Lbx1 expression and frog limb development

In order to identify prospective limb muscle cells in a frog, we cloned Lbx1 from the direct developing frog Eleutherodactylus coqui. Like in embryos of the frog Xenopus laevis but unlike in other vertebrates, EcLbx1 is expressed in all trunk somites. Like in embryos of chick, mouse, and zebrafish, cells expressing EcLbx1 are then found in limb buds, consistent with migration of those cells from somites. EcLbx1 is also expressed in the dorsal spinal cord as in other vertebrates.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6

Mechanisms coupling heart function and cardiac morphogenesis can be accessed in lower vertebrate embryos that can survive to swimming tadpole stages on diffused oxygen. Forward genetic screens in Xenopus tropicalis have identified more than 80 mutations affecting diverse developmental processes, including cardiac morphogenesis and function. In the first positional cloning of a mutation in X. tropicalis, we show that non-contractile hearts in muzak (muz) embryos are caused by a premature stop codon in the cardiac myosin heavy chain gene myh6. The mutation deletes the coiled-coil domain responsible for polymerization into thick filaments, severely disrupting the cardiomyocyte cytoskeleton. Despite the lack of contractile activity and absence of a major structural protein, early stages of cardiac morphogenesis including looping and chamber formation are grossly normal. Muz hearts subsequently develop dilated chambers with compressed endocardium and fail to form identifiable cardiac valves and trabeculae.     

Expression of cyclin D1, cyclin D2, and N-myc in embryos of the direct developing frog Eleutherodactylus coqui,with a focus on limbs

Species of frogs that develop directly have removed the tadpole from their ontogeny and form adult structures precociously. To see whether cell cycle regulators could be involved in this altered embryogenesis, we examined the expression of ccnd1, ccnd2, and mycn in embryos of the direct developing frog, Eleutherodactylus coqui. Notable differences compared to embryos of Xenopus laevis, a species with a tadpole, included prominent expression of ccnd2 in the midbrain and ccnd1 in the mandibular neural crest. The former may contribute to the precocious appearance of the adult-type visual system and the latter to the adult-type jaw. Large domains of ccnd2 and mycn presage the early appearance of limb buds, and ccnd1 and mycn are implicated in digit development.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

Identification of genes associated with regenerative success of <Emphasis Type="Italic">Xenopus laevis</Emphasis>hindlimbs

Background  

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.     

Thyroid hormone induces synthesis and accumulation of tropomyosin and myosin heavy chain in limb buds of premetamorphic tadpoles

Myosin heavy chain (MHC) and tropomyosin (Tm) have been isolated from limb muscles of the North American bullfrog, Rana catesbeiana, and injected into rabbits to raise monospecific antibodies. These antibodies were used to study the localization and synthesis of myosin heavy chain and tropomyosin in the limb buds of premetamorphic (stage VI-VII) tadpoles treated with triiodothyronine (T3) to induce metamorphosis. Indirect immunofluorescence localization detects the accumulation of both MHC and Tm in the developing thigh region within 24 h of T3 treatment. During the subsequent 48 h, the accumulation of these proteins is enhanced in the thigh and progresses from thigh to the distal regions of the limb. Quantitative immunochemical determinations indicate that within 24 h of T3 treatment, synthesis of Tm and MHC are increased 23-fold and 6-fold, respectively. Following 5 days of T3 treatment, the synthetic rates of Tm and MHC are 266 and 70 times the control values, respectively. Both methods suggest that Tm is synthesized and accumulated at a greater rate than myosin heavy chain. These observations suggest that T3 promotes the differentiation of muscle in the limb buds of premetamorphic tadpoles and that limb development promoted by T3 in tadpoles is similar to that described during the embryonic development of higher vertebrates.     

Opercular development and ontogenetic re-organization in a direct-developing frog

The direct-developing frog, Eleutherodactylus coqui, has eliminated the tadpole stage from its ontogeny, and lacks many larval characters. We demonstrate that the dermal folds of E. coqui are homologous with the opercular folds of metamorphosing frogs. In both E. coqui and its metamorphic counterparts the opercular folds grow over the developing forelimb before perforating to free the entrapped limb. Opercular perforation in E. coqui occurs even in the absence of the forelimb but shows no signs of thyroid hormone dependence. The condensation of E. coqui development appears due to the excision of the extended larval period of developmental stasis. Analysis of opercular development, when viewed in conjunction with other developmental characters, suggests the ontogenetic period in the ancestral Eleutherodactylus life-history from which the tadpole was likely eliminated. Received: 7 October 1999 / Accepted: 17 January 2000     

Gene expression profiles in Rana pirica tadpoles following exposure to a predation threat

Background

Rana pirica tadpoles show morphological changes in response to a predation threat: larvae of the dragonfly Aeshna nigroflava induce heightened tail depth, whereas larval salamander Hynobius retardatus induce a bulgy morphology with heightened tail depth. Although both predators induce similar tail morphologies, it is possible that there are functional differences between these tail morphs.

Results

Here, we performed a discriminant microarray analysis using Xenopus laevis genome arrays to compare tail tissues of control and predator-exposed tadpoles. We identified 9 genes showing large-scale changes in their expression profile: ELAV-like1, methyltransferase like 7A, dolichyl-phosphate mannosyltransferase, laminin subunit beta-1, gremlin 1, BCL6 corepressor-like 1, and three genes of unknown identity. A further 80 genes showed greater than 5 fold differences in expression after exposure to dragonfly larvae and 81 genes showed altered expression after exposure to larval salamanders. Predation-threat responsive genes were identified by selecting genes that reverted to control levels of expression following removal of the predator. Thirteen genes were induced specifically by dragonfly larvae, nine others were salamander-specific, and sixteen were induced by both. Functional analyses indicated that some of the genes induced by dragonfly larvae caused an increase in laminins necessary for cell adhesion in the extracellular matrix. The higher expression of gremlin 1 and HIF1a genes after exposure to dragonfly larvae indicated an in vivo hypoxic reaction, while down-regulation of syndecan-2 may indicate impairment of angiogenesis. Exposure to larval salamanders caused down-regulation of XCIRP-1, which is known to inhibit expression of adhesion molecules; the tadpoles showed reduced expression of cα(E)-catenin, small muscle protein, dystrophin, and myosin light chain genes.

