The Carboxyl-terminal Domain of Connexin43 Is a NegativeModulator of Neuronal Differentiation*□S

Received for publication, August 25, 2009, and in revised form, February 11, 2010 Published, JBC Papers in Press, February 17, 2010, DOI 10.1074/jbc.M109.058750

Marcelo F. Santiago‡§, Pepe Alcami‡¶, Katharine M. Striedinger‡�, David C. Spray‡, and Eliana Scemes‡1

From the ‡Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, the§Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro, Brazil, the ¶SelectionInternationale, Departement de Biologie, Ecole Normale Superieure, 46 rue d’Ulm, 75005 Paris, France, and �Accuray, Inc.,Sunnyvale, California 94089

Connexin43 (Cx43) is widely expressed in embryonic brain,and its expression becomes restricted mainly to astrocytes asthe central nervous systemmatures. Recent studies have indi-cated that Cx43 plays important, nonchannel, roles duringcentral nervous system development by affecting neuronalcell migration. Here, we evaluated the effects of Cx43 on neu-ronal differentiation. For that we used an in vitro model ofneural cell development (neurospheres) to evaluate, throughimmunocytochemistry, electrophysiology, andmolecularbio-logy, the degree of neuronal maturation from neurospheresderived fromwild-type (WT) and Cx43-null mice. Our resultsindicate that Cx43 is a negative modulator of neuronal differ-entiation. The percent neurospheres containing differenti-ated neurons and the number of cells displaying inward cur-rents were significantly higher in Cx43-null than in WTlittermate neurospheres. Knockdown of Cx43 with smallinterfering RNA increased the number of WT neurospheresgenerating differentiated neurons. Blockade of gap junc-tional communication with carbenoxolone did not induceneuronal differentiation in WT neurospheres. Transfectionof Cx43-null neurospheres with Cx43 mutants revealed thatCx43 carboxyl terminus prevents neuronal maturation. Inagreement with these in vitro data, in situ analysis of embry-onic day 16 brains revealed increased �-III-tubulin expres-sion in germinal zones of Cx43-null compared with that ofWT littermates. These results indicate that Cx43, and specif-ically its carboxyl terminus, is crucial for signaling mecha-nisms preventing premature neuronal differentiation duringembryonic brain development.

Multiple distinct mechanisms contribute to neocortical de-velopment. Diffusible and nondiffusible molecules have beenimplicated in neural cell proliferation, migration, and differen-tiation during brain development (1–4). Connexins (Cx),2 theproteins forming gap junctions, are prominent in neural pro-

genitor cells and play important roles in neural cell prolifera-tion, migration, and differentiation (5–9). Among the centralnervous system gap junction proteins, Cx43 and Cx26 are thetwo most abundantly expressed in neural stem cells, althoughtranscripts for other connexins (Cx29, Cx30, Cx30.2, Cx32,Cx36, and Cx47) are also present in embryonic states (10–12).The expression pattern of connexin proteins dramaticallychanges as the central nervous system matures, becomingrestrictedwith respect to cell type. For instance, following post-natal development, Cx43 together with Cx30 are mainly foundin astrocytes, whereas Cx29, Cx32, and Cx47 form the oligo-dendrocyte group of gap junction proteins, and Cx36, Cx45,and Cx30.2 are expressed in neurons (for review, see Ref. 13).The dramatic decrease in Cx43 expression during central

nervous system development, specifically during neuroblastmaturation, has raised the possibility that concurrent loss ofgap junctional communicationmay play an important role dur-ing neuronal differentiation. Indeed, initial studies (14, 15) indi-cating an inverse relation between the degree of electrical anddye coupling and neuronal differentiation suggested that cou-pling among neuroblasts, by favoring homogeneity among theirintracellular compartment, was sufficient to maintain the neu-ral progenitors in an undifferentiated state (16). However, incontrast with the idea that reduced coupling favors neuronaldifferentiation, treatment of P19 andNT2 “stem cell lines” withthe gap junction channel blockers carbenoxolone or 18-�-glyc-errhetinic acid was shown to reduce the expression of neuronaldifferentiation markers induced by retinoic acid treatment (17,18). The fact, however, that expression levels of Cx43 in NT2cells dramatically decreases as cells differentiate (17, 18) andthat carbenoxolone and 18-�-glycerrhetinic were shown toreduce the expression of Cx43 in NT2 cells (19) raises thepossibility that the expression of Cx43 itself rather than thereduced gap junctional communication may modulate neuro-nal differentiation.In the present study, we investigated the mechanisms by

which Cx43 modulates neuronal differentiation using neuralstem cells derived from wild-type (WT) and Cx43-null mice.We show here for the first time that, at embryonic day (E) 16,there is an early onset of neuronal differentiation in brains ofmice lacking Cx43. Moreover, using an in vitromodel of neuralcell development we show that this early onset of neuronal dif-

* This work was supported, in whole or in part, by NINDS/National Institutesof Health Grants NS052245 (to E. S.) and NS041282 (to D. C. S.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1S–7S.

