Comparative analysis of amphibian somite morphogenesis: cell rearrangement patterns during rosette formation and myoblast fusion.
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Detailed SEM observations of the changes in cellular morphology, arrangements, and contacts that occur during the process of somite formation were made in two species of urodele amphibians, Ambystoma mexicanum and Pleurodeles waltlii, and one species of anuran amphibian, Rana sphenocephala. After fixation, embryos were fractured transversely, horizontally, and parasagittally, and the intrasomitic cellular arrangement pattern was examined with the SEM. It was found that Ambystoma and Pleurodeles embryos followed exactly the same development sequence in rosette formation and myoblast fusion. Rana somites did not, however, appear to form rosettes. Those myotomal cells underwent fusion immediately after a few segmentations occurred. Patterns of cellular rearrangement were also described during urodele rosette formation at the time of somite segmentation and during myoblast fusion. Extensive changes in cell shape and orientation appeared to occur during those processes. When cells changed their orientation, they often exhibited a triangular configuration. Probable roles of these triangular-shaped cells in rosette formation and myoblast fusion are discussed. During the initial period of myoblast or myotomal cell fusion, cells first send out specialized cell processes and then establish their cell-cell contacts. The establishment of such contacts eventually leads to tight membrane appositions and fusion. Since myoblast fusion appeared to occur between two cells which were tandemly arranged in a rosette, the origin of multi-nuclearity in the fused cells is discussed. Finally, comparative analyses of the pattern of somite formation and subsequent muscle development were made between different species of amphibians. The possibility is discussed that patterns of somitogenesis may provide useful indicators for determining how different families of amphibians evolved. (+info)
Development of amphibian thymus. I. Morphological differentiation, multiplication, migration and lysis of thymocytes in the urodele Pleurodeles waltlii.
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Different stages of thymus morphogenesis and thymocyte differentiation have been studied at the ultrastructural level in the urodele amphibian Pleurodeles waltlii Michah. From stage 38 to 42 the undifferentiated lymphoid stem-cells colonize the epithelial thymic buds. From stage 43 to 45 the lymphoid stem-cells differentiate into lymphoblasts and then transform into typical lymphocytes. A clasmatosis phenomenon seems to be involved in this transformation. From stage 46 to 52, a phase of intense proliferation occurs and relations between dense reticular epithelial cells and lymphocytes are described. At stage 53, numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. At the same time, lymphocytes undergo migrations through the intra- and peri-thymic blood vessels. These lymphocytes should populate the peripheral lymphoid organs, according to the previous finding that stage 52 was the last developmental step for an efficient abrogation of cell-mediated immunity by thymectomy in Pleurodeles. (+info)
Endogenous DHP-sensitive Ca(2+) channels in Pleurodeles oocytes.
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The double electrode voltage-clamp technique was used to study voltage-dependent Ca(2+) channels in Pleurodeles oocytes. From a holding potential of -80 mV, Ba-current (IBa) (recorded in Cl-free solution, Ba(2+ = 40 mM) activated at -36.7 +/- 4 mV, peaked at -11.6 +/- 4 mV and reversed at 55 +/- 7 mV (n = 24). This current activated slowly (rise time was 0.98 +/- 0.2 s;n = 14 at -10 mV) and was not inactivated. Cadmium (Cd(2+), 500 microM) completely inhibited I(Ba). The effect of Cd(2+) was dose-dependent (EC(50) = 37 +/- 5 microM; n = 5). Moreover, IBa was insensitive to omega-conotoxin (10 microM) but interestingly this I(Ba) displayed dihydropyridine (DHP) sensitivity. Bay K 8644 (5 microM), a DHP activator, increased the peak current amplitude in a dose-dependent manner (EC(50) = 5.9 +/- 0.6 microM; n = 10) and shifted the threshold and the maximum of current/voltage relationship towards negative potentials by -10 mV. Nifedipine (5 microM), a DHP antagonist, decreased I(Ba) by 80% at HP of -80 mV (EC(50) = 1.2 +/- 0.2 microM; n = 6). We concluded that Pleurodeles oocytes possess High-Voltage Activated Ca(2+) channels with properties similar to L-type Ca(2+) channels. (+info)
Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians.
