Local generation of glia is a majorastrocyte source in
Woo-Ping Ge1, Atsushi Miyawaki2, Fred H. Gage3, Yuh Nung Jan1& Lily Yeh Jan1
Glial cells constitute nearly 50% of the cells in the human brain1.
the regulation of synaptic connectivity during postnatal develop-
ment2. Because defects in astrocyte generation are associated with
to understand how astrocytes are produced. Astrocytes reportedly
arise from two sources4–6: radial glia in the ventricular zone and
progenitors in the subventricular zone, with the contribution from
each region shifting with time. During the first three weeks of
postnatal development, the glial cell population, which contains
predominantly astrocytes, expands 6–8-fold in the rodent brain7.
Little is known about the mechanisms underlying this expansion.
Here we show that a major source of glia in the postnatal cortex in
progenitors in the subventricular zone, differentiated astrocytes
with blood vessels, couple electrically to neighbouring astrocytes,
and take up glutamate after neuronal activity.
Most radial glia have finished producing their share of astrocytes
and have begun to disappear shortly after birth4–6; astrocytes are
therefore thought to derive mainly from progenitors in the subventri-
cular zone (SVZ) at later stages8. Themassiveexpansionof glia within
the first three postnatal weeks presents a daunting task for their pre-
sumed SVZ progenitors. This task is rendered even more challenging
by the thickening of the cortex compounded by the disappearance of
radial glia, which provides the migratory tracks for newly formed
astrocytes9. We used electroporation to transfect green fluorescent
protein (GFP) plasmids into SVZ/radial glial cells of mice at postnatal
age (about 3%) of the astrocytes derived postnatally from SVZ/radial
glial cells reached cortical layers I–IV; most were left behind in SVZ/
white matter (75%) and layers V–VI (22%) (Fig. 1b, c). It therefore
seems that huge numbers of cortical astrocytes generated postnatally
might arise from a more efficient process, such as local cell prolifera-
cell division within the cortex was reported half a century ago, on the
basis of studies involving [3H]thymidine incorporation into DNA10,
the extent of the contribution of local glial division to postnatal astro-
cyte production remained unknown, owing to the difficulty in distin-
this study, we have obtained evidence to support the hypothesis that
source of glia.
leukaemia retrovirus (MLV) to express GFP in infected dividing cells
infects proliferating cells and has been used for cell-fate tracing in SVZ
and the hippocampal subgranular zone (SGZ) in vivo8,11. We injected
and Dynamics, ExploratoryResearch for AdvancedTechnology,JapanScience and TechnologyAgency,and Brain ScienceInstitute, RIKEN,Wako-city, Saitama,351-0198, Japan.3Laboratoryof Genetics,
The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
+ + +
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Figure 1 | Locally generated glia as a major source of astrocytes.
a, Procedure to label SVZ/radial glia-derived astrocytes by electroporation.
b, The distribution of astrocytes (arrows) 2weeks after electroporation. VZ,
ventricular zone. c, Percentages of astrocytes at different locations. WM, white
matter. d, Proliferating cells (Ki671, red) in a cortical section of P3 mouse.
Nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI, blue).
e, Procedure to label locally proliferating cells by retrovirus. f, Cells labelled by
retrovirus (green). g, Image of infected astrocytes. Astrocytes (BLBP1, red)
(dashed line) were included for analysis in h. RV, retrovirus. h, Percentages of
astrocytes labelled by retrovirus injected locally, calculated as
1003(BLBP1GFP1cells/BLBP1cells). Scale bars, 200mm (b), 50mm
(d), 500mm (f) and 40mm (g).
3 7 6 | N A T U R E | V O L 4 8 4 | 1 9 A P R I L 2 0 1 2
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Animals and tamoxifen administration. The CAG-Fucci-Green transgenic line
was from A.M.’s laboratory, the hGFAP-CreER line was from K.D. McCarthy’s
laboratory (UNC), and the NG2BacDsRed transgenic line was from A.
