A decreasing gradient of Id1 levels in the subventricular neurogenic lineages
Immunohistochemistry and tyramide signal amplification (TSA) immunofluorescence analyses of adult mouse brains with a highly specific rabbit monoclonal anti-Id1 antibody (Perk et al., 2006
) revealed Id1 immunoreactivity (IR) in the parenchyma and in the subventricular region (data not shown). At least two morphologically distinguishable types of Id1+ nuclei, round and elongated, were evident.
Further TSA immunofluorescence and confocal microscopic analyses with the anti-Id1 and anti-GFAP antibody revealed predominantly round Id1 IR in the GFAP+subventricular region (). Immunostaining of dissociated SVZ cells confirmed the expression of Id1 and GFAP in a single cell, indicating that some of the SVZ cells with the Id1+ nuclei are GFAP+ astrocytes (, left inset).
A decreasing gradient of Id1 levels in the subventricular neurogenic lineages
TSA immunofluorescence of Id1 and Mash1, a TA cell marker of the neuronal lineage, revealed co-localization of the two nuclear antigens in some SVZ cells (, top inset). However, Mash1 IR was evident in only a minority of the Id1+ cells. Cells that were Id1+ Mash1− (, bottom left inset), as well as Id1− Mash1+ (not shown), were evident. Interestingly, co-expression of Id1 and Mash1 appeared to be limited to cells expressing comparatively lower levels of Id1 (, compare levels in Id1high
cells at bottom left inset and in Id1intermediate
cells at bottom right inset). Thus, a gradient of Id1 and Mash1 levels was apparent in the subventricular Id1-expressing cells. The cells expressing lower levels of Id1, Id1int
cells, co-expressed Mash1, a marker of a more differentiated phenotype. Moreover, TSA immunofluorescence revealed similar results with Id1 and Olig2, a TA cell marker of the oligodendroglial lineage (Supplementary Figure 5
Consistent with a gradually decreasing gradient of Id1 levels along the subventricular neurogenic lineages, PSA-NCAM+ neuroblasts or NeuN+ neurons never expressed Id1 (). Moreover, S100b+ ependymal cells also never expressed Id1 (). Importantly, S100b+ parenchymal astrocytes were Id1−, suggesting subventricular astrocyte-specific Id1 expression.
Finally, subventricular and parenchymal cells with elongated Id1+ nuclei were CD31+ endothelial cells ( and Supplementary Fig. 2C
middle box and Supplementary Fig. 12
). In the SVZ, the two morphologically discernable Id1+ cell types were often located nearby ( top inset), suggesting that the GFAP+ Id1high
astrocytes reside in or near the perivascular stem cell niche (Shen et al., 2008
; Tavazoie et al., 2008
We further quantitated the Id1-expressing populations by flow cytometry using an Id1VenusYFP
knock-in allele. In the Id1VenusYFP
mouse, a fusion protein of Id1 and Venus fast-maturing yellow fluorescent protein is synthesized from the endogenous Id1
locus (Supplementary Fig. 1
). Subventricular tissue from Id1V/V
mice was microdissected from ~1 mm coronal slices (~0.7 mm to ~−0.3 mm relative to Bregma), dissociated with papain, and analyzed by flow cytometry (Supplementary Fig. 6
). Flow cytometric quantitation of at least 20,000 cells indicated 3.1 ± 0.64% of the cells of the microdissected tissue expressed Id1 (mean ± standard deviation, n = 14, 3 mice per n). Of these Id1-expressing cells, 16% were GFAP+
(). Thus, the Id1-expressing subventricular astrocytes constituted only 0.49% of the cells of the microdissected subventricular tissue, close to the 0.4% of subventricular cells estimated to be stem cells. Of the Id1-expressing cells, 24% were Mash1+
(), though the majority of Mash1+
cells did not express Id1. Consistent with the immunohistological analyses, only one Id1high
cell in ~20,000 subventricular cells dissociated from three mice expressed Mash1 ( inset). Moreover, most Id1intermediate-high
cells expressed lower levels of Mash1 than Id1intermediate-low
cells. Finally, PSA-NCAM+
cells did not express Id1 (), consistent with the immunohistochemical analyses. In sum, of all subventricular Id1-expressing cells, 16% were GFAP+ astrocytes, 24% were Mash1+ cells, 10% were Olig2+ cells (data not shown), and 44% were CD31+ endothelial cells (Supplementary Fig. 12
), with 6% unclassified.
