The correlation between patterns of dye transfer junctions and future developmental fate in Xenopus: u.v. irradiation and lithium treatment

Development ◽  
1989 ◽  
Vol 105 (4) ◽  
pp. 747-752 ◽  
Author(s):  
D.J. Nagajski ◽  
S.C. Guthrie ◽  
C.C. Ford ◽  
A.E. Warner
Keyword(s):  
Lucifer Yellow ◽  
Lithium Treatment ◽  
Cell Communication ◽  
Embryonic Axis ◽  
Axis Formation ◽  
Dye Transfer ◽  
Cell Stage ◽  
The Future ◽  
Vegetal Pole ◽  
Cell Embryo

The correlation between cell-to-cell communication junctions at the 32-cell stage and the subsequent embryonic axis has been examined in Xenopus laevis Disturbances of embryonic axis formation were u.v. irradiation at the vegetal pole before 0.6 in the which generates embryos with dorsal axial embryos were treated with 100mM-lithium chloride 32-cell stage, which generates embryos with ventral The cell-to-cell transfer of Lucifer Yellow was used junctional permeability. Injections were made into cells, lying in tiers 1 and 2 of the 32-cell embryo, relative to the future dorsoventral axis of the embryo on the basis of differences in pigmentation. The Yellow transfer in the future dorsal half of the compared with that in the future ventral half for u.v.-irradiated and Li-treated embryos. Injected subsequently scored for axial developmenf for transfer frequencies. In control embryos at the 32- Yellow transfer was both more frequent and more dorsal regions than in future ventral regions, as In embryos that had been u.v. irradiated before 0.6 in cycle, Lucifer transfer was the same in both light and the animal hemisphere and at the low level ventral regions in normal embryos. These embryos reductions in dorsal axial structures. Embryos the first cell cycle, when u.v. irradiation no longer cytoplasmic movements initiated at fertilization, dorsoventral difference in Lucifer Yellow transfer and normal dorsoventral polarity. Embryos exposed to

The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle

Development ◽  
1996 ◽  
Vol 122 (7) ◽  
pp. 2207-2214 ◽  
Author(s):  
M. Sakai
Keyword(s):  
Cell Cycle ◽  
Formation Process ◽  
Lithium Treatment ◽  
Axis Formation ◽  
Cell Stage ◽  
Spemann Organizer ◽  
Dorsal Axis ◽  
Vegetal Pole ◽  
Organizing Center ◽  
Cell Embryo

Embryos with no dorsal axis were obtained when more than 15% of the egg surface was deleted from the vegetal pole of the early 1-cell embryo of Xenopus laevis. The timing of the deletion in the first cell cycle was critical: dorsal-deficient embryos were obtained when the deletion began before time 0.5 (50% of the first cell cycle) whereas normal dorsal axis usually formed when the deletion was done later than time 0.8. The axis deficiency could be restored by lithium treatment and the injection of vegetal but not animal cytoplasm. Bisection of the embryo at the 2-cell stage, which is known to restore the dorsal structures in the UV-ventralized embryos, had no effect on the vegetal-deleted embryos. These results show clearly that, in Xenopus, (1) the dorsal determinants (DDs) localized in the vegetal pole region at the onset of development are necessary for dorsal axis development and (2) the DDs move from the vegetal pole to a subequatorial region where they are incorporated into gastrulating cells to form the future organizing center. A model for the early axis formation process in Xenopus is proposed.

