The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29).

The potential gap junction forming mouse connexin29 (Cx29) protein is concomitantly expressed with connexin32 (Cx32) in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47) in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harboring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29-mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

Expression of connexin genes in the human retina.

BACKGROUND: Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown. METHODS: Northern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina. RESULTS: Here we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina. CONCLUSION: In contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

Connexin 30 is expressed in the mouse sino-atrial node and modulates heart rate.

AIMS: This study aimed at characterizing expression and the functional role of the Gjb6 gene, encoding for connexin 30 (Cx30) protein, in the adult mouse heart. METHODS AND RESULTS: The expression of the Gjb6 gene in the mouse heart was investigated by RT-PCR and sequencing of amplified cDNA fragments. The sites of Gjb6 expression were identified in the adult heart using transgenic mice with reporter genes (Cx30(LacZ/LacZ) and Cx30(LacZ/LacZ)/Cx40(EGFP/EGFP) mice), as well as anti-HCN4 (hyperpolarization activated cyclic nucleotide-gated potassium channel 4) or anti-connexin antibodies. Cine-magnetic resonance imaging and telemetric ECG recordings were used to evaluate the impact of Cx30 deficiency on cardiac physiology. Gjb6 was shown to be expressed in the sinoatrial (SA) node of the adult mouse heart. Eighty from 100 nuclei on average were LacZ-positive in the SA node of Cx30(LacZ/LacZ) mice. No significant LacZ expression was seen in other cardiac tissues. Cx30 protein was identified in low abundance in the SA node of wild-type mice, as indicated by immunofluorescence experiments. Telemetric ECG recordings indicated that Cx30-deficient mice displayed a mean daily heart rate (HR) that was 9% faster than that measured in control mice (572 +/- 38 b.p.m. vs. 524 +/- 23, P < 0.05). This moderate tachycardia was still observed after inhibition of the autonomic nervous system, demonstrating that Cx30 deficiency resulted in changes in the intrinsic electrical properties of the SA node. Consistent with this hypothesis, Cx30(LacZ/LacZ) displayed a significant reduction of SDNN (standard deviation of the interbeat interval) compared with control mice. Increase of both the cardiac index (20%) and the end-diastolic volume to body weight ratio (16%) with no deficiency in ejection fraction or stroke volume were observed in mutant mice. An increase in cardiac index was interpreted as being a direct consequence of high HR, whereas large end-diastolic volume may be an indirect consequence of prolonged high HR. CONCLUSION: Cx30 is functionally expressed, in low abundance, in the SA node of the adult mouse heart where it participates in HR regulation.

Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells.

In the mouse genome, 20 connexin genes have been detected that code for proteins of high sequence identity in the two extracellular loops, especially six conserved cysteine residues. The mouse connexin23 (Cx23) gene (Gje1) differs from all other connexin genes in vertebrates, since it codes for a protein that contains only 4 instead of 6 cysteine residues in the extracellular loops. Recently, two zebrafish connexin genes (Cx23a and Cx23b) have been identified, and a mouse mutant in the Gje1 gene has been described that exhibits a developmental defect in the lens. Here, we have compared the Cx23 gene in different mammalian species and found no transcripts in cDNA libraries of primates. Furthermore, all primate genomes analyzed contain stop codons in the Cx23 sequence, indicating inactivation of the orthologous primate GJE1 gene. No Cx23 mRNA was found in human eye. In order to analyze the properties of mouse Cx23 channels, we isolated HeLa cell clones stably expressing wild-type mCx23 or mCx23 fused to eGFP. Cells expressing Cx23-eGFP demonstrated its insertion in the plasma membrane but no punctate staining in contacting membranes characteristic for junctional plaques. In addition, we tested whether Cx23 forms functional gap junction channels electrophysiologically in cell pairs as well as by microinjection of neurobiotin and found that mouse Cx23 did not form gap junction channels in HeLa cells. However, there was a significant release of ATP from different Cx23 HeLa cell clones, even in the presence of normal culture medium with high calcium ion concentration, suggesting a hemichannel-based function of Cx23. Therefore, Cx23 seems to share functional properties with pannexin (hemi) channels rather than gap junction channels of other connexins.

