Pathologic and phenotypic alterations in a mouse expressing a connexin47 missense mutation that causes Pelizaeus-Merzbacher-like disease in humans.

Gap junction channels are intercellular conduits that allow diffusional exchange of ions, second messengers, and metabolites. Human oligodendrocytes express the gap junction protein connexin47 (Cx47), which is encoded by the GJC2 gene. The autosomal recessive mutation hCx47M283T causes Pelizaeus-Merzbacher-like disease 1 (PMLD1), a progressive leukodystrophy characterized by hypomyelination, retarded motor development, nystagmus, and spasticity. We introduced the human missense mutation into the orthologous position of the mouse Gjc2 gene and inserted the mCx47M282T coding sequence into the mouse genome via homologous recombination in embryonic stem cells. Three-week-old homozygous Cx47M282T mice displayed impaired rotarod performance but unchanged open-field behavior. 10-15-day-old homozygous Cx47M282T and Cx47 null mice revealed a more than 80% reduction in the number of cells participating in glial networks after biocytin injections into oligodendrocytes in sections of corpus callosum. Homozygous expression of mCx47M282T resulted in reduced MBP expression and astrogliosis in the cerebellum of ten-day-old mice which could also be detected in Cx47 null mice of the same age. Three-month-old homozygous Cx47M282T mice exhibited neither altered open-field behavior nor impaired rotarod performance anymore. Adult mCx47M282T expressing mice did not show substantial myelin alterations, but homozygous Cx47M282T mice, additionally deprived of connexin32, which is also expressed in oligodendrocytes, died within six weeks after birth and displayed severe myelin defects accompanied by astrogliosis and activated microglia. These results strongly suggest that PMLD1 is caused by the loss of Cx47 channel function that results in impaired panglial coupling in white matter tissue.

Peripheral lymphangiogenesis in mice depends on ectodermal connexin-26 (Gjb2).

In order to study the specific function of connexin-26 (Cx26, also known as gap junction beta-2 protein; Gjb2), we generated knockin mice that expressed either a floxed lacZ reporter or, after Cre-mediated deletion, connexin-32 (Cx32)-coding DNA, both driven by the endogenous Cx26 promoter. Heterozygous Cx26knock-inCx32 (Cx26KICx32) embryos developed normally until embryonic day 14.5 but died before birth with severe lymphedemas. Although the jugular lymph sacs were normally developed, these embryos had a strongly reduced dermal lymphatic capillary network. By analyses of ß-galactosidase reporter protein expression and lymphatic or blood endothelial-specific marker proteins, we demonstrated that Cx26 expression is temporally closely linked to lymphangiogenesis. No obvious phenotypic abnormalities were observed in Cx26KICx32 mice when Cre-mediated recombination was directed to mesenchyme or blood endothelium using the Prx1-Cre or Tie2-Cre mouse strains, respectively. By contrast, keratin-5-Cre-mediated replacement of Cx26 with Cx32 or deletion of both Cx26 alleles revealed severe lymphedemas similar to the general Cx26KICx32 phenotype. Thus, conditional ablation of Cx26 (loss of function) in ectoderm leads to partial disruption of lymphatic capillaries and embryonic death. We conclude that appropriate development of dermal lymphatic vessels in mice is dependent on the expression of Cx26 in the ectoderm.

Loss of connexin43-mediated gap junctional coupling in the mesenchyme of limb buds leads to altered expression of morphogens in mice.

