Early Amphibian Embryos Research Articles

The organizer: What it meant, and still means, to developmental biology.

This article is about how the famous organizer experiment has been perceived since it was first published in 1924. The experiment involves the production of a secondary embryo under the influence of a graft of a dorsal lip from an amphibian gastrula to a host embryo. The early experiments of Spemann and his school gave rise to a view that the whole early amphibian embryo was "indifferent" in terms of determination, except for a special region called "the organizer". This was viewed mainly as an agent of neural induction, also having the ability to generate an anteroposterior body pattern. Early biochemical efforts to isolate a factor emitted by the organizer were not successful but culminated in the definition of "neuralizing (N)" and "mesodermalizing (M)" factors present in a wide variety of animal tissues. By the 1950s this view became crystallized as a "two gradient" model involving the N and M factors, which explained the anteroposterior patterning effect. In the 1970s, the phenomenon of mesoderm induction was characterized as a process occurring before the commencement of gastrulation. Reinvestigation of the organizer effect using lineage labels gave rise to a more precise definition of the sequence of events. Since the 1980s, modern research using the tools of molecular biology, combined with microsurgery, has explained most of the processes involved. The organizer graft should now be seen as an experiment which involves multiple interactions: dorsoventral polarization following fertilization, mesoderm induction, the dorsalizing signal responsible for neuralization and dorsoventral patterning of the mesoderm, and additional factors responsible for anteroposterior patterning.

Effects of nitrate on development and thyroid hormone signaling pathway during Bufo gargarizans embryogenesis

Nitrate is known to disrupt the thyroid hormone, which is essential for the metamorphosis of amphibians. However, few studies are focused on the effects of nitrate on the maternal thyroid hormone in early amphibian embryos. We aimed to determine the impact of nitrate on maternal thyroid hormone signaling pathway in Bufo gargarizans embryos. B. gargarizans embryos were exposed to different concentrations of nitrate-nitrogen (NO3-N) for 7 days. High concentration of NO3-N (50, 100, and 200 mg/L) could induce embryonic malformation and influence the development of embryos. In addition, maternal T4 and components of the thyroid hormone (TH) signaling pathway were detected by ELISA and RNA-seq, respectively. The expression levels of mRNA related to thyroid hormone and oxidative stress were affected in the early developing embryos in all NO3-N treatment groups. However, the T4 levels and the spatial expression patterns of type II iodothyronine deiodinase (D2), type III iodothyronine deiodinase (D3), thyroid hormone receptor α (TRα), and thyroid hormone receptor β (TRβ) mRNA were not changed by nitrate. In conclusion, the results of our study highlight the crucial role of the maternal thyroid hormone signaling pathway in normal embryonic development, and the adverse effects of nitrate on the expression levels of mRNA related to thyroid hormone signaling pathway and oxidative stress in B. gargarizans embryos.

Einsteck Transplants.

Einsteck procedure refers to a method whereby the experimenter inserts material into the blastocoel cavity of an early amphibian embryo. This procedure is simpler to perform than other types of grafts, such as Spemann-Mangold, and with practice yields a sizable amount of data suitable for statistical analysis. This protocol for Einsteck transplantation in Xenopus describes the insertion of the gastrula-stage blastopore lip into the blastocoel cavity of a host embryo.

P.2.d.043 - Effect of the inflammatory biomarker C-reactive protein on response to lurasidone treatment in patients with bipolar depression

Nitrate is known to disrupt the thyroid hormone, which is essential for the metamorphosis of amphibians. However, few studies are focused on the effects of nitrate on the maternal thyroid hormone in early amphibian embryos. We aimed to determine the impact of nitrate on maternal thyroid hormone signaling pathway in Bufo gargarizans embryos. B. gargarizans embryos were exposed to different concentrations of nitrate-nitrogen (NO3-N) for 7 days. High concentration of NO3-N (50, 100, and 200 mg/L) could induce embryonic malformation and influence the development of embryos. In addition, maternal T4 and components of the thyroid hormone (TH) signaling pathway were detected by ELISA and RNA-seq, respectively. The expression levels of mRNA related to thyroid hormone and oxidative stress were affected in the early developing embryos in all NO3-N treatment groups. However, the T4 levels and the spatial expression patterns of type II iodothyronine deiodinase (D2), type III iodothyronine deiodinase (D3), thyroid hormone receptor α (TRα), and thyroid hormone receptor β (TRβ) mRNA were not changed by nitrate. In conclusion, the results of our study highlight the crucial role of the maternal thyroid hormone signaling pathway in normal embryonic development, and the adverse effects of nitrate on the expression levels of mRNA related to thyroid hormone signaling pathway and oxidative stress in B. gargarizans embryos.