1 To whom correspondence should be addressed: 1410 Pelham Pkwy.,Kennedy Center, Rm. 203. Bronx, NY 10461. Tel.: 718-430-3303; E-mail:scemes@aecom.yu.edu.

2 The abbreviations used are: Cx, connexin; WT, wild-type; E, embryonic day;DMEM, Dulbecco’s modified Eagle’s medium; CT, carboxyl-terminal; siRNA,small interfering RNA; CBX, carbenoxolone; ANOVA, analysis of variance;

CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; SVZ, subven-tricular zone; shRNA, short hairpin RNA.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 16, pp. 11836 –11845, April 16, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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ferentiation in the Cx43-null brain is unrelated to gap junc-tional coupling but can be prevented by exogenous expressionof the carboxyl-terminal domain of the Cx43 molecule.

EXPERIMENTAL PROCEDURES

Embryonic Brain Sections—Pregnant Cx43 heterozygoteC57BL/6J mice (Gja1Tm1/Kdr, initially purchased from Jack-son Laboratory and maintained for 12 years in our animalfacility) were anesthetized, and E16 embryos were removed.After decapitation, the embryonic brains were fixed in 4%p-formaldehyde overnight and then transferred to a cryo-protective solution (30% sucrose in phosphate-bufferedsaline). Coronal sections (14 �m thick) ofWT and Cx43-nullbrains were cut in a cryostat (Leica-CM1510S; Leica Micro-systems) and processed for immunohistochemitry (seebelow). The Albert Einstein College of Medicine AnimalCare and Use Committee approved all experimental proce-dures performed.Neurosphere Cultures—Neurospheres were prepared as

described previously (20, 21). Briefly, neural progenitor cellswere obtained by aspiration of forebrain tissues of E14 wild-type and Cx43-null C57BL/6J mouse embryos and mechani-cally dissociated into single cells in ice-cold Hanks’ balancedsolution (Ca2�- and Mg2�-free). Cells were transferred to tis-sue culture dishes containing Dulbecco’s modified Eagle’smedium nutrient mixture F12 (DMEM-F12; Invitrogen) sup-plemented with 5% B27 (Invitrogen), 1% antibiotics, and 20ng/ml human recombinant epidermal growth factor (Sigma)and allowed to grow into floating neurospheres. Culturemedium was changed twice a week, and neurospheres weremechanically dissociated into smaller neurospheres once aweek. Neurosphere cultures were maintained for no longerthan 2 months. For in vitro cell differentiation, floating neuro-spheres were plated on glass bottom microwells (MatTek Co.,Ashland, MA) coated with poly-D-lysine- (10 �g/ml; Sigma)and fibronectin (10 �g/ml; Sigma-Aldrich) and bathed inDMEM-F12 in the absence of epidermal growth factor.About 10–15 neurospheres were seeded/MatTek dish.Electrophysiology—Whole cell patch clamp recordings were

performed at room temperature on progenitor cells emigratedfrom 2–3-day adherent neurospheres bathed in a solution con-taining 140 mM NaCl, 2 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 5mM Hepes, 4 mM KCl, 5 mM glucose, 2 mM sodium pyruvate,and 1 mM BaCl2, pH 7.4 (using NaOH). The pipette solutioncontained 130 mM CsCl, 10 mM EGTA, 0.5 mM CaCl2, 3 mM

MgATP, 2 mM Na2ATP, and 10 mM Hepes, pH 7.3 (CsOH).Whole cell membrane currents were measured using the patchclamp technique by applying 10-mV voltage steps from a hold-ing potential of �100 mV to final values of �80 mV. All datawere recorded through an Axopatch 200B amplifier and digi-tized using an Axon Instruments digitizer (Molecular Devices).Clampex 6.0 was used for recording and the Clampfit 9.0 pro-gram for analysis.Immunostaining—Cryosections of E16 WT and Cx43-null

brains (see above) were washed three times with 0.3% TritonX-100 in phosphate-buffered saline (PBST) and incubated withphosphate-buffered saline containing 10% normal goat serumfor 30min at room temperature. Brain sections were incubated

with mouse anti-�-III-tubulin (1:500; Chemicon, Millipore) at4 °C for 72 h after which they were washed several times withPBST prior to incubation with goat anti-mouse Alexa Fluor488-conjugated secondary antibodies. Following severalwashes, slides were mounted in VectaShield containingDAPI (Vector Laboratories, Inc., Burlingame, CA).2–3-day adherent neurospheres were fixed for 5minwith 4%

p-formaldehyde and then treated for 30 min with a solutioncontaining 0.4% Triton X-100 and 10% goat serum prior toovernight incubation with mouse anti-�-III-tubulin (1:500;Chemicon, Millipore). After several washes, cells were incu-bated with goat anti-mouse-conjugated Alexa Fluor 488 sec-ondary antibodies and mounted using VectaShield containingDAPI.Morphometric Analyses of Neurite Extensions—Confocal

z-series (0.5-�m optical section) images of �-III-tubulin-posi-tive cells derived from 2–3-day adherent WT and Cx43-nullneurospheres were acquired using an upright confocal micro-scope (Olympus BX61WI) equipped with an argon/kryptonlaser and 20� dry objective. Images were captured using Fluo-view 4.3 software (Olympus). Neurite lengths of reconstructedimageswere calculated usingNeuronJ (an ImageJ plugin) imagesoftware.Transfection and Carbenoxolone Treatments—Floating Cx43-

