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G M Kidder

Publications and source records attributed to G M Kidder.

At least 19 recordsLinked to original sources
PubMed1992

Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein.

To examine the role of gap-junctional intercellular communication in controlling cell proliferation, we have transfected C6 glioma cells with connexin 43 cDNA. The growth of transfected clones was dramatically reduced compared with nontransfected glioma cells. To further characterize the role of gap junctions in controlling proliferation, we have examined the growth of C6 cells cocultured with transfected cells overexpressing connexin 43. Although C6 cells grew at their normal rate when cocultured with nontransfected C6 cells, when cocultured with connexin 43-overexpressing cells they displayed a dramatic reduction in growth rate. Furthermore, a significant, dose-dependent reduction in cell proliferation was noted when C6 cells were cultured in medium conditioned by transfected cells. This effect correlated with the level of connexin 43 expression. These results suggest that the decreased cell proliferation rate of transfected cells and C6 cells cultured with them is due to the secretion of a growth inhibitory factor(s) and that the secretion of this factor may be linked to the level of gap junctional intercellular communication.

Animals↗
PubMed1992

The genetic program for preimplantation development.

This review summarizes information on accumulation profiles of individual gene transcripts in preimplantation development. Most of the information is from the mouse, but some data from other species are reviewed as well. The principal finding is that the transcription of most genes is not temporally linked with any of the three morphogenetic transitions (compaction, cavitation, and blastocoel expansion) that characterize this period. Most genes that are expressed during preimplantation development of the mouse are already being transcribed in the 4-cell stage, and some clearly begin as early as the 2-cell stage. Once activated, a gene continues to be transcribed at least into the blastocyst stage, resulting in continuous mRNA accumulation. Thus the pattern of gene transcription established at the time of genomic activation in the 2-cell stage is perpetuated into the blastocyst, with a few additions along the way. This information is interpreted in light of previous findings concerning the sensitivity of morphogenetic transitions to inhibition of gene expression. The lack of a clear relationship between the timing of expression of most genes and the schedule of morphogenesis leads one to conclude that temporal regulation is imposed downstream of transcription and translation. This conclusion is substantiated by a consideration of factors controlling the events of compaction.

Animals↗
PubMed1992

Characteristics of C6 glioma cells overexpressing a gap junction protein.

1. C6 glioma cells transfected with connexin43 cDNA display a dramatic increase in the level of connexin43 mRNA and protein. 2. This overexpression of connexin43 is evident at the cellular level, as revealed with in situ hybridization and immunocytochemistry. Transfection with connexin43 cDNA also induced actin stress fibers in these glioma cells. 3. Although we observed up to a 50-fold increase in the level of connexin43 mRNA following transfection, virtually all of this mRNA was present in the polysomal fraction. 4. Overexpression of connexin43 mRNA did not appear to compete with other cellular mRNAs for access to the translational machinery. 5. It is likely that the reduced proliferation rate of the transfected cells, reported earlier, is due to enhanced connexin43 expression and intercellular coupling.

Animals↗
PubMed1992

Intercellular calcium signaling via gap junctions in glioma cells.

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.

Calcium↗
PubMed1991

Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation.

C6 glioma cells express low levels of the gap junction protein connexin 43 and its mRNA and display very weak dye coupling. When implanted into the rat cerebrum, these cells quickly give rise to a large glioma. To investigate the role of gap junctions in the tumor characteristics of these cells, we have used Lipofectin-mediated transfection to introduce a full-length cDNA encoding connexin 43. Several transfected clones were obtained that exhibited various amounts of connexin 43 mRNA transcribed from the inserted cDNA. Immunocytochemical analysis revealed an increase in the amount of connexin 43 immunoreactivity in the transfected cells, being localized at areas of intercellular contact as well as in the cytoplasm. The level of dye coupling was also assessed and found to correlate with the amount of connexin 43 mRNA. When cell proliferation was followed over several days, cells expressing the transfected cDNA grew more slowly than non-transfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.

Animals↗
PubMed1991

Expression of gap junction genes during postnatal neural development.

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.

Animals↗
PubMed1991

Zygotic expression of the connexin43 gene supplies subunits for gap junction assembly during mouse preimplantation development.