Activation of dorsal development by contact between the cortical dorsal determinant and the equatorial core cytoplasm in eggs of Xenopus laevis

Development ◽  
1997 ◽  
Vol 124 (8) ◽  
pp. 1543-1551 ◽  
Author(s):  
H. Kageura
Keyword(s):  
Xenopus Laevis ◽  
Dorsal Side ◽  
Equatorial Region ◽  
Normal Embryo ◽  
Dorsal Region ◽  
The Core ◽  
Dorsal Axis ◽  
The Future ◽  
Vegetal Pole ◽  
Cell Embryo

In eggs of Xenopus laevis, dorsal development is activated on the future dorsal side by cortical rotation, after fertilization. The immediate effect of cortical rotation is probably the transport of a dorsal determinant from the vegetal pole to the equatorial region on the future dorsal side. However, the identity and action of the dorsal determinant remain problematic. In the present experiments, individual isolated cortices from various regions of the unfertilized eggs and embryos were implanted into one of several positions of a recipient 8-cell embryo. The incidence of secondary axes was used not only to locate the cortical dorsal determinant at different times but also to locate the region of the core competent to respond to the dorsal determinant. The dorsal axis-inducing activity of the cortex occurred around the vegetal pole of the unfertilized egg. During cortical rotation, it shifted from there to a wide dorsal region. This is apparently the first evidence for the presence of a dorsal determinant in the egg cortex. The competence of the core of the 8-cell embryo was distributed in the form of gradient with the highest responsiveness at the equator. These results suggest that, in the normal embryo, dorsal development is activated by contact between the cortical dorsal determinant and the equatorial core cytoplasm, brought together through cortical rotation.

Expression of a dominant negative inhibitor of intercellular communication in the early Xenopus embryo causes delamination and extrusion of cells

Development ◽  
1995 ◽  
Vol 121 (2) ◽  
pp. 371-381
Author(s):  
D.L. Paul ◽  
K. Yu ◽  
R. Bruzzone ◽  
R.L. Gimlich ◽  
D.A. Goodenough
Keyword(s):  
Gap Junction ◽  
Intercellular Communication ◽  
Lucifer Yellow ◽  
Single Cells ◽  
Positional Information ◽  
Embryonic Cell ◽  
Dominant Negative ◽  
Dye Transfer ◽  
Cell Stage ◽  
The Right

A chimeric construct, termed 3243H7, composed of fused portions of the rat gap junction proteins connexin32 (Cx32) and connexin43 (Cx43) has been shown to have selective dominant inhibitory activity when tested in the Xenopus oocyte pair system. Co-injection of mRNA coding for 3243H7 together with mRNAs coding for Cx32 or Cx43 completely blocked the development of channel conductances, while the construct was ineffective at blocking intercellular channel assembly when coinjected with rat connexin37 (Cx37). Injection of 3243H7 into the right anterodorsal blastomere of 8-cell-stage Xenopus embryos resulted in disadhesion and delamination of the resultant clone of cells evident by embryonic stage 8; a substantial number, although not all, of the progeny of the injected cell were eliminated from the embryo by stage 12. A second construct, 3243H8, differing from 3243H7 in the relative position of the middle splice, had no dominant negative activity in the oocyte pair assay, nor any detectable effects on Xenopus development, even when injected at four-fold higher concentrations. The 3243H7-induced embryonic defects could be rescued by coinjection of Cx37 with 3243H7. A blastomere reaggregation assay was used to demonstrate that a depression of dye-transfer could be detected in 3243H7-injected cells as early as stage 7; Lucifer yellow injections into single cells also demonstrated that injection of 3243H7 resulted in a block of intercellular communication. These experiments indicate that maintenance of embryonic cell adhesion with concomitant positional information requires gap junction-mediated intercellular communication.

Patterns of junctional communication during development of the early amphibian embryo

Development ◽  
1988 ◽  
Vol 103 (4) ◽  
pp. 769-783 ◽  
Author(s):  
S. Guthrie ◽  
L. Turin ◽  
A. Warner
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Dorsal Side ◽  
Cell Stage ◽  
Animal Pole ◽  
Amphibian Embryo ◽  
Grey Crescent ◽  
The Future ◽  
Junctional Permeability ◽  
Side Gap

