during chicken craniofacial development. cFHF-4 is expressed in the mesenchyme of the frontonasal process, and in the mesenchyme and ectoderm of the mandibular processes. The expression of cFHF-4 and other genes implicated in facial patterning have been analyzed in talpid(2) embryos or in the presence of exogenous retinoic acid. Talpid(2) mutants show abnormal patterns of gene expression, including up-regulation of cFHF-4 in the developing face, which correlate with defects in cartilage formation. By contrast, expression of cFHF-4 in the developing face is strongly downregulated by teratogenic doses of all-trans retinoic acid in a dose-dependent manner. Low levels of retinoic acid that produce distal upper beak truncations do not affect cShh, c-Patched-1, or c-Bmp-2 expression in the face, but downregulate cFHF-4 in the frontonasal process.

Hind limb malformations in free-living northern leopard frogs (Rana pipiens) from Maine, Minnesota, and Vermont suggest multiple etiologies.

BACKGROUND: Reports of malformed frogs have increased throughout the North American continent in recent years. Most of the observed malformations have involved the hind limbs. The goal of this study was to accurately characterize the hind limb malformations in wild frogs as an important step toward understanding the possible etiologies. METHODS: During 1997 and 1998, 182 recently metamorphosed northern leopard frogs (Rana pipiens) were collected from Minnesota, Vermont, and Maine. Malformed hind limbs were present in 157 (86%) of these frogs, which underwent necropsy and radiographic evaluation at the National Wildlife Health Center. These malformations are described in detail and classified into four major categories: (1) no limb (amelia); (2) multiple limbs or limb elements (polymelia, polydactyly, polyphalangy); (3) reduced limb segments or elements (phocomelia, ectromelia, ectrodactyly, and brachydactyly; and (4) distally complete but malformed limb (bone rotations, bridging, skin webbing, and micromelia). RESULTS: Amelia and reduced segments and/or elements were the most common finding. Frogs with bilateral hind limb malformations were not common, and in only eight of these 22 frogs were the malformations symmetrical. Malformations of a given type tended to occur in frogs collected from the same site, but the types of malformations varied widely among all three states, and between study sites within Minnesota. CONCLUSIONS: Clustering of malformation type suggests that developmental events may produce a variety of phenotypes depending on the timing, sequence, and severity of the environmental insult. Hind limb malformations in free-living frogs transcend current mechanistic explanations of tetrapod limb development.

Expression and regulation of chicken fibroblast growth factor homologous factor (FHF)-4 at the base of the developing limbs.

Fibroblast growth factor homologous factors (FHFs) have been implicated in limb and nervous system development. In this paper we describe the expression of the cFHF-4 gene during early chicken development. cFHF-4 is expressed in the paraxial mesoderm, lateral ridge, and, most prominently, in the posterior-dorsal side of the base of each limb bud. The expression pattern of cFHF-4 at the base of the limbs is not altered by tissue grafts containing the zone of polarizing activity (ZPA), by implants of Shh-expressing cells, or by implants of beads containing retinoic acid, nor does it depend on the distal growth of the limb as it is not altered in limb buds that are surgically truncated. In three chicken mutants affecting limb patterning - talpid(2), limbless, and wingless - altered patterns of cFHF-4 expression are correlated with abnormal nerve plexus formation and altered patterns of limb bud innervation. Similarly, ectopic expression of cFHF-4 is correlated with a local induction of limb-like innervation patterns when beads containing FGF-2 are implanted in the flank. In these experiments, both ectopic innervation and ectopic expression of cFHF-4 in the flank were observed regardless of the size of the FGF-2-induced outgrowths. By contrast, ectopic expression of Shh and HoxD13 are seen only in the larger FGF-2-induced outgrowths. Taken together, these data suggest that cFHF-4 regulates or is coregulated with early events related to innervation at the base of the limbs.

Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling.

The developmental mechanisms specifying digital identity have attracted 30 years of intense interest, but still remain poorly understood. Here, through experiments on chick foot development, we show digital identity is not a fixed property of digital primordia. Rather, digital identity is specified by the interdigital mesoderm, demonstrating a patterning function for this tissue before its regression. More posterior interdigits specify more posterior digital identities, and each primordium will develop in accordance with the most posterior cues received. Furthermore, inhibition of interdigital bone morphogenetic protein (BMP) signaling can transform digit identity, suggesting a role for BMPs in this process.

Hedgehog-regulated processing of Gli3 produces an anterior/posterior repressor gradient in the developing vertebrate limb.

