Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling

The adult mouse subependymal zone (SEZ) harbours adult neural stem cells (aNSCs) that give rise to neuronal and oligodendroglial progeny. However it is not known whether the same aNSC can give rise to neuronal and oligodendroglial progeny or whether these distinct progenies constitute entirely separate lineages. Continuous live imaging and single-cell tracking of aNSCs and their progeny isolated from the mouse SEZ revealed that aNSCs exclusively generate oligodendroglia or neurons, but never both within a single lineage. Moreover, activation of canonical Wnt signalling selectively stimulated proliferation within the oligodendrogliogenic lineage, resulting in a massive increase in oligodendrogliogenesis without changing lineage choice or proliferation within neurogenic clones. In vivo activation or inhibition of canonical Wnt signalling respectively increased or decreased the number of Olig2 and PDGFR- α positive cells, suggesting that this pathway contributes to the fine tuning of oligodendrogliogenesis in the adult SEZ. Adult mouse subependymal neural stem cells (aNSCs) give rise to neuronal and oligodendroglial progeny. Berninger and colleagues use continuous live imaging and single-cell tracking to demonstrate that single aNSCs isolated from the mouse brain generate exclusively either oligodendrocytes or neurons. They also show that Wnt activation stimulates oligodendrogenic progenitor proliferation without affecting neurogenic clones.

rise to neuronal and oligodendroglial progeny. Pathologies such as demyelinating diseases or genetic manipulations altering bone morphogenic protein (BMP) signalling have strongly suggested that neuro-and oligodendrogliogenesis are indeed competing cell fates 14,15 . In both instances, the oligodendrogliogenic transcription factor Olig2 was upregulated, resulting in the suppression of a neurogenic fate 14 or even conversion of cells already committed to a neuronal identity 15 . Nonetheless, whether NSCs can give rise to both lineages under physiological conditions remains an unresolved question.
We have recently used a primary culture of the adult SEZ to study the lineage progression of aNSCs by continuous live imaging 16,17 . Adult NSCs in these cultures were characterized by a prolonged cell cycle and marked cell growth before division when compared with transit-amplifying precursors (TAPs). Moreover, these cells exhibit an active glial fibrillary acidic protein (GFAP) promoter characteristic of their astro-/radial glial identity 7,13,18,19 . Finally, aNSCs derived from the ventrolateral aspect of the SEZ gave rise to asymmetric lineage trees consisting of both neuronal and astroglial progeny 17 . However, in these cultures we encountered very few oligodendroglial cells 16 , consistent with the overall scarce production of oligodendroglia in the ventrolateral SEZ (refs 4,5).
In the present study we show that oligodendrogliogenic aNSCs are enriched in the dorsal aspect of the SEZ. Continuous live imaging in vitro revealed that neuro-and oligodendrogliogenic lineages arise from aNSCs exhibiting a protracted cell cycle and an active GFAP promoter. However, we never observed the genesis of neurons and oligodendroglia within the same lineage tree. Moreover, we found that the oligodendrogliogenic lineage is selectively responsive to canonical Wnt signalling, known to be particularly prominent in the dorsal SEZ in vivo 20 .

Oligodendroglial progeny is enriched in primary cultures containing the dorsal SEZ
Although the adult SEZ is known to serve as a source for new oligodendroglial cells 3 , in previous studies we observed rather low numbers of oligodendroglial progeny in cultures of aNSCs isolated from the lateral SEZ. However, we noticed that cultures containing cells derived from both the dorsal and the lateral aspect of the SEZ contained considerably more NG2-immunoreactive oligodendroglial cells 21 (Fig. 1a-c; NG2 is also known as CSPG4, chondroitin sulfate proteoglycan 4). This suggests that the dorsal SEZ has a higher rate of oligodendrogliogenesis than the lateral SEZ. Consistent with this interpretation, 7 days following retroviral birth-dating in vivo we observed more progeny exhibiting an oligodendroglial morphology or expressing the oligodendrocyte progenitor marker PDGFRα (ref. 22) in the dorsal part than the lateral ( Fig. 1d and Supplementary Fig. S1a). Likewise, we noted a significant difference in the number of green fluorescent protein (GFP)-and NG2-double-positive cells between these two SEZ areas in mice expressing a tamoxifen-inducible Cre recombinase under the oligodendroglial lineage-specific Sox10 promoter, crossed to a reporter line (Sox10-iCreERT2 /GFP; refs 23,24 and Supplementary Fig. S1b,c).

