Amphibian Embryos Research Articles

Uncorking gastrulation: the morphogenetic movement of bottle cells

Bottle cell-driven blastopore lip formation externally marks the initiation of gastrulation in amphibian embryos. The blastopore groove is formed when bottle cells undergo apical constriction and transform from cuboidal to flask-shaped. Apical constriction is sufficient to cause invagination and is a highly conserved mechanism for sheet bending and folding during morphogenesis; therefore, studying apical constriction in Xenopus bottle cells could provide valuable insight into this fundamental shape change. Initially described over a century ago, the dramatic shape change that occurs in bottle cells has long captured the imaginations of embryologists. However, only recently have investigators begun to examine the cellular and molecular mechanisms underlying bottle cell apical constriction. Bottle cell apical constriction is driven by actomyosin contractility as well as by endocytosis of the apical membrane. The Nodal signaling pathway, Wnt5a, and Lgl1 are all required for bottle cell formation, but how they induce subcellular changes resulting in apical constriction remains to be elucidated. Xenopus bottle cells now represent an excellent vertebrate system for the dissection of how molecular inputs can drive cellular outputs, specifically the cell shape change of apical constriction.

Junctophilin 1 and 2 Proteins Interact with the L-type Ca2+ Channel Dihydropyridine Receptors (DHPRs) in Skeletal Muscle

Junctophilins (JPs) anchor the endo/sarcoplasmic reticulum to the plasma membrane, thus contributing to the assembly of junctional membrane complexes in striated muscles and neurons. Recent studies have shown that JPs may be also involved in regulating Ca2+ homeostasis. Here, we report that in skeletal muscle, JP1 and JP2 are part of a complex that, in addition to ryanodine receptor 1 (RyR1), includes caveolin 3 and the dihydropyridine receptor (DHPR). The interaction between JPs and DHPR was mediated by a region encompassing amino acids 230-369 and amino acids 216-399 in JP1 and JP2, respectively. Immunofluorescence studies revealed that the pattern of DHPR and RyR signals in C2C12 cells knocked down for JP1 and JP2 was rather diffused and characterized by smaller puncta in contrast to that observed in control cells. Functional experiments revealed that down-regulation of JPs in differentiated C2C12 cells resulted in a reduction of intramembrane charge movement and the L-type Ca2+ current accompanied by a reduced number of DHPRs at the plasma membrane, whereas there was no substantial alteration in Ca2+ release from the sterol regulatory element-binding protein. Altogether, these results suggest that JP1 and JP2 can facilitate the assembly of DHPR with other proteins of the excitation-contraction coupling machinery.

Sublethal concentrations of azinphos-methyl induce biochemical and morphological alterations in Rhinella arenarum embryos

Considering that amphibians are good sentinels of environmental conditions, Rhinella arenarum embryos were used to investigate the effects of sublethal concentrations of the organophosphorus insecticide azinphos-methyl, focusing on its anticholinesterasic or pro-oxidant actions and its possible connection with the appearance of morphological alterations. Early amphibian embryos exposed to azinphos-methyl displayed a protective response through glutathione S-transferase induction, along with superoxide dismutase inhibition. At intermediate embryonic stages, embryos exposed to azinphos-methyl displayed superoxide dismutase inhibition and morphological alterations, although cholinesterase activity was not altered, suggesting that molecular targets other than cholinesterase were involved in the development of morphological alterations. At the end of embryonic development, decreases in reduced glutathione and cholinesterase inhibition were observed, along with a significant increase in the number of malformed embryos. The connection between biochemical alterations and the appearance of malformations was not evident in R. arenarum embryos. However, increased glutathione S-transferase and decreased superoxide dismutase activities could be considered as early markers of exposure to azinphos-methyl. The results obtained demonstrate that sublethal concentrations of azinphos-methyl are a serious threat to toad embryos in their natural habitats because biochemical and morphological alterations could impair their ability to deal with environmental stresses.