Functional quiescence of the subventricular Id1high astrocytes
The analysis above identified two distinct neural cell types with round Id1+ nuclei: Id1high
astrocytes and Id1int
Mash1+ or Olig2+ TA cells. We characterized the cell cycle status of these cells with morphologically distinguishable round IR Id1+ nuclei, thereby excluding the endothelial cells. First, 68% of the neural Id1+ cells were Id1int
Mash1+ or Olig2+ TA cells. Some of these TA cells are expected to be actively cycling in S-phase as identified by a one-hour pulse of thymidine analog. Indeed, 32% of the round Id1+ cells were EdU+ by immunofluorescence (). The majority (95%) of the EdU+ S-phase cells, however, were Id1−. Ki67 and Mcm2 label a larger population of cycling cells than a one-hour pulse of thymidine analog: Ki67 labels cells in G1-, S-, G2-, and M-phase; Mcm2 specifically labels cells in G1-phase and as well as relatively quiescent cells (Maslov et al., 2004
). Accordingly, 73% and 75% of the cells with round Id1+ nuclei were Ki67+ and Mcm2+, respectively (). These results suggested that the 68% of Id1+ cells co-expressing TA cell markers, Mash1 or Olig2, are indeed actively cycling.
Id1high astrocytes are relatively quiescent but can be activated to enter cell cycle
Second, 16% of neural Id1+ cells were Id1high GFAP+ astrocytes. Immunofluorescence analyses indicated a majority of the Id1high astrocytes are EdU− ( and see below), and thus not synthesizing DNA at detectable levels. Nevertheless, 73% of the Id1high astrocytes expressed Ki67. Based on Ki67 or Mcm2 expression but lack of detectable DNA synthesis, we suggest that Id1high astrocytes may be "paused" in various phases of the cell cycle, including G1. Consistent with the relative quiescence of Id1high astrocytes, a 7 d infusion of EdU by miniosmotic pump labeled a greater percentage of Id1high cells than a 1h pulse (, compare to ).
The quantitation above suggested that some Id1high
astrocytes may express proliferation markers Ki67 or Mcm2. Nevertheless these astrocytes are not actively synthesizing DNA and thus may persist after infusion of an antimitotic, Ara-C. This "functional quiescence" of the Id1high
astrocytes was directly tested by 6 d infusion of Ara-C, which ablates rapidly dividing C and A cells but spares quiescent B cells and non-stem-cell astrocytes (Doetsch et al., 1999a
). Indeed, the GFAP+ Id1high
astrocytes persisted after Ara-C ablation ( inset), consistent with their relative quiescence. Concomitantly, the number of round Id1+ nuclei (i.e.neural Id1+ cells) decreased by 83% (compare ); most remaining Id1+ cells were Id1high
(). This reduction is roughly consistent with the quantitation in which 68% of the neural Id1+ cells were Mash1+ or Olig2+ Id1int
TA cells (see above) and therefore expected to be ablated by Ara-C infusion. Thus, this experiment showed directly that the Id1high
astrocytes are indeed functionally quiescent despite expression of Ki67 or Mcm2. At 12 and 48 hours after Ara-C infusion, EdU was injected to label the B cells activated to enter cell cycle. No EdU+ cells were observed immediately after Ara-C infusion (). At 12 and 48 h after the infusion, the majority of the rare EdU+ cells were Id1+ (Id1+ EdU+ / all EdU+ = 63% at 48 h), although not all Id1+ cells were EdU+ (Id1+ EdU+ / all Id1+ = 37% at 48 h). Thus, the Id1high
astrocytes are functionally quiescent but some entered S-phase upon activation. More than half of the cells that entered S-phase after Ara-C infusion were Id1high
Subventricular GFAP+ Id1high astrocytes are B1 type cells
That high Id1 expression defined a functionally quiescent population of subventricular astrocytes capable of entering S-phase raised the possibility that Id1high
astrocytes are B1 type stem cells. Id1-expressing cells were fate-mapped using the Id1IRES-creERT2
and various reporter alleles (Supplementary Figs. 2, 3, and 8
). In mice heterozygous for the Id1IRES-creERT2
allele, Id1-expressing cells express the tamoxifen-inducible cre recombinase, creERT2, from a bicistronic messenger RNA by means of an encephalomyocarditis virus internal ribosome entry site. Because of the low creERT2 expression level and thus activity in the Id1IRES-creERT2/+
mice, cre-mediated recombination most likely occurs in a small number of cells expressing comparatively higher levels of Id1. Fortuitously, this low-level creER activity enabled selective mapping of the Id1high
