In vivo analysis of Ascl1 defined progenitors reveals distinct developmental dynamics during adult neurogenesis and gliogenesis - PubMed (original) (raw)

Comparative Study

In vivo analysis of Ascl1 defined progenitors reveals distinct developmental dynamics during adult neurogenesis and gliogenesis

Euiseok J Kim et al. J Neurosci. 2007.

Abstract

In the adult mammalian brain, new neurons and glia are continuously generated but molecular factors regulating their differentiation and lineage relationships are largely unknown. We show that Ascl1, a bHLH (basic helix-loop-helix) transcription factor, transiently labels neuronal and oligodendrocyte precursors in the adult brain. Using in vivo lineage tracing with inducible Cre recombinase, we followed the maturation of these precursors in four distinct regions. In the hippocampus, Ascl1 mostly marks type-2a progenitor cells with some late stage type-1 stem cells. Thirty days after Ascl1 expression, although a majority of the cells matured to granule neurons, a few cells remained as immature progenitors. By 6 months, however, essentially all Ascl1 lineage cells were granule neurons. In contrast, in the olfactory bulb neuronal lineage, Ascl1 is restricted to transit amplifying cells, and by 30 d all cells matured into GABAergic interneurons. Ascl1 also broadly marks oligodendrocyte precursors in subcortical gray and white matter regions. In the corpus callosum, Ascl1 defines a ventral layer of early oligodendrocyte precursors that do not yet express other early markers of this lineage like PDGFRalpha and Olig2. By 30 d, most had transitioned to mature oligodendrocytes. In contrast, Ascl1 expressing oligodendrocyte precursors in gray matter already coexpressed the early oligodendrocyte markers, but by 30 d they mostly remained as precursors. Our results reveal that Ascl1 is a common molecular marker of early progenitors of both neurons and oligodendrocytes in the adult brain, and these Ascl1 defined progenitors mature with distinct dynamics in different brain regions.

PubMed Disclaimer

Figures

Figure 1.

Ascl1-GFP expression in the adult brain. A, Ascl1-GFP knock-in mice have replaced one allele of Ascl1 coding sequence with that of a nuclear-localized GFP reporter (Leung et al., 2007). B–H″, Images from immunofluorescence of Ascl1 GFP/+ mice (Ascl1, red; GFP, green). B–B″, Transverse section of a mouse E11.5 neural tube. C, Higher magnification of B″ illustrating that GFP persists longer than Ascl1. Ascl1 is restricted to the ventricular zone (VZ) whereas GFP is also present in the mantle zone (MZ). D, Sagittal view of subcortical regions in mouse forebrain at P60 showing Ascl1-GFP expressing cells are enriched in adult neurogenic regions. D', Diagram of adult mouse brain indicating where images in D–H originated. Ascl1-GFP expression in dentate gyrus (E), SVZ (F) around the lateral ventricle (LV), and thalamus (TH) (G). H–H″, GFP and Ascl1 are coexpressed in SGZ of adult hippocampus (arrows). CC, Corpus callosum; HI, hippocampus; OB, olfactory bulb; RMS, rostral migratory stream. Scale bars: 40 μm.

Figure 2.

Ascl1-GFP cells identified as type-2 cells in the adult hippocampal neurogenic lineage. A–E, G, The dentate gyrus from P60 Ascl1 GFP/+ mice was examined by immunofluorescence for GFP (A–E; green), GFAP (A, red), BLBP (B, red), Sox2 (C, red), Dcx (D, red), NeuroD (E, red) and NeuN (B, D, E; blue) stage-specific markers for cells undergoing neurogenesis in the adult hippocampus (F, diagram). Higher magnification showing overlap of GFP with Sox2 (C–C″), Dcx (D–D″), and NeuroD (E–E″) are shown. F, Characterization of the Ascl1-GFP cells places the majority at the Type-2a cell stage according to a current hippocampal neurogenesis model (Steiner et al., 2006). Dashed line indicates SGZ of the DG. Scale bars: 10 μm.

