Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.     

Activated mutants of SHP-2 preferentially induce elongation of Xenopus animal caps

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Previously, using a dominant negative mutant approach, we showed that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, we now have generated novel, activated mutants of SHP-2. Here, we show that expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. Our results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. Our activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems.     

Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps

Treatment of Xenopus animal pole tissue with activin results in the induction of mesodermal cell types and a dramatic elongation of the tissue. The morphogenetic movements involved in the elongation appear similar to those in normal gastrulation, which is driven by cell rearrangement and cell intercalations. We have used this system to explore the potential regulation of cell-cell adhesion and cadherin function during morphogenesis. Quantitative blastomere aggregation assays revealed that activin induction reduced the calcium-dependent adhesion between blastomeres. Activin-induced blastomeres formed smaller aggregates, and a greater proportion of the population remained as single cells compared to uninduced blastomeres. The aggregation was mediated by C-cadherin because C-cadherin was present in the blastomeres during the aggregation assay, and monoclonal antibodies against C-cadherin inhibited the calcium-dependent aggregation of blastomeres. E-cadherin was not detectable until after the completion of the assay and, therefore, does not explain the adhesive differences between induced and uninduced blastomeres. L cells stably expressing C- cadherin (LC cells) were used to demonstrate that C-cadherin activity was specifically altered after activin induction. Blastomeres induced with activin bound fewer LC cells than uninduced blastomers. L cells not expressing C-cadherin did not adhere to blastomeres. The changes in C-cadherin-mediated adhesion occurred without detectable changes in the steady-state levels of C-cadherin or the amount of C-cadherin present on the surface of the cell. Immunoprecipitation of C-cadherin and its associated catenins revealed that the ratio of C-cadherin and the catenins was not altered by activin induction. These results demonstrate that activin decreases the adhesive function of existing C- cadherin molecules on the surface of blastomeres and suggest that decreased cadherin mediated cell-cell adhesion is associated with increased morphogenetic movement.     

Tissue slices from living root caps as a model system in which to study cytodifferentiation of polar cells

Tissue slices of living root caps of cress (Lepidium sativum L.), two to three cell layers in thickness, were prepared by a microsurgical procedure. The viability, cellular structures and cytoplasmic movement of the cells were examined in the light microscope. Nuclei, amyloplasts, vacuoles and endoplasmic reticulum were identified and their positions confirmed after fixation and observation of the same cells in the electron microscope. The distribution of microtubules was shown by immunocytochemistry. During germination, microtubules appear first at the distal edges of the statocytes, while in mature statocytes a distal domain of criss-crossed microtubules could be distinguished from a proximal domain with transversally oriented microtubules. Microfilaments in young statocytes form a nuclear enclosure; in mature statocytes bundles of microfilaments fan out into the cell cortex. The transition from statocytes to secretion cells is accompanied by a more pronounced cortical network of microfilaments, while the nucleus-associated microfilaments remain visible. It is suggested that these microfilaments play a role in the positioning of the nucleus and the translocation of endoplasmic reticulum.Abbreviations ER endoplasmic reticulum - MF microfilament - MT microtubule     

Mesoderm differentiation in early amphibian embryos depends on the animal cap

Embryos of Ambystoma mexicanum from the late morula to the late blastula stage were dissected and cultivated in varying combinations. The marginal zone (presumptive mesoderm) when isolated together with the vegetal region differentiated to notochord after dissection from early blastulae, but did not differentiate to other tissues. When isolated from middle to late blastulae, in addition myoblasts and mesenchyme were formed. The marginal zone isolated together with the animal region (presumptive ectoderm) differentiated to notochord, muscle, mesenchyme, renal tubules and mesothelium irrespective of the stage of dissection. Combination of isolated animal and vegetal regions did lead to the induction of mesodermal organs. The experiments suggest that further steps in the differentiation of mesodermal organs after the induction of mesoderm by the vegetalizing factor depend on factors from the animal region, which are involved in pattern formation.     

Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.

