Connexin45 modulates the proliferation of transit-amplifying precursor cells in the mouse subventricular zone

Abstract

Connexins have been implicated in the regulation of precursor cell migration and proliferation during embryonic development of the mammalian brain. However, their function in postnatal neurogenesis is unclear. Here we demonstrate that connexin (Cx) 45 is expressed in transit-amplifying cells and neuroblasts in the postnatal subventricular zone (SVZ) and modulated the proliferation of SVZ-derived precursor cells in vivo. Thus, overexpression of Cx45 by retroviral injections increased the proliferation of Mash-1–positive transit-amplifying precursor cells in the SVZ. Conversely, conditional deletion of Cx45 in precursor cells decreased proliferation. Finally, we established that Cx45 positively influences cell cycle reentry via ATP signaling that involves intracellular calcium stores and ERK1/2 signaling.

In the adult brain, two neurogenic regions generate new neurons, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampus (1, 2). In the SVZ, neural stem cells give rise to transit-amplifying cells, which after several divisions differentiate into neuroblasts (2, 3). Neuroblasts migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB), where the majority mature into granule cells or periglomerular cells (2, 4). Likewise, in the SGZ of the hippocampus, neural stem cells divide slowly producing precursor cells that differentiate into neuroblasts and mature into glutamatergic granule cells (1, 3).

In the developing embryonic cortex, an important functional role was attributed to connexin (Cx) 43 that was shown to be required for neuroblast migration (5, 6) and for the structural organization of the SVZ (7). In the SGZ of the hippocampus, expression of Cx43 and Cx30 was found to be important for neural stem cell proliferation (8). Connexin isomers, which differ in their molecular weight and tissue specificity, form gap junctions, thus building hydrophilic pores between cells that allow the diffusion of small molecules such as ions and second messengers (9, 10). In addition, there is evidence that connexins also assemble as hemichannels that mediate a number of physiological functions, including development, cell survival, and cell death (11).

Recently, Cx45 was shown to be expressed in the SVZ and rostral migration stream (RMS) (12) and in neurospheres derived from the SGZ (13), but the function of Cx45 in postnatal neurogenic regions has remained unknown. Here we used Cx45 conditional knockout and overexpression to investigate Cx45 function in postnatal neurogenesis. Proliferation of neural precursors in the SVZ was reduced after Cx45 knockout, whereas the opposite effect was obtained upon retroviral overexpression of Cx45 in precursor cells. Overexpression of Cx45 increased proliferation and cell cycle reentry of transit-amplifying precursors, but not of other proliferating cell types in the SVZ. Finally, block of ATP signaling abolished Cx45-dependent precursor cell proliferation.

Results

Cx45 Is Expressed in Neurogenic Niches of the Adult Brain.

Based on microarray analysis of posterior and anterior RMS (pRMS and aRMS) neuroblasts (14), we identified two connexin genes, namely Gja1 coding for Cx43 protein and Gjc1 coding for Cx45 protein, whose expression was down-regulated >50% in the aRMS (Fig. S1 A and B).

To determine Cx45 expression in the neurogenic zones, we used postnatal day (P) 15 genetically modified mice in which Cx45 was deleted in Nestin-positive cells, i.e., Cx45fl/+:Nestin-Cre (15). Deletion of one Cx45 allele is associated with EGFP reporter gene expression (Fig. 1A). In accordance with previously published data (12, 16), we found EGFP expression in a subpopulation of NeuN-positive neurons in the thalamus (Fig. 1B). In the SVZ, Cx45/EGFP expression was confined to Mash1-positive transit-amplifying cells (Fig. 1D) and DCX-positive neuroblasts (Fig. 1E), and some GFAP-positive cells, which might comprise both neural stem cells and mature astrocytes (Fig. 1C). In line with our microarray data that showed a down-regulation of Cx45 expression from pRMS to aRMS, we also detected a fainter EGFP reporter gene signal in the aRMS (Fig. 1A).