On the diversity of the primary steps of embryonic development in the caudate amphibians

Literary data on the peculiarities of the egg cleavage process in various representatives of the order of caudate amphibians consisting of 10 families have been considered. It has been recognized that in considerable number of species of Plethodontidae, Cryptobranchidae, and some other families, the synchrony of divisions is lost already after the 8-celled stage of the cleavage in large, yolk-rich and unpigmented eggs. A"standard" cleavage of early embryos of caudate amphibians, which had been described in the text-books on developmental biology and consists approximately of 10 synchronous divisions of comparatively small eggs, is characteristic only of the families Ambystomatidae and Salamandridae including 19.3% of species within the order Caudata. However, within each of these families there seems to be a number of species with a "nonstandard" type of early cleavage. The evolutionary relationships between two main types of early embryogenesis within the order Caudata are discussed.

Teratogenic effects of triphenyltin on embryos of amphibian ( Xenopus tropicalis): A phenotypic comparison with the retinoid X and retinoic acid receptor ligands

Triphenyltin (TPT) has high binding affinity with the retinoid X receptor (RXR) in animals. The natural ligand of RXR, 9-cis-retinoic acid (RA), is known to induce featured malformations in vertebrate embryos by disrupting RA signal. Limited information is available on the TPT effects on amphibians. We exposed embryos of amphibian ( Xenopus tropicalis) to TPT, 9-cis-RA, all- trans-RA (ligand of retinoic acid receptor, RAR), and LGD1069 (a selective ligand of RXR). The 72 h LC50 of TPT was 5.25 μg Sn/L, and 72 h EC50 was 0.96 μg Sn/L. TPT induced multiple malformations including enlarged proctodaeum and narrow fins. TPT at 5 μg Sn/L inhibited the differentiation of skins and muscles. The reduced brain, loss of external eyes and bent axis were observed in RXR and RAR ligands treatments. TPT and tributyltin (TBT) inhibited the mRNA expression of RXRα and increased that of TRβ. The phenotypes of malformations induced by TPT were similar to those by TBT and were much different from those by the RXR and RAR ligands. These results indicated that TPT was acute toxic and had high teratogenicity to amphibian embryos, and that TPT induced phenotypes of malformations. TPT and TBT might have a similar teratogenic mechanism, which seems not to be mainly mediated through RA signal.

Nipbl regulates organ laterality and Kupffer's vesicle development in Zebrafish

Nipbl regulates organ laterality and Kupffer's vesicle development in Zebrafish

A Framework for Connecting Gene Expression to Morphogenetic Movements in Embryos

Although much has been learned about genetic networks, cell mechanics, and whole-embryo mechanics through experimental and computational studies, the challenge of connecting these separate bodies of knowledge into an integrated whole remains. Here, we offer a multiscale biochemical-mechanical framework from which such integration might proceed. We identify components of the framework for which quantitative descriptions are currently available, and use the framework to gain insight into convergent extension and gastrulation--crucial tissue movements that occur in early stage amphibian embryos.

Social Immunity in Amphibians: Evidence for Vertical Transmission of Innate Defenses

Amphibian embryos are at risk of microbial infection. Here we find evidence that innate immune defenses, both antimicrobial skin peptides and mutualistic microbiota, of adult glass frogs, Hyalinobatrachium colymbiphyllum, can be transmitted to embryos deposited on leaves above rain forest streams in Panama and can inhibit the fungal pathogen Batrachochytrium dendrobatidis. Abstract in Spanish is available at http://www.blackwell-synergy.com/loi/btp.

The Importance of Perivitelline Fluid Convection to Oxygen Uptake ofPseudophryne bibroniiEggs

The ciliated epithelium of amphibian embryos produces a current within the perivitelline fluid of the egg that is important in the convective transfer of oxygen to the embryo's surface. The effects of convection on oxygen uptake and the immediate oxygen environment of the embryo were investigated in Pseudophryne bibronii. Gelatin was injected into the eggs, setting the perivitelline fluid and preventing convective flow. Oxygen consumption rate (M(.)o₂) and the oxygen partial pressure (Po₂) of the perivitelline fluid were measured in eggs with and without this treatment. M(.)o₂ decreased in eggs without convection at Gosner stages 17-19 under normoxia. The lack of convection also shifted embryos from regulators to conformers as environmental Po₂ decreased. A strong Po₂ gradient formed within the eggs when convection was absent, demonstrating that the loss of convection is equivalent to decreasing the inner radius of the capsule, an important factor in gas exchange, by 25%. M(.)o₂ also declined in stage 26-27 embryos without cilia-driven convection, although not to the extent of younger stages, because of muscular movements and a greater skin surface area in direct contact with the inner capsule wall. This study demonstrates the importance of convective flow within the perivitelline fluid to gas exchange. Convection is especially important in the middle of embryonic development, when the perivitelline space has formed, creating a barrier to gas exchange, but the embryos have yet to develop muscular movements or have a large surface area exposed directly to the jelly capsule.