Nishiyama’s laboratory. Both NG2-CreBac31and NG2-CreER were generated in
mice was from H. Zeng’s laboratory. Tamoxifen inductions were as described18.
For induction in hGFAP-CreER;Ai14 transgenic mice, an intraperitoneal or sub-
cutaneous injection of tamoxifen (dissolved in a 1:10 mixture of ethanol and
sunflower oil) at 3mg per 40g of body weight was administered once at the time
indicated. All animals were treated in accordance with protocols approved by the
Institutional Animal Care and Use Committee at UCSF.
In vivo electroporation. Newborn to 2-day-old pups (P0–2) were anaesthetized
by hypothermia (about 4min) and fixed to a support with a sticking plaster. GFP
complementary DNAs were cloned into the chicken b-actin CMV promoter-
in 10mM Tris-HCl pH8.0, with 0.04% trypan blue, was injected into the lateral
ventricle with a pulled-out glass capillary (diameter 50–100mm)32. Animals were
subjected to five electric stimuli of 50V, each lasting 50ms, at 950-ms intervals
using a square-pulse electroporator BTX830.
Retroviral preparation and in vivo infection. pCAG-GFP-PRE contains
replication-defective murine leukaemia virus (MLV)-based retroviral elements
designed to carry and express enhanced GFP under CMV promoter and CAG
promoter (modified chicken b-actin promoter with enhanced sequences from
CMV) with control of the MLV long terminal repeat. We followed the detailed
GFP-PRE, pCMV-gp and CMV-vsvg) were transfected to HEK293T cells with
Lipofectamine 2000. Viruses containing supernatants were harvested 2days after
transfection by centrifugation twice at 65,000g for 2h (Discovery 90SE; Sorvall).
Final virus titres were about 106–107colony-forming unitsml21as measured by
infecting HEK293T cells. Viruses with the GFP reporter gene were injected (1ml)
in vivo infection, pups were anaesthetized with ice for 3–5min, and the injection
was performed as described34. After injection, the pups were put back in a cage
Immunocytochemistry. Mice were perfused with 4% paraformaldehyde in
13PBS. Brains were cut into sections 25–50mm thick with a cryostat (model
CM3050S; Leica). Floating sections were permeabilized with 0.25% Triton
X-100 in 13PBS and then blocked for 2h with 5% BSA and 3% normal goat
serum with 0.25% Triton X-100 in 13PBS. Primary antibodies for Ki67 (1:200
Invitrogen) or Laminin (1:500, rabbit, polyclonal; Sigma) were applied to sections
alone or in combination and left to incubate for 24–48h at 4uC. Together with
DAPI or Hoechst 33342 (1mgml21; Invitrogen), secondary antibodies conjugated
with Alexa488, 555, 568 or 633 (1:750) were applied for 2h at room temperature
(22–25uC). To identify apoptotic astrocytes, sections were incubated for 15min
with 1mgml21propidium iodide after the treatment with 0.2 mgml21RNase
(DNase-free) in 13PBS for 30 min at 37uC as described previously35,36.
Slice preparation. Slices were prepared as described previously15. In brief, after
decapitation, mouse brains were dissected rapidly and sliced with a vibratome
(VT-1000S; Leica) in ice-cold oxygenated (95% O2 and 5% CO2) artificial
cerebrospinal fluid solution (aCSF) containing (inmM): 119 NaCl, 2.5 KCl, 2.5
CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3and 11 glucose. Transverse slices
(250mm inthickness) were then maintained inan incubationchamberforatleast
Electrophysiology and live cell nuclear labelling. Whole-cell recordings from
mouse brain slices were conducted with the aid of markers (GFP or
Hoechst33342) to identify infected cells or dividing cells. Astrocytes in hGFAP-
GFP transgenic mice were identified by bright green fluorescence under the
microscope. For live nuclear labelling, slices were incubated with Hoechst33342
(diluted to 2mgml21in aCSF) at room temperature for 30 min as described
previously14. Recording pipettes were routinely filled with a solution containing
(inmM): 125 potassium gluconate, 15 KCl, 10 HEPES, 3 MgATP, 0.3 Na-GTP, 5
Na-phosphocreatine and 0.2 EGTA (pH7.2–7.4, 290–300 mosM). For glutamate
transporter currents, pipette solution contained (inmM): 125 caesium gluconate,
5 CsCl, 10 HEPES, 3 MgATP, 0.3 Na-GTP, 0.2 EGTA and 5 Na-phosphocreatine
(pH7.2–7.4, 290–300 mosM). Membrane potential in voltage-clamp mode was
held at 280mV. Current pulses (20–60mA, 0.1 ms, 0.05Hz) were delivered
being recorded to induce transporter current.
