scholarly journals Gap Junctional Communication in the Early Xenopus Embryo

2000 ◽  
Vol 150 (4) ◽  
pp. 929-936 ◽  
Author(s):  
Yosef Landesman ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Synergistic Effects ◽  
Xenopus Embryo ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Whole Mount ◽  
Gap Junctional ◽  
Whole Mounts

In the Xenopus embryo, blastomeres are joined by gap junctions that allow the movement of small molecules between neighboring cells. Previous studies using Lucifer yellow (LY) have reported asymmetries in the patterns of junctional communication suggesting involvement in dorso-ventral patterning. To explore that relationship, we systematically compared the transfer of LY and neurobiotin in embryos containing 16–128 cells. In all cases, the junction-permeable tracer was coinjected with a fluorescent dextran that cannot pass through gap junctions. Surprisingly, while LY appeared to transfer in whole-mount embryos, in no case did we observe junctional transfer of LY in fixed and sectioned embryos. The lack of correspondence between data obtained from whole-mounts and from sections results from two synergistic effects. First, uninjected blastomeres in whole-mounts reflect and scatter light originating from the intensely fluorescent injected cell, creating a diffuse background interpretable as dye transfer. Second, the heavier pigmentation in ventral blastomeres masks this scattered signal, giving the impression of an asymmetry in communication. Thus, inspection of whole-mount embryos is an unreliable method for the assessment of dye transfer between embryonic blastomeres. A rigorous and unambiguous demonstration of gap junctional intercellular communication demands both the coinjection of permeant and impermeant tracers followed by the examination of sectioned specimens. Whereas LY transfer was never observed, neurobiotin was consistently transferred in both ventral and dorsal aspects of the embryo, with no apparent asymmetry. Ventralization of embryos by UV irradiation and dorsalization by Xwnt-8 did not alter the patterns of communication. Thus, our results are not compatible with current models for a role of gap junctional communication in dorso-ventral patterning.

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

2001 ◽  
Vol 280 (6) ◽  
pp. H2441-H2450 ◽  
Author(s):  
Andrew T. Chaytor ◽  
Patricia E. M. Martin ◽  
David H. Edwards ◽  
Tudor M. Griffith
Keyword(s):  
Gap Junctions ◽  
Hepatic Artery ◽  
Lucifer Yellow ◽  
Dye Transfer ◽  
Cell Coupling ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
A7r5 Cells ◽  
Gap Junctional

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26,43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 μM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 μM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.

scholarly journals Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment

2005 ◽  
Vol 288 (4) ◽  
pp. C795-C804 ◽  
Author(s):  
Lucia Formigli ◽  
Fabio Francini ◽  
Alessia Tani ◽  
Roberta Squecco ◽  
Daniele Nosi ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Myoblast Differentiation ◽  
Cell Contact ◽  
Cx43 Expression ◽  
Gap Junctional Communication ◽  
Skeletal Myoblasts ◽  
Junctional Communication ◽  
Direct Cell ◽  
Gap Junctional

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

scholarly journals Asymmetric patterns of gap junctional communication in developing chicken skin

Development ◽  
1993 ◽  
Vol 119 (1) ◽  
pp. 85-96 ◽  
Author(s):  
F. Serras ◽  
S. Fraser ◽  
C.M. Chuong
Keyword(s):  
Lucifer Yellow ◽  
Single Cells ◽  
Dye Coupling ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Chicken Skin ◽  
Preferential Pathways ◽  
Gap Junctional ◽  
Feather Development

To study the pattern of gap junctional communication in chicken skin and feather development, we injected Lucifer Yellow into single cells and monitored the transfer of the fluorescent dye through gap junctions. Dye coupling is present between cells of the epithelium as well as between cells of the mesoderm. However, dye transfer did not occur equally in all directions and showed several consistent patterns and asymmetries, including: (1) no dye coupling between mesoderm and epithelium, (2) partial restriction of dye coupling at the feather bud/interbud boundary during early feather bud development, (3) preferential distribution of Lucifer Yellow along the anteroposterior axis of the feather placode and (4) absence of dye coupling in some epithelial cells. These results suggest the presence of preferential pathways of communication that may play a role in the patterning of chicken skin.

Implication of gap junction coupling in amphibian vitellogenin uptake

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 149-157 ◽  
Author(s):  
M.E. Mónaco ◽  
E.I. Villecco ◽  
S.S. Sánchez
Keyword(s):  
Gap Junctions ◽  
Connexin 43 ◽  
Physiological Role ◽  
Follicle Cell ◽  
Pcr Analysis ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Endocytic Activity ◽  
Gap Junctional

SummaryThe aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

Reduced gap junctional communication is associated with the lethal condition characteristic of DDK mouse eggs fertilized by foreign sperm

Development ◽  
1987 ◽  
Vol 101 (3) ◽  
pp. 449-459 ◽  
Author(s):  
M. Buehr ◽  
S. Lee ◽  
A. McLaren ◽  
A. Warner
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Blastocyst Stage ◽  
Weak Base ◽  
Cell Stage ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Condition Characteristic ◽  
Gap Junctional ◽  
Mouse Eggs