Cell-cell communication through gap junctions was examined in Xenopus laevis embryos between the 16-cell and early blastula stages using Lucifer Yellow, Fluorescein, lead EDTA and dicyanoargentate as probes of junctional permeability. Injections were made into cells whose position was identified with respect to the primary cleavage axis and the grey crescent. FITC dextrans revealed cytoplasmic bridges between the injected cell and its sister only. In the animal pole at the 16-cell stage at the future dorsal side of the embryo, Lucifer Yellow was frequently and extensively transferred between cells through gap junctions. At the future ventral side gap junctional transfer of Lucifer Yellow was significantly less frequent and less extensive. The asymmetry of transfer between future dorsal and ventral sides of the animal pole was more marked at the 32-cell stage. In the vegetal pole also at the 32-cell stage, a dorsoventral difference in junctional permeability to Lucifer Yellow was observed. At the 64-cell stage the transfer of Lucifer Yellow was relatively frequent between cells lying in the same radial segment in the animal pole; transfer into cells outside each segment was infrequent, except at the grey crescent. At the 128-cell stage, Lucifer transfer between future dorsal or future ventral cells in the equatorial region was infrequent. A high incidence of transfer was restored at the future dorsal side at the 256-cell stage. At the 32-cell stage, fluorescein was infrequently transferred between animal pole cells although lead EDTA moved from cell to cell with high, comparable frequency in future dorsal and ventral regions. Dicyanoargentate always transferred extensively, both at the 32- and 64-cell stages. Treatment of embryos with methylamine raised intracellular pH by 0.15 units, increased the electrical conductance of the gap junction and produced a 10-fold increase in the frequency of Lucifer Yellow transfer through gap junctions in future ventral regions of the animal pole at the 32-cell stage.

Gap-junctional permeability in early and cleavage-arrested ascidian embryos

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 153-160 ◽  
Author(s):  
B. Dale ◽  
L. Santella ◽  
E. Tosti
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Cell Voltage ◽  
Spatial Location ◽  
Cell Stage ◽  
Junctional Conductance ◽  
Junctional Permeability ◽  
Cell Embryo ◽  
Ascidian Embryos ◽  
Two Stages

Using the whole-cell voltage clamp technique, we have studied junctional conductance (Gj), and Lucifer Yellow (LY) coupling in 2-cell and 32-cell ascidian embryos. Gj ranges from 17.5 to 35.3 nS in the 2-cell embryo where there is no passage of LY, and from 3.5 to 12.2 nS in the later embryo where LY dye spread is extensive. In both cases, Gj is independent of the transjunctional potential (Vj). Manually apposed 2-cell or 32-cell embryos established a junctional conductance of up to 10 nS within 30 min of contact. Furthermore, since we did not observe any significant number of cytoplasmic bridges at the EM and Gj is sensitive to octanol, it is probable that blastomeres in the 2-cell and 32-cell embryos are in communication by gap junctions. In order to compare Gj in the two stages and to circumvent problems of cell size, movement and spatial location, we used cytochalasin B to arrest cleavage. Gj in cleavage-arrested 2-cell embryos ranged from 25.0 to 38.0 nS and remained constant over a period of 2.5 h. LY injected into a blastomere of these arrested embryos did not spread to the neighbour cell until they attained the developmental age of a 32- to 64-cell control embryo. Our experiments indicate a change in selectivity of gap junctions at the 32-cell stage that is not reflected by a macroscopic change in ionic permeability.

Occurrence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation

Development ◽  
1993 ◽  
Vol 118 (1) ◽  
pp. 163-170 ◽  
Author(s):  
M. Fujisue ◽  
Y. Kobayakawa ◽  
K. Yamana
Keyword(s):  
Cell Cycle ◽  
Close Association ◽  
Dorsal Side ◽  
Equatorial Region ◽  
Cell Stage ◽  
Assay Procedure ◽  
Axis Specification ◽  
Vegetal Pole ◽  
Cell Embryo ◽  
Dorsal Cells

