Patterns of junctional communication during development of the early amphibian embryo

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.     

Communication compartments in the gastrulating mouse embryo

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.     

Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer

Dye coupling and cell lineages of blastomeres that participate in the formation of the yolk syncytial layer (YSL) in the zebrafish Brachydanio rerio have been examined. The YSL is a multinucleate layer of nonyolky cytoplasm underlying the cellular blastoderm at one pole of the giant yolk cell. It forms at the time of the 10th (sometimes 9th) cleavage by a collapse of a set of blastomeres, termed marginal blastomeres, into the yolk cell. Marginal blastomeres possess cytoplasmic bridges to the yolk cell before the YSL forms, and injections of fluorescein-dextran into the cells revealed that bridges between the yolk cell and blastoderm do not persist after this time. Injections of Lucifer yellow revealed that shortly after the YSL forms the yolk cell and blastoderm are dye coupled, presumably by gap junctions, and that this coupling disappears gradually during early gastrulation. Lineage analyses revealed that not all of the progeny of early marginal blastomeres participate in YSL formation. Although some descendants of marginal blastomeres remained on the margin during successive cleavages, neither "compartment" nor "strict lineage" models are sufficient to explain the origin of the YSL. It is proposed that the position of a cell on the blastoderm margin, and not the cell's lineage, determines YSL cell fate.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Gap junctional communication in the post-implantation mouse embryo.

We studied the extent of cell-to-cell communication via junctional channels in in vitro-implanted mouse blastocysts by monitoring ionic coupling and the spread of two injected low molecular weight dyes, fluorescein and Lucifer yellow. In the early attached embryos, both trophoblasts and cells of the inner cell mass (ICM) were ionically coupled to one another. Dye injections in either trophoblasts or ICM cells resulted in spread to the entire embryo. As older and more developed embryos were examined, the spread of injected dye was progressively more limited. In the most developed embryos examined, dye injected into a cell in the ICM region resulted in spread throughout the ICM but not into the surrounding trophoblast cells, while dye injected into a trophoblast cell did not spread to any other cell in the embryo. Simultaneous monitoring of ionic coupling and dye injections in embryos of intermediate stages in this transition revealed that the trophoblast and ICM cells were ionically coupled, even across the apparent boundary where no dye was observed to pass. In the latest stage embryos examined in which no injected dye was observed to move out of the ICM, ionic coupling was still observed between the cells of the ICM and the trophoblasts. Furthermore, in the more developed embryos, dye injected into the ICM region frequently was not transferred to all the cells of the ICM, thus suggesting a further compartmentalization of due spread within the ICM. Our observations that ionic coupling is more extensive than the detectable spread of injected dyes may perhaps reflect a reduced number of junctional channels. With fewer channels less dye would pass between cells, so that, together with continuous quenching, the transfer of injected dye would not be detectable. This partial segregation of cell-to-cell communication as indicated by the limited dye spread may parallel specific differentiation processes, in particular that of giant trophoblast, embryonic ectoderm and extraembryonic endoderm differentiation.     

Reduced junctional permeability at interrhombomeric boundaries.

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.     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Estimation of the junctional resistance between electrically coupled receptor cells in Necturus taste buds

Junctional resistance between coupled receptor cells in Necturus taste buds was estimated by modeling the results from single patch pipette voltage clamp studies on lingual slices. The membrane capacitance and input resistance of coupled taste receptor cells were measured to monitor electrical coupling and the results compared with those calculated by a simple model of electrically coupled taste cells. Coupled receptor cells were modeled by two identical receptor cells connected via a junctional resistance. On average, the junctional resistance was approximately 200-300 M omega. This was consistent with the electrophysiological recordings. A junctional resistance of 200-300 M omega is close to the threshold for Lucifer yellow dye-coupling detection (approximately 500 M omega). Therefore, the true extent of coupling in taste buds might be somewhat greater than that predicted from Lucifer yellow dye coupling. Due to the high input resistance of single taste receptor cells (> 1 G omega), a junctional resistance of 200-300 M omega assures a substantial electrical communication between coupled taste cells, suggesting that the electrical activity of coupled cells might be synchronized.     