Figure 3.

Ascl1 lineage cells become mature granule cells in the adult hippocampus. A, Diagram illustrating the strategy for in vivo fate mapping of the Ascl1-expressing cells in adult brain. P60 mice carrying alleles Ascl1 CreERTM (a tamoxifen inducible Cre recombinase expressed from the Ascl1 locus in a BAC transgene) and R26R-stop-YFP were treated with tamoxifen (TM) and brains were harvested 5, 30, or 180 d later. B–E, Five days after tamoxifen treatment YFP (green) is detected in the SGZ of the dentate gyrus (DG) (B). Only few YFP cells incorporate BrdU after a 2 h BrdU pulse (C). In contrast, most YFP cells colabel with Dcx (D) and NeuroD (E), both Type-2b/Type-3 markers. F–H, 30 d after tamoxifen treatment YFP cells are found in the granule cell layer in the DG, have morphology of mature granule neurons, and colabel with the neuronal marker NeuN (G, arrow). A few YFP cells elicit stem cell like morphology and colabel with GFAP (H, arrow). I, J, One hundred and eighty days after tamoxifen treatment YFP cells are still present in the granule cell layer, have extensive dendritic arborization, and colabel with mature granule cell markers NeuN (blue) and Calbindin (red) (J, arrows). K, Quantification of the percentage of YFP cells identified as immature precursors (NeuN−) or mature neurons (NeuN+) at the three different time points after TM. Dashed line indicates the SGZ of the DG. Scale bars: 40 μm.

Figure 4.

Ascl1-GFP cells are transit amplifying cells or neuroblasts in the SVZ and rostral migratory stream. A–H, Immunofluorescence in P60 Ascl1 GFP/+ brains showing GFP (green) colabeled with Ascl1 (A, D), Sox2 (B), GFAP (B), Ki67 (C), NeuN (E), Olig2 (F), Dlx (G), and Dcx (H). GFP and Ascl1 overlap in the SVZ and RMS (A–A‴, D; yellow cells). GFP also overlaps with Ki67, Sox2, Olig2, Dlx, and Dcx, but not GFAP or NeuN identifying Ascl1-GFP cells as transit amplifying/neuroblast cells in this neurogenic region. I, Ascl1-GFP is placed in transit amplifying cell or neuroblasts using a current model for gene expression staging of olfactory bulb neurogenesis (Doetsch, 2003). Diagrams depict the SVZ in a coronal view, or the SVZ and RMS in a sagittal view of an adult brain showing the location of the GFP cells and regions imaged in A–H. LV, Lateral ventricle. Scale bar: 20 μm.

Figure 5.

Ascl1 lineage cells become interneurons in granule and periglomerular layers of the olfactory bulb. Immunofluorescence of Ascl1 CreERTM;R26R-stop-YFP mouse brain sections harvested 5 or 30 d after tamoxifen administration at P60. A–D, Five days after tamoxifen treatment some YFP cells (green) are located near the SVZ (A) and colabel with Sox2 (A, inset; red), but most YFP cells were found within the RMS (B, C) and colabel with the neuroblast marker Dcx (D, arrows). In contrast, 30 d after tamoxifen treatment no YFP cells were detected around the SVZ or RMS (A', B'). At this time the YFP cells were found in the olfactory bulb (C') and express the mature neuronal marker NeuN (E, arrows). F–I, Most YFP cells in the olfactory bulb 30 d after tamoxifen treatment localized to the GCL where newly generated neurons are homogenously GABAergic shown here as overlap with GAD67 (F). A much smaller proportion of YFP cells were located near the GL in diverse interneuron types overlapping with calretinin (G), TH (H), or calbindin (I). Diagrams depict SVZ in a coronal section, or SVZ and RMS, or olfactory bulb in a sagittal section of an adult mouse brain. Boxes indicate the location where images were taken. LV, Lateral ventricle; TH, tyrosine hydroxylase; TM, tamoxifen. Scale bars: 20 μm.

Figure 6.

