Using a human glial fibrillary acidic protein (hGFAP) promoter-driven cre transgene, we have achieved efficient inactivation of a floxed connexin43 (Cx43) gene in astrocytes of adult mice. The loss of Cx43 expression was monitored in a cell-autonomous manner via conditional replacement of the Cx43-coding region by a lacZ reporter gene. In this way, we bypassed the early postnatal lethality previously reported for Cx43 null mice and characterized the phenotypic consequences of Cx43 deficiency in the CNS. Mice lacking Cx43 in astrocytes were viable and showed no evidence of either neurodegeneration or astrogliosis. Spreading depression (SD) is a pathophysiological phenomenon observed in the CNS that is characterized by a propagating wave of depolarization followed by neuronal inactivation. Inhibitors of gap junctional communication have previously been shown to block initiation and propagation of SD. In contrast, we observed an increase in the velocity of hippocampal SD in the stratum radiatum of mice lacking Cx43 in astrocytes. In the same brain subregion, dye-coupling experiments revealed a reduction in overall astrocytic intercellular communication by approximately 50%. This strongly suggests separate and different neuronal and glial contributions of gap junctional intercellular communication to SD. Concomitant with increased velocity of spreading depression, we observed enhanced locomotory activity in mice lacking Cx43 in astrocytes.

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.

Gap junctions are characteristically increased in the myometrium during term and preterm delivery and are thought to be essential for the development of uterine contractions during labour. Expression of connexin43 (Cx43), the major myometrial gap junction protein, is increased during delivery. We have generated a mouse mutant (Cx43fl/fl:SM-CreERT2), in which the coding region of Cx43 can be specifically deleted in smooth muscle cells at any given time point by application of tamoxifen. By this approach, we were able to study long-term effects on myometrial functions that are necessary for parturition as well as gap junction intercellular communication in primary myometrial cell cultures. We found a prolongation of the pregnancy in 82% of tamoxifen-treated Cx43fl/fl:SM-CreERT2 mice as well as decreased dye coupling in cultured primary myocytes of these animals. Other parturition-specific parameters such as the regulation of oxytocin receptor, prostaglandin F receptor or progesterone remained unchanged. Our results indicate the important function of Cx43 during parturition in the living animal and suggest further strategies to investigate the role of connexins in uterine contractility in transgenic mice.

Transcripts of three connexin isoforms (Cx36, Cx43 and Cx45) have been reported in rodent pancreatic islets, but the precise distribution of the cognate proteins is still unknown. We determined expression of Cx36 in a cell-autonomous manner using mice with a targeted replacement of the Cx36 coding region by a lacZ reporter gene. For cell-autonomous monitoring of Cx43 expression, we used the Cre/loxP system: Mice carrying the Cx43 coding region flanked by loxP sites (floxed) also carried an embedded lacZ gene that is activated after Cre-mediated recombination in cells with transcriptional activity of the Cx43 gene. Deletion of the Cx43 coding region in beta-cells did not result in the activation of the embedded lacZ reporter gene. Instead, Cx43 expression was found in endothelial cells of the islets of Langerhans in mice with endothelium-specific deletion. Ubiquitous deletion of Cx43 led to a similar endothelial lacZ expression, but again, activity of the reporter gene was not detected in beta-cells. Mice with targeted replacement of the Cx45 coding region by lacZ showed a vascular expression similar to Cx43. The data show that native insulin-producing cells express a connexin isoform (Cx36) which differs from those (Cx43 and Cx45) expressed by vascular islet cells.

Using the Cre/loxP system, we have circumvented early postnatal lethality and possible pleiotropic effects of general Cx43 gene deletion, in order to determine the expression and function of connexin43 (Cx43) in defined cell types. General or cell type-specific, Cre-mediated deletion of the floxed (i.e. flanked by loxP sites) Cx43-coding region led to activation of the inserted lacZ reporter gene in cells with transcriptional activity of the Cx43 gene. As deduced from lacZ expression in mice with general deletion, transcriptional activity of the Cx43 gene was not only found in a broad range of cell types known to a express Cx43, but also inpancreatic duct cells and vascular cells of the gut and skeletal muscle. Cre-mediated deletion restricted to defined cell types led to lacZ activation highlighting corresponding subsets of cells expressing Cx43, such as vascular endothelial cells, hepatic duct cells and putative neural crest cells, which were otherwise masked by strong Cx43 expression in neighbouring cells. In Cx43 expressing cell types, the floxed Cx43 allele was useful as a Cre-excision reporter for the characterization of Cre transgenes.