Developmental expression patterns of connexin26 and -30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.     

Developmental expression patterns of connexin26 and ‐30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre‐ and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and ‐30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22‐week‐old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear. Dev. Genet. 25:306–311, 1999. © 1999 Wiley‐Liss, Inc.     

Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26-EYFP fusion products was comparable to that of hCx26-pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30-EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26-EYFP and hCx30-EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26-pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg(j)) displayed significantly smaller values (28.8+/-1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5+/-3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (G(j)) on transjunctional voltage (V(j)). The half-activation voltage for G(j) was close to +/-95 and +/-58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg(j)= 45.2 +/- 3.5 nS and V(0)= +/- 34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.     

Expression of connexins 26, 32 and 43 in the human colon--an immunohistochemical study

Gap junctional intercellular communication (GJIC) is a mechanism for direct cell-to-cell signalling and is mediated by gap junctions (GJs), which consist of proteins called connexins (Cxs). GJIC plays a critical role in tissue development and differentiation and is important in maintenance of tissue homeostasis. The purpose of the study was to evaluate the expression of Cx26, Cx32 and Cx43 in the human colon. Surgical specimens were obtained from patients who underwent surgical resection of colorectal tumours. Tissue samples (50 cases) were collected from normal colon, at the maximum distance from the tumor. Using antibodies for Cx26, Cx32 and Cx43, immunohistochemical detection was made. In epithelial cells, strong Cx26 immunoreactivity was found, whereas Cx32 and Cx43 were sparsely distributed. Strong Cx43 immunostaining in muscularis mucosae was observed. In the circular layer of muscularis externa, expression of Cx43 and Cx26 was seen, but only in the portion closest to the submucosa. No immunoreactivity was found in the longitudinal muscle layer. Small vessels stained positively only for Cx43. Furthermore, there was no difference in staining between samples derived from various sections of the colon. This study showed immunohistochemically for the first time the expression of Cx26 in human colon mucosa.     

Differential induction of connexins 26 and 30 in skin tumors and their adjacent epidermis.

Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of CK6, a marker for hyperproliferation, and CK17, a marker for trauma, we discuss that the induction of these gap junctional proteins exceeds a reflection of reactive hyperproliferative or traumatized epidermis. We further discuss the putative roles of these gap junctional proteins in tumor progression.     

Connexins 26 and 30 are co-assembled to form gap junctions in the cochlea of mice

The importance of connexins (Cxs) in the cochlear functions has been indicated by the finding that mutations in connexin genes cause a large proportion of sensorineural deafness cases. However, functional roles of connexins in the cochlea are still unclear. In this study, we compared the relative expression levels of 16 different subtypes of mouse connexins in the cochlea. cDNA macroarray hybridizations identified four most prominently expressed connexins (listed in descending order): Cxs 26, 29, 30, and 43. Two of these connexins (Cx26 and Cx30), both belonging to the beta-group, were investigated for their molecular assemblies in the cochlea. Co-immunostaining showed expressions of Cxs 26 and 30 in the same gap junction plaques and their co-assembly was confirmed by co-immunoprecipitation of proteins extracted from the cochlear tissues. The heterologous molecular assembly of connexins is expected to produce gap junctions with biophysical characteristics appropriate for maintaining ionic homeostasis in the cochlea.     

Expression of gap junction protein connexin43 in the adult rat cochlea: comparison with connexin26.

To elucidate whether the two different gap junction proteins connexin43 (Cx43) and connexin26 (Cx26) are expressed and localized in a similar manner in the adult rat cochlea, we performed three-dimensional confocal microscopy using cryosections and surface preparations. In the cochlear lateral wall, Cx43-positive spots were localized mainly in the stria vascularis and only a few spots were present in the spiral ligament, whereas Cx26-positive spots were detected in both the stria vascularis and the spiral ligament. In the spiral limbus, Cx43 was widely distributed, whereas Cx26 was more concentrated on the side facing the scala vestibuli and in the basal portion. In the organ of Corti, Cx43-positive spots were present between the supporting cells but they were fewer and much smaller than those of Cx26. These data demonstrated distinct differences between Cx43 and Cx26 in expression and localization in the cochlea. In addition, the area of overlap of zonula occludens-1 (ZO-1) immunolabeling with Cx43-positive spots was small, whereas it was fairly large with Cx26-positive spots in the cochlear lateral wall, suggesting that the differences are not associated with the structural difference between carboxyl terminals, i.e., those of Cx43 possess sequences for binding to ZO-1, whereas those of Cx26 lack these binding sequences.     