Fig. 1.

Cx45 expression in the mouse brain. (A) An overview illustrating Cx45/EGFP expression in a sagittal brain section of a P15 Cx45fl/+:Nestin-Cre mouse, indicating that Cx45 is expressed in the thalamus (Th), SVZ, RMS, and olfactory bulb (OB). (B) Cx45/EGFP is expressed in thalamic NeuN⁺ neurons. (C and D) In the SVZ, Cx45/EGFP is expressed in GFAP⁺ neural stem cells/astrocytes, and Mash1⁺ transit-amplifying precursors, respectively. (E) In the RMS, Cx45/EGFP is expressed in DCX⁺ neuroblasts. Arrowheads mark double positive cells. (Scale bars: B, 50 µm; C–E, 20 µm.)

Cx45 Augments Transit-Amplifying Precursor Cell Proliferation in Vivo.

To elucidate the impact of Cx45 on postnatal neurogenesis in vivo, we overexpressed Cx45 in SVZ-derived neural progenitor cells. We injected two retroviruses in a 1:1 ratio into the SVZ of P5 wild-type mice, one expressing Cx45 and EGFP, the other serving as a control, expressed red fluorescent protein (tdTomato) only (Fig. 2A). Because retroviruses infect only fast-dividing precursor cells in the SVZ (17, 18), overexpression of Cx45 is limited to neural precursor cells and their progeny. Eight days after injection, the ratio of green:red (Cx45 overexpression:control) cells was increased along the migrational route in the RMS. Thus, the ratio of green:red cells was ∼1 in the SVZ, but was >2 in the RMS and the OB (Fig. 2B). At 12 d after injection, the ratio of green:red cells was 1.5 in the SVZ and further increased to 4.1 in the RMS and the OB (Fig. 2B and Fig. S2A). This effect became more prominent 18 d after injection (Fig. 2B). It most likely reflects an increase in precursor cell proliferation rather than enhanced neuroblast migration because the ratio of all green:red cells (i.e., counting cells in the SVZ, RMS, and OB) increased between days 8 and 12 after injection from 1.3 ± 0.5 (n = 6) to 3.3 ± 1.4 (n = 7). Furthermore, if green cells migrated faster than control red cells, eventually more red cells would arrive in the OB and the ratio of green:red cells would be 1, which was not the case (Fig. 2B). Indeed, live imaging experiments indicated that neuroblasts infected with control or Cx45 overexpressing virus, migrated at comparable speed (36.4 ± 9.5 µm/h for control n = 5; 31.8 ± 13.9 µm/h for Cx45 overexpression n = 5; Fig. S3 and Movie S1).

Fig. 2.

Cx45 is involved in SVZ precursor cell proliferation in vivo. (A) Wild-type P5 mice were injected with Cx45 overexpressing (Cx45OE, green) and control retrovirus (red) at a 1:1 ratio and were killed 8 (Cx45OE n = 6, control n = 6), 12 (Cx45OE n = 7, control n = 7), or 18 (Cx45OE n = 9, control n = 9) days after injection. (B) At all analyzed timepoints, the ratio of green (Cx45OE):red (control) cells in the RMS and OB was significantly increased in comparison with the SVZ, indicating an involvement of Cx45 in precursor cell proliferation. (C) Wild-type P5 mice were injected with Cx45OE (green) and control retrovirus (red) at a 1:1 ratio and were killed 8 d after injection. Two hours before killing the mice, BrdU was injected to label proliferating cells. (D) For direct comparison, all images shown in E, H, and Fig. S2B were taken from the indicated SVZ area (red square). (E and F) There was a higher percentage of BrdU⁺ cells in the Cx45OE cell population in comparison with controls (Cx45OE n = 7, control n = 7). Arrowheads mark EGFP/BrdU double-positive cells. (G) Cx45 overexpression did not affect cell survival in the SVZ, as evidenced by activated caspase-3 labeling (n Cx45OE = 5, control n = 5). (H–J) Cx45 overexpression increased the number of proliferating Mash1⁺ transit-amplifying precursors (H and I; n Cx45OE = 6, n control = 6), but not DCX⁺ neuroblasts (J; Cx45OE n = 6, control n = 6). Arrowheads mark EGFP/BrdU/Mash1-triple positive cells. Student’s t test was used for statistics (mean ± SD). For F and G, n is number of analyzed hemispheres; for I and J, n is number of analyzed brains. (Scale bars: 20 µm.)