Communication through gap junctions was examined in 8-cell zygotes generated by fertilization of eggs of the DDK inbred strain of mice with spermatozoa of the C3H strain. These zygotes spontaneously begin to extrude cells at the late 16-cell stage and 95% die by the blastocyst stage. The transfer of Lucifer Yellow between cells of DDK/C3H zygotes that had not yet begun to express the defect was significantly slower than in DDK/DDK controls or in controls from other strains. Treatment with the weak base methylamine, to raise intracellular pH, speeded the transfer of Lucifer in all strains; transfer between cells of DDK/C3H zygotes became as fast as that between cells of control zygotes. DDK/C3H zygotes cultured in methylamine either from the 4- to 8-cell stage to the early 16-cell stage (19h) or from the early to the late 16-cell stage (6 h) showed significant rescue to the blastocyst stage. Once spontaneous decompaction of cells from DDK/C3H zygotes had begun (the late 16-cell stage onwards) methylamine treatment was no longer able to bring about rescue. We conclude that zygotes developed from eggs of the DDK strain fertilized by foreign spermatozoa are characterized physiologically by defective gap junctional communication. Improving gap junctional communication is sufficient to allow many zygotes to maintain the compacted state, suggesting a link between compaction and communication through gap junctions.

The role of gap junctions in patterning of the chick limb bud

Development ◽  
1990 ◽  
Vol 108 (4) ◽  
pp. 623-634 ◽  
Author(s):  
F. Allen ◽  
C. Tickle ◽  
A. Warner
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Limb Bud ◽  
Limb Patterning ◽  
Posterior Limb ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Mesenchyme Cells ◽  
Gap Junctional

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.

scholarly journals Regulation of gap junction coupling in bovine ciliary epithelium

2010 ◽  
Vol 298 (4) ◽  
pp. C798-C806 ◽  
Author(s):  
Zhao Wang ◽  
Chi Wai Do ◽  
Virginijus Valiunas ◽  
Chi Ting Leung ◽  
Angela K. W. Cheng ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Quantitative Polymerase Chain Reaction ◽  
Donor Cell ◽  
Ciliary Epithelium ◽  
Dibutyryl Camp ◽  
Dye Transfer ◽  
Qrt Pcr ◽  
Junctional Communication ◽  
Gap Junctional

Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 ( n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 ( n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 ( n = 15) to 0.17 ± 0.05 ( n = 20) after 10 min. Junctional currents were lowered by ∼50% ( n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP ( n = 6); thereafter, heptanol abolished the currents ( n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction ( n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.

scholarly journals Gap Junctional Communication Modulates Gene Expression in Osteoblastic Cells

1998 ◽  
Vol 9 (8) ◽  
pp. 2249-2258 ◽  
Author(s):  
Fernando Lecanda ◽  
Dwight A. Towler ◽  
Konstantinos Ziambaras ◽  
Su-Li Cheng ◽  
Michael Koval ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gene Transcription ◽  
Lucifer Yellow ◽  
Bone Matrix ◽  
Osteoblastic Cells ◽  
Expression Vectors ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Umr 106

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106–01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter–luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2.8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106–01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

scholarly journals Communication compartments in the gastrulating mouse embryo.

1988 ◽  
Vol 107 (1) ◽  
pp. 241-255 ◽  
Author(s):  
G H Kalimi ◽  
C W Lo
Keyword(s):  
Mouse Embryo ◽  
Histological Analysis ◽  
Lucifer Yellow ◽  
Primitive Streak ◽  
Dye Transfer ◽  
Germ Layers ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Ionic Coupling

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.

Reduced junctional permeability at interrhombomeric boundaries

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1069-1076 ◽  
Author(s):  
S. Martinez ◽  
E. Geijo ◽  
M.V. Sanchez-Vives ◽  
L. Puelles ◽  
R. Gallego
Keyword(s):  
Lucifer Yellow ◽  
Single Cells ◽  
Electrical Coupling ◽  
Dye Transfer ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Neuroepithelial Cells ◽  
Junctional Permeability ◽  
Pattern Specification ◽  
Gap Junctional

Intercellular communication is considered to have a role during pattern specification processes in early embryonic development. This report analyzes the changing gap junctional communication properties of chick neuroepithelial cells depending on their position relative to the segmental partitions of the rhombencephalon. Intercellular electrical coupling and dye transfer were studied with microelectrode techniques. Neuroepithelial cells were electrically coupled irrespective of their location relative to interneuromeric boundaries. Iontophoretic injection of biocytin or Lucifer Yellow into single cells inside the rhombomeres was followed by transjunctional diffusion to the surrounding cells. In contrast, dye transfer was strictly limited when the diffusion zone contacted the cells forming the interneuromeric limits. Label injected into the boundary cells did not spread to other cells at all. Avian interrhombomeric boundaries are thus sites of reduced junctional permeability during early morphogenesis.

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