Embryonic Amphibian Cells Research Articles

An adaptive numerical scheme for a partial integro-differential equation

One method of modelling cell-cell adhesion gives rise to a partial integro-differential equation. While non-adaptive techniques work in the numerical modelling of such an equation, there are also many opportunities for optimisation. The studied partial integro-differential equation has a tendency to produce aggregations leaving large regions where both the function value and derivative are equal to zero, leading to a higher resolution than needed and lower than desired resolution where the aggregations form. In order to overcome this we develop an adaptive scheme in both space and time using a modified form of Matlab's ode45 and finite volume methods to more efficiently simulate the studied partial integro-differential equation. We use our numerical scheme to simulate the problem presented by Armstrong et al. [J. Theor. Biol. 243 (2006), pp. 98--113] and compare results. 
 
 References N. J. Armstrong, K. J. Painter, and J. A. Sherratt. A continuum approach to modelling cell-cell adhesion. J. Theor. Biol., 243:98113, 2006. doi:10.1016/j.jtbi.2006.05.030. R. J. LeVeque. Finite volume methods for hyperbolic problems. Cambridge Texts in Applied Mathematics. Cambridge University Press, 2002. doi:10.1017/CBO9780511791253. C. F. Van Loan. Introduction to scientific computing: A matrix vector approach using MATLAB. MATLAB Curriculum. Prentise Hall, 1997. J. A. Sheratt, S. A. Gourley, N. J. Armstrong, and K. J. Painter. Boundedness of solutions of a non-local reaction-diffusion model for adhesion in cell aggregation and cancer invasion. Eur. J. Appl. Math., 20(1):123144, 2009. doi:10.1017/S0956792508007742. M. S. Steinberg. On the mechanism of tissue reconstruction by dissociated cells, I. Population kinetics, differential adhesiveness and the absence of directed migration. P. Natl Acad. Sci. USA, 48(9):15771582, 1962a. doi:10.1073/pnas.48.9.1577. M. S. Steinberg. Mechanism of tissue reconstruction by dissociated cells, II: Time-course of events. Science, 137(3532):762763, 1962b. doi:10.1126/science.137.3532.762. M. S. Steinberg. On the mechanism of tissue reconstruction by dissociated cells, III. Free energy relations and the reorganisation of fused, heteronomic tissue fragments. P. Natl Acad. Sci. USA, 48(10):17691776, 1962c. doi:10.1073/pnas.48.10.1769. P. L. Townes and J. Holtfreter. Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool., 128(1):53120, 1955. doi:10.1002/jez.1401280105.

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40–60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca2+-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both—indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.

Biophysical Properties of Cadherin Bonds Do Not Predict Cell Sorting

Differential binding between cadherin subtypes is widely believed to mediate cell sorting during embryogenesis. However, a fundamental unanswered question is whether cell sorting is dictated by the biophysical properties of cadherin bonds, or by broader, cadherin-dependent differences in intercellular adhesion or membrane tension. This report describes atomic force microscope measurements of the strengths and dissociation rates of homophilic and heterophilic cadherin (CAD) bonds. Measurements conducted with chicken N-CAD, canine E-CAD, and Xenopus C-CAD demonstrated that all three cadherins cross-react and form multiple, intermolecular bonds. The mechanical and kinetic properties of the heterophilic bonds are similar to the homophilic interactions. The thus quantified bond parameters, together with previously reported adhesion energies were further compared with in vitro cell aggregation and sorting assays, which are thought to mimic in vivo cell sorting. Trends in quantified biophysical properties of the different cadherin bonds do not correlate with sorting outcomes. These results suggest that cell sorting in vivo and in vitro is not governed solely by biophysical differences between cadherin subtypes.

Preferential Adhesion Mediated by Hibris and Roughest Regulates Morphogenesis and Patterning in the Drosophila Eye

Cell adhesion is essential for morphogenesis; however, the mechanisms by which cell adhesion coordinates precisely regulated morphogenesis are poorly understood. Here we analyze the morphogenetic processes that organize the interommatidial precursor cells (IPCs) of the Drosophila pupal eye. We demonstrate that the Drosophila immunoglobulin superfamily members Hibris and Roughest are essential for IPC morphogenesis in the eye. The two loci are expressed in complementary cell types, and Hibris and Roughest proteins bind directly in vivo. Primary pigment cells employ Hibris to function as organizers in this process; IPCs minimize contacts with neighboring IPCs and utilize Roughest to maximize contacts with primaries. In addition, we provide evidence that interactions between Hibris and Roughest promote junction formation and that levels of Roughest in individual cells determine their capacity for competition. Our results demonstrate that preferential adhesion mediated by heterophilic interacting cell-adhesion molecules can create a precise pattern by minimizing surface free energy.