Gsx transcription factors repress Iroquois gene expression

We have previously shown that the Gsx family homeobox gene Gsh2 is part of the regulatory network specifying dorsoventral pattern of primary neurons in the developing amphibian embryo. Here, we investigate the role of Gsx transcription factors in regulating the transcription of Iroquois family homeobox genes in the amphibian neural plate. Iroquois genes are key regulators of neural patterning and their expression is coincident with that of the Gsx genes during open neural plate stages. We show that Gsx proteins repress Iroquois expression in the embryo and conversely, inhibition of Gsx activity with either antisense morpholino oligos or an anti-morphic Gsx protein up-regulates Iroquois expression. These data indicate that Gsx factors act as negative regulators of Iroquois gene expression in the amphibian neural plate and support a model in which the Gsx proteins promote neuronal differentiation by repressing the expression of known inhibitors of neuronal differentiation such as Iro3.

Realistic Levels of a Fertilizer Impair Iberian Newt Embryonic Development

Abstract: A wide variety of agricultural chemicals with potential to affect amphibian health are released into the environment daily. Most of these chemicals are xenobiotic compounds that are highly toxic to embryos, tadpoles, and terrestrial stages. Other substances that occur in pristine environments at harmless concentrations, such as inorganic nitrogenous compounds, may reach potentially toxic levels as a consequence of certain human activities, including the application of fertilizers. Most of the studies that analyze the effects of inorganic nitrogen on amphibian embryos are conducted with anurans, whereas little information exists regarding urodeles. Embryos of newts and salamanders usually exhibit longer times to hatch than frogs and toads. A longer hatching time results in a longer exposure of embryos to diffuse environmental pollution and therefore a higher risk of suffering toxic effects during the embryonic stage. We demonstrate that ammonium nitrate, a widely used nitrogenous fertilizer, at ...

The RNA-binding protein Xp54nrb isolated from a Ca2+-dependent screen is expressed in neural structures during Xenopus laevis development

In amphibian embryos, calcium (Ca(2+)) signalling is a necessary and sufficient event to induce neural fate. Transient elevations of [Ca(2+)]i are recorded in neural tissue precursor cells in whole embryos during gastrulation. Using a subtractive cDNA library between control ectoderm (animal caps) and ectoderm induced toward a neural fate by Ca(2+) release, we have isolated several Ca(2+)-induced target genes. Among the isolated genes, Xp54nrb encodes a protein which exhibits the RRM domains characteristic of RNA binding proteins, and is implicated in pre-mRNA splicing steps. Here we show that the Xp54nrb transcripts are expressed throughout early developmental stages, specifically in the neural and sensorial territories and that Xp54nrb could be involved in anterior neural patterning.

Between Biochemists and Embryologists – The Biochemical Study of Embryonic Induction in the 1930s

The discovery by Hans Spemann of the "organizer" tissue and its ability to induce the formation of the amphibian embryo's neural tube inspired leading embryologists to attempt to elucidate embryonic inductions' underlying mechanism. Joseph Needham, who during the 1930s conducted research in biochemical embryology, proposed that embryonic induction is mediated by a specific chemical entity embedded in the inducing tissue, surmising that chemical to be a hormone of sterol-like structure. Along with embryologist Conrad H. Waddington, they conducted research aimed at the isolation and functional characterization of the underlying agent. As historians clearly pointed out, embryologists came to question Needham's biochemical approach; he failed to locate the hormone he sought and eventually abandoned his quest. Yet, this study finds that the difficulties he ran into resulted primarily from the limited conditions for conducting his experiments at his institute. In addition, Needham's research reflected the interests of leading biochemists in hormone and cancer research, because it offered novel theoretical models and experimental methods for engaging with the function of the hormones and carcinogens they isolated. Needham and Waddington were deterred neither by the mounting challenges nor by the limited experimental infrastructure. Like their colleagues in hormone and cancer research, they anticipated difficulties in attempting to establish causal links between complex biological phenomena and simple chemical triggering.