Ci/Gli zinc finger proteins mediate the transcriptional effects of Hedgehog protein signals. In Drosophila, Ci action as transcriptional repressor or activator is contingent upon Hedgehog-regulated, PKA-dependent proteolytic processing. We demonstrate that PKA-dependent processing of vertebrate Gli3 in developing limb similarly generates a potent repressor in a manner antagonized by apparent long-range signaling from posteriorly localized Sonic hedgehog protein. The resulting anterior/posterior Gli3 repressor gradient can be perturbed by mutations of Gli3 in human genetic syndromes or by misregulation of Gli3 processing in the chicken mutant talpid2, producing a range of limb patterning malformations. The high relative abundance and potency of Gli3 repressor suggest specialization of Gli3 and its products for negative Hedgehog pathway regulation.

Limbiting outgrowth: BMPs as negative regulators in limb development.

Rapid progress is being made in understanding how integrated signaling pathways direct patterned outgrowth of the vertebrate limb. In contrast, the mechanisms that constrain limb outgrowth, and thus delimit adult morphology, remain poorly understood. Two recent pioneering reports have implicated bone morphogenetic proteins (BMPs) in negatively regulating the function of the apical ectodermal ridge (AER), an inductive structure required for continued proximodistal specification of limb elements. These studies provide the first insights into how the termination of a limb bud signaling center is accomplished, and intriguingly suggest how distinct aspects of limb morphogenesis are regulated.

Constitutive activation of sonic hedgehog signaling in the chicken mutant talpid(2): Shh-independent outgrowth and polarizing activity.

We have examined the developmental properties of the polydactylous chicken mutant, talpid(2). Ptc, Gli1, Bmp2, Hoxd13, and Fgf4 are expressed throughout the anteroposterior axis of the mutant limb bud, despite normal Shh expression. The expression of Gli3, Ihh, and Dhh appears to be normal, suggesting that the Shh signaling pathway is constitutively active in talpid(2) mutants. We show that preaxial talpid(2) limb bud mesoderm has polarizing activity in the absence of detectable Shh mRNA. When the postaxial talpid(2) limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid(2) limb bud (regulate). However, a Shh-expressing region (zone of polarizing activity) does not reform; nevertheless Fgf4 expression in the apical ectodermal ridge is maintained. Such reformed talpid(2) limb buds develop complete talpid(2) limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not regulate, and a truncated anteroposterior deficient limb forms. In talpid(2) limbs, distal outgrowth is independent of Shh and correlates with Fgf4, but not Fgf8, expression by the apical ectodermal ridge. We propose a model for talpid(2) in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand.

The recombinant limb as a model for the study of limb patterning, and its application to muscle development.

The recombinant limb is a model system that has proved fruitful for analyzing epithelial-mesenchymal interactions and understanding the functional properties of the components of the limb bud. Here we present an overview of some of the insights obtained through the use of this technique. Among these are the understanding that fore or hind limb identity is inherent to the limb bud mesoderm, that the apical ectodermal ridge (AER) is a permissive signaling center and that the limb bud ectoderm plays a central role in the control of dorsoventral polarity. Recombinant limb studies have also allowed the identification of the affected tissue component in several limb mutants. More recently this model has been applied to the study of regulation of gene expressions related to patterning. In this report we use recombinant limbs to analyze pattering of the Pax3 expressing limb muscle cell lineage in the early stages of limb development. In recombinant limbs made without the zone of polarizing activity (ZPA), myoblasts appear intermingled with other mesodermal cells at the beginning of the recombinant limb development. Rapidly thereafter, the muscle precursors segregate and organize around the central forming chondrogenic core of the recombinant. Although this segregation is reminiscent of that occurring during normal development, the myoblasts in the recombinant fail to proliferate appropriately and also fail to migrate distally. Consequently, the muscle pattern in the recombinant limb is defective indicating that normal patterning cues are absent. However, recombinant limbs polarized with a ZPA exhibited a larger mass of muscle cells and a more normal morphogenesis, supporting a role for this signaling center in limb muscle development. Finally, we have ruled out host somite contributions to recombinant limbs by grafting chick recombinant limbs to quail hosts. This initial report demonstrates the value of the recombinant limb model system for dissecting the environmental cues required for normal muscle limb patterning.

Expression of chicken fibroblast growth factor homologous factor (FHF)-1 and of differentially spliced isoforms of FHF-2 during development and involvement of FHF-2 in chicken limb development.