OPCs identified by Ascl1-GFP outside neurogenic niches in adult brain. A–L, Immunofluorescence in P60 Ascl1 GFP/+ brains showing GFP (green) in corpus callosum with NeuN (A), Ascl1 (B), BrdU incorporation (C), Sox2 (D), PDGFRα (E), Olig2 (F), and APC (G), or in thalamus with Olig2 (H, J), PDGFRα (I), Sox10 (K), and APC (L). In the corpus callosum, Ascl1-GFP cells are largely restricted to a narrow line on the ventral side that reliably coexpress Ascl1, mostly colabel with neural progenitor markers such as Sox2, have minor overlap with OPC markers PDGFRα, Olig2, and Sox10, and rarely overlap with the mature oligodendrocyte marker APC. Ascl1-GFP cells in the thalamus colabel with the OPC markers PDGFRα, Olig2, and Sox10 but not APC. CC, Corpus callosum; CTX, neocortex; PCL, pyramidal cell layer of hippocampus. Scale bars: 40 μm.

Figure 7.

Ascl1 -lineage OPCs become mature oligodendrocytes in the adult brain. Immunofluorescence of Ascl1 CreERTM;R26R-stop-YFP mouse brain sections harvested 5 or 30 d after tamoxifen administration at P60. A–G, Five days after tamoxifen administration, YFP cells (green) in corpus callosum no longer colabel with Ascl1 and they appear to spread from the ventral surface (A, B).YFP cells are often found as doublets, indicating they were recently dividing, and illustrated here in thalamus (C, arrows). In both the corpus callosum and thalamus, YFP cells colabel with the OPC markers Olig2 (D), PDGFRα (E), and Sox10 (F), but not the mature oligodendrocyte marker APC (G). H–N, Thirty days after tamoxifen administration, in both corpus callosum (H–J) and thalamus (K–M), YFP cells colabel with OPC markers PDGFRα (H, H', K, K') and Sox10 (I, L), but also with the mature oligodendrocyte marker APC (J, M). N, Quantification of the percentage of YFP cells colabeled with the OPC marker PDGFRα or the mature oligodendrocyte marker APC in both corpus callosum (CC) and thalamus (Th) 30 d after tamoxifen. Diagram depicts the CC and Th in adult coronal brain where images were taken. Scale bars: 20 μm.

Figure 8.

Model of the dynamics of maturation of Ascl1 lineage cells during adult neurogenesis and oligodendrogenesis. In both adult neurogenesis and oligodendrogenesis, Ascl1 expression is transient. Although developmental stages of cells expressing Ascl1 are slightly different, the onset of Ascl1 expression parallels the transition from quiescent stem cells or precursors to immature progenitors. In addition, the rates at which each of these lineages mature are varied. For adult neurogenesis, the terminal differentiation of all Ascl1 lineage cells from the SVZ is completed within 30 d, whereas the maturation of all Ascl1 lineage cells from the SGZ in the hippocampus takes longer. In oligodendrogenic lineages, Ascl1 expressing precursors in white matter mature more rapidly than Ascl1 expressing precursors in gray matter.

References

1. Ahn S, Joyner AL. In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature. 2005;437:894–897. - PubMed
1. Altman J. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J Comp Neurol. 1969;137:433–457. - PubMed
1. Alvarez-Buylla A, Lim DA. For the long run: maintaining germinal niches in the adult brain. Neuron. 2004;41:683–686. - PubMed
1. Battiste J, Helms AW, Kim EJ, Savage TK, Lagace DC, Mandyam CD, Eisch AJ, Miyoshi G, Johnson JE. Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord. Development. 2007;134:285–293. - PubMed
1. Bertrand N, Castro DS, Guillemot F. Proneural genes and the specification of neural cell types. Nat Rev Neuroscience. 2002;3:517–530. - PubMed

In vivo analysis of Ascl1 defined progenitors reveals distinct developmental dynamics during adult neurogenesis and gliogenesis - PubMed (original) (raw)