Different regulatory pathways of endometrial connexin expression: preimplantation hormonal-mediated pathway versus embryo implantation-initiated pathway.

Transformation of the endometrium into the receptive phase is under the control of ovarian steroid hormones and is modulated by embryonic signals during implantation. We have previously shown that this differentiation process is accompanied by a suppression of gap junction connexins (Cx) 26 and 43 before implantation followed by a local induction of both connexins in the implantation chamber. In the present study, we demonstrate that connexin gene expression in the rodent endometrium is regulated via two distinct signaling pathways during these different stages of early pregnancy. During preimplantation, transcription of connexins can be induced by estrogen via an estrogen receptor (ER)-dependent pathway. Additionally, Cx26 and Cx43 are induced by embryonic signals during implantation and delayed implantation as well as during artificially induced decidualization. In contrast to the estrogen-induced expression, this embryonic/decidual-associated induction of Cx26 and Cx43 could not be blocked by antiestrogen, thus pointing to another regulatory pathway independent of the ER. Studies in ERalpha and ERbeta knockout mice confirmed these different pathways, demonstrating that in the endometrium, estrogen-mediated Cx26 gene induction, but not induction during decidualization, is dependent on functional ERalpha. To evaluate potential embryonic signals regulating Cx26 expression, uteri of pseudopregnant animals were incubated with different mediators in an organ-culture model, showing that catechol estrogen and mediators of the inflammatory cascade such as prostaglandin F(2alpha) and interleukin-1beta are able to induce Cx26 expression through the ER-independent pathway. Thus, the present study demonstrates that endometrial expression of Cx26 and Cx43 is induced via estrogen and ERalpha during preimplantation but then utilizes an ER-independent signaling pathway during embryo implantation and decidualization.

Gap junction formation and connexin distribution in pig trophoblast before implantation.

This study describes the gap junctions in extraembryonic cell layers of the preimplantation pig embryo (trophectoderm and endoderm constituting the trophoblast). Using specific antibodies against connexins 31, 32 and 43, we found these connexins in embryos by immunodetection using Western blot and immunofluorescence analysis. By immunofluorescence, the first foci of connexin 31 were detected in the four-cell stage blastomeres, and the first diffuse gap junctions appeared at the eight-cell stage. Intercellular communication was observed with Lucifer yellow transfer to start also at the eight-cell stage around the onset of compaction. Typical gap junctions developed in the trophectoderm of blastocysts, as observed by transmission electron microscopy of thin sections and freeze-fracture replicas. Connexin proteins were differently expressed in time and space: connexin 31 was continuously present in trophectoderm, connexin 32 was essentially found in endoderm during elongation; connexin 43 was distributed in both trophectoderm and endoderm during blastulation and expansion. Connexin 43 was also found in two isoforms, phosphorylated or not, at day 14. Such developmentally regulated connexin expression may be essentially useful to control the exponential growth of trophoblast in preimplantation pig blastocysts.

Modulation of connexin expression in sheep endometrium in response to pregnancy.

The expression pattern of two typical gap junction channel proteins, connexin 43 and connexin 26 (Cx43 and Cx26), was identified in the endometrium of sheep, a species with epitheliochorial type of implantation, by indirect immunohistochemistry during the cyclic phases, early and late pregnancy, and immediately after birth. The extent of Cx43 immunoreaction bound to endometrial stromal cells of the early implantation stage (day 15 p.c.) was comparable to the situation observed in oestrus. The subsequent intensification of feto-maternal contact correlated with a striking increase of stromal Cx43 in the intercaruncular and caruncular regions of the uterus (days 18 and 21 p.c.) and the induction of Cx26 in the glandular epithelium of late implantation (day 21 p.c.). In contrast, both gap junction proteins, coexpressed in the stroma of placentomes and interplacentomal sections on days 131 and 145 p.c., decreased during late pregnancy, while an intense and augmenting staining for Cx26 was detected at the cell borders of the glandular and luminal epithelium. The spatial and temporal distribution of both connexins suggests that, under embryonal and hormonal influences, gap junctional communication is involved in the implantation process and the regulation of endometrial tissue functions during sheep pregnancy and indicates further, that this connexin expression path resembles more the invasive type of implantation.

Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice.