Members of the fibroblast growth factor (FGF) family have been identified as signaling molecules in a variety of developmental processes, including important roles in limb bud initiation, growth and patterning. This paper reports the cloning and characterization of the chicken orthologues of fibroblast growth factor homologous factors-1 and -2 (cFHF-1/cFGF-12 and cFHF-2/cFGF-13, respectively). We also describe the identification of a novel, conserved isoform of FHF-2 in chickens and mammals. This isoform arises by alternative splicing of the first exon of the FHF-2 gene and is predicted to encode a polypeptide with a distinct amino-terminus. Whole-mount in situ hybridization reveals restricted domains of expression of cFHF-1 and cFHF-2 in the developing neural tube, peripheral sensory ganglia and limb buds, and shows that the two cFHF-2 transcript isoforms are present in non-overlapping spatial distributions in the neural tube and adjacent structures. In the developing limbs, cFHF-1 is confined to the posterior mesoderm in an area that encompasses the zone of polarizing activity and cFHF-2 is confined to the distal anterior mesoderm in a region that largely overlaps the progress zone. Ectopic cFHF-2 expression is induced adjacent to grafts of cells expressing Sonic Hedgehog and the zone of cFHF-2 expression is expanded in talpid2 embryos. In the absence of the apical ectodermal ridge or in wingless or limbless mutant embryos, expression of cFHF-1 and cFHF-2 is lost from the limb bud. A role for cFHF-2 in the patterning and growth of skeletal elements is implied by the observation that engraftment of developing limb buds with QT6 cells expressing a cFHF-2 isoform that is normally expressed in the limb leads to a variety of morphological defects. Finally, we show that a secreted version of cFHF-2 activates the expression of HoxD13, HoxD11, Fgf-4 and BMP-2 ectopically, consistent with cFHF-2 playing a role in anterior-posterior patterning of the limb.

Fibroblast growth factors as multifunctional signaling factors.

The fibroblast growth factor (FGF) family consists of at least 15 structurally related polypeptide growth factors. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like growth factor, transforming growth factor-beta, bone morphogenetic protein, and vertebrate homologs of Drosophila wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton.

Limb initiation and development is normal in the absence of the mesonephros.

With rapid progress in understanding the genes that control limb development and patterning interest is becoming focused on the factors that permit the emergence of the limb bud. The current hypothesis is that FGF-8 from the mesonephros induces limb initiation. To test this, the inductive interaction between the Wolffian duct and intermediate mesoderm was blocked rostral to the limb field, preventing mesonephric differentiation while maintaining the integrity of the limb field. The experimental outcome was monitored by following expression of cSim1 and Lmx1, molecular markers for the duct and the mesonephros, respectively. Evidence is presented that the intermediate mesoderm undergoes apoptosis when the inductive interaction with the Wolffian duct is blocked. fgf-8 expression was undetectable in the mesonephric area of embryos with confirmed absence of mesonephros; nevertheless, limb buds formed and limb development was normal. The mesonephros in general, and specifically its fgf-8 expression, was shown to be unnecessary for limb initiation and development; the hypothesis linking the mesonephros and limb development is not supported. Further studies of axial influences on limb initiation should now concentrate on medial structures such as Hensen's node and paraxial mesoderm; the alternative that no axial influences are required should also be examined.

The origin and evolution of animal appendages.

Animals have evolved diverse appendages adapted for locomotion, feeding and other functions. The genetics underlying appendage formation are best understood in insects and vertebrates. The expression of the Distal-less (Dll) homeoprotein during arthropod limb outgrowth and of Dll orthologs (Dlx) in fish fin and tetrapod limb buds led us to examine whether expression of this regulatory gene may be a general feature of appendage formation in protostomes and deuterostomes. We find that Dll is expressed along the proximodistal axis of developing polychaete annelid parapodia, onychophoran lobopodia, ascidian ampullae, and even echinoderm tube feet. Dll/Dlx expression in such diverse appendages in these six coelomate phyla could be convergent, but this would have required the independent co-option of Dll/Dlx several times in evolution. It appears more likely that ectodermal Dll/Dlx expression along proximodistal axes originated once in a common ancestor and has been used subsequently to pattern body wall outgrowths in a variety of organisms. We suggest that this pre-Cambrian ancestor of most protostomes and the deuterostomes possessed elements of the genetic machinery for and may have even borne appendages.

Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation.

The apical ectodermal ridge of the vertebrate limb bud lies at the junction of the dorsal and ventral ectoderm and directs patterning of the growing limb. Its formation is directed by the boundary between cells that do and cells that do not express the gene Radical fringe. This is similar to the establishment of the margin cells at the Drosophila wing dorsoventral border by fringe. Radical fringe expression in chick-limb dorsal ectoderm is established in part through repression by Engrailed-1 in the ventral ectoderm.

Morphogenetic potential of the chick leg interdigital mesoderm when diverted from the cell death program.