null neurospheres were transfected with 6 �g/ml cDNA encod-ing either full-length Cx43, Cx43 truncated at position 257(Met257), or with Cx43CT-(255–382) using Lipofectamine2000reagent (Invitrogen). After 18–19 h, transfection reagents wereremoved, and neurospheres were plated in polylysine/fibronec-tin-coatedMatTek dishes containing DMEM-F12 for 24–30 h.Cx43 constructs were originally obtained from Dr. Eric Beyer(full-length Cx43) and Dr. Mario Delmar (Cx43M257 andCx43CT). In some experiments, WT neurospheres were trans-fected with Cx43 siRNA (sequence CAATTCCTCCTGCCG-CAAT) (22, 24). Floating wild-type neurospheres were treatedwith the gap junction channel blocker carbenoxolone (CBX;100 �M) for 18–19 h and then seeded on poly-D-lysine/fibronectin-coated MatTek dishes containing DMEM-F12 for24–30 h in the continuous presence of CBX. Quantification ofneuronal differentiation from transfectants and CBX-treatedneurospheres was performed after immunostaining by mea-suring the percent neurospheres displaying at least four �-III-tubulin-positive cells with neurite projections extending �60�m out of the neurospheres (referred to as neurospheres withdifferentiated neurons).Statistical Analyses—All data are expressed as mean � S.E.

GraphPad Prism version 5 was used for statistical analysis, con-sisting of one-way analysis of variance (ANOVA) followed byNewman-Keulsmultiple comparison test. Unpaired t test com-parison was also employed in some cases.

RESULTS

Disruption of Cerebral Cortex Layer Formation in the Cx43-null Mice—To evaluate whether global deletion of Cx43 affectsthe lamination of the cerebral cortex during the development ofthe nervous system, we stained brain coronal sections of E16WT and Cx43-null mice with the nuclear marker DAPI. Theanatomical dispersion of cells was the criterion to identify dif-

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ferent sublayers of the neocortical wall (CP, IZ, and VZ/SVZ),and the thickness of each layerwasmeasured andnormalized tothe total cortical thickness (referred to as fractional thickness).For that, 3 WT and 3 Cx43-null E16 brains derived from threeseparate litters were employed and measurements performedon 20 and 17 coronal sections ofWT and Cx43-null forebrains,respectively. Fig. 1A shows the profile of cortical lamination ofa WT animal stained with DAPI. In the Cx43-null mice (Fig.1B), the fractional thickness of sublayer VZ/SVZ is evidentlylarger (0.40� 0.01, n� 43measurements from3 animals) com-pared with that of WT animals (0.33 � 0.01, n � 58 measure-ments from 3 animals; p � 0.0001, unpaired t test; Fig. 1C).Moreover, we found a significant difference between the frac-tional CP thickness of the WT (0.33 � 0.01, n � 58 measure-ments) andCx43-nullmice (0.23� 0.01, n� 43measurements;p � 0.0001, unpaired t test; Fig. 1C). In contrast to the substan-tial and reciprocal thickness of CP and VZ/SVZ sublayers, thefractional thickness of the IZ sublayer was the same in the twogenotypes (WTIZ: 0.36 � 0.01, n � 58 measurements; Cx43-nullIZ: 0.35 � 0.01, n � 43 measurements; p � 0.72, unpaired ttest; Fig. 1C). Similar results in terms of fractional VZ/SVZ andCP sublayer thicknesseswere obtainedwhen analyseswere per-formed for each animal individually (Fig. 1D). Interestingly, wefound that one set ofWT andCx43-null brains had a thinner IZsublayer compared with the other two sets of animals (withinthe same genotype) (Fig. 1D), but they did not significantly dif-fer when compared with each other (between genotypes).These data showing reduction of theCP sublayer thickness in

the Cx43-null E16 brains are in agreement with previousreports (23, 24) of reduced neuronal progenitor cells in the cor-tical plate of E18 and E21 brains, which was attributed todelayed neuronal cell migration in the absence of Cx43. How-ever, in contrast to the reported accumulation of cells at the IZsublayer of E21 brains following knockdown of Cx43 by shRNA(23) and in E18 brains of Cx43f/f:nestin-Cremice (24), we foundan enlargement of the VZ/SVZ sublayers in E16 brains of theglobal Cx43-null mice.The Deletion of Cx43 Induces Early Neuronal Differentiation—

To determine whether the accumulation of cells in the prolif-erative zones could result in premature neuronal differentia-tion, we used the neuronalmicrotubulemarker�-III-tubulin toanalyze the neuronal phenotype in the E16 neocortex of WTand Cx43-null mice. In theWTmice, we observed high expres-sion of �-III-tubulin in the CP and a few cells and/or cell pro-cesses in the IZ (Fig. 2A). Also, there was a rare occurrenceof �-III-tubulin-positive cells in the proliferative sublayersVZ/SVZ at this embryonic age (Fig. 2,A andC). By contrast, the