De novo assembly of gap junctions begins during compaction in the eight-cell stage of mouse development, and intercellular coupling mediated by gap junctions appears to be required for maintenance of the compacted state. We have begun to explore the expression of the family of genes encoding the connexins, the proteins that form the gap junction channels. We recently reported that a protein with antigenic and size similarity with connexin32, the rat liver gap junction protein, is inherited as an oogenetic product by the mouse zygote, but its gene appears not to be transcribed prior to implantation (Barron et al., Dev Genet 10:318-323, 1989). Here we report that another member of this gene family, connexin43, is transcribed by the embryonic genome from shortly after the time of genomic activation. As revealed by Northern blotting, connexin43 mRNA is absent from ovulated oocytes, becomes detectable in the 4-cell stage, and accumulates steadily thereafter to reach a maximum in blastocysts. In contrast, no transcripts of connexin26 could be detected in any preimplantation stage. A protein with antigenic and size similarity with connexin43 from rat heart was found by Western blotting to accumulate from the four-cell stage onward. Immunofluorescence analysis with embryo whole mounts was used to demonstrate that this protein is incorporated into punctate interblastomeric foci during compaction, consistent with its assembly into gap junction plaques. We conclude that connexin43 is one member of the connexin gene family whose zygotic expression is critical for preimplantation morphogenesis.

Animals↗
PubMed1991

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.

Animals↗
PubMed1990

Cell polarity and development of the first epithelium.

In the 4 1/2 to 5 days between fertilization and implantation, the mouse conceptus must gain the abilities to implant and produce an embryo. Each of these is the sole developmental responsibility of one of two cell types forming the blastocyst, trophectoderm and inner cell mass (ICM), respectively. Trophectoderm is a polarized transporting epithelium while the ICM is an aggregate of non-epithelial pluripotent stem cells. These two cell types originate from the division of polar blastomeres when their cleavage furrows parallel their apical surfaces. Blastomeres polarize in response to asymmetric cell--cell contact, and understanding the mechanism of this induction is regarded as the key to understanding the origin of trophectoderm and ICM. Here we propose a model based on transcellular ion current loops for the induction of cell polarity during the development of the first epithelium, trophectoderm.

Animals↗
PubMed1990

Expression of Na,K-ATPase alpha and beta subunit genes during preimplantation development of the mouse.

Na,K-ATPase is a plasma membrane enzyme that plays a critical role in eutherian blastocoel formation (cavitation) by pumping Na+ into the extracellular space enclosed by the trophectoderm. Previous experiments with the mouse had shown that the alpha (catalytic) subunit of the enzyme becomes detectable by immunocytochemistry in the late morula, just prior to the onset of cavitation. In the present study we have used cDNAs corresponding to three mRNA isoforms of the alpha subunit and a beta subunit to determine which genes are expressed during preimplantation development and to explore the timing of their expression. Of the three alpha subunit cDNAs tested by Northern blot hybridization with blastocyst RNA, only alpha 1 produced a hybridization signal, recognizing a single mRNA about 4 kb in length. This mRNA is relatively abundant in zygotes but barely detectable by the 2-cell stage and then accumulates steadily thereafter to reach its preimplantation maximum in blastocysts. The beta 1 cDNA detected mRNA of about 2.6-2.8 kb. This mRNA is present in zygotes but could not be detected in 2-, 4-, or 8-cell stages; it is present at a low level in late morulae and is abundant in blastocysts. The temporal profile of accumulation of beta 1 mRNA thus matches more closely than does alpha 1 the timing of appearance of the catalytic subunit. This suggests that the beta subunit may regulate production of the holoenzyme and hence the timing of cavitation.

Animals↗
PubMed1990

Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm.

Na+,K(+)-ATPase is a marker of the basolateral plasma membrane domain of polarized epithelial cells, including the mural trophectoderm of the mammalian blastocyst (Watson and Kidder (1988). Dev. Biol. 126, 80-90). We have used this marker to explore the factors governing the establishment and maintenance of apical/basolateral polarity during differentiation of trophectoderm. A polyclonal antiserum (anti-GP80) against human cell-CAM 120/80, a homolog of the mouse cell-cell adhesion protein, uvomorulin, was used to prevent cell flattening (compaction) and formation of the epithelial junctional complex. The majority of treated embryos failed to develop a blastocoel; instead their blastomeres developed fluid-filled cavities that expanded while untreated control embryos were cavitating. Immunocytochemistry revealed that the catalytic subunit of Na+,K(+)-ATPase was contained within the membranes lining these cavities, as well as within numerous punctate foci in the cytoplasm. The down-regulation of expression of the enzyme that normally occurs in the ICM and polar trophectoderm did not take place, since the immunoreactivity remained equally strong in all blastomeres. The enzyme could not be detected in plasma membranes. We conclude that uvomorulin-mediated cell adhesion is involved in spatially restricting the expression of the catalytic subunit and is a prerequisite for the insertion of enzyme-laden vesicles into plasma membranes, but not for expression of the catalytic subunit gene. When fully developed blastocysts were treated with cytochalasins to disrupt the epithelial junctional complex, the catalytic subunit shifted from the basolateral to the apical plasma membrane. This finding suggests a primary role for the apical plasma membrane in the process of polarization, and implies that tight junctions are a manifestation of polarity that serve to maintain the separation between apical and basolateral markers.