Forces driving cell sorting in the amphibian embryo.

Adhesion differences are the main driver of cell sorting and related processes such as boundary formation or tissue positioning. In the early amphibian embryo, graded variations in cadherin density and localized expression of adhesion-modulating factors are associated with regional differences in adhesive properties including overall adhesion strength. The role of these differences in embryonic boundary formation has not been studied extensively, but available evidence suggests that adhesion strength differentials are not essential. On the other hand, the inside-out positioning of the germ layers is correlated with adhesion strength, although the biological significance of this effect is unclear. By contrast, the positioning of dorsal mesoderm tissues along the anterior-posterior body axis is essential for axis elongation, but the underlying sorting mechanism is not correlated with adhesion strength, and may rely on specific cell adhesion. Formation of the ectoderm-mesoderm boundary is the best understood sorting related process in the frog embryo. It relies on contact-induced cell repulsion at the tissue interface, driven by Eph-ephrin signaling and paraxial protocadherin-dependent self/non-self recognition.

Sebox regulates mesoderm formation in early amphibian embryos.

Mix/Bix genes are important regulators of mesendoderm formation during vertebrate embryogenesis. Sebox, an additional member of this gene family, has been implicated in endoderm formation during early embryogenesis in zebrafish. However, it remains unclear whether Sebox plays a unique role in early Xenopus embryos. In this study, we provide evidence that Sebox is uniquely required for the formation of mesoderm during early Xenopus embryogenesis. Sebox is dynamically expressed in the involuted mesoderm during gastrulation. It is activated by Nodal/Activin signaling and modulated by zygotic Wnt/β-catenin signaling. Overexpression of Sebox perturbs movements during convergent extension and inhibits the expression of mesodermal, but not endodermal, genes induced by Nodal/Activin signaling. Depletion of Sebox using a specific morpholino increases the expression of noncanonical wnt5a, wnt5b, and wnt11b. Depletion of Sebox also up-regulates the expression of pcdh8.2, a paraxial mesoderm-specific protocadherin, in a Wnt11B-dependent manner. Sebox morphants display reduced development of the head and notochord. Our findings illustrate that Sebox, a unique member of the Mix/Bix gene family, functions downstream of Nodal/Activin signaling and is required for the proper expression of noncanonical Wnt ligands and the normal development of mesoderm in Xenopus.

An evolutionary procedure for inferring MP systems regulation functions of biological networks

Metabolic P systems are a modeling framework for metabolic, regulatory and signaling processes. The key point of MP systems are flux regulation functions, which determine the evolution of a system from a given initial state. This paper presents important improvements to a technique, based on genetic algorithms and multiple linear regression, for inferring regulation functions that reproduce observed behaviors (time series datasets). An accurate analysis of three case studies, namely the mitotic oscillator in early amphibian embryos, the Lodka---Volterra predator-prey model and the chaotic logistic map show that this methodology can provide, from observed data, significant knowledge about the regulation mechanisms underlying biological processes.

From time series to biological network regulations: an evolutionary approach

In this paper we present a new methodology, based on genetic algorithms and multiple linear regression, for discovering regulation mechanisms responsible for observed time series in biological networks. The modeling framework employed is called Metabolic P systems; they are deterministic and time-discrete dynamical systems proposed as an effective alternative to ordinary differential equations for modeling biochemical systems. Our methodology is here successfully applied to the mitotic oscillator in early amphibian embryos. Starting from the time series of substances involved in this system, we are able to reconstruct an MP system reproducing the observed dynamics, where the regulatory components were discovered by our evolutionary methodology. In particular, genetic algorithms are used as a variable selection technique to identify the best representation of any regulation function in terms of some given primitive functions.