Spemann's organizer has potent neural inducing and mesoderm dorsalizing activities in the Xenopus gastrula. A third activity, the organizer's ability to induce a secondary gut, has been difficult to analyze experimentally due to the lack of early gene markers. Here we introduce endodermin, a pan-endodermal gene marker, and use it to demonstrate that chordin (Chd), a protein secreted by the organizer region, is able to induce endodermal differentiation in Xenopus. The ability of chd, as well as that of noggin, to induce endoderm in animal cap explants is repressed by the ventralizing factor BMP-4. When FGF signaling is blocked by a dominant-negative FGF receptor in chd-injected animal caps, neural induction is inhibited and most of the explant is induced to become endoderm. The results suggest that proteins secreted by the organizer, acting together with known peptide growth factors, regulate differentiation of the endodermal germ layer.     

"Tissue" transglutaminase in animal development

The "tissue" transglutaminase is a multifunctional enzyme that in its cross-linking configuration catalyzes Ca2+ -dependent reactions resulting in post-translational modification of proteins by establishing epsilon(gamma-glutamyl) lysine cross-links and/or covalent incorporation of biogenic amines (di- and poly-amines and histamine) into proteins. Several laboratories have shown that in Vertebrates, "tissue" transglutaminase (tTG) gene expression specifically characterizes cells undergoing apoptosis or programmed cell death (PCD). The Ca2+ -dependent activation of this enzyme leads to the formation of detergent-insoluble cross-linked protein polymers in cells undergoing PCD. This insoluble protein scaffold could stabilize the integrity of the dying cells before their clearance by phagocytosis, preventing the non-specific release of harmful intracellular components (e.g. lysosomal enzymes, nucleic acids, etc.) and consequently inflammatory responses and scar formation in bystander tissues. In this review we attempt to present an overview of the current knowledge on tTG expression and regulation in animal reproduction and development. The data available so far further strengthen the relationship existing between tTG expression and the induction of PCD.     

Regulation of ectodermal differentiation in Xenopus laevis animal caps treated with TPA and ammonium chloride

Animal caps isolated from Xenopus laevis embryos at the blastula stage were treated sequentially with NH₄Cl, a known cement gland inducer, and with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a known neural inducer. The two artificial inducers were also used in reverse order to see if they can mimic the natural inducers acting during the progressive determination of the ectodermal organ. Immunofluorescence and whole-mount in situ hybridization were used to study the expression of tubulin, taken to indicate an early step on the pathway of cell elongation, and neural cell adhesion molecule (N-CAM) taken to indicate an early step in the determination of the nervous system. The expression of XCG-1, a marker of early specification of the cement gland, was also studied. The results showed that the two artificial inducers can mimic the effects of the natural inducers in animal cap explants. The TPA behaves like a neural inducer, reducing the number and the extension of the cement gland when added to the medium in addition to NH₄Cl, before or after NH₄Cl treatment. In the process of cement gland/neural induction, it is possible to redirect the ectoderm already specified as cement gland to neural tissue, but it does not seem possible to respecify the neural tissue as cement gland. Moreover, the animal caps were also cut into dorsal and ventral parts and the two halves were treated separately. The results were similar to those obtained with treatment of the entire animal cap, suggesting that a dorsal-ventral pattern is not yet established before the gastrula stage, and that in normal embryos there are boundaries between the effects of different inducers.     

Isolation of Xenopus HGF gene promoter and its functional analysis in embryos and animal caps

Previously, we isolated Xenopus HGF (hepatocyte growth factor) cDNA and showed in Xenopus embryos that expression of this gene starts at the late gastrula stage mainly in the ventral mesoderm, and furthermore that the expression is induced in animal cap by activin A and bFGF (basic fibroblast growth factor). Here we have cloned the Xenopus HGF gene, covering a 14 kb 5-upstream region and a 0.2 kb 5-coding region. Within about 0.5 kb of the 5-flanking region, the Xenopus HGF gene contained a TATA-like element AATGAAA, one putative NF-1 binding site, two NF-IL-6 binding motif sequences, one putative TGF--dependent inhibitory element (TIE) and one AP-1 binding site. A recombinant circular plasmid consisting of a 1.7 kb HGF promoter region and the bacterial chloramphenicol acetyltransferase (CAT) gene was first expressed at the late gastrula stage in the ventral mesoderm, as was the endogenous HGF gene. The expression of the fusion gene was induced in animal cap cells by activin A and bFGF although induction by the latter was not so strong. Using a series of 5-deletion constructs introduced into animal caps, silencer elements, which seem to be essential for the gene's regionally correct expression, and the element responsible for induction by activin were found. The results show that the HGF gene promoter isolated here contains elements which may endow the gene with the regulative function for its temporally and spatially regulated expression, although the element necessary for induction by bFGF seems to be missing.     