cells (see below and Discussion). In Id1IRES-creERT2/+
mice, the creER activity was not leaky but tamoxifen inducible (Supplementary Fig. 9
3 d after the last of three tamoxifen doses, X-gal histochemistry of Id1IRES-creERT2/+;StLa
ac Z, Supplementary Fig. 3
) mouse sections revealed rare single cells with astrocytic morphology in the SVZ (Supplementary Fig. 2
and Supplementary Fig. 10C
, n = 3). In total, we examined 144 12-micron cryo-sections covering the anterior SVZ (from ~1.8 mm to ~−0.9 mm relative to Bregma), and found 10 single tau-β-gal+ subventricular astrocytic cells. A comparison of the number of X-gal+ cells to Id1-immunoreactive cells indicated <1% labeling efficiency in the brain at the three day time point, consistent with the notion that recombination occurred only in Id1high
Mash1/Olig2− cells. These rare tau-β-gal+ cells in the SVZ with astrocytic morphology were GFAP+ astrocytes (Supplementary Fig. 10D
). As expected, no X-gal+ cells with neuronal morphology nor tau-β-gal+ NeuN+ neurons were found in the OB at this time (Supplementary Fig. 10E, F
At present no single marker specifically identifies the B1 type stem cells, with the possible exception of Tailless (Liu et al., 2008
). Nevertheless, in the SVZ, the B1 type stem cells are organized in a "pinwheel" architecture and extend a characteristic long single basal process within the SVZ (Mirzadeh et al., 2008
). Indeed, immunofluorescence of YFP+ cells in Id1IRES-creERT2/+;R26LSL-YFP
ox YFP) mice revealed that YFP+ cells are Id1high
astrocytes with round nuclei (). Consistent with a B1 type identity of the genetically identified Id1high
astrocytes, immunofluorescence of SVZ whole mounts from Id1IRES-creERT2/+;R26LSL-YFP
mice 3 d post-tamoxifen revealed rare YFP+ GFAP+ cells with the characteristic long single GFAP+ basal process (, n = 7). Moreover, the YFP+ astrocytes were located at the center of the pinwheel () and extended basal processes at least 90 µm long (). B2 type astrocytes were never observed. Finally, all YFP+ astrocytes analyzed were Mash1− (and EdU−, see below), consistent with the data that the Id1high
cells are Mash1−. Thus, the genetically labeled cells in these mice are indeed Id1high
astrocytes, rather than the Id1int
Mash1+ C type TA cells (n = 13 cells).
Subventricular GFAP+ Id1high astrocytes are B1 type astrocytes
Furthermore, combined with the Id1IRES-creER
allele, the GFAPLSL-GFP
transgenic mouse (Casper and McCarthy, 2006
) enabled genetic identification of the GFAP
astrocytes. Immunofluorescence of whole mounts from the Id1IREScreERT2/+
mice 3 d post-tamoxifen revealed rare GFP+ (GFAP+ Id1high
) subventricular astrocytes at the center of a pinwheel (, n = 4). Moreover, immunofluorescence confirmed GFAP expression in these astrocytes and revealed a long basal process, consistent with the data above (, n = 6). Consistent with the cell cycle status of most Id1high
astrocytes, the GFP+ astrocytes did not incorporate EdU during a 1 h pulse (), indicating that these cells are distinct from the Id1int
Mash1+, Dlx2+, or Olig2+ C type TA cells in S phase. Finally, these astrocytes extended a single basal GFAP+ process and contacted the nearby vasculature (data not shown).
Subventricular GFAP+ Id1high B1 type astrocytes are neurogenic stem cells
We then examined whether the genetically identified GFAP+ Id1high
B1 type astrocytes function as stem cells. 1 and 6 mo after the last tamoxifen dose, X-gal histochemistry of Id1IRES-creERT2/+;StLa
mice sections revealed rare β-gal+ cells in the SVZ, RMS, and the OB (Supplementary Fig. 11
). 6 mo after the last tamoxifen dose, in 72 12-micron sections examined, 4 single subventricular X-gal+ cells were found. X-gal+ cells were also found in the RMS and OB. Clusters of X-gal+ cells were never found, consistent with a cell division time longer than the time required for migration out of the SVZ. However, the number of X-gal+ cells in the SVZ increased by 1 mo post-tamoxifen (Supplementary Fig. 9
Some tau-β-gal+ cells in the RMS and OB of the Id1IRES-creERT2/+;StLa mice at 1 mo post-tamoxifen were DCX+ neuroblasts with migratory morphology () and NeuN+ neurons (), respectively. Thus, under normal physiologic conditions subventricular GFAP+ Id1high B1 type astrocytes gave rise to DCX+ neuroblasts that migrated to the OB and differentiated into NeuN+ neurons.
