Gap junctions are clustered channels between contacting cells through which direct intercellular communication via diffusion of ions and metabolites can occur. Two hemichannels, each built up of six connexin protein subunits in the plasma membrane of adjacent cells, can dock to each other to form conduits between cells. We have recently screened mouse and human genomic data bases and have found 19 connexin (Cx) genes in the mouse genome and 20 connexin genes in the human genome. One mouse connexin gene and two human connexin genes do not appear to have orthologs in the other genome. With three exceptions, the characterized connexin genes comprise two exons whereby the complete reading frame is located on the second exon. Targeted ablation of eleven mouse connexin genes revealed basic insights into the functional diversity of the connexin gene family. In addition, the phenotypes of human genetic disorders caused by mutated connexin genes further complement our understanding of connexin functions in the human organism. In this review we compare currently identified connexin genes in both the mouse and human genome and discuss the functions of gap junctions deduced from targeted mouse mutants and human genetic disorders.

Self-renewal of rodent embryonic stem (ES) cells is enhanced by partial inhibition of glycogen synthase kinase-3 (Gsk3)1 2. This effect has variously been attributed to stimulation of Wnt signalling via β-catenin1, stabilisation of cMyc3, and global de-inhibition of anabolic processes4. Here we demonstrate that β-catenin is not necessary for ES cell identity or expansion, but its absence eliminates the self-renewal response to Gsk3 inhibition. Responsiveness is fully restored by truncated β-catenin lacking the C-terminal transactivation domain5. However, requirement for Gsk3 inhibition is dictated by expression of Tcf3 and mediated by direct interaction with β-catenin. Tcf3 localises to many pluripotency genes6 in ES cells. Our findings confirm that Tcf3 acts as a transcriptional repressor and reveal that β-catenin directly abrogates Tcf3 function. We conclude that Gsk3 inhibition stabilises the ES cell state primarily by reducing repressive influence on the core pluripotency network.

Background-Connexin 43 (Cx43) is a major determinant of conduction in the ventricular working myocardium of mammals. We investigated the effect of decreased Cx43 expression on conduction velocity and arrhythmogenesis using adult mice with inducible deletion of Cx43. Methods and Results-Cx43Cre-ER(T)/ϩ mice, in which 1 coding region of the Cx43 gene was replaced by Cre-ER(T), were mated to Cx43 fl/fl mice, generating Cx43 Cre-ER(T)/fl mice. Application of 4-hydroxytamoxifen (4-OHT) induced Cre-ER(T)-mediated deletion of the floxed Cx43 allele. Epicardial ventricular mapping using a 13ϫ19 multiterminal electrode grid (300-m spacing) was performed on Langendorff-perfused hearts from Cx43 fl/fl plus carrier (nϭ10), Cx43 fl/fl plus 4-OHT (nϭ10), Cx43Cre-ER(T)/fl plus carrier (nϭ9), and Cx43Cre-ER(T)/fl plus 4-OHT (nϭ10). Cx43 protein amount in group 3 hearts was decreased by Ϸ50% compared with group 1. 4-OHT did not affect cardiac protein amounts in group 2 but decreased Cx43 expression up to 95% in group 4 compared with group 3. Epicardial activation of both left ventricle (LV) and right ventricle (RV) during sinus rhythm was similar in all groups. Conduction velocity (CV) changed only in group 4 animals. For RV (LV), longitudinal CV decreased from 38 (35) to 31.6 (33.6) and transverse CV from 24.4 (16.8) to 10.1 (11.3) cm/s. Dispersion of conduction in RV (LV) was increased by 91% (38%). Programmed stimulation resulted in ventricular arrhythmias in group 4 (7 of 10 mice) but never in groups 1 through 3. Conclusions-Heterozygous expression of Cx43 did not affect ventricular conduction velocity. Up to 95% decrease of Cx43 protein in 4-OHT-treated Cx43 Cre-ER(T)/fl mice reduced conduction velocity and increased dispersion of conduction and propensity for ventricular arrhythmias.

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye cou- inflammation ͉ macrophage ͉ connexin ͉ cytokines ͉ dye coupling

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

We have previously demonstrated that differentiation of embryonic stem (ES) cells is associated with downregulation of cell surface E-cadherin. In this study, we assessed the function of E-cadherin in mouse ES cell pluripotency and differentiation. We show that inhibition of E-cadherin-mediated cell-cell contact in ES cells using gene knockout (Ecad , EcadRNAi, and CHAVC-treated ES cells to the activin receptor-like kinase inhibitor SB431542 led to differentiation of the cells, which could be prevented by re-expression of E-cadherin. To confirm the role of transforming growth factor b family signaling in the self-renewal of Ecad 2/2 ES cells, we show that these cells maintain an undifferentiated phenotype when cultured in serum-free medium supplemented with Activin A and Nodal, with fibroblast growth factor 2 required for cellular proliferation. We conclude that transhomodimerization of E-cadherin protein is required for LIF-dependent ES cell selfrenewal and that multiple self-renewal signaling networks subsist in ES cells, with activity dependent upon the cellular context.

To analyze the effect of connexin loss on the repair of wounded tail skin,we have studied the following transgenic mouse mutants: connexin30–/–, connexin31–/– and connexin43Cre-ER(T)/fl (for inducible deletion of the connexin43 coding region). Connexin43 and connexin31 are expressed in the basal and spinous layers of wild-type epidermis, whereas connexin31 and small amounts of connexin30, as well as connexin26 proteins,were found in the granulous layer. Connexin43 was downregulated in connexin31-deficient mice, whereas mice with reduced connexin43 exhibited an upregulation of connexin30. During wound healing, connexin30 and connexin26 proteins were upregulated in all epidermal layers, whereas connexin43 and connexin31 protein expression were downregulated. In connexin31–/– mice, reduced levels of connexin30 protein were observed on days 1 and 2 after wounding. The closure of epidermal wounds in mice with decreased amounts of connexin43 protein occurred one day earlier. Under these conditions the expression profiles of connexin30 and connexin31 were also temporarily shifted by one day. Furthermore, dye transfer between keratinocytes in skin sections from connexin43-deficient mice was decreased by 40%. These results suggest that downregulation of connexin43 appears to be a prerequisite for the coordinated proliferation and mobilization of keratinocytes during wound healing.

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