Origin and organization of the zebrafish fate map

We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.     

Galactosyltransferase activity of intact neural retinal cells from the embryonic chicken

Permeability of electrotonic junctions between isolated and reaggregated Fundulus blastomeres was evaluated with a new fluorescent dye, Lucifer yellow CH. The dye is readily shown to pass between coupled cells. It does not enter from the bathing medium, nor does it move between cells via the enlarged extracellular space sometimes seen between them. Thus, we conclude that passage is via a private pathway, presumably provided by the gap junctions described for this tissue. In contrast to previous findings, fluorescein (as the sodium salt, uranine) also passes between coupled Fundulus cells. Although it can enter from the bathing medium, it may be less concentrated in the space between a cell pair than in the uninjected cell. Again, passage via gap junctions is indicated. Molecular models demonstrate that Lucifer yellow and fluorescein are similar in size. Thus, similarity in ability to permeate junctional membranes is to be expected.     

Junctional communication is induced in migrating capillary endothelial cells

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.     

Acetylcholine-evoked uncoupling restricts the passage of Lucifer Yellow between pancreatic acinar cells

Summary Direct cell to cell movement of the fluorescent dye Lucifer Yellow CH (457 daltons) in exocrine acinar tissue is demonstrated by direct observation of living mouse pancreatic segments. Electrical uncoupling of pancreatic acinar cells by local application of a high concentration of acetylcholine significantly restricts cell to cell passage of the fluorescent dye. This result shows that a secretagogue can control direct movement of organic molecules between cells through junctional channels.     

The morphology and coupling of Aplysia bag cells within the abdominal ganglion and in cell culture

The bag cells in the abdominal ganglion of Aplysia californica control egg-laying behavior by releasing a polypeptide (ELH) during an afterdischarge of synchronous action potentials. We have used intracellular injection of Lucifer Yellow to study the morphology and interconnections of the bag cells. These neurosecretory cells are typically multipolar and their processes extend in all directions out from the bag cell clusters into the surrounding connective tissue, where they branch in a complex manner. In some of the dye injection experiments, dye transfer from the injected cell to neighboring cells was observed. Freeze fracture of the bag cell clusters and their surrounding connective tissue revealed numerous gap junctions on bag cell processes within the clusters as well as on more distal processes. We have also examined the morphology and coupling between bag cells in primary culture. As in the intact ganglion, bag cells in culture were found to be multipolar. All pairs of bag cells whose somata or processes had formed contacts in culture were electrically coupled. The strongest coupling was observed between pairs of cells whose somata appeared closely apposed. In these cases transfer of Lucifer Yellow between cells could also be observed. It is therefore likely that the synchrony of bag cell action potentials during a bag cell afterdischarge is a result of coupling between individual cells in the bag cell cluster.     

Dorsal specification in blastoderm at the blastula stage in the goldfish, Carassius auratus

The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180°, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed: the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore, the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.     

Permeability of gap junctions at the segmental border in insect epidermis

We have examined cell-cell communication between epidermal cells of fifth-instar larvae of the milkweed bug Oncopeltus fasciatus and those of maggots of the blowfly Calliphora erythrocephala. Ionic coupling and the transfer of injected Lucifer Yellow (molecular weight 450) and lead-EDTA (molecular weight 374) were used to map the pattern of communication. All epidermal cells, regardless of their position with respect to the segmental border, were ionically coupled. In both species Lucifer Yellow was transferred freely between cells lying in the same segment—that is, in the same developmental compartment as defined by cell lineage. Dye injections close to the segmental border showed that Lucifer Yellow was not transferred between cells in adjacent segments—that is, across the compartmental border. In Calliphora failure of Lucifer Yellow transfer at the segmental border was always observed; in Oncopeltus Lucifer Yellow was not transferred in 90% of preparations examined. Injections of PbEDTA^2? in Calliphora showed that this anion was transferred freely from cell to cell and did not respect the segmental boundary. Previous studies of the distribution of gap junctions at and away from the segmental border make it unlikely that the failure of Lucifer Yellow to cross from segment to segment is due to a reduced number of gap-junctional channels at the border. We conclude that gap junctions at the segmental borders may have different permeability properties from those between cells in the same segment.     