Neuronal and oligodendroglial progeny arise from distinct lineages
Although aNSCs have been proposed to give rise to all major neural lineages, concomitant neuro-and oligodendrogliogenesis from single aNSCs has not been observed directly. To address this question we carried out continuous live imaging of primary cultures of the dorsal and lateral adult SEZ to track the lineage progression of aNSCs (refs 16,17). In this culture aNSCs can be identified by a slow cell cycle, substantial cell growth before division and asymmetric cell division within the lineage tree 16 . We encountered lineage trees originating from aNSCs fulfilling the above criteria and generating large clones of βIII-tubulin-positive neurons and few GFAP-positive astroglia ( Fig. 2a and Supplementary Fig. S2a). In these neurogenic clones none of the progeny was positive for NG2 ( Fig. 2a and Supplementary Fig. S2a). In contrast, few founder cells gave rise to NG2-positive oligodendroglia (Fig. 2b-d and Supplementary Fig. S3a and Video S1). However, these clones never contained any βIII-tubulin-positive progeny (Fig. 2b-d and Supplementary Fig. S3a). We also observed an instance of asymmetric cell division in which the founder cell gave rise to both NG2-positive oligodendroglial and GFAP-positive astroglial progeny ( Fig. 2d and Supplementary Fig. S4a). Many of the clonal founder cells were characterized by a slow cell cycle (Fig. 2e) and exhibited massive cell growth before division (Fig. 2f). Moreover, using a mouse line in which monomeric red fluorescent protein 1 (mRFP1) is driven by the hGFAP promoter 25 we observed the generation of oligodendrogliogenic clones from slow-dividing mRFP1-positive cells ( Supplementary  Fig. S4b). None of the cells that expressed mRFP1 for the first 24 h following plating was found to co-express NG2, indicating that the cells that give rise to oligodendrogliogenic clones are not oligodendrocyte precursor cells (OPCs) right from the beginning, but rather aNSCs ( Supplementary Fig. S2d). Finally, clones containing both NG2-and GFAP-positive progeny from GFP-reporter positive cells could be observed following fate mapping using the GLAST :: CreERT2 /GFP mouse line 24,26 (Fig. 2g). These data indicate that the SEZ harbours aNSCs that are capable of, and restricted to, generating NG2-positive oligodendroglial and GFAP-positive astroglial progeny. Strikingly, in no instance (n > 110 clones) did we observe the generation of both neuronal and oligodendroglial progeny from a single clonal founder cell, strongly supporting the notion that these two lineages remain strictly separated. To further characterize the progenitors giving rise to the neuro-or oligodendrogliogenic lineages we used SEZ cultures isolated from hGFAP-RFP mice and analysed the expression of different fate determinants 24 h after plating. We observed that 18 ± 1% of the RFPpositive cells expressed Olig2, a marker of the oligodendrogliogenic lineage, whereas 65 ± 12% expressed Pax6, indicative of commitment to the neuronal lineage. Finally, 75 ± 5% of the RFP-positive cells were immunoreactive to Mash1, known to be expressed in both lineages. Given that 85% of the clones are neurogenic and 9% of the clones are oligodendrogliogenic, with the remaining 6% consisting of GFAP-positive cells only, these data suggest that the expression of Pax6 and Olig2 is indicative of a neuronal and oligodendroglial fate commitment as early as the aNSC stage ( Supplementary Fig. S2c-e).