Analysis of connexin subunits required for the survival of vestibular hair cells.

Mutations in connexin (Cx) genes are responsible for a large proportion of human inherited prelingual deafness cases. The most commonly found human Cx mutations are either Cx26 or Cx30 deletions. Histological observations made in the organ of Corti of homozygous Cx26 and Cx30 gene knockout mice show that cochlear hair cells degenerate after the onset of hearing. However, it is unclear whether vestibular hair cells undergo similar degeneration in connexin knockout mice. To address this question, we first examined expression patterns of Cx26 and Cx30 in the saccule, utricle, and ampulla by immunolabeling. In wild-type mice, Cx26 and Cx30 immunoreactivity was found extensively in vestibular supporting cells and connective tissue cells, and the two Cxs were co-localized in most gap junction (GJ) plaques. Targeted deletion of the Cx30 gene, which caused little change in Cx26 expression pattern, resulted in a significant and age-related loss of vestibular hair cells only in the saccule. dUTP nick end labeling (TUNEL) staining also revealed on-going apoptosis specifically in saccular hair cells of Cx30(-/-) mice. These results indicated that hair cell survival in the utricle and ampulae does not require Cx30. Importantly, over-expressing the Cx26 gene from a modified bacterial artificial chromosome in the Cx30(-/-) background rescued the saccular hair cells. These results suggest that it is the reduction in the total amount of GJs rather than the specific loss of Cx30 that underlies saccular hair cell death in Cx30(-/-) mice. Hybrid GJs co-assembled from Cx26 and Cx30 were not essential for the survival of saccular hair cells.

Restoration of connexin26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin30-linked deafness.

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a Cx30-/- background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf Cx30-/- mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations.

Keratin transgenic and knockout mice: functional analysis and validation of disease-causing mutations.

The intermediate filament (IF) cytoskeleton of mammalian epithelia is generated from pairs of type I and type II keratins that are encoded by two large gene families, made up of 54 genes in humans and the mouse. These genes are expressed in a spatiotemporal and tissue-specific manner from the blastocyst stage onward. Since the discovery of keratin mutations leading to epidermolysis bullosa simplex, mutations in at least 18 keratin genes have been identified that result in keratinopathies of the epidermis and its appendages. Recently, noncanonical mutations in simple epithelial keratins were associated with pancreatic, liver, and intestinal disorders, demonstrating that keratins protect epithelia against mechanical and other forms of stress. In recent years, animal models provided novel insight and significantly improved understanding of IF function in tissue homeostasis and its role in disease. Pathological phenotypes detected in mutant mice generated so far range from embryonic lethality to tissue fragility to subtlety, which often depends on their genetic background. This range implies at least a partial influence of yet unidentified modifier genes on the phenotype after the ablation of the respective keratin. To date, nearly all available keratin mouse models were generated by taking advantage of conventional gene-targeting strategies. To reveal their cell type-specific functions and the mechanisms by which mutations lead to disease, it will be necessary to use conditional gene-targeting strategies and the introduction of point-mutated gene copies. Furthermore, conditional strategies offer the possibility to overcome embryonic or neonatal lethality in some of the keratin-deficient mice.

Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells.

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.

Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.

In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from 'sniffer cells', a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the 'sniffer cell' population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.

Functional properties of mouse connexin30.2 expressed in the conduction system of the heart.