Animals↗
PubMed1989

Connexin32, a gap junction protein, is a persistent oogenetic product through preimplantation development of the mouse.

Gap junctions appear de novo during compaction in the eight-cell stage of mouse development. This is a critical event in the life of the embryo, because gap junctional intercellular communication is an essential requirement for maintaining compaction and, hence, for development of the blastocyst. Recently, a family of genes encoding gap junction proteins (connexins) has been identified and cloned, and we have taken advantage of the availability of antibodies and cDNA probes to investigate the expression of these genes in early development. We found that a protein with antigenic and size similarity to the "liver" gap junction protein, connexin32, is present throughout preimplantation development from the zygote through the late morula. Connexin32 mRNA, however, could not be detected in any preimplantation stage. This, and the presence of connexin32 in zygotes before activation of embryonic transcription, leads us to conclude that this protein is inherited as an oogenetic product that persists well beyond the transition from the oogenetic to embryonic program of gene expression. Furthermore, we found that mRNA for another gap junction protein, connexin43, is fairly abundant in preimplantation embryos. We conclude that it is more likely connexin43, and not connexin32, that is used to assemble new connexons as the level of intercellular coupling increases after compaction.

Animals↗
PubMed1988

Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis.

We have employed immunofluorescence with a rat kidney Na+/K+-ATPase polyclonal antibody to investigate the cellular distribution and timing of appearance of this enzyme during preimplantation development. The enzyme is first detected in the late morula within the cytoplasm of each blastomere. When cavitation begins this distribution changes dramatically to a ring encircling the blastocoel, restricted to the basolateral cell margins. Using this enzyme as a marker for cavitation, we examined its expression in embryos that had been treated with wheat germ agglutinin (WGA), which causes cleavage arrest and was reported to trigger premature compaction- and cavitation-like events in early cleavage stages (L. V. Johnson, 1986, Dev. Biol. 113, 1-9). Although WGA-treated 2-,4-, and 8-cell embryos quickly underwent compaction- and cavitation-like events, no Na+/K+-ATPase expression was observed. Thus the WGA effect does not likely involve acceleration of the developmental program for cavitation. Embryos arrested at the 8-cell stage but cultured overnight to Day 4, however, expressed the enzyme in the typical blastocyst pattern (around each fluid-filled cavity). We conclude that Na+/K+-ATPase expression is initiated or increases dramatically in the late morula and is independent of cytokinesis. The enzyme assumes a distribution during cavitation consistent with its presumed role in transtrophectodermal fluid transport.

Animals↗
PubMed1987

Gap junction assembly in the preimplantation mouse conceptus is independent of microtubules, microfilaments, cell flattening, and cytokinesis.

Gap junctions first appear during compaction in the eight-cell stage of mouse development. Their assembly can be initiated in the near absence of transcription and protein synthesis from the four-cell stage, indicating the existence of preformed precursors. We have investigated the temporal control of this event, focusing on the possible involvement of the cytoskeleton, cell flattening, and cytokinesis. Embryos in various cleavage stages were treated with cytochalasins, to disrupt microfilaments and block cell flattening, cytokinesis, or both, or nocodazole, to promote microtubule depolymerization. To assess their capacity to initiate gap junction assembly after such treatments, the embryos were then aggregated with communication-competent, compacted embryos that had been labeled with carboxyfluorescein diacetate. Passage of the fluorescent dye, carboxyfluorescein, from labeled to unlabeled embryo was taken as evidence that interembryonic junction formation had occurred. The capacity to assemble gap junctions was acquired at the normal time by embryos prevented by cytochalasin treatment from undergoing cell flattening or any cytokinesis from fertilization onward. Likewise, treatment with nocodazole beginning in the four-cell or early eight-cell stage did not interfere with gap junction assembly. Neither drug affected the inability of four-cell embryos to assemble gap junctions prematurely. We conclude that intact microfilament or microtubule networks are not required for gap junction assembly in this system, nor do they restrain junctional precursors from assembling prematurely. Furthermore, the timing of gap junction assembly is not linked to cell flattening, cytokinesis, or cell number.