Specification of the dorsoventral axis is a subject of great importance in amphibian embryogenesis. We have found that cytoplasm of the vegetal dorsal cells of a 16-cell embryo of Xenopus laevis, when injected into the ventral vegetal cells of a recipient at the same stage, can induce formation of a second axis. In the present experiments, using the same assay procedure, we found that the cytoplasm around the vegetal pole of an egg before cortical rotation is also active in inducing a second axis, that the activity decreases throughout the second half of the cell cycle and appears in a presumptive dorsal equatorial region at the 2- to 16-cell stages. This is the first demonstration of the localization of dorsal forming activity in any specific region of an egg. After UV irradiation, a treatment that is known to block cortical rotation and thereby inhibit axis specification, the activity remains near the vegetal pole beyond the first cell cycle and does not appear in an equatorial region, at least at the 16-cell stage. This suggests that cortical rotation or a related force is in some way involved in changes in distribution of the activity. We also found that UV-irradiated 8-cell embryos can rescue dorsal development when they are cut into halves along the first cleavage plane. Histological examination revealed that the rescued embryos have a neural tube and notochord. In the half embryo, the animal and vegetal regions came into contact during wound healing, an event that enables the activity to localize in the new equator of an embryo. Therefore this rescue suggests that, if the activity is distributed only in the equatorial region, dorsal specification occurs. In fact, the dorsal side of the rescued embryos seems to correspond to the plane through which the embryos have been cut. Based on our results, we propose (1) that a determinant that carries axis-inducing activity is first present around the vegetal pole, (2) that the determinant shifts from the vegetal pole to an equatorial region by or in close association with cortical rotation and (3) that occurrence of the determinant in the equatorial region is a prerequisite for axis specification.

Overexpression of cardiac connexin45 increases susceptibility to ventricular tachyarrhythmias in vivo

2006 ◽  
Vol 290 (1) ◽  
pp. H163-H171 ◽  
Author(s):  
Tetsuo Betsuyaku ◽  
Nkiruka S. Nnebe ◽  
Rune Sundset ◽  
Sushmitha Patibandla ◽  
Charles M. Krueger ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Ventricular Arrhythmias ◽  
Ventricular Myocytes ◽  
Lucifer Yellow ◽  
Cell Communication ◽  
Dye Transfer ◽  
Cx43 Expression ◽  
Altered Cell ◽  
And Control

Electrophysiological remodeling involving gap junctions has been demonstrated in failing hearts and may contribute to intercellular uncoupling, delayed conduction, enhanced arrhythmias, and vulnerability to sudden death in patients with heart failure. Recently, we showed that failing human hearts exhibit marked increases in connexin45 (Cx45) expression in addition to previously documented decreases in connexin43 (Cx43) expression. Each of these changes results in reduced gap junction coupling. The objective of the present study was to examine functional consequences of increased Cx45 in cardiac gap junctions. Transgenic mice with cardiac-selective overexpression of the developmentally downregulated cardiac connexin, connexin45 (Cx45OE mice) were subjected to in vivo electrophysiology studies in which an intracardiac catheter was used to induce ventricular arrhythmias in anesthetized mice, and in which ambulatory ECG monitoring was used to detect spontaneous arrhythmias in unanesthetized mice. Hearts were analyzed by TaqMan RT-PCR, immunostaining, immunoblotting, and echocardiography. Lucifer yellow and neurobiotin dye transfer was used to assess coupling in transgenic and control myocyte cultures. Cx45 mRNA was two orders of magnitude greater in Cx45OE mice. Cx45-immunoreactive signal at gap junctions increased twofold and total Cx45 protein by immunoblotting increased 25% in Cx45OE mice compared with nontransgenic littermate controls. Functionally, Cx45OE mice exhibited more inducible ventricular tachycardia than controls but did not exhibit any other functional or structural derangements as assessed by echocardiography. Ventricular myocytes isolated from Cx45OE mice exhibited diminished intercellular transfer of Lucifer yellow dye and increased transfer of neurobiotin, consistent with altered cell-to-cell communication. Thus increased myocardial expression of Cx45 results in remodeling of intercellular coupling and greater susceptibility to ventricular arrhythmias in vivo.

