A–C, LacZ staining of 50 µm brain slices obtained from Cx47^+/M282T mice. The β-gal activity is restricted to nuclear localisation. A, β-Gal positive cells in the corpus callosum show typical oligodendrocytic chain-like organization and coexpression of the oligodendrocytic marker CNPase. LacZ expression was not detectable in GFAP-positive astrocytes (B) or NeuN-positive neurons (C). D, Immunostaining with Cx47 antibodies (green) revealed signals in close proximity of Syto-61 stained nuclei in the corpus callosum of Cx47^+/+ mice. Weaker signals were also found more distal to the nuclei. E, Cx47 antibody stainings on heterozygous Cx47^+/M282T mice resulted in weaker, but similarly localized immunosignals compared to those obtained on wildtype brain tissue. Cx47 gap junction immunosignals were predominantly localized close to β-gal positive nuclei indicated by antibody staining. F, Homozygous Cx47^M282T/M282T mice showed apparent β-gal immunoreactivity, but robust Cx47 gap junction immunosignals were not detected in the perikarya. Very weak Cx47 signals were noticed in brain tissue of Cx47^M282T/M282T mice. G, Immunoblot staining against β-gal on whole brain lysates of 40-day-old mice yielded strong signals with Cx47^M282T/M282T, weaker signals with Cx47^+/M282T and no signals with Cx47^+/+ tissue. H, Immunoblot analysis on Cx47 protein resulted in unspecific bands at 50 kDa of whole brain lysates obtained from wildtype, Cx47^M282T/M282T and Cx47 deleted (Cx47^−/−) tissue. Immunoprecipitated Cx47 was detected in wildtype and Cx47^M282T/M282T tissue. Lysates of Cx47-eGFP fusion protein expressing HeLa cells yielded signals at 75 kDa as expected and additional weaker signals at approximately 40 kDa after immunoprecipitation of Cx47 and with crude HeLa cell lysate. Additional signals at 40 kDa in these lanes may represent the cleaved C-terminal region of Cx47. Scale bars: A–C, 50 µm; D–F, 20 µm.

Semi-thin sections of cerebellar white matter of Cx47^+/+ (A, C) Cx47^+/M282T (B, E, I) and Cx47^M282T/M282T (C, F, G, J) mice are shown after staining with toluidine blue/pyronin g. Cystic spaces in white matter are indicated by asterisks in C and can be delineated from blood vessels (BV) due to their irregular lining and absence of endothelia. At higher magnification (F, G) cysts can be shown to contain cellular debris (arrows in G). No alterations were seen in cerebella from heterozygous mice (B, E). In the prechiasmatic optic fascicle of Cx47^M282T/M282T mice, similar cysts are evident (arrows in J, compared to blood vessles (BV) in the WT control shown in H). Scale bars: A–C, 50 µm; D–J, 20 µm.

A1 and B1, Membrane currents of oligodendrocytes were recorded in response to a series of voltage steps ranging from −170 and + 50 mV (50 ms, 10 mV increments) from a holding potential of −70 mV. A2 and B2, Oligodendrocytes were identified by their typical morphology as revealed by dialysis with the fluorescent dye Alexa 594. A3 and B3, Superpositions of sequential confocal stacks indicating biocytin/streptavidin-Cy3 labeled networks. C–E, Quantification of networks in homozygous and heterozygous mCx47M282T as well as Cx47^−/− mice compared to wildtype animals. C, Percentage of injected cells which were coupled to at least one other cell and thereby formed a network (dark gray) versus injected uncoupled cells (light gray). Note that in Cx47^+/M282T mice only 27% of injected oligodendrocytes were found to be coupled. Asterisks indicate statistical significance (Chi Square Test, *** p<0.001). D, Boxplot indicating median (dark line), 25^th and 75^th percentiles (upper and lower hinges, respectively) of the number of coupled cells per network in Cx47^M282T/M282T and Cx47^+/M282T compared to wildtype mice. Similarly to Cx47^−/− mice, Cx47^M282T/M282T animals displayed a significant reduction in the number of cells participating in the oligodendrocytic network (6%; 4%–13%; Kruskal-Wallis test, p = 0.001, Mann-Whitney U-test, *** p<0.001). E, Extent of tracer spread as defined by the largest distance between two somata within the coupled network. No significant difference was observed between homozygous and heterozygous mCx47M282T mice compared to wildtype animals. In Cx47^−/− mice, the extent of tracer spread was significantly reduced compared to wildtype animals (132±11 average, One way ANOVA p<0.05, Bonferroni post hoc test, p<0.05).

