Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an Nethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

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 nontransfected cells. These transfected cells will be useful in examining the role of gap junctions in tumorigenesis.Intercellular communication by gap junctions has been implicated in the control of cell growth and differentiation (1, 2). These intramembranous channels, or connexons, are composed of hexameric assemblies of the gap junction proteins connexins. Isolation ofconnexins and cloning oftheir cDNAs indicate that they are encoded by a family of genes that exhibit some degree of cellular and tissue specificity (3, 4).C6 glioma cells exhibit a low level of intercellular communication by gap junctions, as shown by morphological and functional studies (5-7). Consistent with these observations, these cells contain low levels of the same gapjunction mRNA and protein that is expressed in astrocytes, namely connexin 43 (6, 8). Since tumorigenicity has been correlated with a low level or absence of intercellular communication by gap junctions (1, 9), the low level of connexin 43 mRNA and protein in C6 glioma cells may relate to the invasive growth of these cells in vivo (10).As an initial step to understand the role of gap junctions in normal and abberant cellular growth, we sought to alter the expression of connexin 43 in C6 glioma cells. To this end, we transfected these cells with a full-length cDNA for connexin 43 (11). We have obtained several clones that transcribe the transfected cDNA at moderate to high levels. Whereas connexin 43-like immunoreactivity is not readily detectable in nontransfected cells, such reactivity is prevalent in these transfected cells. The level of intercellular dye coupling correlates with the relative level of connexin 43 mRNA. Furthermore, the growth rate of the glioma cells expressing the transfected connexin 43 cDNA was significantly lower than that of nontransfected C6 cells. MATERIALS AND METHODSExpression Vector. The plasmid used for expression of the connexin 43 cDNA in rat C6 glioma cells was derived from pLTR ( Fig. 1), a simian virus 40 (SV40)-based vector, which includes the enhancer portion of the Harvey sarcoma virus long terminal re...

The ovarian follicle in mammals is a functional syncytium, with the oocyte being coupled with the surrounding cumulus granulosa cells, and the cumulus cells being coupled with each other and with the mural granulosa cells, via gap junctions. The gap junctions coupling granulosa cells in mature follicles contain several different connexins (gap junction channel proteins), including connexins 32, 43, and 45. Connexin43 immunoreactivity can be detected from the onset of folliculogenesis just after birth and persists through ovulation. In order to assess the importance of connexin43 gap junctions for postnatal folliculogenesis, we grafted ovaries from late gestation mouse fetuses or newborn pups lacking connexin43 (Gja1(-)/Gja1(-)) into the kidney capsules of adult females and allowed them to develop for up to 3 weeks (this was necessitated by the neonatal lethality caused by the mutation). By the end of the graft period, tertiary (antral) follicles had developed in grafted normal (wild-type or heterozygote) ovaries. Most follicles in Gja1(-)/Gja1(-) ovaries, however, failed to become multilaminar, with the severity of the effect depending on strain background. Dye transfer experiments indicated that intercellular coupling between granulosa cells is reduced, but not abolished, in the absence of connexin43, consistent with the presence of additional connexins. These results suggest that coupling between granulosa cells mediated specifically by connexin43 channels is required for continued follicular growth. Measurements of oocyte diameters revealed that oocyte growth in mutant follicles is retarded, but not arrested, despite the arrest of folliculogenesis. The mutant follicles are morphologically abnormal: the zona pellucida is poorly developed, the cytoplasm of both granulosa cells and oocytes is vacuolated, and cortical granules are absent from the oocytes. Correspondingly, the mutant oocytes obtained from 3-week grafts failed to undergo meiotic maturation and could not be fertilized, although half of the wild-type oocytes from 3-week grafted ovaries could be fertilized. We conclude that connexin43-containing gap junction channels are required for expansion of the granulosa cell population during the early stages of follicular development and that failure of the granulosa cell layers to develop properly has severe consequences for the oocyte.

The connexins are a family of at least 15 proteins that form the intercellular membrane channels of gap junctions. Numerous connexins, including connexin43 (Cx43), have been implicated in reproductive processes by virtue of their expression in adult gonads. In the present study, we examined the gonads of fetal and neonatal mice homozygous for a null mutation in the Gja1 gene encoding Cx43 to determine whether the absence of this connexin has any consequences for gonadal development. We found that in both sexes at the time of birth, the gonads of homozygous mutants were unusually small. This appears to be caused, at least in part, by a deficiency of germ cells. The germ cell deficiency was traced back as far as Day 11.5 of gestation, implying that it arises during early stages of germ line development. We also used an organ culture technique to examine postnatal folliculogenesis in the mutant ovaries, an approach necessitated by the fact that Gja1 null mutant offspring die soon after birth because of a heart abnormality. The results demonstrated that folliculogenesis can proceed to the primary (unilaminar) follicle stage in the absence of Cx43 but that subsequent development is impaired. In neonatal ovaries of normal mice, Cx43 could be detected in the somatic cells as early as Day 1, when primordial follicles begin to appear, supporting the conclusion that this connexin is required for the earliest stages of folliculogenesis. These results imply that gap junctional coupling mediated by Cx43 channels plays indispensable roles in both germ line development and postnatal folliculogenesis.

Abstract. 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. E Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36

Female fertility is determined to a large extent by the quality (developmental competence) of the oocyte as reflected in its ability to undergo meiosis, be fertilized, and give rise to a healthy embryo. Growth of the mammalian oocyte is coordinated with that of the follicle that encloses it by the actions of signals that pass in both directions between the germline and somatic components. This review summarizes what is known about the roles played by two different modes of intrafollicular signalling in oogenesis: paracrine factors activating receptors on the opposite cell type, and direct sharing of small molecules throughout the follicle via gap junction channels. Recent evidence indicates that these two modes of signalling interact to regulate oocyte growth and granulosa cell proliferation, and that defects in either can contribute to female infertility.

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.