Townes and Holtfreter (1955): Directed movements and selective adhesion of embryonic amphibian cells

Townes and Holtfreter (1955): Directed movements and selective adhesion of embryonic amphibian cells

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells.

The temporal appearance and subcellular distribution of mitsugumin29 (MG29), a 29-kDa transmembrane protein isolated from the triad junction in skeletal muscle, were examined by immunohistochemistry during the development of rabbit skeletal muscle. MG29 appeared in the sarcoplasmic reticulum (SR) in muscle cells at fetal day 15 before the onset of transverse tubule (T tubule) formation. In muscle cells at fetal day 27, in which T tubule and triad formation is ongoing, both SR and triad were labeled for MG29. In muscle cells at newborn 1 day, the labeling of the SR had become weak and the triads were well developed and clearly labeled for MG29. Specific and clear labeling for MG29 was restricted to the triads in adult skeletal muscle cells. When MG29 was expressed in amphibian embryonic cells by injection of the cRNA, a large quantity of tubular smooth-surfaced endoplasmic reticulum (sER) was formed in the cytoplasm. The tubular sER was 20-40 nm in diameter and appeared straight or reticular in shape. The tubular sER was formed by the fusion of coated vesicles [budded off from the rough-surfaced endoplasmic reticulum (rER)] and vacuoles of rER origin. The present results suggest that MG29 may play important roles both in the formation of the SR and the construction of the triads during the early development of skeletal muscle cells.

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells

Immunolocalization of mitsugumin29 in developing skeletal muscle and effects of the protein expressed in amphibian embryonic cells

Cell-cycle-dependent nuclear translocation of HSP70 in amphibian embryonic cells.

Using immunocytochemical methods, we analyzed the localization of the HSP70 protein constitutively expressed during embryogenesis in the amphibian Pleurodeles waltl. Our results provide evidence for nuclear transfer of the protein during gastrulation, and particularly for predominant nuclear labeling in gastrula internalized cells. Using two inhibitors of DNA replication -hydroxyurea (HUA) and aphidicolin- or/and an inhibitor of transcription -actinomycin D- applied to embryos, we demonstrated that nuclear transfer of HSP70 is related to the transcriptional activity of the cells during the early S phase of the cell cycle.

The RGD-dependent and the Hep II binding domains of fibronectin govern the adhesive behaviors of amphibian embryonic cells

In early amphibian development, interactions between fibronectin and both ectoderm and mesoderm cells are critical in the progression of gastrulation movements. In the Pleurodeles waltl embryo, it has been established that ectoderm cells of the animal hemisphere organize a fibrillar-extracellular matrix containing fibronectin. Mesoderm cells migrate along the blastocoel roof using these fibronectin fibrils as substratum. Fibronectin is an adhesive glycoprotein which possesses multiple cell-binding domains. From previous studies, it is clear that amphibian ectoderm and mesoderm cells interact with fibronectin in an RGD-dependent manner, whereas the contributions of RGD-independent domains in the adhesive behaviors of gastrula cells has not been defined. To study this question, we have used bacterially expressed Pleurodeles waltl fibronectin-fusion proteins. The approach consisted of in vitro adhesion assays with either isolated cells or tissue fragments of embryos dissected at the onset of gastrulation. Tissues were obtained from regions of the embryo which represent presumptive ectoderm cells or from the dorsal-marginal zone which contains cells of the presumptive cephalic, chordal and somitic mesoderm. The results show that both the RGD-dependent and the Hep II domains of fibronectin mediate attachment and spreading of isolated cells. Both regions cooperate to control the proper expansion of a sheet of dorsal mesoderm cells. The Hep II domain promotes the migration of cells ahead of the mesoderm-cell sheet.

Introduction: Johannes Holtfreter, pioneer in experimental embryology.