A, D, G and J, Immunohistochemical analysis on 25 µm sagittal brain slices obtained from 10-day-old animals revealed fewer myelinated, MBP-positive fine fibers pervading the cerebellar granular layer of Cx47^+/M282T, Cx47^M282T/M282T and Cx47^−/− mice compared to Cx47^+/+ littermates. B, E, H and K, Delayed myelin formation is accompanied by elevated GFAP expression, indicating astrogliosis in the cerebellar white matter which appeared minor in Cx47^+/M282T tissue but was pronounced in cerebella of P10 Cx47^M282T/M282T and Cx47^−/− mice. Arrows indicate the transition of white matter to the granule cell layer. Compared to wildtype tissue fewer GFAP-positive cells were localized in the granular layer of Cx47^M282T/M282 cerebella. Note the GFAP-positive radial processes of Bergmann glia cells pervading the molecular layer. Signals of GFAP-positive Begmann glia processes were used as internal control for staining intensities. C, F, I and L, Microglia were constantly distributed throughout the whole brain. Activated microglia displayed increased levels of Iba1. In P10 cerebella microglia with elevated Iba1 expression were found to a greater extent in brain slices obtained from Cx47^+/M282T, Cx47^M282T/M282T and Cx47^−/− animals relative to Cx47^+/+ tissue. Auxiliary lines (MBP and Iba1 stainings) indicate the granular and Purkinje layer transition. Scale bars: A–L, 200 µm; insets, 50 µm.

Immunoblots were performed with cerebellar lysates. At least three animals per group were analyzed. For each oligodendrocytic marker, protein levels were normalized to β-tubulin expression as internal standard. Expression levels of Cx47^+/+ mice were set to 100% per group. A, Densitometric quantification of immunoblot analyses revealed significantly decreased CNPase expression in P10, P16 and P40 Cx47^M282T/M282T mice compared to their wildtype littermates (** p<0,05; *** p<0,001; ANOVA). Cx47^+/M282T mice showed a significant reduction of CNPase expression on P16 in comparison to Cx47^+/+ animals. However, at an age of 90 days both Cx47^M282T/M282T and Cx47^+/M282T mice displayed significantly higher CNPase expression levels compared to Cx47^+/+ littermates. Similarly, 90-day-old homozygous mCx47M282T expressing mice additionally deficient for Cx32 (Cx47^M282T/M282T/Cx32^Y/−), displayed significantly increased CNPase levels in immunoblot analysis. B, Relative CNPase expression in Cx47^+/+ mice during postnatal development was determined via densitometric analysis of immunoblots. CNPase quantification analyses (A) showed the development-dependent expression of CNPase in cerebella of Cx47^+/+, Cx47^+/M282T and Cx47^M282T/M282T mice. In the cerebellum, Cx47^+/+ mice displayed an expression peak of CNPase around P40, while the CNPase levels in Cx47^+/M282T and Cx47^M282T/M282T cerebellar tissue augmented on P90 of postnatal development. C and D, Analogous illustration of MBP expression: C, At each developmental stage, MBP levels in Cx47^+/M282T and Cx47^M282T/M282T cerebella were normalized to the MBP expression level of Cx47^+/+ animals (100%). In Cx47^+/M282T and Cx47^M282T/M282T cerebella a significant MBP reduction was observed between P10 and P40 compared to wildtype littermates, with strongest impact on P10. Three-month-old Cx47^M282T/M282T mice did not show significantly altered MBP expression any longer, while MBP signals were significantly enhanced in Cx47^+/M282T mice on P90. Immunoblots on lysates obtained from P90 Cx47^M282T/M282T/Cx32^Y/− mice revealed a clear reduction of MBP expression. D, Development-dependent MBP expression illustrates early hypomyelination and compensation during adolescence in Cx47^+/M282T and Cx47^M282T/M282T animals. Error bars indicate SEM. E, Representative immunoblot analyses on cerebellar tissue obtained from Cx47^+/+, Cx47^+/M282T and Cx47^M282T/M282T mice during postnatal development (40 µg proteins/lane). Cx47^M282T/M282T/Cx32^Y/− animals analyzed on P90 exhibited disorders during movement.

