Norton B. Gilula

Gap junctional communication in the preimplantation mouse embryo

In this study, we examined cell-to-cell communication via gap junctional channels between the cells of the early mouse embryo from the 2-cell stage to the preimplantation blastocyst stage. The extent of communication was examined by monitoring for the presence of ionic coupling, the transfer of injected fluorescein (molecular weight 330) and the transfer of injected horseradish peroxidase (molecular weight 40,000). In the 2-cell, 4-cell and precompaction 8-cell embryos, cytoplasmic bridges between sister blastomeres were responsible for ionic coupling and the transfer of injected fluorescein as well as the transfer of injected horseradish peroxidase. In contrast, no communication was observed between blastomeres from different sister pairs. Junction-mediated intercellular communication was unequivocably detected for the first time in the embryo at the early compaction stage (late 8-cell embryo). At that stage, ionic coupling was present and fluorescein injected into one cell spread to all eight cells of the embryo. Injected horseradish peroxidase was passed to only one other cell, however, again indicating the presence of cytoplasmic bridges between sister blastomeres. Junctional communication with respect to both ionic coupling and dye transfer was retained between all the cells throughout compaction. At the blastocyst stage, trophoblast cells of the blastocyst were linked by junctional channels to other trophoblast cells as well as to cells of the inner cell mass, as indicated by the spread of injected fluorescein. In addition, the extent of communication between the cells of the inner cell mass was examined in inner cell masses isolated by immunosurgery; both ionic coupling and the complete spread of injected fluorescein were observed.

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.

Modulation of cell-to-cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH.

Prior to ovulation, cell-to-cell communication between the oocyte and the cells of the cumulus oophorus is terminated (N. B. Gilula, M. L. Epstein, and W. H. Beers, 1978, J. Cell Biol. 78, 58–75; R. M. Moor, M. W. Smith, and R. M. C. Dawson, 1980, Exp. Cell Res. 126, 15–29). In this paper we report that LH, Bt2-cAMP, and the cyclic nucleotide phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, are all capable of interrupting communication in vitro in rat follicle-enclosed cumulus-oocyte complexes. Moreover, the breakdown of communication appears to be closely correlated with the ability of hyaluronidase to disperse the cumulus cell mass. This observation allows simple screening of the effects of various agents on cumulus-oocyte communication. The in vitro system employed in this study has also been used to investigate the relationship between communication and oocyte maturation. The findings presented indicate that the interruption of communication between the cumulus and the oocyte leads to relief of meiotic arrest, and are consistent with the possibility that cAMP, transmitted from the cumulus to the oocyte, may be the inhibitor of oocyte maturation in vivo.

Modulation of cell junctions during differentiation of the chicken otocyst sensory epithelium.

Abstract The differentiation of sensory and support cells within the embryonic chick otocyst is accompanied by alterations in the distribution of preexisting intercellular junctions. Prior to innervation of this epithelium, tight, gap and adhering junctions exist between all cells. Upon differentiation of the epithelium, apical bands of tight and adhering junctions are maintained throughout, while gap junctions and desmosomes are found only between support cells. Thus, some of the gap junctions that join homogeneous epithelial cells prior to innervation are removed as sensory cells differentiate, and a separate population of very large gap junctions is formed between differentiating support cells. Morphological evidence suggests two possible mechanisms which may be responsible for the observed changes in gap junctional distribution: removal of gap junctions by internalization, and formation of gap junctions by aggregation of precursor particles. The temporal correlation between junctional modulation, cytological differentiation of sensory and support cells, and ingrowth of nerve fibers makes the latter event a likely developmental cue for differentiation of this epithelium.

Synaptogene sis in the vestibular sensory epithelium of the chick embryo

SummaryThe formation of synapses between sensory cells and the terminals of afferent axons has been examined in the embryonic chick labyrinth. Neurites initially cross the otocyst basal lamina and ramify among the undifferentiated epithelial cells by stage 25 of Hamburger and Hamilton. At the same time granular vesicles, with diameters averaging 130 nm, appear in the basal cytoplasm of a few of the epithelial cells. These vesicles often exist in groups at sites of contact with neuntes. By stages 27–28, non membrane-bound densities are frequently found in association with groups of granular vesicles at the plasma membrane. Smaller, clear synaptic vesicles are also a prominent component of these arrangements in presumptive hair cells. Presynaptic ribbons opposite postsynaptic densites are identifiable at about stage 28, and their number increases during subsequent embryonic stages. Specialized appositions, including adherent, postsynaptic and possibly gap junctional contacts, join epithelial cells and nerve terminals throughout this period. The distribution of these junctions is variable, and is not necessarily correlated with the sites of formation of presynaptic ribbons. By stage 32, well-developed chemical synapses consisting of presynaptic ribbons with vesicle halos and postsynaptic densities are common features of hair cell-afferent nerve terminal contact regions. In addition, possible sites of gap junctional contact between adjacent intra-epithelial nerve endings found at stage 32 presage those found in the cristae and maculae of pre-hatch (stage 45) embryos and adults.