Wnt signalling increases oligodendroglial progeny in primary cultures of the adult mouse SEZ
Given that canonical Wnt signalling is strongly activated in the dorsal SEZ (ref. 20), we hypothesized that Wnt signalling may contribute to the regulation of adult SEZ oligodendrogliogenesis. To assess this we first treated primary cultures of the adult SEZ (dorsal + lateral) with recombinant Wnt3a protein, which is known to activate the canonical Wnt signalling pathway 27 . After 6 days of continuous treatment, the number of NG2-positive cells increased eightfold (Fig. 3a,b). This effect was highly specific to the oligodendroglial lineage, as the number of GFAP-positive astroglia and β-III-tubulin-positive neurons remained unchanged (Fig. 3a,b). This lineage-specific effect of Wnt3a stands in striking contrast to the effect of growth factors EGF (refs 3,16,28) and FGF2 (Fig. 3e,f), which concomitantly increase both oligodendroglial and astroglial progenies. The specificity of the Wnt3a effect on the oligodendroglial lineage was further corroborated by the selective increase in oligodendroglial messenger RNA transcripts, including the OPCspecific cell cycle regulator cyclin D1 (ref. 29), but not astroglial or neuronal mRNAs, with the notable exception of NeuroD1 and Eomes (Tbr2; Fig. 3d   with Wnt7a protein, an activator of the non-canonical signalling pathway 27 , was not able to reproduce the effect of Wnt3a (Fig. 3c). The effect of Wnt3a treatment was abolished by co-treatment with recombinant BMP4 (Fig. 3g), consistent with a previous report that BMP signalling in the lateral SEZ suppresses oligodendrogliogenesis in vivo 14 . Moreover, BMP4 treatment reduced the number of NG2positive cells in control cultures containing both the dorsal and lateral SEZ to levels similar to those in lateral SEZ-only cultures (362 BMP4-treated versus 1,273 control-treated cells per 339 mm 2 area). Conversely, treatment of cultures of the lateral SEZ-only cultures with the BMP inhibitor noggin increased the number of NG2-positive cells (694 noggin-treated versus 318 control-treated cells per 339 mm 2 area), suggesting that the low level of oligodendrogliogenesis in lateral SEZ-only cultures is due to endogenous BMP signalling 14 .

Wnt stimulates proliferation by shortening cell cycle length
This massive increase in NG2-positive cells following Wnt3a treatment could be accounted for by either the activation of quiescent aNSCs, an increase in proliferation within the lineage, an increase in survival or a fate switch of neurogenic aNSCs toward the oligodendroglial lineage. To discern between these possibilities we analysed the number of oligodendrogliogenic lineage trees, cell cycle length, number of amplifying rounds of division and the cellular composition of each clone. Figure 4a and Supplementary Video S2 show an example of an oligodendrogliogenic lineage tree originating from a slow-dividing aNSC that was characterized by marked cell growth before cell division. In agreement with the subsequent acquisition of an oligodendroglial identity, mRFP1 expression became downregulated along lineage progression (Supplementary Video S2). Analysis of several experiments revealed a marked increase in the number of cell divisions within the time window of tracking ( Fig. 4a,b; Supplementary Fig. S5b). In Wnt3atreated cultures, oligodendroglial lineage trees showed an average of 5.70 ± 0.41 rounds of division, compared with 3.25 ± 0.46 in control conditions (P < 0.001; Fig. 4e and Supplementary Fig. S3). Accordingly, cell cycle length was significantly decreased when compared with controls ( Fig. 4d). We also observed asymmetric cell divisions within clones generated from mRFP1-positive founder cells generating both oligodendroglial and astroglial progeny ( Fig. 4c and Supplementary  Fig. S5a). Another salient feature of the Wnt3a effect was the enhanced migratory behaviour of cells within oligodendrogliogenic clones (Fig. 4f), including the founder cells (for example see the position of the founder cell between 0-00:05 and 1-15:30 in Fig. 4a and Supplementary Video S2). Finally, we noted that clonal founder cells exhibited massive cell growth before cell division (Fig. 4g). We also noted a 40% increase in the number of oligodendrogliogenic clones ( Supplementary Fig. S3), suggesting that Wnt3a treatment results in the activation of quiescent aNSCs or a switch in cell lineage. Consistent with the above finding that Wnt3a treatment did not alter the number of βIII-tubulin-positive neurons, we observed no changes in the lineage progression of neurogenic clones ( Fig. 4h and Supplementary Fig. S2b).
To distinguish between activation of quiescent aNSCs and a switch from neuronal to oligodendroglial fate, we first monitored adult SEZ cultures under control conditions and subsequently added Wnt3a. Whereas the addition of Wnt3a did not alter the division pattern and cell fate decisions within neurogenic clones (Fig. 5a,b) the same treatment clearly enhanced the rate of proliferation within the oligodendrogliogenic lineage by decreasing cell cycle length (Fig. 5a,c and Supplementary Video S3).