To confirm that Cx45 overexpression increases SVZ cell proliferation, we again injected the retroviral mix (1:1 ratio) into the SVZ of P5 wild-type mice (Fig. 2 C and D), and 2 h before killing the mice, we injected BrdU to label proliferating cells. Because we killed mice already 2 h after injection, only fast-proliferating cells in the SVZ and RMS were labeled. There was a significant increase in BrdU-labeled cells after Cx45 overexpression (green cells) in comparison with control cells (red) in the SVZ (Fig. 2 E and F), but not in the RMS (Fig. S2C), indicating that Cx45 expression levels affected transit-amplifying cell proliferation (i.e., precursor cells residing in the SVZ). We also showed that the increase in the number of transit-amplifying precursor cells was not due to cell survival, because the number of activated caspase-3–labeled cells in the vicinity of the SVZ was comparable in Cx45 overexpressing and control conditions (Fig. 2G).

To further corroborate that Cx45 overexpression affected only transit-amplifying cell proliferation, the BrdU-labeled cell population was analyzed in more detail. Upon Cx45 overexpression, the number of BrdU-labeled Mash1-positive (a marker of transit amplifying precursors) cells increased compared with the control condition (Fig. 2 H and I), whereas the number of DCX-positive (a marker of neuroblasts) cells was similar in the two conditions (Fig. 2J). Also the number of GFAP-positive (a marker of neural stem cells and astrocytes) cells was not affected by overexpression of Cx45. Thus, in the virus-infected BrdU-labeled cell population the number of GFAP-positive cells was negligible (1/103 for Cx45, mice n = 11; and 1/25 for tdTomato, mice n = 11). This result is not too surprising given that retroviruses infect by and large fast-dividing cells, which are transit-amplifying precursors and neuroblasts in the SVZ (17, 18). Thus, Cx45 overexpression enhanced the proliferation of transit-amplifying precursor cells in the SVZ that contributed more cells to the OB.

The striking feature of Cx45 overexpressing cells (green) was their cluster formation. Groups of 3–20 green fluorescent cells were often found in a close proximity, often adjoining each other (Fig. 2E and Fig. S2B). Red fluorescent cells very rarely formed such clusters (Fig. 2E). Many clustered cells were labeled by BrdU (Fig. 2 E and H and Fig. S2B) and were BrdU and Mash1-double positive (Fig. 2H). Thus, adjoining Cx45 overexpressing transit-amplifying cells were characterized by increased proliferation. Here the obvious question comes to mind whether the Cx45-mediated effect on proliferation depends on gap junctions or hemichannels. Both substrates have been shown for other connexins to play a functional role in different cell types and organs, during embryonic development and in the adult organism (10, 11). Thus, we patched transit-amplifying precursor cells in clusters of Cx45 overexpressing cells in acute brain slices 7–13 d after injection (Fig. S4A). One cell out of each cluster was patched to confirm that, based on electrophysiological parameters, it was a transit-amplifying precursor cell. Their input resistance was 2.13 ± 0.5 GΩ (n = 5), similar to the input resistance of transit amplifying precursor cells reported in a previous study (19), but distinct from the input resistance of stem cells and ependymal cells (30–40 MΩ) and from the input resistance of neuroblasts in the SVZ and RMS (∼4 GΩ) (20). The patched cell was filled with neurobiotin and/or sulforhodamine, i.e., dyes known to pass gap junctions. Both neurobiotin and sulforhodamine did not diffuse into other cells of the cluster (Fig. S4 B and C), suggesting that the above described Cx45 effect on cell proliferation is not mediated by gap junctions.