Characterization of the expression pattern of connexins in neural tissue is a necessary prerequisite for understanding the functional relevance of the corresponding gap junction channels in brain. Here we describe the cell type-specific expression of connexin45 in the CNS and the spatiotemporal expression pattern from embryonic day 19.5 to adult brain using a recently described connexin45 LacZ-reporter mouse. The connexin45 gene is highly expressed during embryogenesis and up to 2 weeks after birth in nearly all brain regions. Afterward its expression is restricted to the thalamus, the CA3 region of hippocampus and the cerebellum. In adult mouse brain, the pattern of LacZ-staining in combination with the analysis of different neuronal and glial marker proteins strongly suggests that connexin45 is expressed in neurons, but presumably not in astrocytes or mature oligodendrocytes. Expression of the LacZ/connexin45 reporter gene in subsets of neurons, such as cerebral cortical, hippocampal and thalamic neurons as well as basket and stellate cells of cerebellum should be corroborated by functional investigations of connexin45 protein in electrical synapses. Based on its expression pattern during development, we suggest that the connexin45-containing gap junction channels have a rather ubiquitous role during brain development and may contribute to functional specification in certain subsets of neurons in the adult brain.

Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens.

Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action.

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Intracellular domains of mouse connexin26 and -30 affect diffusional and electrical properties of gap junction channels.

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

Expression of the gap junction connexins Cx43, Cx45 and Cx26 in human uterine leiomyomata.

Uterine leiomyomata of 34 premenopausal women undergoing leiomyomectomy or hysterectomy, and in four cases the corresponding myometrium, were collected at laparotomy or laparoscopy to investigate the ability of these benign smooth muscle cell tumors to express different connexins. Immunohistochemical and Northern blot analyses were performed for the characterization of the expression of connexins Cx43, Cx45, Cx26 and Cx32. Immunofluorescence revealed the presence of Cx43 in most leiomyomata. Only seven leiomyomata lacked Cx43 expression. Cx45 was expressed in 13, a weak Cx26 immunostaining was found in seven cases, whereas Cx32 could not be detected. No correlations between the 17 beta-estradiol or progesterone serum levels and the expression patterns of the connexins Cx43, Cx45 and Cx26 could be observed. Gonadotropin-releasing hormone (GnRH)-agonist or progestin treatment did not influence the connexin expression pattern. Northern blot analyses confirmed these results; however, transcripts of Cx26 were not detectable. Connexin transcripts between myomata and the corresponding myometrium showed no obvious differences. Our data show that uterine leiomyomata are capable of expressing different connexins comparable to the corresponding myometrium, but do not respond to different hormonal conditions. The ability to express the appropriate connexins could explain why these tumors, though developing independently of hormonal levels, are still differentiated benign smooth muscle tumors.

Connexin31-deficiency in mice causes transient placental dysmorphogenesis but does not impair hearing and skin differentiation.

Mutations in the human GJB3 gene that codes for Connexin31 (Cx31), a protein subunit of gap junction channels, have recently been reported to cause deafness and the skin disorder erythrokeratodermia variabilis. To study the function of this gene in mice, we generated animals with targeted replacement of the Cx31 gene (Gjb3) by a lacZ reporter gene. Although homozygous Cx31-deficient adult mice (Gjb3(-/-)) were found among the offspring of heterozygous Cx31-deficient parents (Gjb3(+/-)), 60% of the animals expected according to Mendelian inheritance were lost between ED 10.5 and 13.5. Placentas of Gjb3(-/-) embryos at ED 9.5 were smaller than controls as a result of severely reduced labyrinth and spongiotrophoblast size. From ED 10.5 onward, placentas of surviving Gjb3(-/-) embryos recovered progressively and reached normal size and morphology by ED 18.5. This corresponds to a time period in which another connexin isoform, Connexin43, is upregulated in spongiotrophoblast cells of Cx31-deficient and control placentas. No morphological or functional defects of skin or inner ear were observed in surviving adult Gjb3(-/-) mice. We conclude that Cx31 is essential for early placentation but can be compensated for by other connexins in the embryo proper and adult mouse.

Functional rescue of defective mutant connexons by pairing with wild-type connexons.