Gap junction channels composed of connexin (Cx) 40, Cx43, and Cx45 proteins are known to be necessary for impulse propagation through the heart. Here, we report mouse connexin30.2 (mCx30.2) to be a new cardiac connexin that is expressed mainly in the conduction system of the heart. Antibodies raised to the cytoplasmic loop or the C-terminal regions of mCx30.2 recognized this protein in mouse heart as well as in HeLa cells transfected with wild-type mCx30.2 or mCx30.2 fused with enhanced green fluorescent protein (mCx30.2-EGFP). Immunofluorescence analyses of adult hearts yielded positive signals within the sinoatrial node, atrioventricular node, and A-V bundle of the cardiac conduction system. Dye transfer studies demonstrated that mCx30.2 and mCx30.2-EGFP channels discriminate poorly on the basis of charge, but do not allow permeation of tracers >400 Da. Both mCx30.2 and mCx30.2-EGFP gap junctional channels exhibited weak sensitivity to transjunctional voltage (Vj) and a single channel conductance of approximately 9 pS, which is the lowest among all members of the connexin family measured in HeLa cell transfectants. HeLa mCx30.2-EGFP transfectants when paired with cells expressing Cx40, Cx43, or Cx45 formed functional heterotypic gap junction channels that exhibited low unitary conductances (15 to 18 pS), rectifying open channel I-V relations and asymmetric Vj dependence. The electrical properties of homo- and hetero-typic junctions involving mCx30.2 may contribute to slow propagation velocity in nodal tissues and directional asymmetry of excitation spread in the AV nodal region.

Loss of connexin36 channels alters beta-cell coupling, islet synchronization of glucose-induced Ca2+ and insulin oscillations, and basal insulin release.

Normal insulin secretion requires the coordinated functioning of beta-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of beta-cells with extracellular signals and neighboring cells. In particular, adjacent beta-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36(-/-)) featured beta-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36(-/-) mice did not display the regular oscillations of intracellular calcium concentrations ([Ca(2+)](i)) seen in controls due to the loss of cell-to-cell synchronization of [Ca(2+)](i) changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of beta-cells, particularly for the pulsatility of [Ca(2+)](i) and insulin secretion during glucose stimulation.

Emerging complexities in identity and function of glial connexins.

Recent research results indicate that glial gap-junction communication is much more complex and widespread than originally thought, and has diverse roles in brain homeostasis and the response of the brain to injury. The situation is far from clear, however. Pharmacological agents that block gap junctions can abolish neuron-glia long-range signaling and can alleviate neuronal damage whereas, intriguingly, opposite effects are observed in mice lacking connexin43, a major gap-junction subunit protein in astrocytes. How can the apparently contradictory results be explained, and how is specificity achieved within the glial gap-junction system? Another key issue in understanding glial connexin function is that oligodendrocytes and astrocytes, each of which express distinct connexin isotypes, are thought to participate in brain homeostasis by forming a panglial syncytium. Molecular analysis has revealed a surprising diversity of connexin expression and function, and this has led to new hypotheses regarding their roles in the brain, which could be tested using new approaches.

Expression and functions of neuronal gap junctions.

Gap junctions are channel-forming structures in contacting plasma membranes that allow direct metabolic and electrical communication between almost all cell types in the mammalian brain. At least 20 connexin genes and 3 pannexin genes probably code for gap junction proteins in mice and humans. Gap junctions between murine neurons (also known as electrical synapses) can be composed of connexin 36, connexin 45 or connexin 57 proteins, depending on the type of neuron. Furthermore, pannexin 1 and 2 are likely to form electrical synapses. Here, we discuss the roles of connexin and pannexin genes in the formation of neuronal gap junctions, and evaluate recent functional analyses of electrical synapses that became possible through the characterization of mouse mutants that show targeted defects in connexin genes.

New insights into the expression and function of neural connexins with transgenic mouse mutants.

Gap junctions represent direct intercellular conduits between contacting cells. The subunit proteins of these conduits are called connexins. To date, 20 and 21 connexin genes have been described in the mouse and human genome, respectively, many of them represent sequence-orthologous pairs. Targeted deletion of connexin genes in the mouse genome opened new insights into the biological function of these channel forming proteins, which, in some cases, could be correlated to phenotypic abnormalities in humans, suffering from inherited diseases caused by mutations in the corresponding orthologous connexin gene. Replacing the connexin coding DNA by an appropriate reporter gene has clarified in several cases its cell type specific expression in mouse brain. Various studies demonstrated that connexin36 is mainly expressed in interneurons of retina and brain. Targeted deletion of connexin36 evoked a loss of electrical signal transduction and interferes with synchrony which probably leads to defects in visual transmission and memory. Deletion of connexin43 in astrocytes of mouse brain resulted in increased spreading depression consistent with the notion of altered "spatial buffering" of K(+) ions and glutamate secreted by active neurons. General connexin30-deficiency led to hearing impairment and apoptosis of hair cells, similar to that observed in mice with cochlea specific deletion of connexin26. Reporter gene expression in connexin30-deficient mice indicated that astrocytes in certain brain regions and leptomeningeal as well as ependymal cells are labelled. Reporter gene expression in connexin45- and connexin47-deficient mice was used to reassign connexin45 expression to certain CNS neurons and connexin47 expression to oligodendrocytes.