There is evidence that the interdigital mesoderm may be in an undifferentiated state. For example, under experimental manipulation in vivo it may be diverted from cell death to digit formation. In the present work we wanted to analyze the maximum morphogenetic potential of the interdigital cells. To do this we made recombinant limbs of three types, the first using dissociated-reaggregated leg interdigital mesoderm, the second using the same tissue but without dissociation and the third adding a piece of polarizing region to the dissociated interdigit. In all three the massive cell death of the interdigit failed to occur. The first type of recombinant formed a small nodule of cartilage while the other two formed a well-developed digit. Our data indicate that the maximum morphogenetic potential of the interdigital tissue appears constrained to form digits and that dissociation of the tissue decreased this ability; polarizing region restores the ability of dissociated cell recombinants to form a digit. We also analyzed in these recombinants the expression of a battery of genes implicated in interdigital cell death or in digital morphogenesis. The pattern of expression of each gene analyzed was identical in the three types of recombinant limbs. The expression of Msx1 and Msx2 genes was maintained under the ridge indicating a good interaction between the interdigital cells, both dissociated and undissociated, and the apical ridge. The expression of Hoxd-12, Hoxd-13 and Hoxa-13 genes was maintained in the recombinants, indicating that these cells carry information about their autopodial origin, and this correlates well with their distal restricted morphogenetic potential. Finally, the patterns of expression of the Bmp-2, Bmp-4 and Bmp-7 genes indicated that they are independently regulated in the recombinants and that Bmp-4 and Bmp-7 have wider expression domains than the areas of cell death that were only detected under the regressing apical ridge during day 3 of the experiment.

Surgical removal of limb bud Sonic hedgehog results in posterior skeletal defects.

Using Sonic Hedgehog (Shh) as a marker for polarizing region cells we have repeated the experiments of MacCabe et al. (1973) and Fallon and Crosby (1975) in an attempt to reexamine the question of a continuous role for the polarizing region during limb development. We report that the earlier experiments probably left Shh-expressing cells after surgery. Our results show that Shh-expressing cells do not regenerate and complete removal of the polarizing region results in truncations along the anteroposterior (A-P) axis; further, A-P patterning cannot be restored when a bead soaked in FGF is implanted in the limb bud mesenchyme to maintain outgrowth after extirpation of the polarizing region. However, in order to reproducibly remove all Shh-positive cells, it is possible that cells with posterior limb skeletal fate also must be removed. Therefore, microsurgical approaches do not permit an unequivocal answer to the question raised in this and the earlier papers and it remains a reasonable possibility that at least up to stage 20-21 the polarizing region plays a continuous role in patterning of the limb bud during its development.

The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions.

We have analyzed the pattern of expression of several genes implicated in limb initiation and outgrowth using limbless chicken embryos. We demonstrate that the expressions of the apical ridge associated genes, Fgf-8, Fgf-4, Bmp-2 and Bmp-4, are undetectable in limbless limb bud ectoderm; however, FGF2 protein is present in the limb bud ectoderm. Shh expression is undetectable in limbless limb bud mesoderm. Nevertheless, limbless limb bud mesoderm shows polarization manifested by the asymmetric expression of Hoxd-11, -12 and -13, Wnt-5a and Bmp-4 genes. The posterior limbless limb bud mesoderm, although not actually expressing Shh, is competent to express it if supplied with exogenous FGF or transplanted to a normal apical ridge environment, providing further evidence of mesodermal asymmetry. Exogenous FGF applied to limbless limb buds permits further growth and determination of recognizable skeletal elements, without the development of an apical ridge. However, the cells competent to express Shh do so at reduced levels; nevertheless, Bmp-2 is then rapidly expressed in the posterior limbless mesoderm. limbless limb buds appear as bi-dorsal structures, as the entire limb bud ectoderm expresses Wnt-7a, a marker for dorsal limb bud ectoderm; the ectoderm fails to express En-1, a marker of ventral ectoderm. As expected, C-Lmx1, which is downstream of Wnt-7a, is expressed in the entire limbless limb bud mesoderm. We conclude that anteroposterior polarity is established in the initial limb bud prior to Shh expression, apical ridge gene expression or dorsal-ventral asymmetry. We propose that the initial pattern of gene expressions in the emergent limb bud is established by axial influences on the limb field. These permit the bud to emerge with asymmetric gene expression before Shh and the apical ridge appear. We report that expression of Fgf-8 by the limb ectoderm is not required for the initiation of the limb bud. The gene expressions in the pre-ridge limb bud mesoderm, as in the limb bud itself, are unstable without stimulation from the apical ridge and the polarizing region (Shh) after budding is initiated. We propose that the defect in limbless limb buds is the lack of a dorsal-ventral interface in the limb bud ectoderm where the apical ridge induction signal would be received and an apical ridge formed. These observations provide evidence for the hypothesis that the dorsal-ventral ectoderm interface is a precondition for apical ridge formation.