To investigate whether Cx45 hemichannels are involved in SVZ precursor cell proliferation, we injected P5 wild-type mice with two retroviruses in a 1:1 ratio into the SVZ, one expressing Cx45 and EGFP, the other expressing only tdTomato (Fig. S4 D and E). Ten days after injection, organotypic slice cultures were prepared from injected brains, and slices were incubated in the presence or absence of Lanthanum, a connexin hemichannel inhibitor that was shown not to affect gap junctions (21). Two hours before fixation, BrdU was added to the culture media to label proliferating cells. Corroborating the in vivo data, in control slices, the number of BrdU-positive Cx45 overexpressing cells was twice that of BrdU-positive control cells (Fig. S4 F and G). However, addition of Lanthanum completely blocked the Cx45-dependent increase in precursor cell proliferation.

Next we studied the effect of cell-type specific Cx45 ablation on postnatal neurogenesis in vivo. We injected BrdU into P15 Cx45fl/fl:Nestin-Cre and Cx45fl/fl control mice (Fig. 3A), and the number of transit-amplifying cell in the BrdU-positive SVZ cell population was quantified 2 h after injection. Ablation of Cx45 in Nestin-positive cells led to a ∼30% reduction of Mash1/BrdU-double positive cells, indicating that Cx45 deletion in transit-amplifying cells impaired proliferation (Fig. 3B).

Fig. 3.

Cx45 knockout impairs transit-amplifying precursor cell proliferation. (A) P15 Cx45fl/fl:Nestin-Cre and Cx45fl/fl mice were injected with BrdU and analyzed 2 h after injection. (B) Proliferation of Mash1⁺ precursors in the SVZ was reduced in Cx45fl/fl:Nestin-Cre (n = 8) compared with Cx45fl/fl (n = 8) mice. (C) P5 Cx45fl/fl mice were injected with Cre overexpressing (green) and control retrovirus (red) at a 1:1 ratio and killed 2 (Cx45KO n = 7, control n = 7), 8 (Cx45KO n = 7, control n = 7), 12 (Cx45KO n = 6, control n = 6), or 18 (Cx45KO n = 7, control n = 7) days after injection. Two hours before killing the mice, BrdU was injected to label proliferating cells. (D and E) Eight days after injection and later there were fewer BrdU⁺/Cx45KO cells (green) in comparison with BrdU⁺/control cells (red) in the SVZ. For each time-point, data were analyzed as relative changes in the number of Cx45KO cells compared with controls. Student’s t test was used for statistics (mean ± SD). n is number of analyzed hemispheres. Lv, lateral ventricle. (Scale bar: 20 µm.)

To exclude a detrimental effect of Cx45 ablation on postnatal neurogenesis that might result from an inadvertent modification during embryonic development, we injected a mix of EGFP-Cre–expressing and red fluorescent protein (control)–expressing retroviruses (1:1 ratio) into the SVZ of P5 Cx45fl/fl mice (Fig. 3C). Because retroviruses infect only fast-dividing precursor cells in the SVZ (17, 18), expression of Cre and deletion of Cx45 is limited to neural precursor cells and their progeny. Mice were analyzed 2, 8, 12, and 18 d after injection, and BrdU was injected 2 h before sacrificing the mice (Fig. 3C). Beginning at 8 d after injection, there was a decrease in BrdU-labeled Cre-expressing cells (green cells) in comparison with control cells (red) in the SVZ (25%; Fig. 3 D and E). This difference was even more apparent 12 and 18 d after injection (Fig. 3E; 30% for 12 d and 50% for 18 d). Hence cell-specific Cx45 knockout in fast-dividing precursor cells resulted in a decrease in cell proliferation, thus confirming the data obtained in Cx45fl/fl:Nestin-Cre mice.