FIGURE 1. Altered sublayer thickness of embryonic forebrains from Cx43-null mice. A and B, epifluorescence images of embryonic (day 16) forebraincryosections (14 �m) showing the distribution of nuclei of progenitorsstained with DAPI in three cortical layers (CP, cortical plate; IZ, intermediatezone; VZ/SVZ, ventricular zone/subventricular zone) of wild-type (A) and

Cx43-null (B) mice. Scale bar, 16 �m. C, quantitative analyses of layer thickness(fraction of total), based on DAPI staining, shown as bar histograms of mean �S.E. (error bars) values that were obtained from 58 – 43 hemicoronal sec-tions of 3 WT and 3 Cx43-null animals, respectively. The fractional thick-ness of VZ/SVZ of Cx43-null brains was larger whereas the CP was smallerthan those of WT littermates. D, mean � S.E. (error bars) fractional thick-ness values obtained for the three cortical layers measured from each ofthe 3 wild-type (white bars) and Cx43-null (black bars) animals individually.Images were acquired using a cooled-CCD HQ2 camera (Photometrics)attached to an epifluorescence inverted microscope (TE2000-E; Nikon)equipped with a 10� dry objective and UV filter set. Three different litterswere used.

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expression of �-III-tubulin was markedly higher in the Cx43-null mice and spread through all neocortical layers (Fig. 2B). Ofnote is the higher incidence of expression of�-III-tubulin in the

VZ/SVZ sublayer of the Cx43-null mice (Fig. 2, B andD) as wellasmore intense�-III-tubulin immunoreactivity in the IZ (com-pare Fig. 2, A and B). Quantification of �-III-tubulin-positivecells in the germinal zone (VZ/SVZ) indicates a 6-fold increasein the number of neurons in Cx43-null E16 brains comparedwith those ofWT (Fig. 2E; Cx43-null� 37.05� 1.7 cells, n� 20hemicoronal sections from 3 animals; WT � 6.24 � 0.6 cells,n � 25 hemicoronal sections from 3 animals).

These results suggest that global deletion of Cx43 promotesearly neuronal differentiation in the E16mouse neocortex. Theappearance of �-III-tubulin-positive cells at the VZ/SVZ alsosuggests that the early postmitotic neuroblasts differentiateprior to their migration into the cortical plate and that theiraccumulation in the VZ/SVZ thereby promotes the thickeningof this sublayer (Fig. 1; for DAPI staining and demarcation ofsublayer boundaries, see supplemental Fig. 1S). The presence ofmore �-III-tubulin-positive tangential processes in the IZ (Fig.2) also suggests an early differentiation of the tangentiallymigrating neurons originating from the ganglionic eminence.Early Neuronal Differentiation from Cx43-null Neurospheres—

To evaluate the mechanisms by which Cx43 modulates neuro-nal differentiation, we used the neurosphere assay as an in vitromodel of cell development. Neural progenitor cells derivedfromWTandCx43-null E14 brains grown into embryonic bod-ies (neurospheres) were seeded in poly-D-lysine and fibronec-tin-coated dishes for 2–3 days to allow cell differentiation andthen immunostained for �-III-tubulin. Quantification of per-centCx43-null neurospheres that after 3 days of adhesion to thesubstrate generated differentiated neurons (�-III-tubulin-pos-itive cells) revealed that 70.7� 5.9% (n� 70 neurospheres from16 animals derived from 7 litters) of Cx43-null neurospheresdisplayed neurons with neurites whereas only 21.7� 6.6% (n�99 neurospheres from 20 animals derived from 9 litters) ofWTneurospheres generated such differentiated �-III-tubulin-pos-itive cells (Fig. 3, A–C). Although the percent neurosphereswith differentiated neurons from Cx43-null brains was high,the absolute number of �-III-tubulin-positive cells generatedfrom each neurosphere was quite small; nevertheless, there wasa significantly higher percentage of cells expressing the neuro-nal marker in Cx43-null neurospheres compared with those ofWT littermates (WT: 1.6 � 0.3%, n � 14 neurospheres from 5animals derived from 3 litters; Cx43-null: 3.9�.0.8%; n � 14neurospheres from 5 animals derived from 3 litters; p � 0.011,unpaired t test; Fig. 3D).Besides this increased number, Cx43-null-derived �-III-

tubulin-positive cells displayed a more mature phenotypethan those fromWTneurospheres. Quantification of neuritelength and number of primary and of secondary brancheswere obtained from confocal images of neurospheres con-taining �-III-tubulin-positive cells using NeuronJ mor-phometry software. These analyses revealed that all threeparameters of morphological complexity were higher in neu-rons derived from Cx43-null than those derived from WTneurospheres (Fig. 3, E–G and supplemental Fig. 2S). �-III-Tubulin-positive cells derived from Cx43-null neurospheresdisplayed neurites that were three times longer than those ofWT cells (Cx43-null: 563.1 � 95.8 �m; WT: 166.3 � 37.4�m; n � 12–13 cells from 3 WT and 2 Cx43-null animals