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.

Forming and Interpreting Gradients in the Early Xenopus Embryo

The amphibian embryo provides a powerful model system to study morphogen gradients because of the ease with which it is possible to manipulate the early embryo. In particular, it is possible to introduce exogenous sources of morphogen, to follow the progression of the signal, to monitor the cellular response to induction, and to up- or down-regulate molecules that are involved in all aspects of long-range signaling. In this article, I discuss the evidence that gradients exist in the early amphibian embryo, the way in which morphogens might traverse a field of cells, and the way in which different concentrations of morphogens might be interpreted to activate the expression of different genes.

The mechanism and pattern of yolk consumption provide insight into embryonic nutrition inXenopus

Little is known about how metabolism changes during development. For most animal embryos, yolk protein is a principal source of nutrition, particularly of essential amino acids. Within eggs, yolk is stored inside large organelles called yolk platelets (YPs). We have gained insight into embryonic nutrition in the African clawed frog Xenopus laevis by studying YPs. Amphibians follow the ancestral pattern in which all embryonic cells inherit YPs from the egg cytoplasm. These YPs are consumed intracellularly at some point during embryogenesis, but it was not known when, where or how yolk consumption occurs. We have identified the novel yolk protein Seryp by biochemical and mass spectrometric analyses of purified YPs. Within individual YPs, Seryp is degraded to completion earlier than the major yolk proteins, thereby providing a molecular marker for YPs engaged in yolk proteolysis. We demonstrate that yolk proteolysis is a quantal process in which a subset of dormant YPs within embryonic cells are reincorporated into the endocytic system and become terminal degradative compartments. Yolk consumption is amongst the earliest aspects of differentiation. The rate of yolk consumption is also highly tissue specific, suggesting that nutrition in early amphibian embryos is tissue autonomous. But yolk consumption does not appear to be triggered by embryonic cells declining to a critically small size. Frog embryos offer a promising platform for the in vivo analysis of metabolism.

Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure.

Physical forces drive the movement of tissues within the early embryo. Classical and modern approaches have been used to infer and, in rare cases, measure mechanical properties and the location and magnitude of forces within embryos. Elongation of the dorsal axis is a crucial event in early vertebrate development, yet the mechanics of dorsal tissues in driving embryonic elongation that later support neural tube closure and formation of the central nervous system is not known. Among vertebrates, amphibian embryos allow complex physical manipulation of embryonic tissues that are required to measure the mechanical properties of tissues. In this paper, we measure the stiffness of dorsal isolate explants of frog (Xenopus laevis) from gastrulation to neurulation and find dorsal tissues stiffen from less than 20 Pascal (Pa) to over 80 Pa. By iteratively removing tissues from these explants, we find paraxial somitic mesoderm is nearly twice as stiff as either the notochord or neural plate, and at least 10-fold stiffer than the endoderm. Stiffness measurements from explants with reduced fibronectin fibril assembly or disrupted actomyosin contractility suggest that it is the state of the actomyosin cell cortex rather than accumulating fibronectin that controls tissue stiffness in early amphibian embryos.

The use of antibodies to gap junction protein to explore the role of gap junctional communication during development.

Antibodies raised against the major 27 kDa protein electrophoretically eluted from isolated gap junctions and affinity purified against the antigen have been used to explore the role of communication through gap junctions in the early amphibian and mouse embryos. In both species, injection of the antibodies into one cell completely blocks both dye transfer and electrical coupling between cells connected by gap junctions. In the amphibian embryo the generation of a communication-incompetent clone of cells leads to patterning defects in the region derived from the antibody-injected cell. In the mouse embryo, blocking cell-to-cell communication leads to decompaction of the communication-incompetent cells. The possible significance of these findings in relation to development in general and to the organization of the first transporting epithelia to appear during development is discussed.