We subsequently investigated whether the effect of Cx45 on cell proliferation resulted from an alteration of the cell cycle. To this end, we injected a retrovirus expressing Cx45 and EGFP or only EGFP into the SVZ of P5 wild-type mice. Two days later, virus-infected cells were labeled with BrdU and analyzed after another 22 h by using the proliferation marker Ki67 (Fig. 4A). After Cx45 overexpression, the proportion of cells reentering the cell cycle, namely BrdU/Ki67 double-positive cells, was increased by 30% (Fig. 4 B and C). Thus, Cx45 enhanced proliferation of transit-amplifying precursor cells in the SVZ by increasing the fraction of cells reentering the cell cycle.

Fig. 4.

Cx45 promotes cell cycle reentry of SVZ precursor cells. (A) P5 wild-type mice were injected either with Cx45 overexpressing (Cx45OE, n = 7) or control EGFP (n = 6) retrovirus. Two days thereafter, mice were injected with BrdU and analyzed 22 h later. (B and C) In the BrdU⁺ population, the fraction of BrdU⁺Ki67⁺ cells was higher in the Cx45OE cells compared with controls, indicating that Cx45OE cells reenter cell cycle more often than control cells. Open arrowheads point to EGFP/BrdU double-positive cells that were negative for Ki67, filled arrowheads indicate Cx45OE/BrdU/Ki67 triple-positive cells. Student’s t test was used for statistics (mean ± SD). n is number of analyzed hemispheres. (Scale bar: 20 µm.)

Cx45-Mediated Intracellular Signaling That Is Involved in SVZ Precursor Cell Proliferation.

It was shown that Cx43 regulates proliferation of neural precursors in developing retina via ATP (22). Because ATP might be released via Cx45 gap junctions/hemichannels, we analyzed whether ATP signaling is involved in SVZ cell proliferation. We generated organotypic cultures from brains of P15 mice and incubated them with the ATP analog 2-MeSATP (a chemical that stimulates P2X and P2Y purinergic receptors), the P2X/P2Y receptor inhibitor suramin, or the P2X receptor inhibitor TNP-ATP (Fig. S5A). BrdU was added 2 h before fixation to quantify SVZ cell proliferation. The addition of suramin and TNP-ATP significantly inhibited proliferation in the SVZ, whereas 2-MeSATP increased cell proliferation (Fig. S5 B and C). Thus, ATP signaling via P2X receptors is involved in SVZ cell proliferation.

Finally, we investigated whether the altered ATP signaling occurred downstream of Cx45. P5 wild-type mice were injected with two retroviruses in a 1:1 ratio into the SVZ, one expressing Cx45 and EGFP, and the other expressing only tdTomato (Fig. 5A). Ten days after injection, organotypic slice cultures were prepared from injected brains and slices were incubated in the presence or absence of inhibitors. Two hours before fixation, BrdU was added to the culture media to label proliferating cells. Although in control slices Cx45 overexpression resulted in increased cell proliferation (Fig. 5 C and D), upon addition of suramin or TNP-ATP in the culture media, the enhancement of cell proliferation was completely abolished (Fig. 5 C and D). To probe for the contribution of Ca²⁺ stores downstream of connexin-mediated ATP signaling as was reported for radial glial cells (23), we depleted intracellular Ca²⁺ stores by using cyclopiazonic acid (CPA). This treatment blocked Cx45-dependent cell proliferation (Fig. 5D). The effect was most likely due to IP3-sensitive Ca²⁺ stores, because 2-APB (IP3-sensitive Ca²⁺ release inhibitor), but not ryanodine (ryanodine-sensitive Ca²⁺ release inhibitor) blocked, at least partially, Cx45-dependent cell proliferation. Furthermore, the chelation of extracellular Ca²⁺ with EGTA almost completely abrogated precursor cell proliferation in the SVZ (Fig. 5D).