Wnt signalling stimulates oligodendrogliogenesis in vivo
Consistent with a role of canonical Wnt signalling in the adult SEZ in vivo, we could detect various components of the canonical Wnt signalling pathway (dishevelled 1, Lrp5 co-receptors and Wnt3 ligand). Moreover, these signalling components were differentially expressed between the dorsal and the ventral wall of the SEZ, in agreement with the differences in oligodendrogliogenesis in these regions (Fig. 6e). Thus, we next addressed the question of whether canonical Wnt signalling augments oligodendrogliogenesis in the adult SEZ using a lentivirus for local expression of Wnt3 (LV-Wnt3-iresGFP; ref. 31). We confirmed in vitro that lentivirus-expressed Wnt3 is indeed secreted from cells isolated from the adult SEZ and exerts a similar effect as recombinant Wnt3a protein ( Supplementary Fig. S6). Next we proceeded to inject LV-Wnt3-iresGFP or a lentivirus expressing GFP only (LV-GFP) as control into the adult SEZ at lateral and dorsal locations. We counted the number of cells expressing Olig2 and PDGFRα within the area defined by GFP expression 14 days after lentivirus injection, and normalized it to the respective areas. lineage, because the neuronal and astroglial populations were not affected by local overexpression of Wnt3 (Fig. 7a,b). To corroborate the effect of Wnt signalling on the oligodendroglial lineage, we infused Wnt3a or artificial cerebrospinal fluid over 7 days using osmotic minipumps into the lateral ventricle, followed by 7 days without infusion before killing the animals. As expected, direct infusion markedly increased the number of Olig2-positive cells in the lateral SEZ (99 Olig2-positive cells 0.075 mm −2 in controls versus 589 cells 0.240 mm −2 following Wnt3a infusion (n = 2 animals); Fig. 7c). Carrying out these experiments in GLAST :: CreERT2 /GFP mice revealed that part of the effect of Wnt3a treatment comprised oligodendroglia derived from reporter-positive cells, supporting the notion that canonical Wnt signalling enhances oligodendrogliogenesis from aNSCs (Fig. 7c). Conversely, we carried out injections of a lentivirus encoding for a dominant-negative form of TCF4 (dnTCF4; ref. 32), a downstream target of the canonical Wnt signalling pathway regulator β-catenin, to inhibit Wnt signalling in the dorsal SEZ. After 14 days, we observed a significant reduction in the percentage of reporter-and Olig2-double-positive cells (from 54 ± 11% control to 17 ± 6% with dnTCF4) as well as in the reporter-and PDGFRαdouble-positive cells (from 38 ± 7% in controls to 20 ± 7% with dnTCF4) (Fig. 6g-i). To identify the source of Wnt3 in the adult SEZ, we carried out fluorescence-activated cell sorting (FACS) analysis to isolate fractions containing specific cell populations following a protocol previously described 33 . Quantitative PCR following reverse transcription (RT-PCR) revealed that Wnt3 is expressed mainly by the O4 + /EGFR + (OPC) fraction in the adult SEZ (Fig. 6f). of the Wnt3-encoding lentivirus (Fig. 8). Consistent with the in vitro live imaging data, the number of proliferating cells within the ectopic Wnt3-expression domain incremented to approximately 250% (2,796 Wnt3-treated versus 1,147 control-treated cells mm −2 ; Fig. 8b). These data support the notion that canonical Wnt signalling exerts its effect on the oligodendroglial lineage through an enhancement of proliferation.

DISCUSSION
Although the adult SEZ contains stem cells capable of generating neurons and oligodendroglia, single-cell tracking in vitro provides evidence that these two neural lineages have distinct pedigrees. Both arise from aNSCs of astro-/radial glial identity, but in no instance did we observe a clonal founder cell generating simultaneously neuronal and oligodendroglial offspring. Moreover, canonical Wnt signalling promoted proliferation selectively within the oligodendrogliogenic lineage without affecting the neuronal lineage.