FIGURE 2. Premature neuronal differentiation in embryonic brains ofmice lacking Cx43. A and B, confocal images of embryonic (day 16) fore-brains of WT (A) and Cx43-null (B) mice showing the distribution of �-III-tubu-lin-positive neuronal cells in the three cortical layers (VZ/SVZ, IZ, and CP); LV,lateral ventricle. Immunohistochemstry revealed increased number of cellsexpressing anti-�-III-tubulin in the VZ/SVZ and IZ layers of the forebrains ofCx43-null mice compared with those of WT littermates. Note the tangentialdistribution of �-III-tubulin-positive fibers (*) in the IZ of Cx43-null brain.C and D, confocal images of WT and Cx43-null VZ/SVZ layers amplified 2�from regions delimited by the squares in A and B, respectively. Arrows indicatecell bodies of �-III-tubulin-positive cells, and arrowhead in C shows part of ablood vessel. Images were acquired using a laser scanning confocal invertedmicroscope (Zeiss Duo 510 Meta) equipped with a 20� dry objective andfluorescein isothiocyanate filter sets using an argon ion laser (488 nm line).Antibodies used were mouse anti-�-III-tubulin (1:500; Chemicon) and goatanti-mouse Alexa Fluor-conjugated 488 nm antibodies (1:2000; Invitrogen).E, bar histograms showing mean � S.E. (error bars) number of �-III-tubulin-positive cells/hemicoronal section present in the VZ/SVZ of WT and Cx43-null(KO) E16 brains. DAPI stain of sections shown in this figure that were used todefine sublayers boundaries are displayed in supplemental Fig. S1.

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derived from the same litter; Fig. 3E); furthermore, eachCx43-null-derived neuron had twice as many primary neu-rites (3.75 � 2.18, n � 12 cells; Fig. 3F) as the WT-derived

cells (1.85 � 0.19, n � 13 cells) and 12 times more secondaryneurites (2.75 � 0.82, n � 12 cells; Fig. 3G) thanWT-derivedneurons (0.23 � 0.17, n � 13 cells).

FIGURE 3. More substantial neuronal differentiation from Cx43-null-derived neurospheres than WT. A and B, examples of epifluorescence images of WT (A) andCx43-null (B) neurospheres showing the expression of the neuronal marker �-III-tubulin in 3-day adherent progenitors. Note the extensive neurite projections fromCx43-null neurospheres and their complete absence in that of a WT littermate. Insets in B illustrate a �-III-tubulin-positive cell with three neurites (arrowhead)emanating from the cell body. Inset on the right was magnified twice that of the left. Scale bar, 50 �m. C and D, bar histograms of the mean � S.E. (error bars) values ofpercent neurospheres with differentiated neurons (�-III-tubulin-positive cells with neurite projections) (C) and of percent neurons/ neurosphere (D) obtained from WT(white bars) and Cx43-null (black bars) mice. E–G, bar histograms of the mean � S.E. (error bars) values of neurite length (E) and of the number of primary (F) andsecondary (G) neurites measured from �-III-tubulin-positive progenitor cells derived from WT (white bars) and Cx43-null (black bars) neurospheres. Morphometricanalyses of neurites from �-III-tubulin-positive cells were performed on reconstructed images of confocal z-sections (supplemental Fig. 2S) using NeuronJ software.

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To extend the analysis of neuronal maturation to functionalfeatures, we performed whole cell patch clamp recordings oncells emigrated from WT and Cx43-null adherent neuro-spheres to investigate the presence and characteristics of theiractive conductances. In agreement with the higher number andadvanced morphology of �-III-tubulin-positive cells found inCx43-null neurospheres, electrophysiological recordings (Fig.4, A and B) also evidenced a higher number of cells displayingneuronal-like inward currents in Cx43-null-derived cells thanin WT littermates (Cx43-null: 73.5 � 6.4%, WT: 27.2 � 14.1%;n � 29–34 cells from 5 or 6 animals derived from 4 litters; p �0.02, unpaired t test; Fig. 4C). Analyses of inward current ampli-tudes recorded fromWT and Cx43-null cells in which currentswere present indicated that both displayed similar currentamplitudes (Cx43-null: 542.2 � 111.7 pA, n � 14 cells; WT:849.9 � 269.7 pA, n � 8 cells, p � 0.05 unpaired t test; Fig. 4D).None of the cells displaying inward currents was electricallycoupled to neighboring cells, as measured by dual-whole cellvoltage clamp recordings (data not shown). Thus, our morpho-logical and electrophysiological findings strongly indicate thatdeletion of Cx43 favors neuronal differentiation. No significantdifference in ratio of nestin to glial fibrillary acidic proteinexpression levels was observed by Western blot analyses of1–7-day adherentWT andCx43-null neurospheres, suggestingthat these markers are not affected by Cx43 expression(supplemental Fig. 3S).Neuronal Differentiation Is Independent of Gap Junctional