Subventricular GFAP+ Id1high B1 type astrocytes are neurogenic stem cells
As some of the genetically labeled cells in the Id1IRES-creERT2/+;StLa
mouse brains were endothelial cells (Supplementary Figs. 2 and 12
), we utilized a postmitotic neuron-specific reporter allele, TauLSL-mGFP-IRES-nLacZ
(Hippenmeyer et al., 2005
). In this mouse, the endogenous Tau
) promoter specifically labels the postmitotic neuronal progeny of the Id1high
cells (Supplementary Fig. 8
). This enabled facile identification of the neuronal progeny and flow cytometric quantitation of the neuronal output at the steady-state from subventricular Id1high
B1 astrocytes to the OB's using X-gal histochemistry and flow cytometry.
X-gal histochemistry of OB's at six months post-tamoxifen revealed nuclear-β-gal+ postmitotic neurons in the granular cell layer and in the glomerular layer (). Granular neurons were more numerous than periglomerular neurons. Flow cytometric quantitation at 2, 6, and 24 w post-tamoxifen indicated 6068 ± 2480, 28179 ± 4866, and 67195 ± 2087 GFP+ neurons, respectively (mean ± SEM, n = 3 per time point, ). The increase in neuronal output was statistically significant between 2 and 6 w (P < 0.05, unpaired two-tailed Student's t-test) and 6 and 24 w (P < 0.01, unpaired two-tailed Student's t-test). Immunofluorescence of OB sections from 1 mo post-tamoxifen confirmed the neuronal morphology of GFP+ cells in the granular cell layer () and GABA immunoreactivity ( inset).
We then directly demonstrated the quiescence, cell cycle entry, and neurogenic potential of the subventricular Id1high astrocytes with the Id1IRES-creERT2/+;TauLSL-mGFP mice. These mice were gavaged with tamoxifen, then infused with Ara-C. This infusion ablates the rapidly-dividing Id1int Mash1/Olig2int C type cells as well as the A type neuroblasts, while relatively quiescent Id1high B type cells persist (see inset). 12 h after Ara-C infusion, when some of the normally quiescent B type cells enter cell cycle, EdU was injected to label B type cells in S-phase. Finally, after a 2 w chase, analyses of the sections revealed GFP+ EdU+ neurons in the olfactory bulb (). This experiment directly demonstrated that 1) the genetically-identified Id1high B1 type astrocytes are functionally quiescent and thus can persist through the antimitotic treatment, 2) these genetically-identified astrocytes can enter S-phase, and 3) these genetically identified astrocytes are undifferentiated and give rise to postmitotic neurons whose identity is unambiguously reported by the TauLSL-mGFP knock-in allele.
Next, we examined whether the GFAP+ Id1high B1 type astrocytes persist in the niche. In Id1IRES-creERT2/+;StLa mice 3 mo after tamoxifen administration, whole mount immunofluorescence revealed tau- -gal+ GFAP+ astrocytes with B1 type morphology (). Finally, in Id1IRES-creERT2/+;R26LSL-YFP mice 6 mo after tamoxifen administration, YFP+ cells (that once expressed high levels of Id1) continued to express Id1 ().
Cultured subventricular Id1high stem cells can self-renew asymmetrically
The data described above strongly suggested that Id1high
astrocytes are stem cells. Potentially, the Id1high
B1 type stem cell could self-renew asymmetrically and produce an asymmetric pair of progeny cells in vivo, i.e., an Id1high
B1 type stem cell and an Id1int
C type TA cell which differ in Id1 protein levels. Experiments indicated that the Id1 and Id1-Venus expression levels are heterogeneous in NS adherent adult SVZ cell cultures (Glaser et al., 2007
; Pollard et al., 2006
), as shown in and Supplementary Fig. 7B
). These adherent radial glia-like tripotent cells derived from adult SVZ neurospheres were ~100% nestin+, and importantly, free of non-neural cell types, e.g., endothelial cells (Supplementary Fig. 7
Cultured subventricular Id1high stem cells can self-renew asymmetrically
Based on their in vitro functional activity (see below and ) and transcriptional profile (see Supplementary Information
), we surmised that the Id1high
NS cells are functionally and molecularly distinct B-like stem and A-like neuroblast cells, respectively. Most B or A cells do not express C cell markers, Mash1
, and Olig2
, and accordingly, these were not differentially expressed in Id1high
B-like and Id1low
A-like cells in microarray analyses. To determine whether Id1intermediate
NS cells correspond to C-like cells, four populations were FAC sorted () and then stained with Mash1 or Olig2 antibodies. Indeed, the progression of Mash1 or Olig2in Id1high
, to Id1low
cells was consistent with that observed in B, C, to A cells in vivo, respectively (). Thus, the Id1-Venus
mouse and the adherent culture system provided a unique experimental system to examine the self-renewal behavior of unperturbed Id1high
Id genes are necessary for self-renewal, a characteristic of stem cell identity, but dispensable for proliferation
We examined the progeny of the Id1high B-like stem and Id1low A-like neuroblast cells in the adherent adult SVZ cell cultures by culturing the purified cells individually and measuring the percentage of Id1high, Id1int, and Id1low cells flow cytometrically. In serum-free medium supplemented with FGF-2 and EGF, the Id1high (Mash1− or Olig2−) stem cells generated Id1high (Mash1− or Olig2−) and Id1int (Mash1+ or Olig2+) cells (as evidenced by the leftward shift of the histogram concomitant with increase in cell density), but the Id1low neuroblast cells generated only Id1low cells and did not generate Id1high or Id1int cells ().