Actin Cytoskeleton↗
PubMed1986

Intercellular junctional coupling in preimplantation mouse embryos: effect of blocking transcription or translation.

Gap junction formation in cleavage stage mouse embryos was examined by testing for ionic coupling or by observing the intercellular movement of the fluorescent dye, Lucifer yellow CH. Our results confirm that embryo-wide cell coupling, mediated by cell-to-cell membrane channels (gap junctions), is acquired in the 8-cell stage after compaction has begun. However, not all partially compacted embryos were found to be ionically or dye coupled, suggesting that the initiation of gap junction assembly is not necessarily triggered by the onset of cell flattening. The rate of fluorescent dye movement throughout the embryo was found to increase as embryos proceed through compaction and beyond, indicating that the number of gap junctional channels between blastomeres increases as development progresses. The inhibitors alpha-amanitin and cycloheximide were used to assess the requirement of new transcription and protein synthesis, respectively, for the onset of intercellular coupling and its progressive increase during compaction. Treatment conditions were chosen to bring about suppression of mRNA and protein synthesis within 2 hr. Ionic coupling was detected in almost all compacted 8-cell embryos treated with either inhibitor from the 4-cell stage. On the other hand, dye coupling was weak or undetectable in such embryos. We propose that a limited supply of junctional components is present by the 4-cell stage to serve as a pool of precursors for the first gap junctions to be assembled in the 8-cell stage. However, it is apparent that continued embryonic gene expression is required for the full extent of junctional coupling to be established.

Amanitins↗
PubMed1985

On the use of alpha-amanitin as a transcriptional blocking agent in mouse embryos: a cautionary note.

We have tested the effect of alpha-amanitin at 10, 50 and 100 micrograms/ml, on precursor uptake and incorporation into poly(A)+ RNA and poly(A)- RNA of mouse embryos on days 2, 3 and 4 of gestation. Embryos were pretreated with the inhibitor for 2 hr, then labeled for 2 hr in its continued presence. RNA fractions were separated by affinity chromatography on oligo(dT)-cellulose. alpha-Amanitin did not suppress uptake of RNA precursors at any of the concentrations tested in any stage. At 10 micrograms/ml, we could not detect any effect on incorporation into either RNA fraction in any stage. Only the highest concentration tested, 100 micrograms/ml, was effective in all stages in substantially suppressing incorporation into poly(A)+ RNA within 2 hr. Longer treatments increased the level of suppression to a maximum of about 80%. Incorporation into poly(A)- RNA was suppressed to roughly the same extent. Despite previously reported data, it cannot be assumed that alpha-amanitin at concentrations less than 100 micrograms/ml brings about a quick interruption of mRNA synthesis in preimplantation mouse embryos.

Adenosine↗
PubMed1985

Timing of transcription and protein synthesis underlying morphogenesis in preimplantation mouse embryos.

During preimplantation development of the mouse, embryos pass through a series of morphogenetic events: compaction, fluid accumulation to form the blastocoele (cavitation), and escape from the zona pellucida (hatching). We have used the inhibitors alpha-amanitin and cycloheximide to investigate the timing of transcriptional and translational events underlying these morphogenetic stages. Groups of embryos were transferred from a common pool into medium containing one or the other inhibitor at regular time intervals, and then were scored over the ensuing 24 or more hours for their ability to reach a particular morphogenetic end point. By comparing the time when the control population reached an end point with the time at which embryos had to be transferred into the inhibitor in order to prevent them from reaching that end point, we could determine when in advance of each event the necessary transcription or protein synthesis has been completed. Our results suggest that compaction (as well as cleavage to the eight-cell stage) is an embryonically, rather than maternally, programmed event, although the necessary transcription is completed well in advance, at least by the early four-cell stage. The transcriptional and translational events underlying fluid accumulation, on the other hand, appear to be completed within a few hours of the start of this process. For hatching, there is once again a long delay between the apparent time of completion of the necessary transcriptional events and the process itself, with protein synthesis being completed just a few hours in advance. Our results raise the possibility that post-transcriptional regulatory mechanisms play an important role in the timing of morphogenetic events in early mouse embryos.

Amanitins↗