Centrosome Aurora A gradient ensures single polarity axis in C. elegans embryos

2020 ◽  
Vol 48 (3) ◽  
pp. 1243-1253 ◽  
Author(s):  
Sukriti Kapoor ◽  
Sachin Kotak
Keyword(s):  
Symmetry Breaking ◽  
Cell Fate ◽  
Cell Cortex ◽  
Axis Formation ◽  
Aurora A Kinase ◽  
Aurora A ◽  
C Elegans ◽  
Cell Embryo ◽  
Polarity Establishment

Cellular asymmetries are vital for generating cell fate diversity during development and in stem cells. In the newly fertilized Caenorhabditis elegans embryo, centrosomes are responsible for polarity establishment, i.e. anterior–posterior body axis formation. The signal for polarity originates from the centrosomes and is transmitted to the cell cortex, where it disassembles the actomyosin network. This event leads to symmetry breaking and the establishment of distinct domains of evolutionarily conserved PAR proteins. However, the identity of an essential component that localizes to the centrosomes and promotes symmetry breaking was unknown. Recent work has uncovered that the loss of Aurora A kinase (AIR-1 in C. elegans and hereafter referred to as Aurora A) in the one-cell embryo disrupts stereotypical actomyosin-based cortical flows that occur at the time of polarity establishment. This misregulation of actomyosin flow dynamics results in the occurrence of two polarity axes. Notably, the role of Aurora A in ensuring a single polarity axis is independent of its well-established function in centrosome maturation. The mechanism by which Aurora A directs symmetry breaking is likely through direct regulation of Rho-dependent contractility. In this mini-review, we will discuss the unconventional role of Aurora A kinase in polarity establishment in C. elegans embryos and propose a refined model of centrosome-dependent symmetry breaking.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
W. J. D. Reeve ◽  
C. A. Ziomek
Keyword(s):  
Ligand Binding ◽  
Single Cells ◽  
Progressive Increase ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Characteristic Distribution ◽  
Surface Polarization ◽  
Cell Embryo ◽  
Fluorescent Ligand ◽  
Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Gap junction expression and cell proliferation in differentiating cultures of Cx43 KO mouse hepatocytes

2001 ◽  
Vol 281 (4) ◽  
pp. G1004-G1013 ◽  
Author(s):  
Takashi Kojima ◽  
Alfredo Fort ◽  
Mingyuan Tao ◽  
Masao Yamamoto ◽  
David C. Spray
Keyword(s):  
Lucifer Yellow ◽  
Primary Cultures ◽  
Mrna Levels ◽  
Wild Type ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Junctional Conductance ◽  
Mouse Hepatocytes

Primary cultures of adult mouse hepatocytes are shown here to reexpress differentiated hepatocyte features following treatment with 2% DMSO and 10−7 M glucagon. To examine the roles of gap junctional communication during hepatocyte growth and differentiation, we have compared treated and untreated hepatocytes from connexin (Cx)32-deficient [Cx32 knockout (KO)] and wild-type mice. In untreated cultures, DNA replication of Cx32 KO hepatocytes was markedly higher than of wild types. Although Cx26 mRNA levels remained high at all time points in wild-type and Cx32 KO hepatocytes, Cx32 mRNA and protein in wild-type hepatocytes underwent a marked decline, which recovered in 10-day treated cultures. Increased levels of Cx26 protein and junctional conductance were observed in Cx32 KO hepatocytes at 96 h in culture, a time when cell growth rate was high. Treatment with DMSO/glucagon highly reinduced Cx26 expression in Cx32 KO hepatocytes, and such treatment reinduced expression of both Cx32 and Cx26 expression in wild types. Dye transfer was not observed following Lucifer yellow injection into DMSO/glucagon-treated Cx32 KO hepatocytes, whereas the spread was extensive in wild types. Nevertheless, high junctional conductance values were observed in treated cells from both genotypes. These studies provide a method by which the differentiated phenotype can be obtained in cultured mouse hepatocytes and provide in vitro evidence that expression of gap junctions formed of Cx32 are involved in the regulation of growth of mouse hepatocytes.

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