Developmental DynamicsVolume 205, Issue 3 p. 214-216 Free Access Introduction: Johannes Holtfreter, pioneer in experimental embryology Viktor Hamburger, Corresponding Author Viktor Hamburger Department of Biology, Washington University, St. Louis, Missouri 63110Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, MO 63110Search for more papers by this author Viktor Hamburger, Corresponding Author Viktor Hamburger Department of Biology, Washington University, St. Louis, Missouri 63110Department of Anatomy and Neurobiology, Washington University Medical School, St. Louis, MO 63110Search for more papers by this author First published: March 1996 https://doi.org/10.1002/(SICI)1097-0177(199603)205:3<214::AID-AJA2>3.0.CO;2-LCitations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References Holtfreter, J. (1939) Tissue affinity, a means of embryonic morphogenesis. Archiv für Experimentelle Zellforschung 23: 169– 209. Townes, P. L., and Holtfreter, J. (1955) Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool. 123: 53– 120. Trinkaus, J. P. (1984) Cells Into Organs. 2nd ed. Englewood Cliffs, NJ: Prentice Hall Inc. Citing Literature Volume205, Issue3March 1996Pages 214-216 ReferencesRelatedInformation

Rat HPC-1/Syntaxin 1A and Syntaxin 1B Interrupt Intracellular Membrane Transport and Inhibit Secretion of the Extracellular Matrix in Embryonic Cells of an Amphibian

HPC-1/syntaxin 1A and syntaxin 1B are proteins that have been implicated in the docking and/or fusion of synaptic vesicles to the presynaptic plasma membrane in neural cells. Capped RNAs (cRNAs) for rat HPC-1 and syntaxin 1B were injected into embryonic cells of an amphibian, the Japanese newt. The effects of the proteins translated from the injected cRNAs on intracellular membrane transport and secretion of the extracellular matrix (ECM) were then investigated. Immunoblotting and immunoelectron microscopy showed that the HPC-1 synthesized in the embryonic cells was localized on the membranes of Golgi complexes and vacuoles and on the plasma membrane. Electron microscopy revealed the morphological deformation of Golgi complexes, an appearance of large number of vacuoles, and the disappearance of the ECM from the cell surface in the cRNA-injected embryos. The results showed that HPC-1 and syntaxin 1B interrupt the pathways of intracellular membrane transport and inhibit the secretion of ECM by amphibian embryonic cells. Similar mechanisms may be involved in regulation of the secretory process of synaptic vesicles in mammalian neural cells and in regulation of intracellular membrane transport and constitutive secretion of ECM in amphibian embryonic cells.

Electric-field-induced permeabilization and fusion of embryonic amphibian cells.

The technique of electropulsation has been shown to be highly efficient in promoting penetration of exogenous molecules into living cells, transfection, and cell fusion in different animal, vegetal, and bacterial cell systems. Introduction of such exogenous compounds, i.e., plasmids, into living cells is of great interest for embryological studies. Embryonic amphibian ectodermal cells from Pleurodeles waltl gastrulae, either freshly dissociated or cultured for 5 days, can be permeabilized when submitted to an external electric field of sufficient intensity: 500 V/cm for isolated spherical cells and 150-200 V/cm for plated cultured cells. Permeabilization was indicated by both the leakage of metabolites (ATP) from the cells and the uptake of exogenous compounds (pyranin) into the cells. With the use of higher field intensities (600 V/cm for freshly dissociated cells and 300 V/cm for cultured cells) cell fusion and syncytial structures could also be obtained. Isolated spherical cells had 100% viability immediately after being pulsed at intensities up to 600 V/cm. Cell lysis was observed above this value, although the nonlysed cells were observed to spread on a substrate and differentiate normally. For the cultured plated cells, cell viability fell with increasing electric-field strength, and for a given electric field value, cell viability decreased with the age of the culture after pulsing. Nevertheless, for electric-field intensities less than or equal to 300 V/cm, 100% of the cells remained attached to the substrate and differentiated normally over the following 5 days.

Sensitivity of gap junctional conductance to H ions in amphibian embryonic cells is independent of voltage sensitivity.

In vertebrate embryos gap junctional conductance (gj) is reduced by transjunctional voltage (Vj) and by cytoplasmic acidification; in each case sensitivity is comparable to those of other channels gated by voltage and ligand-receptor binding. We show here that the mechanisms by which Vj and intracellular pH (pHi) gate gj are apparently independent. Partial reduction of gj by lowering pHi neither attenuates nor enhances further reduction by Vj. Certain drugs irreversibly (glutaraldehyde, 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) or reversibly (retinoic acid) abolish dependence of gj on pHi without appreciably affecting kinetic properties of voltage dependence or the shape of the steady-state Vj-gj relation. These findings suggest that the mechanisms by which pHi and Vj act on the gap junction are at least partially distinct and presumably involve separate regions of the junctional macromolecules.

