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.
+ + +
- - -
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).
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viruses locally into layers I–IV of the barrel or motor cortex of wild-
type mice at P0–6 (Fig. 1e, f) and examined GFP expression 1week
later in samples that were also stained with antibodies against brain
lipid-binding protein (BLBP) (Fig. 1g), which labels radial glia during
embryonic development and astrocytes in the postnatal brain12.
Whereas about 30% of infected cells were NG2 glia (27.6%, n5662
infected cells, Supplementary Fig. 2), 55–70% of infected cells were
astrocytes (BLBP1, 56.9%, n5369 GFP1cells, Fig. 1g; GFAP1,
68.6%, n5662 GFP1cells, Supplementary Fig. 2), indicating that
these astrocytes originated locally in vivo.
To determine whether a major astrocyte source was derived from
the local generation of glia, we injected retroviruses with higher titre
(1ml, (1–3)3107) into the cortex of P0–2 mice and compared the
number of GFP-expressing astrocytes (BLBP1GFP1) with the total
number of astrocytes (BLBP1) within an infected region 7–10 days
labelled (GFP1BLBP1, 46.863.8%, n55 mice; Fig. 1f–h). Because
the half-life of infectivity of MLV retrovirus is 5–8h at 37uC,
the doubly labelled astrocytes probably correspond to astrocytes
undergoing division in the time window of 5–8h plus the progeny
differences in the morphology of astrocytes (Fig. 1g), the density of
dividing cells (Supplementary Fig. 3) or the percentage of GFAP-
occupied area(Supplementary Fig. 4)in brainregions with or without
retroviral infection. Our observations therefore suggest that local pro-
liferation is a major source of astrocytes in the postnatal cortex.
To test the possibility that multiple dividing cell types infected by
retroviruses, including astrocytes, NG2 glia and perhaps some
unknown progenitors in the cortex, gave rise to these GFP-expressing
astrocytes, we labelled acute brain slices with the nuclear marker
bc d e
Area (×1000 μm2)
NG2 glia (D)
SVZ cell (D)
Astro (ND) Astro-like (D)
NG2 glia (D)
SVZ cell (D)
SVZ cell (D)
Figure 2 | Properties of dividing cells within the cortex. a, Nuclei
(arrowheads, Hoechst33342) of dividing cells at different mitotic stages in
prometaphase (Hoechst33342, arrowhead). c–e, Voltage responses from an
Astro-like-D cell (c), a dividing NG2 glia (d) and a dividing SVZ cell
(e). f–i, Current responses from a non-dividing (ND) astrocyte (f), an Astro-
voltages (inset, g). j, Current–voltage curves in f–i (the circles indicate the
at metaphase) stained with anti-GFAP (red). l, m, Morphology of a non-
and dividing cells (arrowheads) in the SVZ (m) of a P8 hGFAP-CreER;Ai14
transgenic mouse. n, Summary of the area covered by the processes of non-
by a Bonferroni post-hoc test; two asterisks, P,0.01). o, A non-dividing
astrocyte (arrow) was loaded with biocytin. A dividing astrocyte is labelled by
biocytin (arrowhead). tg, transgenic. Scale bars, 5mm (a) and 10mm
(b, k–m, o). Error bars indicate s.e.m.