Astro-/radial glial NSCs give rise to oligodendroglial progeny
Consistent with previous reports 4, 34 , we find that the SEZ contains aNSCs generating oligodendroglia. First, we observed that oligodendrogliogenic clones were initiated by cells with an active hGFAP promoter, a defining feature of aNSCs (refs 4,13,18,19). GFAP expression distinguishes oligodendroglia-producing cells of the SEZ from those of other forebrain regions such as the corpus callosum and the cerebral cortex 4 . Furthermore, genetic fate-mapping experiments corroborated the aNSC origin of SEZ oligodendroglia (Fig. 2g). Moreover, oligodendrogliogenic lineage trees frequently exhibited asymmetric cell division generating both oligodendroglia and astroglia, in contrast to OPCs of other forebrain regions 35 . Finally, none of the cells characterized by an active GFAP promoter expressed NG2 at the onset. Thus, these data indicate that the oligodendroglial progeny observed in the adult SEZ derives from aNSC rather than pre-differentiated OPCs.

Oligodendrogliogenic lineage progression
Continuous live imaging of aNSCs and their progeny in vitro revealed that oligodendrogliogenic lineage trees are characterized by amplification of the clone through proliferation of fast-dividing TAPs. Oligodendrogliogenic TAPs are characterized by a high degree of migratory behaviour, whereas neurogenic TAPs typically remain closely associated before reaching the neuroblast stage, when the propensity for migratory behaviour markedly increases 16 . This suggests that siblings within a neurogenic clone are likely to exert stronger influences on one another through cell-to-cell signalling than those of oligodendrogliogenic clones. The differences in migratory properties between these two lineages may reflect their distinct behaviour in vivo 2,4,36-38 .

The neurogenic and the oligodendrogliogenic progenies constitute distinct lineages
One of the key findings of this study is the observation that clonal founder cells give rise selectively to neuronal (and astroglial) or oligodendroglial (and astroglial) progenies, but never mixed ones. The analysis of the molecular profile of the clonal founder cells suggested that Pax6 and Olig2 are expressed by neurogenic and oligodendrogliogenic aNSCs, respectively. This strict separation between the neuronal and oligodendroglial lineages observed here seems to be at odds with the fact that during development neurons and oligodendroglia are often generated in a sequential manner within the same progenitor domain 39 , and is even more surprising because a classical hallmark of aNSCs is their supposed ability to generate neurons, astroglia and oligodendroglia on a clonal level using the neurosphere assay 28,40 . However, this assay requires the stimulation of aNSCs with EGF and FGF2. Studying the clonogenic competence of spinal cord neurospheres, FGF-2 has been shown to confer trilineage differentiation capacity on cells that are only bipotent in vivo 41 . Likewise, EGF was previously found to induce massive astro- 16,28 and oligodendrogliogenesis 34,42 from SEZ aNSCs. The fact that the dorsal SEZ harbours more oligodendrogliogenic aNSCs than the lateral SEZ is consistent with the notion of a mosaic organization of the SEZ (ref. 11). According to this notion, different germinative domains within the SEZ are of distinct embryonic origins along the dorsoventral axis of the forebrain 12 , which in turn accounts for their respective competence in generating distinct progeny. Future studies are required to define the regional and temporal origins of the various neuro-and oligodendrogliogenic aNSCs in the adult SEZ during embryogenesis.