The novel mouse connexin39 gene is expressed in developing striated muscle fibers.

The recently identified mouse connexin39 (mCx39) gene encodes a peptide of 364 amino acids that shows only 61% sequence similarity to its putative human orthologue connexin40.1 (hCx40.1). The coding regions of mCx39 and hCx40.1 are located on two different exons as described for murine and human connexin36. Northern blot and RT-PCR analyses revealed that mCx39 is expressed after embryonic day (ED) 13.5 up to birth and is absent from the adult stage. Polyclonal antibodies raised to a peptide corresponding to the 16 C-terminal amino acid residues detected a protein band of about 40 kDa apparent molecular mass in lysates of several embryonic tissues. In sections of ED14.5, ED16.5 and neonatal (P0) tissues, immunofluorescent signals were prominent between myotubes in the developing diaphragm, within the intercostal muscle, in the region around the occipital bone, as well as in muscles of the limb, tongue and connective tissue around the eye. These antibodies yielded punctate signals on apposed plasma membranes of HeLa cells transfected with Cx39 cDNA but did not react with wild-type cells. Furthermore, no intercellular permeation of microinjected neurobiotin and other tracers could be detected in Cx39 transfected HeLa cells. However, after microinjection of Alexa488 into myotubes of dissected neonatal diaphragm, we found spreading of this dye into neighbouring cells. As expression of no other known connexin could be verified in these cells, intercellular dye transfer might result from functional expression of Cx39 in developing striated muscle fibers.

Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes.

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(-)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(-) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(-) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.

Functional expression of connexin57 in horizontal cells of the mouse retina.

Horizontal cells are interneurons of the vertebrate retina that exhibit strong electrical and tracer coupling but the identity of the channel-forming connexins has remained elusive. Here we show that horizontal cells of the mouse retina express connexin57 (Cx57). We have generated Cx57-deficient mice by replacing the Cx57 coding region with a lacZ reporter gene, expressed under control of the endogenous Cx57 promoter. These mice were fertile and showed no obvious anatomical or behavioural abnormalities. Cx57 mRNA was expressed in the retina of wild-type littermates but was absent from the retina of Cx57-deficient mice. Previously reported results that the Cx57 gene was very weakly expressed in several other mouse tissues turned out to be unspecific. Cx57 mRNA is abundantly expressed in the retina and weakly in the thymus of adult mice but absent in all other adult tissues tested, including brain. Furthermore, Cx57 is expressed in embryonic kidney at E16.5 to E18.5 days post-conception, as indicated by the pattern of lacZ expression. Within the retina, lacZ signals were assigned exclusively to horizontal cells based on co-localization with cell-type-specific marker proteins. Microinjection of Neurobiotin into horizontal cells of isolated retinae revealed less than 1% of tracer coupling in Cx57-deficient retinae compared with wild-type controls. Cx57 is the first connexin identified in mammalian horizontal cells and the first connexin whose expression is apparently restricted to only one type of neuron.

Gap junctions and the connexin protein family.