Transegmental electrical coupling between adjacent intersegemental muscles in the tobacco hawkmoth (Manduca sexta)

The lateral intersegmental muscles of pharate adult tobacco hawkmoths (Manduca sexta), exhibited electrical coupling across the segmental boundary. The degree of electrical coupling was constant throughout adult development. These muscle fibres did not appear to be dye coupled in that neither cobalt ions nor the flourescent dye Lucifer Yellow CH passed between cells. Electrical coupling was unaffected by cellular acidification with CO₂. Data are presented which suggest that this electrical coupling may be through the extracellular space rather than some membrane specialization. It is further speculated that many invertebrates may show this form of electrical coupling due to the metameric architecture of certain skeletal muscles.     

Changes in junctional communication associated with cell cycle arrest and differentiation of trochoblasts in embryos of Patella vulgata

In early embryos of molluscs, different clones of successively determined trochoblasts differentiate into prototroch cells and together contribute to the formation of a ciliated ring of cells known as the prototroch. Trochoblasts differentiate after cell cycle arrest, which occurs two cell cycles after the commitment of their stem cell. To study the changes of junctional communication in embryos of Patella vulgata in relation to commitment, cell cycle arrest, and differentiation of the trochoblasts, we have monitored electrical coupling as well as transfer of fluorescent dyes. The appearance of dye coupling in embryos of Patella occurs after the fifth cleavage (at the 32-cell stage), when the cell cycles of all embryonic cells become asynchronous and longer. At the 32- and 64-cell stages all cells are well coupled. However, after the 72-cell stage dye transfer to or from any cell of the four interradial clones of four primary trochoblasts becomes abruptly reduced, whereas electrical coupling between these cells and the rest of the embryo can still be detected. From scanning electron microscopical analysis of the cell pattern we conclude that this change in gap junctional communication coincides with cell cycle arrest and with the development of cilia in all four clones of primary trochoblasts. Similarly, after the 88-cell stage the four radial clones of accessory trochoblasts stop dividing, reduce cell coupling, and become ciliated. By the formation of the prototroch, the embryo becomes subdivided into an anterior (pretrochal) and a posterior (posttrochal) domain which will develop different structures of the adult. At the 88-cell stage, the cells within each of these two domains remain well coupled and form two different communication compartments that are separated from each other by the interposed ring of uncoupled trochoblasts. The relations among control of cell cycle, changes in junctional communication, and differentiation are discussed.     

Distinct effects of ectopic expression of Wnt-1, activin B, and bFGF on gap junctional permeability in 32-cell Xenopus embryos.

A polarity in gap junctional permeability normally exists in 32-cell stage Xenopus embryos, in that dorsal cells are relatively more coupled than ventral cells, as measured by transfer of Lucifer yellow dye. The current study extends our analysis of whether gap junctional permeability at this stage can be modulated by secreted factors, and whether the polarity in gap junctional permeability correlates with the effects of ectopic expression of these secreted factors on the subsequent phenotype of the developing embryo. Following ectopic expression of activin B or Wnt-1, but not bFGF, the transfer of Lucifer yellow between ventral animal pole cells is detected in a greater percentage of 32-cell stage embryos. This increased incidence of dye transfer between ventral cells correlates with axial duplications later in development. However, there are differences in the extent of Lucifer yellow transfer between animal and vegetal hemisphere blastomeres which is dependent on whether activin B or Wnt-1 RNA had previously been injected. These results suggest that enhanced gap junctional permeability between ventral cells of 32-cell Xenopus embryos correlates with subsequent defects in the dorsoventral axis, although there are at present no direct data demonstrating a role for gap junctions in establishment or maintenance of this axis. Moreover, while both activin B and bFGF are mesoderm-inducing growth factors, only activin B has effects on gap junctional permeability in 32-cell embryos following ectopic expression, demonstrating an interesting difference in physiological responses to expression of these factors.