Canonical Wnt signalling selectively stimulates proliferation within the oligodendrogliogenic lineage
Moreover, apart from distinct embryonic origins the fate choice of aNSCs can also be affected by local signalling. Indeed, it has been previously shown that sonic hedgehog signalling determines aNSC positional identity 9 . Here we observed that stimulation with Wnt3/3a results in a massive expansion of oligodendrogliogenic clones without affecting the generation of other cell types both in vitro and in vivo. Canonical Wnt signalling has been reported to be particularly high in the dorsal SEZ (ref. 20). Accordingly, we observed that mRNAs encoding for several components of the Wnt canonical pathway are expressed at higher levels in the dorsal SEZ when compared with the lateral SEZ. These data, together with the fact that gain-or loss-of-function of Wnt signalling increases or decreases oligodendrogliogenesis, respectively, suggest that local Wnt signalling contributes to the enrichment of oligodendroglia in the dorsal SEZ. Conversely, local BMP signalling has been shown to repress an oligodendrogliogenic fate in the lateral SEZ (ref. 14). Thus, largely non-overlapping domains of high Wnt and BMP signalling seem to define the level of oligodendrogliogenesis in the adult SEZ. Of note, mRNA expression analysis suggests that Wnt3 is the physiologically relevant player, whereas Wnt3a was hardly detectable within adult SEZ tissue. Intriguingly, FACS analysis suggests that the cell type expressing the highest amount of Wnt3 mRNA comprises cells of the oligodendroglial lineage. This suggests that, in the adult SEZ, regions of higher density of Wnt3-secreting oligodendroglial precursor cells promote local enhancement of oligodendrogliogenesis through a positive feedback loop.
Whereas canonical Wnt signalling has been implicated in the regulation of adult neurogenesis in the dentate gyrus 31 , we did not observe an obvious effect of Wnt3/3a treatment on SEZ neurogenesis. These data provide support for the notion of the separation of the neuroand oligodendrogliogenic lineages in vivo and may represent distinct cellular targets of intervention in therapeutic contexts. However, Wnt3a treatment of SEZ cultures augmented the expression of fate determinants such as Eomes (tbr2) and neurod1. This indicates that canonical Wnt signalling regulates the differentiation rather than the expansion of neurons belonging to the glutamatergic lineage 43 , also enriched in the dorsal SEZ (ref. 10).
To our knowledge, the present study is the first to report a massive effect of canonical Wnt signalling on the proliferation within the oligodendroglial lineage, although previous studies have shown that Wnt signalling maintains OPCs in an immature state [44][45][46] . Moreover, after traumatic brain injury, the canonical Wnt signal transducer β-catenin translocates to the nucleus of proliferating NG2 progenitor cells in the cerebral cortex 47 , potentially contributing to the increase in NG2 progenitor cells after stab wound injury 48 .
Several lines of evidence suggest that the effect of Wnt signalling on oligodendrogenesis is primarily due to a selective activation of proliferation within the oligodendrogliogenic lineage and less likely to be due to a switch in cell fate. First, whereas Wnt3a stimulated massive expansion of NG2-positive oligodendroglia, no concomitant decrease in neurogenesis was observed. Second, Wnt3a treatment caused a marked reduction in cell cycle length and simultaneous increase in cell divisions selectively within the oligodendrogliogenic lineage. Third, Wnt treatment did not cause the appearance of mixed clones (containing both neurons and oligodendroglia), a finding incompatible with canonical Wnt signalling acting by diverting neurogenic TAPs towards oligodendrogliogenesis.
Thus, in addition to previously described restrictions regarding neuronal subtype specification 11 , our study reveals that aNSCs in the adult SEZ are also lineage restricted with respect to the decision of acquiring a neuronal or an oligodendroglial identity, and that these lineages are differentially regulated by regionalized niche signals such as Wnts.

Time-lapse video microscopy.
Time-lapse video microscopy and single-cell tracking [49][50][51] of primary SEZ cultures was carried out with a cell observer (Zeiss) at a constant temperature of 37 • C and 8% CO 2 . Phase contrast microscopy images were acquired every 5 min for 6-10 days using a ×20 phase contrast microscopy objective (Zeiss), an AxioCamHRm camera and Zeiss AxioVision 4.7 software. Single-cell tracking was carried out using a self-written computer program (TTT; ref. 52). Videos were assembled using ImageJ 1.42q (National Institutes of Health) software and are played at a speed of 1 or 2 frames per second.

RNA extraction and real-time PCR.
Four wells from untreated and Wnt3atreated SEZ cultured cells were lysed in RLT Plus buffer and total RNA was extracted with an RNeasy Plus Micro Kit (Qiagen), according to the manufacturer's instructions. RNA was retro-transcribed using Super-ScriptIII reverse transcriptase (Invitrogen) and random primers (Roche). Each complementary DNA was diluted one to five, and 1 µl was used for each real-time reaction. Real-time PCR was carried out on a LightCycler 480 (Roche) using the LightCycler Probes Master kit (Roche) and Monocolor Hydrolysis Probe/UPL Probe (Roche) according to the manufacturer's instructions (20 µl final volume). The expression of each gene was analysed in triplicate. Data analysis was carried out with the Ct method 53 . Quantification was carried out on two independent experiments. Primers are listed in Supplementary Table S1.