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. 2007 Nov 14;27(46):12623-9.
doi: 10.1523/JNEUROSCI.3812-07.2007.

Dynamic contribution of nestin-expressing stem cells to adult neurogenesis

Diane C Lagace  1 , Mary C Whitman, Michele A Noonan, Jessica L Ables, Nathan A DeCarolis, Amy A Arguello, Michael H Donovan, Stephanie J Fischer, Laure A Farnbauch, Robert D Beech, Ralph J DiLeone, Charles A Greer, Chitra D Mandyam, Amelia J Eisch
Affiliations

Dynamic contribution of nestin-expressing stem cells to adult neurogenesis

Diane C Lagace et al. J Neurosci. .

Abstract

Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreER(T2)/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreER(T2)/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreER(T2)/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreER(T2)/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.

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Figures

Figure 1.
Figure 1.
Recombination in nestin-CreERT2/R26R-YFP mice is induced by TAM and is specific to neurogenic regions. a, The nestin-CreERT2 construct has 5.8 kb of the nestin promoter and exons 1–3, including the second intronic enhancer. b, c, YFP+ cells are evident 12 d after TAM in the SVZ, RMS, OB GCL, and SGZ of nestin-CreERT2/R26R-YFP mice given TAM but not vehicle. d, e, Recombination efficiency in the SGZ is similar in male and female mice 12 d after TAM (male, 3654 ± 278 vs female, 4239 ± 414). f, g, Mice given BrdU 1 d before TAM or vehicle and killed 28 d later have similar numbers of SGZ BrdU cells (vehicle, 2718 ± 278 vs TAM, 2788 ± 238). Scale bars: b, c, 1 mm; d–g, 100 μm. H, Hilus of dentate gyrus.
Figure 2.
Figure 2.
Neurogenesis in the SVZ/RMS/OB following TAM. a, Time course for the appearance of YFP+ cells in the SVZ, RMS, and OB GCL 1, 12, 30, and 65 d after TAM. Recombined cells (YFP+, green) are present 1 d after TAM within the SVZ, whereas fewer YFP+ cells are present in the RMS and virtually no recombined cells are present within the GCL. With increasing time after TAM, there are many recombined cells in each of these areas with a variable proportion of these cells being neuroblasts as demonstrated by coexpression of YFP and DCX (red). b, c, YFP+ cells in the OB GCL (a) and OB GL (b) significantly increased up to 100 d (*p < 0.05 vs 12 d). d, The proportion of YFP+ cells that are migrating neuroblasts (DCX+) increased in the caudal RMS but decreased in the RMS in OB and GCL in OB (*p < 0.05, **p < 0.01, and ***p < 0.005 vs 12 d). e, The proportion of YFP+ cells (black and yellow bars) that are not migrating neuroblasts (black bars) increased in the OB GCL (*p < 0.05 and ****p < 0.005 vs 12 d; ##p < 0.01 and ###p < 0.005 vs 30 d). f–i, Thirty days after TAM, most recombined cells (f, g) in the GCL were NeuN+ (*p < 0.05 vs 30 d) as well as GABAergic interneurons (h, i), as assessed via colabeling with YFP and GAD65/67 (h; counterstain DRAQ5) and CR (i). Scale bar: f, h, i, 50 μm. Error bars indicate SEM.
Figure 3.
Figure 3.
Neurogenesis in the SGZ after TAM. a, At increasing time points after TAM, the YFP+ cell number in the hippocampal SGZ increased up to 30 d (**p < 0.01 vs 1 d; ##p < 0.01 vs 12 d). b, c, The proportion of YFP+ cells in SGZ-expressing immature markers decreased, whereas those expressing mature markers increased (arrows indicate YFP+/Nestin+, YFP+/GFAP+/Sox2+, YFP+/Ki67+, and YFP+/DCX+/CR+; arrowheads indicate YFP+/Nestin− and YFP+/GFAP−/Sox2+). d–f, An increasing percentage of YFP+ cells in the GCL of the dentate gyrus has a mature phenotype with branched processes (d, arrow) extending into the molecular layer and colocalization with the neuronal marker NeuN (**p < 0.01 vs 30 d; ##p < 0.01 vs 65 d) (e; arrows indicate YFP+/NeuN+). g, The YFP+ cell number in the oGCL increased (*p < 0.01 vs 12 d). Error bars indicate SEM.
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