A, Scheme of homologous recombination of the targeting vector into the Cx47 coding region. The resulting transgenic allele (Cx47M282Tneo) includes mCx47M282T coding DNA, an internal ribosome entry site (IRES) followed by a nuclear localization signal (nls) fused to LacZ coding DNA and a neomycin selection cassette flanked by frt-sites. The endonuclease BsmBI restriction site was generated by T to C transition on nucleotide 845 of the Gjc2 coding region, resulting in the mutant mCx47M282T. B, Flp recombinase activity causes deletion of the neomycin selection cassette. Specific primer binding sites and MfeI restriction sites are indicated. C, PCR products specific for wild-type (570 bp) and transgenic loci (730 bp) using DNA obtained from tail tip tissue. D, Presence of the mutant allele was proven by PCR amplification of a 700 bp Cx47 fragment and subsequent BsmBI digestion. Only mutant alleles yielded the 350 bp fragment. E, PCR analysis resulted in amplification of the 1,700 bp fragment of the neomycin selection cassette deprived allele and the 450 bp fragment of the Cx47M282Tneo locus. F, Homologous recombination and different allelic combinations were confirmed by Southern blot hybridization using a probe derived from the 3′ region of Cx47. MfeI digestion of genomic DNA prepared from liver resulted in the of 4.6 kb fragment for the Cx47 wildtype allele, the 10.5 kb fragment for the Cx47M282Tneo allele and the 8.5 kb fragment for the Cx47M282T allele.

Sagittal 50 µm brain sections were X-Gal stained for LacZ expression and counterstained with eosin. The LacZ reporter gene reflects the expression of Cx47. A, In the cerebellum of 7-day-old Cx47^M282T/M282T mice several X-Gal stained oligodendrocytes were already found in the medullary centre. B, Stainings on cerebella of P10 mice revealed an increase in the number of β-gal positive cells in the white matter compared to P7 mice. Single β-gal positive cells were already found in the granular layer. C and D, Cx47 expressing cells further increased in number during development of the cerebellum. In addition to their localisation in the cerebellar white matter, LacZ expressing cells were found to be widespread among the granular layer in the cerebellum of 105 day old mice. E, Compared to the number of X-Gal stained cells in the white matter of P7 cerebellum, only few cells were stained in P7 cerebral white matter, localized in the corpus callosum and the hippocampal fimbria. Single cells were found in the cortex proximal to the corpus callosum. The hippocampal gray matter was almost free of β-gal positive cells at this developmental stage. F, On P10 the number of X-Gal stained cells was remarkably increased in the corpus callosum and the hippocampal fimbria compared to P7. β-Gal positive cells decreased in number from ventral to dorsal in the corpus callosum to the cortex. G, P16 animals showed robust LacZ expression in the white matter of the cerebrum including corpus callosum, hippocampal fimbria and hippocampal alveus. Furthermore, β-gal positive cells were present at the hippocampal fissure and near the hippocampal regions Cornu ammonis 1 (CA1) and CA3. Compared to P10 the number of LacZ expressing cells increased dramatically in cortical layers VI to IV of P16 mice. H, 105 days after birth Cx47 expressing cells represented by LacZ reporter gene stainings were localized all over the telencephalon with strong accumulation and typical oligodendrocytic chain-like organizaiton in the white matter. In gray matter, β-gal positive cells were scatterly distributed with a decreasing gradient ranging from layers VI to I of the neocortex. Scale bar: A–H, 500 µm.

A and B, Circles represent mean ± sem locomotion (distance moved in cm) on indicated trials. C and D, Circles represent mean ± sem running speed (in cm/s) on indicated trials. E and F, Circles represent mean ± sem number of rearings on indicated trials. G and H, Circles represent mean ± sem duration of rearings (in s) on indicated trials. I and J, Motor coordination and balancing on the accelerating rotarod. Circles represent mean ± sem time of active performance (in s) on acquisition and retention trials. ***: Cx47^+/+ versus Cx47^M282T/M282T, p = 0.01; Bonferroni test. *: Cx47^+/+ versus Cx47^+/M282T, p = 0.01; Bonferroni test.