Cell surface properties of amphibian embryonic cells

Gastrula and neurula embryos of Cynops pyrrhogaster were dissociated into cell suspensions with ethylenediaminetetraacetic acid (EDTA), trypsin, and solution. The cells were cultured in Niu-Twitty's balanced salt solution and their aggregates were examined histologically. The results indicated that the capacity of amphibian embryonic cells for aggregate formation, sorting out, and notochord differentiation was not suppressed by EDTA and alkali treatments. Trypsin treatment, however, virtually suppressed the cell's capacity for aggregate formation. When aggregate formation resulted from trypsin-dissociated cells, the aggregates showed the sorting out and no differentiation of notochord.

Lactate — a suppressor of diffenrentiation in embryonic cells

All kinds of visible differentiation in cultured amphibian embryonic cells are suppressed in the presence of lactate. This effect differs in certain respects from that observed with actinomycin, but like the latter lactate inhibits RNA synthesis. In this communication we present some further observations on the effect of lactate.

Lectin-mediated agglutination of amphibian embryonic cells.

Dissociated blastula cells of Xenopus laevis are agglutinated with wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA), concanavalin A (Con A) and, to a lesser extent with soya bean agglutinin (SBA). They are not agglutinated with fucose-binding protein. Neuraminidase treatment of cells enhances their agglutinability with RCA and SBA, but has no effect on Con A- and WGA-mediated agglutinability. Treatment of cells with procaine, or xylocaine, has no effect on the cells' agglutinability or on the extrusion of lobopodia. Treatment with colchicine or cytochalasin B either separately or simultaneously has no effect on lectin-mediated agglutinability. Cells treated with cytochalasin B or colchicine and cytochalasin B simultaneously lack lobopodial extensions, while colchicine alone has no effect on these structures. Phenothiazine tranquillizers inhibit agglutination mediated by all of the above mentioned lectins. Lobopodial extensions are absent in cells treated with these compounds. Glutaraldehyde fixation inhibits RCA and WGA mediated agglutinability and reduces the Con A-mediated agglutinability. Results suggest that in this system microtubules and microfilaments are not involved in lectin-mediated agglutination.

Role of acid phosphatase in the breakdown of yolk platelets in developing amphibian embryos.

Yolk platelets in differentiating embryonic amphibian cells degrade by a membranous "unraveling" or "delamination" process. Acid phosphatase activity was demonstrated at the peripheries of those platelets which were in the process of degrading and thus this lytic enzyme appears to be involved in the breakdown process. We believe that this relationship provides a means by which early cells obtain the nutrients necessary for early differentiation.

Differentiation of the four animal and the four vegetal blastomeres of the eight-cell-stage ofTriturus alpestris

The 4 animal and 4 vegetal blastomeres of the eight-cell-stage ofTriturus alpestris were isolated and cultured for up to 12 days. Because of the difficulty of obtaining intact animal and vegetal blastomeres of the same embryo, we either cut off the vegetal blastomeres or sucked off the animal blastomeres. The culture of early embryonic amphibian cells is improved by the use of 50% Leibovitz-medium with added fetal calf serum providing a stable pH and optimal osmotic pressure.Isolated animal blastomeres differentiated to irregularly shaped ciliated epidermis. 30% of the cases showed small amounts of myotomes, notochord and neuroid cells in addition to irregular epidermis. The vegetal blastomeres formed trunk and tail structures but only 6% of all cases formed nearly complete head structures in addition.From the results we conclude that the vegetal blastomeres as well as the animal blastomeres of the eight-cell-stage are already determined as to their future fate. The possibility of partial regulation and the influence of asymmetric or irregular cleavage on the further development of isolated blastomeres is discussed.

The binding of concanavalin A to dissociated embryonic amphibian cells

Fluorescein isothiocyanate-conjugated concanavalin A (F-ConA) binds to the surface of EDTA-dissociated cells from blastula and gastrula stage Rana pipiens, R. pipiens ♀ × R. catesbeiana ♂, and R. pipiens ♀ × R. esculenta ♂ embryos. This binding is abolished by α-methyl- d-mannopyranoside. Gastrula cells show a ConA-induced clustering or capping of ConA-binding sites not shown by blastula stage cells. There are no striking differences in amount or pattern of ConA binding to cells from normal and hybrid embryos, nor to cells from different regions of normal gastrula stage embryos. Soy bean agglutinin (SBA) binds weakly to normal gastrula cells.