Expression and localization of Tmie in adult rat cochlea

Loss-of function mutations in transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). However, the functional roles of TMIE in the cochlea remain unclear. A primary step toward the understanding of the role of TMIE in hearing and its dysfunction is the documentation of its cellular and sub-cellular location within the cochlea, the auditory organ. In this study, we located and determined the cellular expression of Tmie within the rat cochlea using a polyclonal anti-Tmie antibody. The anti-Tmie antibody identified a specific band of 17 kDa in a variety of rat tissues by using Western blot analyses. The expression products of Tmie were also detected in the spiral limbus, spiral ligament, organ of Corti, and stria vascularis by immunohistochemistry analysis and RT-PCR. Our results point out the presence and localization of Tmie products in the cochlea of rat. Knowledge of spatial distribution of Tmie will provide important insight into the mechanisms that lead to deafness due to mutations in the TMIE gene.     

Immunohistochemical analysis of connexins 26, 32 and 43 in rat uterus during late pregnancy: lack of connexin 26 in the myometrium

The spatial and temporal patterns of expression of connexin 26, connexin 32 and connexin 43 were investigated in rat uterus at days 17, 19 and 22 of pregnancy and during delivery (23 days) by immunocytochemistry, Gap junctions, which are essential for the development of labour, are known to undergo rapid increase in the rat myometrium at the end of pregnancy. The expression of connexin 43, the major myometrial gap junction protein, was low throughout pregnancy but increased immediately before the onset of labour (day 22). It was found predominantly in the myometrium, although limited staining was also apparent in the stroma. Immunolabelling revealed the presence of connexins 26 and 32 in uterine luminal epithelial cells on days 17 and 19 of pregnancy, with a marked increase in connexin 26 expression at days 19, 22 and 23; however, marked expression of connexin 32 was apparent only at day 23. No immunoreactivity for either connexin 26 or 32 was found in myometrial cells at any stage of pregnancy. We conclude, contrary to other recent reports, that connexin 26 is not a gap junction protein of the rat myometrial smooth muscle cell. © 1998 Chapman & Hall     

Expression of connexins during development and following manipulation of afferent input in the rat locus coeruleus

Synchronous activity of locus coeruleus (LC) neurons during early postnatal development is regulated, in part, by electrotonic coupling. Connexin (Cx) proteins that make up gap junction channels are localized to both neurons and glia in the LC during this period. In adult rats, however, synchrony exists only under certain experimental conditions. The expression of Cx proteins was examined using western blot analysis at several developmental time points. Immunoblot analysis revealed little to no expression of Cx26 while Cx32, Cx43 and Cx36 were present at all time points examined. A progressive increase in Cx43 was identified from the first postnatal week through adulthood. Immunocytochemical detection of Cx36 and Cx43 in adult LC showed that Cx36 was associated with neuronal processes while Cx43 was localized to glia. In adult LC, in vitro intracellular recordings combined with neurobiotin injections confirmed the presence of gap junctional communication albeit to a lesser extent than in early postnatal periods. The degree to which synaptic inputs to LC neurons impact on Cx protein expression was also evaluated. Samples of the LC from rats that received an electrolytic lesion of the amygdala were processed for western blot analysis of Cx36 and Cx43. The predominantly neuronal Cx36 exhibited an increase in expression while the glial Cx43 was unchanged. The present results indicate that, despite subtype-specific changes during development, several Cx proteins are expressed in the adult LC. In addition, manipulating afferent input to the LC, in adult rats, results in increases in neuronal Cx protein levels but not in glial Cx levels suggesting that altering synaptic inputs to the LC may alter synchronous activity in noradrenergic neurons.     

Expression of epithelial calcium transport system in rat cochlea and vestibular labyrinth

Background  

The low luminal Ca²⁺ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca²⁺-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium.     

Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication

In theepididymis, Cx43 forms gap junctions between principal and basal cellsbut not between adjacent principal cells. Cx30.3, 31.1, and 32 wereidentified in adult rat epididymis by RT-PCR, whereas Cx26 was presentin young rats. Postnatal development studies indicate that Cx26 mRNAwas detectable only in the caput-corpus region of the epididymis andthat levels increased by fivefold during the first 4 wk postnatally,when epithelial cells differentiate, and decrease to nondetectablelevels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout theepididymis in young rats and began to increase in the second and thirdweeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32were localized to the lateral plasma membranes between adjacentepithelial cells of the epididymis. Colocalization studies indicatethat Cx26 and Cx32 exist either independently of one another or cancolocalize along the lateral plasma membrane of epithelial cells inyoung rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.