Gap junctions (Gj) form conduits between adjacent cells that are composed of connexin (Cx) protein subunits and allow direct intercellular communication. To date, the connexin gene family comprises 20 members in the mouse and 21 members in the human genome, 19 of which can be grouped as sequence-orthologous pairs. The structure of connexin genes is relatively simple. An untranslated exon 1 is separated by an intron of different length from exon 2, containing the uninterrupted coding region and the 3'-untranslated region (3'-UTR). However, in some connexin genes, the untranslated regions and the reading frame are spliced. Among the known "cardiovascular" connexins, Cx37 and Cx40 were demonstrated to be functionally expressed in mouse and human endothelial cells and Cx40, Cx43 as well as Cx45 in cardiomyocytes of both species. Functional properties, like permeabilities, charge selectivity and unitary conductivity were investigated after directed expression of these connexins in cultured cell lines or paired Xenopus oocytes. Targeted deletion of their coding sequence in the mouse genome allowed study of the biological relevance of Cx37, Cx40, Cx43 and Cx45 with regard to cardiovascular morphology and function. After ablation of Cx37 or Cx40, mice were viable and could be used to study defects in the adult cardiovascular system but loss of Cx43 or Cx45 led to neonatal or embryonic lethality, respectively. Conditional and cell-type specific deletion of both connexins in the heart or blood vessels can help to overcome this obstacle. As yet only little is known about mutations in the human genes for Cx37, Cx40, Cx43 and Cx45. Thus, a profound comparison between human and mouse phenotypes is not yet possible.

Expression pattern of lacZ reporter gene representing connexin36 in transgenic mice.

Targeted deletion of the connexin36 (Cx36) gene in the mouse genome leads to visual transmission defects, weakened synchrony of rhythmic inhibitory potentials in the neocortex, and disruption of gamma-frequency network oscillations. We have generated transgenic mice in which a reporter protein consisting of the exon1 coded N-terminal part of Cx36 fused to beta-galactosidase (N36-beta-gal) is expressed instead of Cx36. Here, we have used these mice for a detailed analysis of the reporter gene expression. By beta-gal staining of adult retina, we found expression of the lacZ reporter gene in the ganglion cell layer, in two rows of the inner nuclear layer, and in the photoreceptor layer. In the brain, beta-gal staining was present in gamma-aminobutyric acid (GABA)ergic neurons of the cerebellar nuclei, in non-GABAergic neurons of the inferior olive, in mitral cells of the olfactory bulb, and in parvalbumin-positive cells of the cerebral cortex. Outside the central nervous system, N36-beta-gal signals were detected in insulin producing beta-cells of the pancreas and in the medulla of the adrenal gland of adult Cx36(+/del[LacZ]) mice. This expression pattern suggests that Cx36 fulfills functional roles not only in several types of neurons in the retina and central nervous system but also in excitable cells of the pancreas and adrenal gland.

Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina.

Transgenic technology, immunocytochemistry, electrophysiology, intracellular injection techniques, and reverse transcription PCR were combined to study the expression of neuronal connexin36 (Cx36) in the outer plexiform layer of the mouse retina. Transgenic animals expressed either a fusion protein of full-length Cx36 with enhanced green fluorescent protein (EGFP) attached at the C terminus or exon 2 of Cx36 was replaced bybeta-galactosidase (beta-gal). In the outer nuclear layer,beta-gal-positive cell bodies, which were confined to the most distal region close to the outer limiting membrane, displayed immunoreactivity against S-cone opsin. Cx36-EGFP puncta colocalized with cone pedicles, which were visualized by intracellular injection. In reverse transcriptase PCR experiments, Cx36 mRNA was never detected in samples of rods harvested from the outer nuclear layer. These results strongly suggest expression of Cx36 in cones but not in rods. In vertical sections, Cx36 expression in the vitreal part of the outer plexiform layer was characterized by a patchy distribution. Immunocytochemistry with antibodies against the neurokinin-3 receptor and the potassium channel HCN4 (hyperpolarization-activated cyclic nucleotide-gated potassium channel) displayed clusters of the Cx36 label on the dendrites of OFF-cone bipolar cells. In horizontal sections, these clusters of Cx36 appeared as round or oval-shaped groups of individual puncta, and they were always aligned with the base of cone pedicles. Double-labeling experiments and single-cell reverse transcriptase PCR ruled out expression of Cx36 in horizontal cells and rod bipolar cells. At light microscopic resolution, we found close association of Cx36-EGFP with the AMPA-type glutamate receptor subunit GluR1 but not with GluR2-GluR4, the kainate receptor subunit GluR5, or the metabotropic glutamate receptor mGluR6.