Communication Blockade—To evaluate whether lack of Cx43gap junctional communication interferes with neuronal

maturation, two strategies wereemployed, namely, pharmacologicalblockade of gap junction channelsand acute down-regulation of Cx43using siRNA.Continuous treatment(48 h) ofWT neurospheres with thegap junction channel blocker CBX(100 �M) did not increase the num-ber of WT neurospheres with neu-rons (�-III-tubulin-positive cellswith neurite extensions) (WTCTRL:26.5 � 7.4%, n � 99 neurospheresfrom 9 animals derived from 7 lit-ters; WTCBX: 13.2 � 8.1, n � 43neurospheres from 4 animalsderived from 2 litters; p � 0.5ANOVA followed by Newman-Keuls test; Fig. 5). That CBX treat-ment effectively decreased dye cou-pling among cells was tested usingthe Lucifer Yellow microinjectiontechnique. Lucifer Yellow microin-jection into one cell derived from3-day adherent WT neurosphereresulted in dye diffusion to 8.3� 1.4cells (n� 7 experiments; 3 animals);this spread was reduced to 2.7 � 1.2cells (n � 3 experiments, 2 animals;p� 0.05 ANOVA followed by New-

man-Keuls test) after 48-h treatment with CBX, a value similarto thatmeasured fromCx43-null-derived cells (2.9� 0.6, n� 7experiments, 3 animals; p � 0.05 ANOVA followed by New-man-Keuls test). Differently from CBX, however, knockdownof Cx43 with siRNA significantly increased the percentage ofneurospheres with neurons compared with those mock-treated WT neurospheres (WTMock: 21.1 � 6.6%, n � 55neurospheres from 7 litters; WTCx43siRNA: 70.4 � 8�18.5%,n � 61 neurospheres from 5 animals derived from 3 litters;p � 0.05 ANOVA followed by Newman-Keuls test; Fig. 5).Examples of efficient Cx43 knockdown by siRNA are illus-trated in supplemental Figs. 4S and 5S. Thus, these resultsindicate that early onset of neuronal differentiation seen inCx43-null neurospheres and in Cx43-null brains is inde-pendent of gap junctional communication but dependent onthe expression of Cx43 itself.The Cytoplasmic C-terminal Domain of Cx43 Prevents Neu-

ronal Differentiation—To test whether specific domains ofCx43 modulate neuronal differentiation, we transfected Cx43-null neurospheres with three Cx43 constructs: full-length Cx43(Cx43), Cx43 lacking the carboxyl terminus (M257: truncatedat position 257), and the Cx43 carboxyl terminus itself (CT:amino acids 255–382). Two days after plating, transfected andmock-transfected cultures were immunostained with �-III-tu-bulin. As shown in Fig. 6, both Cx43 and CT constructs but notM257 abrogated the onset of neuronal differentiation seen inCx43-null cells. The percentage of mock-treated Cx43-nullneurospheres with differentiated neurons decreased from65.8� 3.9% (n� 50 neurospheres from7 animals from6 litters)

FIGURE 4. Electrical excitability of WT and Cx43-null progenitor cells. A and B, whole cell recordingsobtained from WT (A) and Cx43-null (B) progenitors showing the presence of (neuronal-like) inward currents incells from both genotypes. C and D, quantitative analyses of number of cells displaying inward currents (C)revealed a significantly higher number of cells with neuronal-like currents (21 of 29 cells) in the Cx43-nullgenotype compared with those of WT (8 of 34) progenitors. The amplitudes (D) of inward currents recordedfrom excitable WT and Cx43-null cells were not significantly different (p � 0.1469, unpaired t test). None of thecells displaying inward currents was electrically coupled to nearby cells, as evaluated by dual whole cell voltageclamp recordings.

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to 6.3 � 6.3% (n � 44 neurospheres from 5 animals derivedfrom 4 litters) and to 9.0 � 9.0% (n � 33 neurospheres from 3animals derived from 3 litters; p � 0.05 ANOVA followed byNewman-Keuls test) in neurospheres transfected with full-length Cx43 and with Cx43CT, respectively. The number ofneurospheres with neurons following transfection with Cx43lacking CT, i.e. transfected with the M257 construct (59.67 �0.9%, n � 74 neurospheres from 5 animals derived from 3 lit-ters) was similar to that recorded frommock-transfectedCx43-null (65.8 � 3.9%, n � 50 neurospheres from 6 litters; p � 0.05ANOVA followed by the Newman-Keuls test) (Fig. 6). Exam-ples of Western blots showing transfection efficiency andimmunofluorescence for cellular distribution of Cx43 and itsmutants are shown in supplemental Figs. 6S and 7S, respec-tively. Thus, these results support the hypothesis that expres-sion of Cx43 itself, and specifically its carboxyl terminus, mod-ulates neuronal differentiation during early stages of braindevelopment.