We have compared the functional and structural integrity of gap junction channels assembled from a Cx45 truncation mutant with those of gap junction channels assembled from wild-type (wt) Cx45 and Cx43. These channel-forming proteins are constitutively expressed in HeLa cells. The truncation mutant lacks the last 26 amino acids of the COOH-terminus, including nine serine phosphorylation sites that are associated with regulatory processes of these channels. We determined the presence of gap junction plaques in these cells with the immunogold freeze fracture technique, which showed that plaque formation is similar in all the clones investigated. Junctional permeability was probed with calcein transfer and flow cytometry analyses and junctional conductance was measured in cell pairs with double whole-cell patch-clamp techniques. For homotypic pairing only the truncated mutant did not form permeable channels. However, coupling was restored for heterotypic channels (pairing wtCx45- or wtCx43- with mutant-connexons), whose junctional communication was not different from that of the homotypic channels. Our results indicate that the presence of gap junction plaques does not warrant functional coupling and that heterotypic trCx45/wtCx45 channels can be regulated by the intact wtCx45 connexons. This dominant-positive effect is also operative when wtCx43 are paired with trCx45 connexons.

Expression of connexin genes in hippocampus of kainate-treated and kindled rats under conditions of experimental epilepsy.

We have analyzed whether the expression of connexin genes is altered in the hippocampus of kindled and kainate-treated rats, i.e., animal models of human temporal lobe epilepsy. We have tested this hypothesis by analyzing mRNA, protein abundance and cellular location of connexins (Cx) 43, 36, 32 and 30. The expression of glial fibrillary acid protein and mRNA was also monitored both in kainate-treated and kindled rats, in order to take into account reactive gliosis under these conditions. We found significantly increased expression of GFAP mRNA (100%) and protein (178%) in kainate-treated rats 4 weeks after kainate application, whereas in kindled rats only moderate increases of GFAP mRNA and protein were detected 2-3 weeks (group 2) or 4-6 weeks (group 1) after the last stage 5 induced seizure. Under gliotic conditions, connexins 43 and 30 mRNA or protein expression in astrocytes of kainate-treated rats were nearly unaffected. Cx36 mRNA expression (presumably in neurons) was significantly reduced (44%), whereas abundance of Cx36 protein was only slightly reduced. In both groups of kindled rats, Cx30 and Cx43 mRNA or protein expression were either slightly decreased or unchanged. Again, Cx36 mRNA and protein expression were reduced by about half in group 2. Immunofluorescence analysis of Cx43, Cx36 and Cx30 expression revealed that 4 weeks after the last kainate administration or kindling, cellular localization of these connexins was indistinguishable from control animals.

Unique and shared functions of different connexins in mice.

BACKGROUND: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two 'knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. RESULTS: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. CONCLUSIONS: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

Defective vascular development in connexin 45-deficient mice.

In order to reveal the biological function(s) of the gap-junction protein connexin 45 (Cx45), we generated Cx45-deficient mice with targeted replacement of the Cx45-coding region with the lacZ reporter gene. Heterozygous Cx45(+/)(-) mice showed strong expression of the reporter gene in vascular and visceral smooth muscle cells. Cx45-deficient embryos exhibited striking abnormalities in vascular development and died between embryonic day (E) 9.5 and 10.5. Differentiation and positioning of endothelial cells appeared to be normal, but subsequent development of blood vessels revealed impaired formation of vascular trees in the yolk sac, impaired allantoic mesenchymal ingrowth and capillary formation in the labyrinthine part of the placenta, and arrest of arterial growth, including a failure to develop a smooth muscle layer surrounding the major arteries of the embryo proper. As a consequence, the hearts of most Cx45-deficient embryos were dilated. The abnormal development of the vasculature in the yolk sac of Cx45(-)(/)(-) embryos could be caused by defective TGFbeta signalling, as the amount of TGF beta1 protein in the epithelial layer of the yolk sac was largely decreased in the E9.5 Cx45(-)(/)(-) embryo, compared with the wild-type embryo. The defective vascular development was accompanied by massive apoptosis, which began in some embryos at E8.5 and was abundant in virtually all tissues of the embryos at E9.5. We conclude that in Cx45(-)(/)(-) embryos, vasculogenesis was normal, but subsequent transformation into mature vessels was interrupted. Development of different types of vessels was impaired to a varying extent, which possibly reflects the complementation by other connexin(s).

Hepatocarcinogenesis in female mice with mosaic expression of connexin32.