Calcium efflux from amphibian sciatic nerve

Eight Xenopus laevis were injected intraperitoneally with 45CaCl2 and 16-18 h later an unbranched section from each sciatic nerve was removed. Efflux measurements of nerve from which the perineurial sheath had been removed could be described by three compartments of approximately equal size with half-lifes of 2.37 +/- 0.76 (SD), 30.3 +/- 17.3 and 196 +/- 61 min, the shortest lived compartment representing diffusion from the extracellular space with a coefficient of diffusion of 2.1 +/- 0.7 X 10(-6) cm2/s. Efflux from nerve in which the perineurium remained intact was characterized by a half-life of 862 +/- 399 min resulting from the sheath acting as a diffusion barrier of permeability 3.4 +/- 1.6 X 10(-7) cm/s. The perineurium was found to bind or sequester a quantity of calcium 1-2 times that contained in an equal volume of plasma.     

Bridging the gap from frog research to human therapy: A tale of neural differentiation in Xenopus animal caps and human pluripotent cells

Over the last decade, much progress has been made toward an understanding of the mechanism of regulation of neural differentiation. In this article, following a brief overview of neural induction research, I would like to discuss the potential contribution of basic embryological research to the progress of human therapeutic development in the present and future, focusing on the medical application of in vitro differentiation of neural tissues. This kind of linkage between basic and medical research will probably be strengthened even more by the recent emergence of human induced pluripotent stem cells. Human pluripotent stem cells are powerful tools for bridging the gap from our accumulated knowledge of embryology to regenerative medicine, as well as to a wide spectrum of medical and pharmaceutical research and development. In this commentary, I describe these issues with a particular emphasis on the contributions made by Japanese scientists.     

Tissue digestion for aluminum determination in experimental animal studies

Four different procedures for the determination of aluminum in tissues by atomic absorption spectrometry (AAS) were investigated. They consisted of conventional acid digestion carried out before and after sample drying, associated or not with fat extraction. Drying was carried out in a conventional oven at 65 °C for 24 h. For fat extraction, different solvents and solvent mixtures were investigated considering both extraction yield and sample adequacy for further AAS measurement. Acid digestion was carried out with pure HNO₃ or with its mixture with HClO₄. After digestion, aluminum was measured by graphite furnace atomic absorption spectrometry. Tissues were collected from Al-exposed and nonexposed mice. The results indicated that drying the sample prior to digestion is advantageous as the amount of acid necessary can be significantly reduced. This procedure does not contribute to increase the aluminum level in the samples providing that careful measures to avoid contamination are taken, as the same procedures carried out without taking any precautions to avoid contamination produced imprecise results. Finally, aluminum was not found in the fatty fraction of any sample, even in exposed mice, demonstrating that aluminum does not accumulate in this part of the tissues.     

Methods for animal cell immobilization using electrostatic droplet generation

Summary A technique for the production of small polymer microbeads (less than 150 m diam.) utilizing electrostatic spraying is presented. Video and image analysis were performed to reveal details of the mechanism of droplet formation. Microbeads coated with poly-L-lysine were prepared and found to be effective as microcarriers for culturing surface immobilized insect cells to a high cell density (1.55 × 10⁷ cells/ml microbeads).     