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Hoechst33342, a dye that can permeate live cell membranes, to dis-
tinguish dividing cells from non-dividing cells (Fig. 2a). In some
experiments we also used slices from CAG-Fucci-Green transgenic
mice to identify dividing cells in the SVZ and cortex (Supplementary
Fig. 5). In this line, the green fluorescent protein mAG accumulates
specifically during the S (synthesis) to M (mitosis) stages of the cell
cycle13, thus facilitating the identification of dividing cells for whole-
cell patch-clamp recordings (Fig. 2b). Excluding cells of the vascular
into two groups: dividing NG2 glia14, with characteristic small sodium
currents and rectifying current–voltage (I–V) curve (Fig. 2d, h, j) and
astrocyte-like dividing cells (Astro-like-D; Fig. 2c, g, j), so named
for their similarity to differentiated astrocytes (Fig. 2f, j). Astrocytes
characteristically displayed large, delayed rectifier potassium currents
(Kdr) and large, inwardly rectifying potassium currents (Kir) but no
sodium currents, and they had a linear I–V curve15(Fig. 2f, j). In con-
trast, dividing cellsrecordedinthe SVZ (Supplementary Fig.5) had no
Kircurrent and a very small Kdr(Fig. 2e, i, j), typical of immature
progenitors16. To further characterize the Astro-like-D cells, our
immunostaining revealed that they were GFAP1but Nestin2(Fig. 2k
and Supplementary Fig. 6). We then compared the morphology of
Astro-like-D cells with that of mature astrocytes or SVZ dividing
progenitors in hGFAP-CreER;Ai14 transgenic mice. Crossing Ai14
transgenic mice17with hGFAP-CreER transgenic mice18allowed
robust expression of the red fluorescent protein tdTomato after
inducible astrocyte-specific Cre-mediated recombination. We admi-
nistered tamoxifen to hGFAP-CreER;Ai14 transgenic mice at P0–2
and assessed the cellular morphology 1week later (Supplementary
logy comparable to that of neighbouring mature astrocytes
(Ki672tdTomato1; Fig. 2l, n and Supplementary Fig. 8). In contrast,
SVZ dividing progenitors had a bipolar/unipolar morphology
(Ki671tdTomato1; Fig. 2m, n and Supplementary Fig. 8). Because
coupling by meansof gapjunctions is ahallmarkfeature ofastrocytes,
we injected biocytin into individual non-dividing astrocytes in brain
slices of hGFAP-GFP transgenic mice, which express GFP under the
Astro-like-D cells (Ki671GFP1) and mature astrocytes (Ki672GFP1;
Fig. 2o) were coupled by means of gap junctions. Thus, unlike SVZ
dividing progenitors (Fig. 2e, i, j) and glioblasts20, Astro-like-D cells
in the cortex are differentiated astrocytes. Taken together with a pre-
vious report21and our tracing results from NG2-CreBac/ERTM;Ai14
Ki67+ GFP+ (%)
0:32 0:36 0:48
In vivo imaging
0:00 0:04 0:080:12
Figure 3 | Time-lapse imaging of local proliferation of astrocytes.
a–c, Proliferating astrocytes (arrowheads) in the cortex of hGFAP-GFP
transgenic mice, at P3 (a), P6 (b) and P14 (c). d, Summarized data for the
percentage of Ki671GFP1cells among GFP1cells with strong GFP signals.
(e, parent cells (arrows); f, daughter cells (arrowheads)). g, Time-lapse images
in vivo. i–k, Images from a P4 triply transgenic hGFAP-CreER;Ai14;CAG-
Fucci-Green mouse (i, combined images; j, tdTomato; k, mAG signal;
arrowheads, dividing astrocytes). l, m, Time-lapse images at 1h35min (l) and
dividing astrocytes). Scale bars, 40mm (a–g) and 100mm (k, m).
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Astro-like-D cells are the parent cells of locally generated astrocytes.
To assess the abundance of proliferating astrocytes within the cor-
tex, weperfusedhGFAP-GFPtransgenic mice forKi67 immunostain-
ing and observed numerous astrocytes in the process of cell division
before P10 (18.9% at P3, n5956 GFP1cells; 13.1% at P6, n5619
GFP1cells; 1.5% at P14, n5269 GFP1cells; 0.30% at P48–52,
monitor local generation of astrocytes, we performed time-lapse
imaging of acute cortical slices from hGFAP-GFP transgenic mice
and found that roughly 2% of astrocytes divided within 3h
(2.060.2%, 16 of 809 cells with strong GFP signals from five mice,
glia are reported to have a very weak GFP signal in another hGFAP-
GFP transgenic line22, we tested whether NG2 glia could have been
among the dividing cells with a strongGFPsignal, byloading biocytin
into cells with a strong GFP signal through a recording pipette. We
found that biocytin diffused only among cells with strong GFP signals
(n55 slices; Supplementary Fig. 10), which is consistent with our
observations from doubly transgenic hGFAP-GFP;NG2BacDsRed
mice (Supplementary Fig. 11). Thus, the dividing cells with strong
GFP expression are astrocytes rather than NG2 glia.