DISCUSSION

About one-third of the 20 mammalian connexins areexpressed in the adult central nervous system, which are devel-

opmentally regulated, with differentneural cell types expressing specificsubsets of gap junction proteins atparticular stages of development.Cx43 is prominent in early embry-onic days (E12–E18) of central nerv-ous system development, beingfound in all neural precursor typeslocated in the VZ and in radial gliaand neuroblasts extending to theCP(10, 12, 25). During postnatal stages,however, Cx43 becomes restrictedto the astrocyte population and isreplaced by Cx36 and Cx30.2 inpostmitotic neuroblasts (13, 16,26–28).Because of the wide distribution

of Cx43 in early stages of braindevelopment, it has been proposedthat Cx43 is involved in importantcentral nervous system functions.Indeed, previous studies have indi-cated that Cx43 promotes cell divi-sion (29, 30) and neural cell migra-tion (6, 8, 23, 24) and that it delaysneuronal differentiation (15, 31).Embryonic brain derived from

Cx43-null mice, labeled with bro-modeoxyuridine, indicated an accu-mulation of bromodeoxyuridine-positive cells within the innermostzones and reduction of cell numberin the cortical plate at particularembryonic stages compared withsimilarly treated brains of WT ani-mals (6). Similar to results obtained

withmice totally lacking Cx43, delayed neuronalmigrationwasalso reported in embryonic brains of the nestin-Cre:Cx43fl/fl(24) and following in utero electroporation of Cx43 shRNA orCx43 siRNA in WT embryonic brain ventricles (23, 24).Our results showing a reduction of CP size in Cx43-null

brains compared with those of WT siblings are in accordancewith previous studies showing delayed neuronal migration inbrains in which Cx43 is deleted or down-regulated (6, 23, 24).However, unlike previous reports showing accumulation ofcells in the IZ (23, 24), we found an enlargement of the VZ/SVZlayers in brains of mice lacking Cx43. Such a difference may berelated to the distinct stages of brain development analyzed(E16 versus E18 and E21) and/or to the different approachesused (total deletion of Cx43 versus shRNA knockdown andCx43 conditional knock-out). Moreover, our results showing,for the first time, increased �-III-tubulin immunoreactivity inthe germinal zones of brains of mouse embryos lacking Cx43suggest that the slowly migrating neuroblasts assume a moremature neuronal phenotype when Cx43 is absent. This earlyonset of neuronal maturation was also observed in Cx43-nullneurospheres, as indicated by the increased number of neuro-spheres producing�-III-tubulin-positive cells, longer andmore

FIGURE 5. Neuronal differentiation is independent of gap junction-mediated coupling. A–C, epifluores-cence images showing �-III-tubulin-positive cells in mock transfected (A), CBX-treated (B), and Cx43 siRNA-transfected WT neurospheres (C). Note the presence of extensive neuronal neurite projections after knock-down of Cx43 with siRNA from WT neurospheres compared with CBX- and mock-treated neurospheres. Scalebar, 50 �m. D, bar histograms showing the mean � S.E. (error bars) values obtained for the percent WT neuro-spheres with differentiated neurons (�-III-tubulin-positive cells with neurites) after 48-h treatment with trans-fection reagents (Mock), gap junction channel blocker CBX (100 �M), and after transfection with Cx43 siRNA.Knockdown of Cx43 with siRNA significantly increased the number of neurospheres with differentiated neu-rons to levels similar to those found in Cx43-null neurospheres; neither mock transfection nor CBX treatmentaltered the number of WT neurospheres with differentiated neurons. For knockdown efficiency, seesupplemental Figs. 4S and 5S.

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numerous neurite extensions, and increased number of cellswith neuronal-like inward currents in cells derived from Cx43-null neurospheres than those fromWT littermates (Figs. 3 and4). We did not detect alteration in the ratio of nestin to glialfibrillary acidic protein expression levels (supplemental Fig. 3S)or number of O4-positive cells (oligodendrocyte progenitors;data not shown) between the two genotypes (which after 2–3days of plating of WT neurospheres represent no more than2–3% of total cell population) (20). Thus, we favor the hypoth-esis that increased neuronal differentiation in Cx43-null brainsis more likely related to faster progression of neuronal progen-itors to amoremature phenotype rather than to increased neu-ronal commitment at the expense of other lineages. Neverthe-less, further studies are necessary to provide evidence in favoror against this hypothesis.Although the reports cited above strongly support a role for

Cx43 during neurogenesis, the mechanism(s) by which gapjunctions, specifically Cx43, regulate brain development is stilldebated. Early studies had emphasized gap junctional couplingas a mechanism by which a cohort of coupled cells migratingalong the radial glial fibers would maintain their identity andtherefore contribute to the specification of the neocorticalcolumnar cytoarchitecture by sharing gap junctional diffusablesignaling molecules and/or by displaying synchronous activity(14, 32–40). This hypothesis was supported by evidence indi-cating an inverse relationship between gap junctional commu-nication and neuronal differentiation. For instance, newbornneurons derived fromcultured immortalized hippocampal pro-genitor cells were shown to be dye and electrically coupled and

to express high levels of Cx43, whereas upon differentiation,Cx43 levels as well as cell coupling were shown to be dramati-cally reduced as the cells became electrically excitable (15).However, more recent studies have emphasized a channel-