Light-induced inhibitors from intact and cultured caps of Zea roots

Growth inhibitors were assayed from extracts of intact (attached) and of excised (cultured) root caps of Zea mays L., cv. Merit, the roots of which show a positive geotropic response only after exposure to light. If caps are intact at the time of illumination, at least two inhibitory substances are produced, an acid inhibitor and a neutral inhibitor, whereas if caps are detached from roots, placed in culture and then illuminated only the neutral inhibitor is formed. Cycloheximide retards inhibitor production in both intact and cultured caps. When [¹⁴C]mevalonic acid is included in the culture medium and the caps are illuminated, 15–25% of the recoverable ¹⁴C cochromatographs with the neutral inhibitor, whereas in caps cultured in the dark, this radiolabelling pattern is not observed. Cyloheximide in the light reduces the incorporation of ¹⁴C into compounds cochromatographing at the R_f of the neutral inhibitor. It is suggested that the neutral inhibitor may be important in the light-induced bending of roots.Abbreviations ABA abscisic acid - CH cycloheximide     

The plasma-membrance IMP pattern as related to animal/vegetal polarity in the amphibian egg

Freeze-fracture electron microscopy of the plasma membrane of the fertilized, uncleaved Xenopus egg shows that intramembranous particles (IMPs) range in size from ca. 50 to 200 Å and that more IMPs are attached to the E-face than to the P-face. The overall IMP densities of the animal and the vegetal hemisphere do not differ significantly. IMP-free regions (?, ca. 0.1 μm) on the tips of surface protrusions were irregularly distributed in the animal and the vegetal half (E-face) occupying ca. 8.5 and 2%, respectively of the free area. The relative densities for 16 different IMP sizes have been compared, on the basis of seven animal and seven vegetal halves, counting (E-faces only) ca. 10,000 IMPs in each hemisphere. For IMP sizes of ≤81 Å, a significant difference (P < 0.0005) was found, more small IMPs being present in the animal half. Some evidence for IMP-associated thin elements was found. These findings are discussed in relation to plasma membrane anisotropy and the morphogenetic role of the egg cortex.     

Tissue engineering: advances in in vitro cartilage generation

Damaged or diseased articular cartilage frequently leads to progressive debilitation resulting in a marked decrease in the quality of life. Tissue engineering, a budding field in modern biomedical sciences, promises creation of viable substitutes for failing organs or tissues. It represents the amalgamation of rapid developments in cellular and molecular biology on the one hand and material, chemical and mechanical engineering on the other. Current tissue engineering approaches are mainly focused on the restoration of pathologically altered tissue structure based on the transplantation of cells in combination with supportive matrices and biomolecules. The ability to manipulate and reconstitute tissue structure and function in vitro has tremendous clinical implications and is likely to have a key role in cell and gene therapies in coming years.     

Tissue strategies as developmental constraints: Implications for animal evolution

A consideration of developmental constraints at the tissue level brings into focus the relationship between genes, cell behavior and morphological evolution. This common framework provides a rationale for phenomena as seemingly divergent as the lack of homeotic appendages in humans and the Cambrian explosion.     

Underestimated ranges and overlooked refuges from amphibian chytridiomycosis

Aim

Accurately documenting and predicting declines and shifts in species’ distributions is fundamental for implementing effective conservation strategies and directing future research; species distribution models (SDM) have become a powerful tool for such work. Nevertheless, much of the data used to create these models are opportunistic and often violate some of their basic assumptions. We use amphibian declines and extinctions linked to the fungus Batrachochytrium dendrobatidis (Bd) to examine how sampling biases in data collection can affect what we know of this disease and its effect on amphibians in the wild.

Location

Queensland, Australia.

Methods

We developed a distribution model for Bd incorporating known locality records for Bd and a subset of climatic variables that should correctly characterize its distribution. We tested this (original) model with additional surveys, recorded new Bd observations in novel environments and reran the distribution model. We then investigated the difference between the original and new models, and used frog abundance and infection status data from two of these new localities to look at the susceptibility of the torrent frog Litoria nannotis to chytridiomycosis.

Results

While largely correct, the original SDM underestimated the distribution of Bd; sampling in ‘unsuitable’ drier environments discovered abundant populations of susceptible frogs with pathogen prevalences of up to 100%. The validation surveys further uncovered a new population of the frog Litoria lorica coexisting with the pathogen; this species was previously believed to be an extinct rain forest endemic.

Main conclusion

Our results indicate that SDMs constructed using opportunistically collected data can be biased if species are not at equilibrium with their environment or because environmental gradients have not been adequately sampled. For disease ecology, the better estimations of pathogen distribution may lead to the discovery of new populations persisting at the edge of their range, which has important implications for the conservation of species threatened by chytridiomycosis.