Biocytin labelling. Glialcells were filledwith 0.1% biocytin(eN-biotinyl-L-lysine;
Vector Lab) by means of a whole-cell recording electrode, as reported previ-
overnight with 4% paraformaldehyde at 4uC before treatment for 2h with block-
ing solution containing5% BSA, 3% normalgoat serum and 0.25% Triton X-100.
Vector Lab). In Fig. 2o, DyLight 549 was added together with Alexa 633 (second
antibodies against anti-Ki67) after washing out excess primary antibody against
Confocal time-lapse imaging of acute brain slices. GFP1cells at cortical slices
from hGFAP-GFP transgenic mice (P3–5) were imaged on a Zeiss LSM510 two-
photon confocal microscope equipped with objective 203/0.5W and 633/0.9W
(Zeiss). Cells were scanned with xyz mode (four optical slices in z, with 8-mm
interval between slices). The frame interval was 4min for 30–100 frames.
Projection images were made from z-stacks that included all individual GFP1
cells. During imaging, slices were kept in a chamber with perfusion of aCSF (see
above) at 32–34uC.
Confocal time-lapse imaging in vivo. The pups (P3–6, hGFAP-CreER;Ai14
transgenic pups; Fig. 3l, m) were anaesthetized by hypothermia: 4–5 min in ice
incision site healed (no bleeding). A high-speed micro-drill was used to thin a
attached to a 1-ml pipette tip that was connected to a tube for inhalation. Pups
were then immobilized with 4% agarose. Imaging was performed using a two-
at 930nm, equipped with one of the following objectives: 103, 0.25 numerical
aperture (NA); 203, 0.8NA collected emission more than 560nm for tdTomato
and 500–550nm for mAG. Sometimes tdTomato was excited with a laser at
543nm. Images were taken every 1.5h for the first 3h, and then the pups were
putback in a boxandallowedto movefreely.Additional images were taken every
9–12h for the following 18–24h. During imaging, pups were fully anaesthetized
session, isoflurane was turned off and oxygen was left on until the animal fully
recovered. For Fucci-Green;hGFAP-CreER;Ai14 pups (Fig. 3i–k) we removed a
31. Zhu, X., Bergles, D. E. & Nishiyama, A. NG2 cells generate both oligodendrocytes
and gray matter astrocytes. Development 135, 145–157 (2008).
32. Li, G. et al. Regional distribution of cortical interneurons and development of
inhibitory tone are regulated by Cxcl12/Cxcr4 signaling. J. Neurosci. 28,
33. Tashiro, A., Zhao, C. & Gage, F. H. Retrovirus-mediated single-cell gene knockout
technique in adult newborn neurons in vivo. Nature Protocols 1, 3049–3055
34. Merkle, F. T., Mirzadeh, Z. & Alvarez-Buylla, A. Mosaic organization of neural stem
cells in the adult brain. Science 317, 381–384 (2007).
35. Barres, B. A. et al. Cell death and control of cell survival in the oligodendrocyte
lineage. Cell 70, 31–46 (1992).
36. Krueger, B. K., Burne, J. F. & Raff, M. C. Evidence for large-scale astrocyte death in
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