Conclusion

The connective tissue of tadpoles exposed to larval salamanders may be looser. The differences in gene expression profiles induced by the two predators suggest that there are functional differences between the altered tail tissues of the two groups of tadpoles.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1389-4) contains supplementary material, which is available to authorized users.     

Secondary coverage of the yolk by the body wall in the direct developing frog, Eleutherodactylus coqui: an unusual process for amphibian embryos

 Eleutherodactylus coqui develops directly from a large 3.5-mm egg to a froglet, without an intervening tadpole stage. We have examined the development of the body wall, a structure whose behavior has been altered in this derived development. In an event that is unusual for amphibian embryos, the yolk mass is secondarily surrounded by the body wall, which originates near the embryo’s trunk. The epidermis of the body wall is marked by melanophores, and the rectus abdominis, which will form the ventral musculature, is near its leading edge. As the body wall expands, the epidermis, melanophores, and rectus abdominis all move from the dorsal side to close over the yolk at the ventral midline. The original ectoderm over the yolk undergoes apoptosis, as it is replaced by body wall epidermis. Intact muscles are not required for ventral closure of the body wall, despite their normal presence near the advancing edge. Comparative examination of embryos of Xenopus laevis and Rana pipiens suggests that ventral closure does not occur in species with tadpoles. The expansion of dorsal tissues over the yolk, as illustrated by E. coqui, may have been important in the origin of amniote embryos. Received: 23 April 1998 / Accepted: 28 June 1998     

An invasive frog, Eleutherodactylus coqui, increases new leaf production and leaf litter decomposition rates through nutrient cycling in Hawaii

A frog endemic to Puerto Rico, Eleutherodactylus coqui, invaded Hawaii in the late 1980s, where it can reach densities of 50,000 individuals ha⁻¹. Effects of this introduced insectivore on invertebrate communities and ecosystem processes, such as nutrient cycling, are largely unknown. In two study sites on the Island of Hawaii, we studied the top-down effects of E. coqui on aerial, herbivorous, and leaf litter invertebrates; herbivory, plant growth, and leaf litter decomposition rates; and leaf litter and throughfall chemistry over 6 months. We found that E. coqui reduced all invertebrate communities at one of the two study sites. Across sites, E. coqui lowered herbivory rates, increased NH₄⁺ and P concentrations in throughfall, increased Mg, N, P, and K in decomposing leaf litter, increased new leaf production of Psidium cattleianum, and increased leaf litter decomposition rates of Metrosideros polymorpha. In summary, E. coqui effects on invertebrates differed by site, but E. coqui effects on ecosystem processes were similar across sites. Path analyses suggest that E. coqui increased the number of new P. cattleianum leaves and leaf litter decomposition rates of M. polymorpha by making nutrients more available to plants and microbes rather than through changes in the invertebrate community. Results suggest that E. coqui in Hawaii has the potential to reduce endemic invertebrates and increase nutrient cycling rates, which may confer a competitive advantage to invasive plants in an ecosystem where native species have evolved in nutrient-poor conditions.     

Immunocytochemical localization of two myosins within the same muslce cells in Caenorhabditis elegans.

Nematodes synthesize two major classes of myosin heavy chains. These heavy chains associate to form only homodimeric myosin molecules, and these myosin homodimers are anti-genically different from one another (Schachat, Garcea and Epstein, 1978). The two myosins may be designated unc-54 myosin, since this species is altered in mutants of the unc-54 locus, and non-unc-54 myosin, since this class is not affected in unc-54 mutants. We present here experiments in which specific anti-myosin IgG and anti-unc-54 myosin IgG are used to locate the two myosins within the same body-wall muscle cells of Caenorhabditis elegans. These results are necessary for further evaluation of the possible functions of the two myosin homodimers in the thick filaments of these muscles.Myosin can be localized to all body-wall and pharyngeal muscle cells using anti-myosin antibody. In longitudinal sections of body-wall muscle, the staining with anti-myosin coincides with the birefringence of A bands that contain thick filaments. Anti-unc-54 myosin stains all body-wall A bands uniformly but does not react with the pharynx. This result demonstrates that unc-54 is located exclusively in body-wall muscle cells of the wild-type strain N2. Non-unc-54 myosin is localized with anti-myosin in all body-wall muscle cells of the unc-54 null mutant E190, as expected; however, unc-54 myosin could not be detected by anti-unc-54 myosin antibody in this mutant.Since we can localize unc-54 myosin and non-unc-54 myosin in all body-wall muscle cells of wild-type and E190, respectively, we conclude that the two myosins must be present in the same muscle cells. In addition, since unc-54 myosin is located in all body-wall A bands, at least some sarcomeres must contain both myosins. This conclusion is consistent with the observations of Garcea, Schachat and Epstein (1978) that wild-type and E190 synthesize similar amounts of non-unc-54 myosin. Within the limits of resolution of our methods, unc-54 myosin is distributed throughout body-wall A bands. We conclude, therefore, that the majority of thick filaments within these A bands must contain unc-54 myosin along their entire length. Possible roles for unc-54 and non-unc-54 myosins in the assembly and organization of thick filaments are discussed.