PCC4azal teratocarcinoma stem cell differentiation in culture: I. Biochemical studies

Abstract PCC4azal embryonal carcinoma cells were observed to spontaneously differentiate under defined culture conditions to endoderm-like cells and subsequently to giant cells. This differentiation was examined by determining the specific activities of several enzymes in the stem and endoderm-like cell populations. With differentiation, the level of alkaline and acid phosphatase activities remained unchanged, plasminogen activator specific activity increased fivefold, and lactate dehydrogenase (LDH) specific activity decreased to 40% of its original level. Isozyme analysis revealed a shift of the LDH isozymes toward LDH1 with the appearance of LDH2 for the first time in the endoderm-like cells. The surface antigen SSEA-1 was detected by indirect immunofluorescence on virtually all of the stem cells. However, the SSEA-1 antigen was not present on many of the endoderm-like cells, and it was completely undetectable on giant cells as assayed by immunofluorescence. The expression of H-2 antigen was examined in a similar manner using anti-H-2b antiserum; this antigen was not detected on the stem, endoderm-like, or giant cells. Thus, there are defined biochemical changes that accompany the differentiation of PCC4azal stem cells in culture.

Cell-to-Cell Communication and Development

Publisher Summary This chapter discusses cell-to-cell communication and development. Cell-to-cell communication can be defined as the transfer of low-molecular weight substances from cell-to-cell via a specialized low-resistance pathway. The low-resistance pathway appears to be a cell surface membrane specialization that is called the gap junction or nexus. In essence, junctional pathways are established between cells to permit the exchange of small cytoplasmic molecules or to facilitate the propagation of an action potential from cell-to-cell. The chapter presents a few examples of this modulation that have been studied in laboratory. There has been considerable progress in defining the property of cell-to-cell communication in many different tissues and organisms. Although most of the information has been descriptive, it provides an overall framework for beginning to understand the potential regulatory role of communication in development and differentiation. Recent studies focused on the communication patterns during specified events in differentiation provide access to understanding the way by which communication might perform its functions and the way by which it may be modulated. Cell-to-cell communication property appears to be expressed by almost all cells in both vertebrate and invertebrate organisms.

PCC4azal teratocarcinoma stem cell differentiation in culture: II. Morphological characterization

Abstract The ultrastructural morphology of the PCC4azal embryonal carcinoma cells and their differentiated counterparts, endoderm-like cells and giant cells, was characterized and compared with that of the cells of embryoid bodies. The ultrastructure of the PCC4azal embryonal carcinoma cells is similar to that of the embryonal carcinoma cells of the embryoid body. These cells are small, with a large nucleus and relatively few cytoplasmic organelles. Gap junctions and modified adherens junctions are formed at some areas of intercellular contact between the embryonal carcinoma cells. The differentiated PCC4azal endoderm-like cells have a more developed cytoplasm, containing an extensive endoplasmic reticulum with large Golgi regions. Most striking is the de novo appearance of epithelial-like junctional complexes which join the apical borders between the endoderm-like cells, thus polarizing the cell monolayer. The zonula occludens junctions of the junctional complex are extensive, consisting of six or more strands of tight junctional ridges. Terminal webs are present in the apical regions that are inserted into the zonula adherens region of the junctional complex. Gap junctions continue to join neighboring cells, and some gap junctions are intercalated within tight junctional ridges. The ultrastructure of the differentiated endodermal cells of the embryoid bodies is very similar to that of the PCC4azal endoderm-like cells. The embryoid body endodermal cells form similar junctional complexes which also contain continuous belts of tight junctions that are intercalated with gap junctions. As the PCC4azal endoderm-like cells are transformed to giant cells, a massive cytoskeleton is formed, consisting of a large complex system of 10-nm filaments, microtubules, and 7-nm microfilaments. The junctional complexes that were present during the endodermal stage are partially disassembled as the giant cells migrate apart. Thus, the differentiation process in this system is characterized by significant and distinctive morphological changes.

PCC4azal teratocarcinoma stem cell differentiation in culture: III. Cell-to-cell communication properties

Abstract The cell-to-cell communication properties of the PCC4azal embryonal carcinoma cells and their differentiated endoderm-like cells and giant cells were characterized by ionic coupling, metabolic coupling, and transfer of an injected fluorescein dye. All PCC4azal cell types communicate well with one another: stem cells with stem cells, endoderm-like cells with endoderm-like cells, and giant cells with giant cells. In addition, the stem cells communicate well with giant cells as assayed by metabolic coupling. The ability of the undifferentiated teratocarcinoma cells to metabolically couple with a variety of heterologous cell types was examined. The stem cells were always efficient donors but very poor recipients in almost all combinations with cells of fibroblast or epithelial morphology that were derived from several different species. In contrast, these same heterologous cell types were both efficient donors and efficient recipients with each other.

Molecular Events in Membrane Fusion Occurring During Mast Cell Degranulation

Mast cells provide an unusually attractive system for considering the molecular events involved in membrane fusion. When either antigen, (1) anti-immunoglobulin (Ig) antibody (2) or concanavalin A (con A) (3,11) bind to and cross link cytophilic IgE (9,11,13) on the surface of sensitized mast cells in the presence of extracellular Ca2+ (7), they induce exocytotic histamine release (degranulation) within seconds (l6). Degranulation involves the fusion of granule membranes with plasma membrane (and subsequently with other granules) followed by the opening of the granule contents to the extracellular space (3,12). Histamine contained in the granules is released by a process of cation exchange; histamine bound to granule matrix exchanges mainly with extra-cellular Na+ (16). Histamine release leads to easily recognisable ultra-structural changes in the granules, including loss of electron density and homogeneity, and an increase in size (3,4,12). Since the cells degranulate all over their surface there is always an extensive amount of membrane interaction and fusion taking place. They are, therefore, an excellent system in which to study the molecular events that occur during membrane fusion.