independent effect of Cx43 on neural cell development, and atleast three mechanisms were proposed, including (i) a rippleeffect ofGja1 gene ablation on other genes such as the puriner-gic receptors involved in cell migration (8); (ii) an adhesivenessproperty provided by the interaction between the extracellularloops of Cx43 from radial glia with those of Cx43 expressed inmigrating neuroblasts (23); and (iii) a scaffolding property ofCx43CT due to its interaction with cytoskeletal elements (24),mediated in part by linker proteins such as ZO-1 andN-cadherins.Results described here are in contrast with the early hypoth-

esis described above that functional gap junction-mediatedcouplingmodulates neuronal differentiation.Our results show-ing that blockade of gap junctional communication with CBXdid not recapitulate the effects seen when Cx43 was deleted(knock-outmice) or knocked down (siRNA) provide strong evi-dence that expression ofCx43 itself but not direct cell-cell com-munication is involved in neuronal differentiation. Further-more, because we found that expression of the Cx43CTfragment alone, but not of Cx43 lacking this domain, was suffi-cient to restore the WT phenotype, as determined by percentneurospheres with �-III-tubulin-positive cells, we favor theinterpretation that the carboxyl-terminal domain of Cx43 is anintegral part of the signal transduction pathways that controlneuronal differentiation and neurite outgrowth, such as the

FIGURE 6. Cx43 carboxyl terminus modulates neuronal differentiation. A–D, examples of epifluorescence images showing �-III-tubulin-positive cells inCx43-null neurospheres mock transfected (A), transfected with full-length Cx43 (B), transfected with Cx43 truncated at position 257 (Cx43-M257) (C), andtransfected with the Cx43 carboxyl terminus itself (Cx43-CT) (D). Scale bar, 50 �m. E, bar histograms showing the mean � S.E. (error bars) values obtained for thepercent Cx43-null neurospheres with differentiated neurons (�-III-tubulin-positive cells with neurite projections) following 48 h treatment with transfectionreagents alone (Mock) and after transfection with full-length Cx43, Cx43 lacking the carboxyl terminus (M257), and with Cx43 carboxyl terminus itself (CT). Asignificant decrease in the percent neurospheres with differentiated neurons was obtained after expression of full-length Cx43 and the CT itself, but not withCx43 constructs lacking the CT. These two effective constructs significantly reduced the number of neurospheres displaying neuronal neurite projections tolevels seen in WT neurospheres. Transfection efficiency and cellular distribution of Cx43 and its mutants are shown in supplemental Figs. 6S and 7S.

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Wnt and Src-tyrosine kinase pathways (41, 42). In this regard,studies have shown that the association of Cx43 with �-cateninat the cell membrane is disrupted following deletion of Cx43,leading to �-catenin translocation into the nucleus where theprotein complexes with transcription factors and modulatesthe expression of specific target genes (43).Moreover, the asso-ciation of Cx43with Src-tyrosine kinases is well known (44, 45),and a recent study indicated that the lack of a Src binding site(SH3 domain) located within amino acids 264–287 of Cx43CT(46) impairs purinergic receptor-mediated calcium rises (47),an important signaling mechanism involved in neural cellmigration (8, 48).Besides interacting with key proteins involved in neural

development, theGja1 gene encoding Cx43 has been proposedto modulate a large number of gene networks (49). Microarrayanalyses of gene expression levels in WT and Cx43-null E19brains revealed that 5% of the spotted genes had their expres-sion levels altered in the null genotype (49). Of these regulatedgenes, we found that calbindin, a calcium-binding proteinexpressed in some pyramidal neurons and in GABAergic inter-neurons originating from the ganglionic eminence, was up-reg-ulated (1.6-fold) in brains of Cx43-null E18 embryos. This is inaccordance with our observation of intense �-III-tubulin in theIZ of Cx43-null brains, where inhibitory interneurons tangen-tially migrate from the ganglionic eminence to the forebrain.Similarly up-regulated (1.9-fold) in the null genotype were�-catenin (plakophillin), a protein complexing with the cad-herin cell adhesion molecule which promotes neurite out-growth, Gli2 (1.5-fold), a transcription factor promoting pre-mature neuronal differentiation, and drebrin (2.3-fold), aneuronal specific actin-binding protein involved in neuronalmorphogenesis.Certainly, further studies are necessary to disclose com-

pletely the intricate mechanisms by which Cx43 influencesneurodevelopment. However, the findings described here indi-cating that the cytoplasmic carboxyl-terminal domain of Cx43is a negativemodulator of neuronal differentiationmay providea new target molecule to study brain malformation anddiseases.

Acknowledgments—We thank Drs. Dominick P. Purpura andMichael V. L. Bennett for comments and suggestions on an earlierversion of this manuscript; Dr. Steve Taffet (SUNY Upstate MedicalUniversity, Syracuse, NY) for the Cx43-M254-FLAG construct;Melissa Aleksey and Aisha Cordero for technical assistance with ani-mal husbandry and genotyping, neurosphere cultures, and initialbrain cryosections; and Kevin Fisher for assistance with imageanalyses.

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ScemesMarcelo F. Santiago, Pepe Alcami, Katharine M. Striedinger, David C. Spray and Eliana

Neuronal DifferentiationThe Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of

doi: 10.1074/jbc.M109.058750 originally published online February 17, 20102010, 285:11836-11845.J. Biol. Chem. 

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