Mutations in the GJA1 gene coding for connexin43 (Cx43) cause oculodentodigital dysplasia (ODDD), a pleiotropic human disorder with characteristic morphologic anomalies of face, teeth, bones and digits. Interdigital webbings, also called syndactylies, are a characteristic phenotype of this disease showing high intra- and interfamilial penetrance. Therefore, we decided to study the molecular basis of syndactylies caused by Cx43 mutations. In order to reveal the impact of Cx43-mediated gap junctional coupling, we used mice expressing the human point mutation Cx43G138R and, in addition, 'knock-out' mice lacking Cx43. Both conditional mouse models developed syndactylies as a consequence of disturbed interdigital apoptosis, which we show to be due to reduced expression of two key morphogens: sonic hedgehog (Shh) and bone morphogenic protein 2 (Bmp2). Diminished levels of Bmp2 and subsequent up-regulation of fibroblast growth factors (Fgfs) lead to an insufficient induction of interdigital apoptosis. Interestingly, the reduction of Shh expression in Cx43 mutants begins on embryonic day 10.5 indicating a disturbance of the Fgf/Shh regulatory feedback loop, and confirming the recently published observation that gap junctions can relay Fgf signals to neighboring cells. Thus, Cx43-mediated gap junctional coupling in the mesenchyme of limb buds after ED11 is essential to maintain Shh expression, which regulates the downstream signaling of Bmp2. Besides diminished interdigital apoptosis, the decreased expression of Bmp2 in Cx43 mutants may also be involved in other morphological alterations in patients suffering from ODDD.

Connexin hemichannel-mediated CO2-dependent release of ATP in the medulla oblongata contributes to central respiratory chemosensitivity.

Arterial PCO2, a major determinant of breathing, is detected by chemosensors located in the brainstem. These are important for maintaining physiological levels of PCO2 in the blood and brain, yet the mechanisms by which the brain senses CO(2) remain controversial. As ATP release at the ventral surface of the brainstem has been causally linked to the adaptive changes in ventilation in response to hypercapnia, we have studied the mechanisms of CO(2)-dependent ATP release in slices containing the ventral surface of the medulla oblongata. We found that CO(2)-dependent ATP release occurs in the absence of extracellular acidification and correlates directly with the level of PCO2. ATP release is independent of extracellular Ca(2+) and may occur via the opening of a gap junction hemichannel. As agents that act on connexin channels block this release, but compounds selective for pannexin-1 have no effect, we conclude that a connexin hemichannel is involved in CO(2)-dependent ATP release. We have used molecular, genetic and immunocytochemical techniques to demonstrate that in the medulla oblongata connexin 26 (Cx26) is preferentially expressed near the ventral surface. The leptomeninges, subpial astrocytes and astrocytes ensheathing penetrating blood vessels at the ventral surface of the medulla can be loaded with dye in a CO(2)-dependent manner, suggesting that gating of a hemichannel is involved in ATP release. This distribution of CO(2)-dependent dye loading closely mirrors that of Cx26 expression and colocalizes to glial fibrillary acidic protein (GFAP)-positive cells. In vivo, blockers with selectivity for Cx26 reduce hypercapnia-evoked ATP release and the consequent adaptive enhancement of breathing. We therefore propose that Cx26-mediated release of ATP in response to changes in PCO2 is an important mechanism contributing to central respiratory chemosensitivity.

Human sFLT1 Leads to Severe Changes in Placental Differentiation and Vascularization in a Transgenic hsFLT1/rtTA FGR Mouse Model.

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1-mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans.

Connexin32 can restore hearing in connexin26 deficient mice.

Functional gap junction channels composed of certain connexin proteins are essential for the function of the cochlea. Homozygous deficiency in the Gjb2 (mice) or GJB2 (human) gene coding for connexin26 (Cx26) in the cochlea leads to hearing impairment in mice and humans, respectively. Here we have studied the functional equivalence of Cx26 and connexin32 (Cx32) isoforms in the cochlea. We analyzed a conditional mouse mutant in which the Gjb2 coding DNA was exchanged by LacZ DNA coding for the reporter protein beta-galactosidase. This allowed us to follow the unrestricted and cell type specific expression of Gjb2 promoter activity. After inner ear specific, Otogelin-Cre recombinase mediated deletion of the loxP-site-flanked LacZ coding DNA, transcription of the Gjb1 gene, coding for Cx32 was activated by the Gjb2 promoter. Interbreeding of these mice with conditional Gjb2 null mice resulted in animals in which Cx32 instead of Cx26 protein is expressed in the non-sensory epithelial network of the cochlea. When we analyzed the auditory function in these mice, we found that the expression of Cx32 protein is sufficient to support hearing in the absence of Cx26. Thus Cx32 can functionally replace Cx26 in the mouse cochlea resulting in almost normal hearing.