     

Expression of gap junctional connexins 26, 32, and 43 mRNA in ovarian preovulatory follicles and corpora lutea in sheep

The objective of the current study was to evaluate the expression of connexins (Cx)26, Cx32, and Cx43 mRNA in granulosa and theca cells during the peri-ovulatory period (experiment 1) and in the corpus luteum (CL) during the estrous cycle (experiment 2) and during prostaglandin F2alpha (PGF)-induced luteal regression (experiment 3) in FSH-treated ewes. In experiment 1, Cx26, Cx32, and Cx43 mRNA was expressed in granulosa and theca cells, and expression of Cx32 and Cx43 mRNA, but not Cx26, was greater (p<0.001) in granulosa than in theca cells throughout the peri-ovulatory period. Expression of Cx43 mRNA in granulosa and theca cells decreased (p<0.01) 24 h after hCG treatment. In experiment 2, expression of Cx26 mRNA in the CL tended to be greater (p<0.06) on day 10 than on days 5 or 15, but expression of Cx43 mRNA was greater (p<0.01) on day 5 than on days 10 and 15 of the estrous cycle. In experiment 3, expression of Cx26, but not Cx32 or Cx43 mRNA decreased (p<0.001) during PGF-induced luteal regression. In all 3 experiments, expression of Cx32 mRNA was much less than Cx26 and Cx43 mRNA. Moreover, Cx32 mRNA expression was unchanged during the peri-ovulatory period or during several stages of luteal development and PGF-induced regression of the CL. Thus, we have shown that the mRNA expression pattern of Cx26 and Cx43 changes during peri-ovulatory period and during several stages of the luteal development. This suggests that Cx26 and Cx43 play a role in ovarian tissue remodeling during the critical time around ovulation and throughout luteal tissue growth, differentiation, and regression in sheep.     

Expression of peripherin in human cochlea

The organ of Corti contains two different types of auditory receptors; the inner (IHCs) and outer (OHCs) hair cells. This dualism is further represented in their innervation, IHCs being innervated by type I neurons, and OHCs by type II neurons (in man, named small ganglion cells). Two efferent systems are also present. Here, we have analyzed the expression of the 57-kDa neuron-specific intermediate filament protein peripherin (PP) in human cochlea. In the human organ of Corti, PP seems to be specifically expressed in OHC afferents. Small or type II spiral ganglion cell bodies also intensely express PP. Thus, PP can be used as a marker for the characterization of the innervation of the OHC system in man.     

Different localizations of 21 and 27 kDa gap-junction proteins in rat salivary glands

Antibodies against 21 and 27 kDa gap-junction proteins from rat liver were used to examine the identification and localization of gap-junction proteins in rat salivary glands. Acinar cells of the submandibular glands and parotid glands stained well for the 27 kDa gap junction protein and less intensely for the 21 kDa protein. Acinar cells of the sublingual glands were stained heavily for the 27 kDa gap junction protein and stained well for 21 kDa gap junction protein. No 27 kDa protein was observed in the ducts of the salivary glands. The 21 kDa gap-junction protein was distributed in some of the intercalated ducts in the parotid and submandibular glands. Immunoblotting of an extract of parotid glands with antibodies against 21 and 27 kDa gap-junction proteins revealed the presence of 21 and 27 kDa proteins in the parotid glands. It is concluded that the 27 kDa gap-junction protein in tistributed as a major component of the gap junctions in the acinar cells of all the salivary glands; the 21 kDa protein is localized as a minor component in the acinar cells and some portions of the intercalated ducts in the salivary glands. It is possible that these gap-junction proteins might contribute to the regulation of function of the salivary glands.     

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.     

Expression and function of connexins in the epidermis, analyzed with transgenic mouse mutants

Eight different connexins are expressed in mouse epidermis with overlapping expression patterns in different epidermal layers. Analyses of mice with deficiency or modifications of distinct connexins yielded insights into the large variety of connexins in the epidermis. Connexin43 (Cx43) deficiency in mouse epidermis resulted in a significant acceleration of wound closure. Truncation by 125 amino acid residues of the Cx43 C-terminal region led to an altered epidermal expression pattern of Cx43 and defective development of the epidermal water barrier in transgenic mice, although the truncated Cx43 protein could still form open gap junctional channels in transfected HeLa cells. Thus, the phenotypic abnormalities observed in mice with truncated Cx43 protein (Cx43K258Stop) are more likely due to defective regulation of this protein rather than the closed Cx43 channel. Our studies of connexin-deficient mice revealed an extensive redundancy of connexins expressed in mouse epidermis. Epidermal connexins seem to form two functional groups in which deficiency of one connexin isoform can be compensated by other connexin isoforms of the same group.