The energy cost of embryonic development in fishes and amphibians, with emphasis on new data from the Australian lungfish, Neoceratodus forsteri

The rate of oxygen consumption throughout embryonic development is used to indirectly determine the 'cost' of development, which includes both differentiation and growth. This cost is affected by temperature and the duration of incubation in anamniote fish and amphibian embryos. The influences of temperature on embryonic development rate, respiration rate and energetics were investigated in the Australian lungfish, Neoceratodus forsteri, and compared with published data. Developmental stage and oxygen consumption rate were measured until hatching, upon which wet and dry gut-free masses were determined. A measure of the cost of development, the total oxygen required to produce 1 mg of embryonic dry tissue, increased as temperature decreased. The relationship between the oxygen cost of development (C, ml mg(-1)) and dry hatchling mass (M, mg) in fishes and amphibians is described by C = 0.30 M(0.22 0.13 (95% CI)), r (2) = 0.52. The scaling exponent indicates that the cost of embryonic development increases disproportionally with increasing hatchling mass. At 15 and 20°C, N. forsteri cost of development is significantly lower than the regression mean for all species, and at 25°C is lower than the allometrically scaled data set. Unexpectedly, incubation of N. forsteri is long, despite natural development under relatively warm conditions, and may be related to a large genome size. The low cost of development may be associated with construction of a rather sluggish fish with a low capacity for aerobic metabolism. The metabolic rate is lower in N. forsteri hatchlings than in any other fishes or amphibians at the same temperature, which matches the extremely low aerobic metabolic scope of the juveniles.

Influence of a Combination of Agricultural Chemicals on Embryos of the Endangered Gold-Striped Salamander (Chioglossa lusitanica)

Pollution from agrochemicals may be contributing to the global decline of amphibian populations. Environmentally relevant concentrations of a fertiliser, ammonium nitrate, and a commercial formulation of the herbicide glyphosate Roundup Plus were tested on the embryonic development of Chioglossa lusitanica. This study introduces new data at three different levels. First, we provide previously unknown information about hatchling traits of C. lusitanica. Second, we present the first ecotoxicological study of this endangered species, to which environmental pollution is considered a major threat. Third, we conduct the first experiment with an amphibian species exposed to a mixture of a glyphosate-based herbicide and a nitrogenous fertiliser. Control individuals hatched with an average (±SD) total length of 18.77 (±2.02) mm and at an average Harrison's developmental stage of 44.58 (±1.24). Mean hatching time among controls was 11.52 (±1.29) weeks. None of the chemicals or their interaction produced lethal effects; however, a significant interaction was found when analysing total length at hatching. Individuals exposed to the herbicide hatched at a larger size than controls, and this effect was especially clear when the fertiliser was added to the water. The absence of pollutant-related mortality or severe sublethal effects is in agreement with most studies indicating a high tolerance of amphibian embryos to agrochemicals. However, further research considering other life stages and additional natural factors (i.e., predators, food availability) is needed to estimate the ecological impact of chemical mixtures on C. lusitanica.

The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos

Pax3 and Pax7 paralogous genes have functionally diverged in vertebrate evolution, creating opportunity for a new distribution of roles between the two genes and the evolution of novel functions. Here we focus on the regulation and function of Pax7 in the brain and neural crest of amphibian embryos, which display a different pax7 expression pattern, compared to the other vertebrates already described. Pax7 expression is restricted to the midbrain, hindbrain and anterior spinal cord, and Pax7 activity is important for maintaining the fates of these regions, by restricting otx2 expression anteriorly. In contrast, pax3 displays broader expression along the entire neuraxis and Pax3 function is important for posterior brain patterning without acting on otx2 expression. Moreover, while both genes are essential for neural crest patterning, we show that they do so using two distinct mechanisms: Pax3 acts within the ectoderm which will be induced into neural crest, while Pax7 is essential for the inducing activity of the paraxial mesoderm towards the prospective neural crest.

