Normal embryonic development and cardiac morphogenesis in mice with Wnt1-Cre-mediated deletion of connexin43.

Mice harboring a null mutation in the gap junction protein connexin43 (Cx43) die shortly after birth due to an obstruction of the right ventricular outflow tract of the heart. These hearts exhibit prominent pouches at the base of the pulmonary outlet, i.e., morphological abnormalities that were ascribed to Cx43-deficiency in neural crest cells. In order to examine the Cx43 expression pattern in neural crest cells and derived tissues and to test whether neural crest-specific deletion of Cx43 leads to the conotruncal defects seen in Cx43null mice, we ablated Cx43 using a Wnt1-Cre transgene. Deletion of Cx43 was complete and occurred in neural crest cells as well as in neural crest-derived tissues. Nevertheless, hearts of mice lacking Cx43 specifically in neural crest cells were indistinguishable from controls. Thus, the morphological heart abnormalities of Cx43 null mice are most likely not caused by lack of Cx43 in neural crest cells.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-gamma and tumor necrosis factor-alpha.

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 coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-gamma (INF-gamma) or tumor necrosis factor-alpha (TNF-alpha) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-gamma plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-alpha antibody, suggesting the release and autocrine action of TNF-alpha. Treatment with INF-gamma plus TNF-alpha also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell-cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18 alpha-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-gamma plus LPS or INF-gamma plus TNF-alpha. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene.

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.