Discrete solutions to differential equations by metabolic P systems

The relationships existing between metabolic P systems and ODE systems are investigated. Formal results show that every MP system determines a structure, called an MP graph, which results in an ODE system whose solution equals, in the limit, the evolution of any non-cooperative MP system that can be derived from the initial one by means of a systematic procedure. Examples based on the model of a mitotic oscillator in early amphibian embryos, the Lotka–Volterra predator–prey population dynamics, and the Lorenz strange attractor are provided, showing the applicability of the proposed computational approach.

Somatic H1 histone accumulation and germ layer determination in amphibian embryos

The induction of mesoderm and/or endoderm from prospective ectoderm and dorsalization of the marginal zone mesoderm may be linked to inhibition of cell cycling and DNA synthesis in early amphibian embryos. In turn, this may lead to reduction of somatic H1 histone accumulation. A greater number of cell cycles and rounds of DNA synthesis characterizes the induction of neural tissue. This is correlated with an increase of somatic H1 histone accumulation. The number of rounds of DNA replication may regulate the level of H1 histone accumulation and this may have a role in germ layer determination.

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis*1

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis . Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by “relamination” (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus , and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by “relamination” (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.

Immunocytochemistry of the amphibian embryo--from overview to ultrastructure.

Amphibian embryos are standard research objects to study pattern formation and morphogenesis. Due to their external development and robust nature, experimental manipulations such as microinjections or transplantations can be easily performed. However, most immunocytochemical approaches addressing the specific localization of proteins are hampered by the fragility of the large and yolky embryonic cells which render high resolution staining difficult. Immunocytochemical data are therefore often restricted to either overall patterns in whole embryo preparations or to immunofluorescent localization with limited resolution on sections. High resolution or ultrastructural protein localization data are rare and can be achieved only with time consuming procedures. Here, a comparative study of immunocytochemical methods suitable for light and electron microscopy using different kinds of plastic resins is presented. Three main approaches are described: preembedding staining of whole embryos, postembedding staining of ultrathin sections and preembedding staining of vibratome sections. All the procedures are designed to study protein expression in early amphibian embryos en gros as well as en detail and the described techniques are suitable to combine two or three levels of resolution on the very same biological specimen. Examples are presented and advantages and disadvantages of the different protocols are discussed.

Interaction between Wnt and TGF-beta signalling pathways during formation of Spemann's organizer.

Members of the Wnt and TGF-beta superfamilies regulate both cell fate and proliferation during development and tissue maintenance. In the early amphibian embryo, the Wnt and TGF-beta superfamily signalling cascades are required for the establishment of a dorsal signalling centre, Spemann's organizer. Intracellular proteins of both pathways, upon activation, translocate to the nucleus to participate in transcription. Here we show that beta-catenin and Lef1/Tcf, which are downstream components of the Wnt signalling cascade, form a complex with Smad4, an essential mediator of signals initiated by members of the TGF-beta growth factor superfamily. In Xenopus, this interaction directly and synergistically affects expression of the twin (Xtwn) gene during formation of the organizer. This is, to our knowledge, the first demonstration of a physical interaction between TGF-beta and Wnt signalling components in vivo.

The first pure embryonic inducing factor.

This is a personal account of the discovery of the mesoderm-inducing activity of fibroblast growth factors. The background is my work on the dorsalising signal in early amphibian embryos that was done at the imperial Cancer Research Fund in London. I became interested in mesoderm induction because of the embryologic work of Nieuwkoop and the partial purification of a "vegetalising factor" by Tiedemann. Although, initially, we expected inducing factors to be novel substances, it gradually became clear that the impure preparations we were studying had properties in common with growth factors. This opened the way to the testing of candidate factors, resulting in the conclusion that some inducing factors and growth factors,in fact, were the same molecules despite being discovered through different routes and assayed using different methods. After a slow start looking at the molecular biology of fibroblast growth factors in Xenopus, we eventually found that their most interesting functions in early development lay not at the stage of mesoderm induction but, rather, in the anteroposterior patterning of the body, which occurs during gastrulation.