A1–A5, Example of cell-type identification in biocytin labeled networks of Cx47^M282T/M282T mice. Biocytin was detected with streptavidin-Cy3 (A1) and oligodendrocytes were identified by CNPase immunostaining (A2). A3, Immunolabeling of the same section for the astrocytic marker GFAP. A4, Overlay of A1 and A2. A5, Overlay of A1 and A3. In this network no coupled cell expressed GFAP. B1–B5, Biocytin labeling with streptavidin-Cy3 (B1) combined with immunostaining for CNPase (B2) and GFAP (B3) in the corpus callosum of Cx47^+/M282T mice. B4, Overlay of B1 and B2. B5, Overlay of B1 and B3. Note the GFAP-positive astrocyte (asterisk) labeled with biocytin. The same cell was CNPase-negative (B4, asterisk). C, Boxplot indicating median (dark line), 25^th and 75^th percentiles (upper and lower hinges, respectively) of the population of cells coupled within networks. Values of specific glial cell types are given as percentage of the number of biocytin-positve cells per network. In both Cx47^M282T/M282T and Cx47^+/M282T mice the majority of coupled cells were CNPase-positive, a small population lacked immunopositive signals for both CNPase and GFAP, and few GFAP-positive cells were found. Cx47^+/M282T expression in comparison to wildtype tissue did not result in significant differences regarding the population of coupled cells per network. In Cx47^M282T/M282T mice, immunostaining revealed an increased amount of CNPase-positive cells in the panglial networks (100%; 86%–100%; Kruskal-Wallis test, p<0.01, Mann-Whitney U-test, **p<0.01). A significantly diminished number of cells negative for both CNPase and GFAP were detected in these networks (0%; 0%–7%; Kruskal-Wallis test, p<0.01, Mann-Whitney U-test, *p<0.05). In Cx47^−/− mice all coupled cells were identified as eGFP-positive, i.e. oligodendrocyes [23]. No significant difference in oligodendrocyte-to-oligodendrocyte or oligodendrocyte-to-astrocytel coupling between Cx47^M282T/M282T and Cx47^+/M282T mice was observed.

Additional deletion of Cx32 in homozygous mCx47M282T expressing mice resulted in severe myelin malformation, astrogliosis and microglia activation. A, Immunohistochemical analysis on 25 µm sagittal cerebellar slices obtained from 90-day-old animals yielded strong myelin staining in the granular layer of wildtype mice. D, G and J, Only few MBP-positive fibers attaining the Purkinje cell layer were found in corresponding regions of Cx47^+/M282T, Cx47^M282T/M282T and Cx47^+/M282T/Cx32^Y/− mice. M, MBP immunohistochemical signals were very weak in Cx47^M282T/M282T/Cx32^Y/− animals and appeared like debris in the granular layer. B, E, H, K and N, GFAP stainig on cerebellar slices of all five genotypes revealed astrogliosis only in Cx47^M282T/M282T/Cx32^Y/− animals (N) whereas Cx47^+/+, Cx47^+/M282T, Cx47^M282T/M282T and Cx47^+/M282T/Cx32^Y/− mice did not display increased GFAP expression in the cerebellar white matter. H inset, Like P10 mice (Figure 8H), adult Cx47^M282T/M282T mice harboured fewer GFAP-positive cells in the granular layer compared to Cx47^+/+ and Cx47^+/M282T mice. Arrows indicate the transition of white matter to the granule cell layer. Signals of GFAP-positive Begmann glia processes were used as internal control for staining intensities. O, Furthermore, considerable numbers of Iba1 expressing activated microglial cells were present in Cx47^M282T/M282T/Cx32^Y/− cerebellar white matter. All other genotypes investigated did not display microglia with increased Iba1 exression in cerebellar tissue. Auxiliary lines indicate the granular and Purkinje cell layer transition. Scale bars: A–O, 200 µm; insets MBP and GFAP stainings, 50 µm; insets Iba1 stainings, 25 µm.