Cell cycle parameters of the adherent Id1high
cells were measured by BrdU pulse-chase and flow cytometric quantitation (data not shown). When the experimental data (shown in ) were modeled with population dynamics based on experimentally obtained cell cycle parameters of the Id1high
populations, the data closely resembled the population dynamics predicted of asymmetric self-renewal in ~ 50% of the Id1high
cells (, also see Supplementary Information
). This mathematical analysis was consistent with the notion that Id1high
cells can self-renew asymmetrically in a population of stem/progenitor cells.
In support of the notion that the Id1high cells can self-renew asymmetrically during serial passages the percentage of the Id1high cells were diluted out by more rapidly proliferating Id1low cells (). Despite the reduction in the frequency of Id1high cells, obvious morphological changes to the predominantly bipolar radial glia-like morphology were absent even after forty passages, or four months, in culture (data not shown). Finally, when Id1high cells were FAC sorted and cultured at clonal density, Id1high cells gave rise to clusters of cells expressing high and lower levels of Id1 (the latter undetectable by confocal microscopy) ().
Id genes are necessary for self-renewal, a characteristic of stem cell identity
The experiments above suggested that in addition to identifying the B1 type astrocyte stem cells, high Id1 expression may be functionally important in neural stem cell identity. We thus assayed neurosphere-forming activity of Id1high and Id1low fractions in early and later passage Id1V/V adult adherent SVZ cell cultures. When Id1high and Id1low cells were FAC sorted and compared in clonal neurosphere assays, Id1high cells generated more and larger spheres (, sphere number, ~2-fold increase, n = 19, P < 0.01; sphere size, ~3.5-fold increase, n = 19, P < 0.0001; unpaired two-tailed Student’s t-test). Upon passaging, only Id1high cell-derived neurospheres formed secondary neurospheres (n = 18). The Id1low cell-derived spheres did not form secondary spheres. When cultured as an adherent monolayer, Id1low cells proliferated more rapidly thanId1high cells (). Thus, only Id1high cells were capable of self-renewing anchorage-independently.
The analyses above suggested high levels of Id1 are required for self-renewal in unperturbed cells, a characteristic of stem cell identity. However, adult Id1−/−
mice are viable and fertile, and neurosphere self-renewal is normal (data not shown), suggesting redundancy or compensation. Thus, we asked whether both Id1
are functionally required for neurosphere formation using the Id1-floxed
conditional allele (Supplementary Fig. 4
). Neurospheres of Id1+/+;Id3+/+
, and Id1fl/fl
genotype were nucleofected with cre recombinase cDNA (~50% efficiency by flow cytometry). Ablation of Id1
, but not Id1
alone, was sufficient to reduce secondary neurosphere formation by ~50% (), a number consistent with near complete absence of self-renewal in the transfected population. Genomic PCR detected the unrecombined floxed allele in neurospheres that formed (data not shown).
Subgranular GFAP+ Id1high astrocytes are also neurogenic stem cells
To determine whether high Id1 expression is a general characteristic of GFAP+ astrocyte stem cells, we examined Id1 expression and the progeny of Id1-expressing cells in the hippocampal dentate gyrus. Id1+ cells were evident in the subgranular layer, and these cells were GFAP+ (), although a clear long apical GFAP+ process wasn't apparent for all Id1+ subgranular cells. These Id1+ cells also expressed nestin, a marker of neural stem/progenitor cells (). Consistent with the Id1 gradient evident in the subventricular neurogenic lineages, subgranular Id1+ cells never expressed DCX and NeuN (). Finally, in Id1IRES-creERT2/+;StLa mice one month post-tamoxifen, tau-β-gal+ NeuN+ cells with neuronal morphology were evident in the granuar cell layer ().
Subgranular Id1high cells are also B type neural stem cells