Fig. 5.

Mechanism underlying Cx45-mediated regulation of SVZ precursor cell proliferation. (A) Wild-type P5 mice were injected with Cx45 overexpressing (Cx45OE, green) and control retroviruses (red) at a 1:1 ratio and were used for organotypic slice cultures 10 d after injection. Slices were cultured for 1 d with the chemicals indicated in D. Two hours before fixation, BrdU was added to the culture medium. (B) For direct comparison, images shown in C were taken from the indicated SVZ area (red square). (C and D) Without inhibitors, the percentage of BrdU⁺ cells in the Cx45OE cell population was 2.5 times higher than in tdTomato control cells. However, upon addition of suramin or TNP-ATP, the Cx45-dependent enhancement of cell proliferation was abolished. Cx45-dependent cell proliferation was also inhibited by cyclopiazonic acid (CPA) that depletes intracellular Ca²⁺ stores and, to some extent, by 2-APB (IP3-sensitive Ca²⁺ release inhibitor), but not by ryanodine (ryanodine-sensitive Ca²⁺ release inhibitor). Moreover, chelation of extracellular Ca²⁺ by EGTA strongly reduced precursor cell proliferation in the SVZ. Finally, the ERK1/2 inhibitor U0126, but not the protein kinase C inhibitor GF109203X, blocked Cx45-dependent proliferation. Filled arrowheads point to EGFP/BrdU double-positive cells, open arrowhead indicates tdTomato/BrdU double-positive cell. Quantification was based on 21 sections per condition (obtained from seven hemispheres, three slices per hemisphere). The number of red cells in control condition was normalized to 1, and all other groups were normalized to this control. Two-way ANOVA, with post hoc Bonferroni (mean ± SEM) was used for statistical analysis. (Scale bars: 50 µm.) (E) Proposed model of Cx45-mediated regulation of transit-amplifying precursor cell proliferation.

To test whether P2X/P2Y receptor signaling recruits downstream the activation of ERK1/2 and PKC, as was shown for other systems (24), we applied U0126, an inhibitor of MEK (the upstream activator of ERK1/2), or the PKC inhibitor GF109203X to organotypic cultures and analyzed the effect on SVZ cell proliferation. Whereas GF109203X did not affect proliferation of SVZ precursor cells, U0126 blocked Cx45-dependent cell proliferation (Fig. 5D).

Discussion

In the present study, we investigated a role of Cx45 in postnatal neurogenesis by using mice with Cx45 conditional knockout or Cx45 overexpression. We showed that knockout of Cx45 reduced proliferation of SVZ-derived neural progenitors, whereas overexpression of Cx45 enhanced progenitor proliferation. Increase in proliferation after Cx45 overexpression was specifically attributed to transit-amplifying precursors. Finally, we showed that Cx45-mediated modulation of precursor cell proliferation relied on ATP and Ca²⁺ signaling.

Gap junction proteins were reported previously to influence embryonic and postnatal neurogenesis. In the embryonic brain, Cx43 was found to affect migration of neural precursor cells (6, 7). In the postnatal brain, application of gap junctional blockers to the SVZ impaired proliferation and migration of neural precursor cells (25). In the present study, we demonstrated that proliferation of transit-amplifying precursor cells in the SVZ is modulated by Cx45 expression levels. Overexpression of Cx45 in transit-amplifying precursors increased the number of cells reentering the cell cycle. Conversely, proliferation of neural stem cells and neuroblasts was not affected by the knockout or overexpression of Cx45.

Cx45 might exert its function in postnatal neurogenesis via gap junction-mediated intercellular communication or via hemichannels. Using retrovirus-mediated Cx45 gene manipulation, we observed that Cx45 overexpressing cells in the SVZ often formed clusters of cells adjoining each other. However, cells in these clusters were not coupled by gap junctions as was indicated by a lack of dye spread from filled cells in the clusters. Thus, Cx45 hemichannels rather than gap junctions potentiate precursor cell proliferation. Moreover, the connexin hemichannel inhibitor Lanthanum, which does not affect gap junctions when applied extracellularly (21), completely abrogated Cx45-dependent SVZ precursor cell proliferation.