Zygotic VegT is required for Xenopus paraxial mesoderm formation and is regulated by Nodal signaling and Eomesodermin

The T-box gene VegT plays a crucial role during mesendoderm specification of the amphibian embryo. While the function of maternal VegT (mVegT) has been extensively investigated, little is known about the function and transcriptional regulation of zygotic VegT (zVegT). In the present study, we used comparative genomics and a knockdown experiment to demonstrate that zVegT is the orthologous gene of zebrafish Spadetail/Tbx16 and chick Tbx6L/Tbx6, and has an essential role in paraxial mesodermal formation. zVegT knockdown embryos show several defects in the patterning of trunk mesoderm, such as abnormal segmentation of somites, a reduction in muscle, and the formation of an abnormal mass of cells at the tail tip. We also identified the cis-regulatory elements of zVegT that are necessary and sufficient for mesoderm-specific expression. These cis-regulatory elements are located in two separate upstream regions of zVegT, corresponding to the first intron of mVegT. The results of in vitro binding and functional assays indicate that Forkhead box H1 (FoxH1) and Eomesodermin (Eomes) are the trans-acting factors required for zVegT expression. Our results highlight the essential role of zVegT in organization of paraxial mesoderm, and reveal that zVegT is regulated by a coherent feedforward loop of Nodal signaling via Eomes.

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.

The ghost of predation future: threat-sensitive and temporal assessment of risk by embryonic woodfrogs

Amphibians are able to learn to recognize their future predators during their embryonic development (the ghost of predation future). Here, we investigate whether amphibian embryos can also acquire additional information about their future predators, such as the level of threat associated with them and the time of day at which they would be the most dangerous. We exposed woodfrog embryos (Rana sylvatica) to different concentrations of injured tadpole cues paired with the odor of a tiger salamander (Ambystoma tigrinum) between 1500 and 1700 hours for five consecutive days and raised them for 9 days after hatching. First, we showed that embryos exposed to predator odor paired with increasing concentrations of injured cues during their embryonic development subsequently display stronger antipredator responses to the salamander as tadpoles, thereby demonstrating threat-sensitive learning by embryonic amphibians. Second, we showed that the learned responses of tadpoles were stronger when the tadpoles were exposed to salamander odor between 1500 and 1700 hours, the time at which the embryos were exposed to the salamander, than during earlier (1100–1300 hours) or later (1900–2100 hours) periods. Our results highlight the amazing sophistication of learned predator recognition by prey and emphasize the importance of temporal considerations in experiments examining risk assessment by prey.

Extracellular matrix assembly and organization during zebrafish gastrulation

Zebrafish gastrulation entails morphogenetic cell movements that shape the body plan and give rise to an embryo with defined anterior–posterior and dorsal–ventral axes. Regulating these cell movements are diverse signaling pathways and proteins including Wnts, Src-family tyrosine kinases, cadherins, and matrix metalloproteinases. While our knowledge of how these proteins impact cell polarity and migration has advanced considerably in the last decade, almost no data exist regarding the organization of extracellular matrix (ECM) during zebrafish gastrulation. Here, we describe for the first time the assembly of a fibronectin (FN) and laminin containing ECM in the early zebrafish embryo. This matrix was first detected at early gastrulation (65% epiboly) in the form of punctae that localize to tissue boundaries separating germ layers from each other and the underlying yolk cell. Fibrillogenesis increased after mid-gastrulation (80% epiboly) coinciding with the period of planar cell polarity pathway-dependent convergence and extension cell movements. We demonstrate that FN fibrils present beneath deep mesodermal cells are aligned in the direction of membrane protrusion formation. Utilizing antisense morpholino oligonucleotides, we further show that knockdown of FN expression causes a convergence and extension defect. Taken together, our data show that similar to amphibian embryos, the formation of ECM in the zebrafish gastrula is a dynamic process that occurs in parallel to at least a portion of the polarized cell behaviors shaping the embryonic body plan. These results provide a framework for uncovering the interrelationship between ECM structure and cellular processes regulating convergence and extension such as directed migration and mediolateral/radial intercalation.