Mice deficient for connexin32 (Cx32), the major gap junction forming protein in liver, are highly susceptible to hepatocarcinogenesis. Because the Cx32 gene is located on the X-chromosome, heterozygous females show mosaicism with respect to Cx32 expression; this enables their use in studying the effect of Cx32-deficiency in a mixed Cx32-plus/Cx32-minus environment in vivo. Female C3H/He mice (Cx32(+/+)) were crossed with Cx32-deficient C57BL/129Sv males (Cx32(Y/-)) to yield F1 females heterozygous with respect to Cx32 (Cx32(+/-)). Patches of hepatocytes were observed in normal liver that either expressed Cx32 or failed to express the protein. The mean fraction of Cx32-negative tissue in liver was about 60% and did not change significantly with age of mice. Neoplastic liver lesions, induced in weanling mice, were identified in serial liver sections by their deficiency in glucose-6-phosphatase staining. Parallel sections were used for immunohistochemical demonstration of Cx32 protein. Smaller lesions were either homogenously Cx32-negative or showed unchanged to slightly elevated levels of Cx32 protein. There were no major differences in number and size distribution between lesions of these 2 phenotypes. In addition, larger lesions were mostly Cx32-negative but often contained embedded patches of Cx32-positive cells. Staining for the proliferation-associated nuclear antigen Ki-67 did not reveal significant differences between Cx32-negative and Cx32-positive hepatocytes in Cx32-mosaic tumors. This suggests that expression of Cx32 within a subpopulation of tumor cells does not negatively regulate their growth nor does it seem to affect the proliferation of their directly neighboring Cx32-negative counterparts.

Expression patterns of connexin genes in mouse retina.

To analyze the molecular basis of gap junctional communication in mouse retina, we examined the expression pattern of the following 13 connexin (Cx) genes: Cx26, Cx30, Cx30.3, Cx31, Cx31.1, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, and Cx50. By using reverse transcriptase-polymerase chain reactions with primer oligonucleotides to murine connexin genes, we detected mRNAs of Cx26, Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, and Cx50. Retinae from heterozygous mice with targeted replacement of most of the Cx45 open reading frame by a lacZ reporter gene showed Cx45 promoter activity in somata of the ganglion cell layer and the inner nuclear layer. Immunoblot and immunofluorescence analyses with antibodies generated to murine connexin epitopes revealed the presence of Cx36, Cx37, Cx43, and Cx45 proteins: The outer and inner plexiform layer were immunopositive for Cx36 and Cx45. Cx37 immunoreactivity was found in blood vessels of the inner retina. Cx43 immunolabeling was detected in the ganglion cell layer and nerve fiber layer where it was largely colocalized with immunostaining of glial fibrillary acidic protein suggesting that Cx43-positive cells could be of glial origin. No Cx26 protein was detected in retina by using Cx26 antibodies for immunoblot analyses or confocal microscopy. Furthermore, comparative immunofluorescence analyses of retinae from mice deficient for Cx31, Cx32, or Cx40 with retinae of wild-type mice revealed no specific immunostaining. Our results demonstrate regional specificity in expression of connexin genes in mouse retina and, thus, provide a basis for future assignments of functional defects in connexin-deficient mice to cells in different regions of the retina.

Acute-phase response and circadian expression of connexin26 are not altered in connexin32-deficient mouse liver.

In mouse hepatocytes, the gap junctional proteins connexin32 (Cx32) and connexin26 (Cx26) are expressed in the same gap junctional plaque. Expression of the major Cx32 protein is downregulated during liver regeneration and cholestasis. Here we have analyzed the acute-phase response (after experimental inflammation) and circadian connexin expression in Cx32-deficient and wild-type mouse liver. Acute-phase response was triggered by intraperitoneal injection of lipopolysaccharide (LPS). Injection of recombinant mouse interleukin-1beta (mIL-1beta), mIL-6 or tumor necrosis factor alpha (mTNF-alpha) had no inflammatory effect. Northern blot analysis of positive and negative acute-phase transcripts following stimulation with cytokine or LPS revealed no difference between Cx32-deficient livers and wild-type controls, suggesting that loss of the Cx32 gene had no effect on experimental liver inflammation. Actin, beta-fibrinogen and Cx26 transcripts were increased after endotoxin stimulation. Under conditions of hepatic acute-phase response, Cx32 transcripts were not detected in LPS-treated livers of wild-type mice. Immunoblot analysis of proteins from inflamed wild-type livers indicated a strongly diminished amount of Cx32 protein, whereas the level of Cx26 protein was increased. Although intraperitoneal injection of mIL-1, mIL-6 as well as mTNF-alpha did not induce an acute-phase response, Cx32 protein expression was diminished, suggesting that post-transcriptional downregulation of Cx32 preceded the acute-phase response. Northern blot hybridization of RNA from wild-type and Cx32-deficient mouse liver revealed a similar circadian regulation of Cx26 and GAPDH transcripts with maximal expression around 2 p.m. and a minimum after midnight.