For Cx43, there is evidence that hemichannels/gap junctions regulate the division of neural progenitor cells in the developing retina and developing cortex via purinergic signaling (22, 23, 26). ATP released via connexin hemichannels activates different purinergic receptors and triggers Ca²⁺ waves resulting in further ATP release (22, 27). Indeed, ATP receptors were shown to be expressed in SVZ neural precursor cells (28, 29). Here, we showed that Cx45-dependent proliferation of SVZ precursor cells involves ATP signaling via P2X receptors. Cx45-dependent cell proliferation furthermore required extracellular Ca²⁺ and Ca²⁺ release from intracellular stores. In astrocytic cultures, P2 receptors evoke Ca²⁺ release and modulate cell cycle reentry via ERK1/2 and cyclin D1 (30). Based on these results and on ours showing that Cx45-dependent proliferation is blocked by the ERK1/2 pathway inhibitor U0126 in transit-amplifying precursors, we propose a model in which Cx45 downstream signaling is linked to cell cycle machinery (Fig. 5E).

In summary, here we demonstrated that the Cx45 expression level is a critical determinant of neural progenitor cell proliferation in the postnatal and adult mouse brain. Because Cx45 is already expressed during embryonic brain development, future studies will have to establish whether Cx45 expression plays a similar role during embryonic neurogenesis.

Materials and Methods

Animals.

For all our experiments, we used wild-type, Cx45fl/fl, and Cx45fl/fl:Nestin-Cre mice (15). All procedures with animals were performed according to the Heidelberg University Animal Care Committee.

Plasmid Cloning.

The Cx45 ORF was PCR amplified from whole mouse brain mRNA and cloned into a shuttle vector. Cx45 ORF was then subcloned into retroviral MMLV plasmid containing RSV promoter, IRES, and EGFP to generate MMLV-RSV-Cx45-IRES-EGFP. As control retroviral plasmids, we used MMLV-RSV-tdTomato or MMLV-RSV-EGFP. MMLV-RSV-EGFP-T2A-Cre was a generous gift from Wolfgang Kelsch (Heidelberg University).

Retrovirus Production and Injections.

Retroviruses were produced and viral titer was measured as described (17). Cx45-EGFP and tdTomato retroviruses or Cre-EGFP and tdTomato retroviruses were mixed at 1:1 titer ratio. The same batch of retrovirus mix was used for all experiments. One microliter of recombinant retrovirus mix was delivered into the SVZ of each hemisphere of P5 C57BL/6 mouse pups as described (17). See SI Materials and Methods for more details.

Organotypic Cultures.

Organotypic cultures were prepared as described (14). Briefly, 250-μm sagittal brain sections were cut with vibratome and used for organotypic cultures. Sections were cultured for 1 d, and different inhibitors were added to the culture medium (SI Materials and Methods). Sections were cultured for one more day, and then BrdU (Sigma-Aldrich) was added into culture media (final concentration 10 μM). Sections were fixed 2–3 h thereafter and processed for immunohistochemistry. See SI Materials and Methods for more details.

BrdU Injections and Proliferation Analysis in Vivo.

To label proliferating cells in the SVZ, mice were injected i.p. with BrdU at a concentration of 30 mg/kg (body weight), and brains were dissected 2 h after injection. To calculate the number of SVZ precursor cells that reenter the cell cycle, we injected P5 mice with either control EGFP expressing retrovirus or Cx45 overexpressing retrovirus. Two days after, BrdU was injected as described above. The number of proliferating cells in the SVZ that reenter cell cycle was determined as the percentage of Ki67-positive cells (a marker of any active phase of the cell cycle) in the BrdU-positive virus infected cell population.