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Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed. (+info)
Integrin alpha v subunit is expressed on mesodermal cell surfaces during amphibian gastrulation.
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Mesodermal cell migration during amphibian gastrulation is dependent on cellular interactions with fibronectin. One mechanism whereby cells bind fibronectin is through alpha v-containing integrin heterodimers. In order to investigate the role of alpha v in amphibian gastrulation, we have cloned the Pleurodeles homologue of the integrin alpha v subunit using homology PCR. The deduced amino acid sequence is 73 and 74% identical with the human and chick homologues, respectively. The 4.8-kb mRNA is expressed during oogenesis and persists throughout development. Messenger RNA and protein are widely expressed in oocytes and embryos while cell surface expression is spatially regulated. The protein first appears on the plasma membrane of fully grown oocytes. Fertilization results in the progressive loss of alpha v membrane localization. Before and during gastrulation, the integrin alpha v subunit is expressed on the surface of mesodermal cells. These data show that alpha v expression is developmentally regulated by a post-translational mechanism which correlates with the onset of mesodermal cell migration at gastrulation. (+info)
Regulation of Na+, K+ ATPase activity during meiotic maturation of Pleurodeles waltl oocytes. Role of calcium.
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Changes in activity of the Na+, K+ ATPase of maturing Pleurodeles waltl were followed by measuring the resting potential in presence or absence of the specific inhibitor dihydroouabain. Corresponding currents were measured in voltage clamp conditions to eliminate the differences in resting potential at the origin and at the end of the meiotic maturation process. Our data confirm previous results obtained on Xenopus, indicating that the Na+,K+ pump activity disappears from the plasma membrane during progesterone-induced maturation and can be reactivated by an increase in internal Ca2+ triggered by ionomycin. Moreover we show by ultrastructural histochemistry that these modulations are likely to depend on the internalization and reinsertion of the transporter into the plasma membrane. (+info)
A putative zinc-binding protein on lampbrush chromosome loops.
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We used mAb A33/22, which recognizes a nuclear protein on the loops of amphibian lampbrush chromosomes, to select cDNA clone PwA33 from an expression library of the newt Pleurodeles waltl. A myc-tagged transcript of clone PwA33 was injected into Pleurodeles oocytes. The translation product localized in the germinal vesicle (GV) and was distributed on the lampbrush loops in a pattern identical to that of the endogenous protein. PwA33 encodes a 71 kDa protein with three distinct domains: a region rich in Cys/His residues that may form zinc fingers, a coiled-coil domain with potential for dimerization and a third 'rfp-like' domain that is shared by several other nuclear proteins. The putative zinc fingers and the coiled-coil domain resemble features in known nucleic acid-binding regulatory proteins. These structures, coupled with a distinctive pattern of expression in embryonic tissues, suggest that A33 may function as a regulatory protein during early development. It is unlikely that the large store of A33 in the GV is bound to DNA. Instead, its association with the nascent transcripts on the lampbrush chromosome loops suggests a role in pre-mRNA synthesis or processing. (+info)
hnRNP G: sequence and characterization of a glycosylated RNA-binding protein.
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The autoantigen p43 is a nuclear protein initially identified with autoantibodies from dogs with a lupus-like syndrome. Here we show that p43 is an RNA-binding protein, and identify it as hnRNP G, a previously described component of heterogeneous nuclear ribonucleoprotein complexes. We demonstrate that p43/hnRNP G is glycosylated, and identify the modification as O-linked N-acetylglucosamine. A full-length cDNA clone for hnRNP G has been isolated and sequenced, and the predicted amino acid sequence for hnRNP G shows that it contains one RNP-consensus RNA binding domain (RBD) at the amino terminus and a carboxyl domain rich in serines, arginines and glycines. The RBD of human hnRNP G shows striking similarities with the RBDs of several plant RNA-binding proteins. (+info)