Connexin expression in the retina.

Here, we review recent results from our laboratory on connexin expression in mouse retina in the context of previous results with other vertebrate species. In mouse retina, four different connexin proteins were detected by immunoblot and immunofluorescence: connexin (Cx)-36, -37, -43 and -45. Cx36 and Cx45 immunoreactive signals were found in the inner and outer plexiform layer, both of which are known to show interneuronal gap junctions. Cx43 was detected in the ganglion cell layer, presumably in astrocytes, where it appeared to be colocalized with glial fibrillary acid protein. Cx37 was expressed in retinal endothelial cells. Additionally, Cx26, -31, -32 and -40 mRNAs were detected in retina by RT-PCR but none of the corresponding proteins were found. In order to exclude cross reactions of the corresponding antibodies, retinae from targeted connexin-deficient mice (Cx31 -/-, Cx32 -/- and Cx40 -/-) were used as negative controls for immunoblot and immunofluorescence analyses of wild-type retina. Further detailed investigation of cell type specific connexin expression in the mouse retina will be necessary for functional analyses of targeted mouse mutants with defects in connexins expressed in retinal neuronal cells.

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.

Connexin expression patterns in human trophoblast cells during placental development.

This study focuses on the gap junction expression pattern in trophoblast cells during human placental development in vivo and in vitro. Investigations of cell-cell communication properties within the subpopulations of trophoblast responsible for invasion, placental growth and feto-maternal transport seem of special interest because the intercellular channels are believed to coordinate proliferation and differentiation processes. From all gap junction connexins (Cx) investigated (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43), Cx40 was the only connexin clearly detected within the cytotrophoblast of human placenta, and was restricted to the extravillous trophoblast of cell islands and cell columns. Most intense staining was found in the juxtastromal area correlated to the proliferating extravillous trophoblast cells. Connexin protein expression was missing during trophoblast migration into the decidua but was re-expressed in trophoblast aggregates within the decidua. Cx40 expression decreased with progressing pregnancy and no connexins could be detected in villous or extravillous trophoblast of mature placentae. In parallel, isolated trophoblast cells of first and second trimester placentae revealed Cx40 expression and, in contrast to the situation in vivo, Cx43 was also found. In isolated cells of mature placentae, expression of both Cx40 and Cx43 transcripts was decreased to low levels and Cx40 immunoreactivity was absent. Cx43 protein, however, was still detectable in trophoblast cultures of term placentae. Our studies suggest that Cx40 is the characteristic channel for the proliferating cell population of cell islands and cell columns of first and second trimester placentae and isolated trophoblast and is probably involved in regulation and coordination of the invasive pathway.

Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation.

Fluid shear stress (flow) modulates endothelial cell function via specific intracellular signaling events. Previously we showed that flow activated ERK1/2 in an integrin-dependent manner (Takahashi, M., and Berk, B. C. (1996) J. Clin. Invest. 98, 2623-2631). p130 Crk-associated substrate (Cas), a putative c-Src substrate, was originally identified as a highly phosphorylated protein that is localized to focal adhesions and acts as an adapter protein. Recent reports have shown that Cas is important in cardiovascular development and actin filament assembly. Flow (shear stress = 12 dynes/cm(2)) stimulated Cas tyrosine phosphorylation within 1 min in human umbilical vein endothelial cells. Phosphorylation peaked at 5 min (3.5 +/- 0.7-fold) and was sustained to 20 min. Tyrosine phosphorylation of Cas was functionally important because flow stimulated association of Cas with Crk in a time- and force-dependent manner. Flow-mediated activation of c-Src, phosphorylation of Cas, and association of Cas with Crk were all inhibited by calcium chelation and pretreatment with the Src family-specific tyrosine kinase inhibitor PP1. To determine the role of c-Src in flow-stimulated phosphorylation of Cas, we transduced cells with adenovirus encoding kinase-inactive Src. Expression of kinase-inactive Src prevented flow-induced Cas tyrosine phosphorylation but not ERK1/2 activation. Calcium-dependent activation of c-Src and tyrosine phosphorylation of Cas defines a new flow-stimulated signal pathway, different from ERK1/2 activation. This pathway may be involved in focal adhesion remodeling and actin filament assembly.