12); however, mature astrocytes could conceivably be induced to
undergo local gliogenesis by means of a stab wound in vivo23. We
therefore performed in vivo imaging with an open skull but an intact
pial surface within 1h after surgery on the triply transgenic hGFAP-
dividing astrocytes (12.4%, tdTomato1mAG1; Fig. 3i–k, P3–6), a
similar observation to that in brain sections (Fig. 3a, b, d). Because
the thinned skull preparation does not cause astrocytic gliosis or
activation of microglia24, we then performed long-term time-lapse
imaging with the thinned skull preparation in hGFAP-CreER;Ai14
generated locally within the cortex (about 8% of progeny, (832)/212
we probably underestimated the percentage of astrocytes produced on
the basis of in vivo time-lapse imaging.
multiple cell types as SVZ cells do8,25, we recorded from their progeny
during and shortly after cytokinesis (Fig. 4a, b). The two daughter cells
shared similar I–V relationships that were characteristic of astrocytes
(Fig. 4c). In addition, we examined the daughter-cell morphology in
P6–8 hGFAP-CreER;Ai14 transgenic mice and found the two daughter
astrocyte marker, BLBP (Fig. 4d and Supplementary Table 1). Thus,
locally dividing astrocytes in the cortex primarily undergo symmetrical
division to generate two daughter astrocytes.
To determine whether the progeny maintained their astrocytic fate
after exiting fromthe cell cycle, we administeredtamoxifen at P0–2 to
hGFAP-CreER;Ai14 transgenic mice to label astrocytes permanently
with tdTomato and examined their locally generated progeny 1week
the astrocyte marker BLBP. Although there were many progenitors or
neurons with tdTomato expression in SVZ and hippocampal dentate
BLBP1or GFAP1in the cortex (99.8%, motor and barrel cortex;
enter programmed cell death in the cortex (Supplementary Fig. 13), it
is most likely that the progeny arising from local astrocyte division
retained astrocytic identity long after exiting from the cell cycle. For
further confirmation, tamoxifen was administered at P0–2 and
retroviruses were injected locally at P3–5 (3 days after tamoxifen) in
the cortex of hGFAP-CreER;Ai14 transgenic mice. The fate of doubly
labelled cells (tdTomato1GFP1) was then assessed 2 weeks later.
Because tdTomato marked cells that had expressed astrocyte markers
cells correspond to the progeny of astrocytes that were infected by
Fig. 4e, f), a characteristic of differentiated astrocytes. These results
demonstrate that locally generated progeny retain astrocytic identity
long after they exit from the cell cycle.
We then asked whether daughter astrocytes arising from local
astrocytedivision integratefunctionally into theexisting glial network
as mature astrocytes. The intercellular communication by means of
GFP+ with endfeet
GFP+ no endfoot
No. of cells coupled
Figure 4 | Symmetricdivisionofproliferatingastrocytesandthefunctionof
their progeny. a, A pair of daughter astrocytes (arrowheads) at late telophase
under differential interference contrast. b, Both cells had GFP signal. Nuclei
were stained with Hoechst33342 (HO, inset). c, Voltage responses of two
daughter cells evoked by step currents (21 to 6nA). d, Two daughter cells in
astrocyte infected by GFP-expressing retroviruses (green) and expressing
in a P19 hGFAP-CreER;Ai14 transgenic mouse.Tamoxifen was injected at P2,
and cells were infected with retrovirus at P5. f, The percentage of progeny cells
with endfeet, blue). g, A retrovirus-infected astrocyte progeny (GFP1, green,
arrow) in the absence (Ctrl, upper) or presence (lower) of 100mM
carbenoxolene (CBX) was injected with biocytin (red). Without CBX, both
GFP1astrocytes (arrowheads) and GFP2astrocytes (asterisks) contained
biocytin (red), as a result of gap-junction coupling with the astrocyte progeny
injectedwithbiocytin. h, The numberofcellscoupled.Twoasterisks,P,0.01,
(unpaired t-test). i, Current responses of uninfected (GFP2) and infected
(GFP1) astrocyte progeny. j, k, Glutamate transporter current (j) and its
summarized data (k) from infected astrocyte progeny before (black) and after
(red) application of blocker TBOA (100mM, 70.465.3%, n57). Two
asterisks, P,0.01 (paired t-test). Scale bars, 10mm (a, b, d, e) and 20mm
(g). Error bars indicate s.e.m.
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in the brain26. At 1–3weeks after viral infection, we loaded biocytin
into infected astrocytes and found that biocytin diffused into
20min (26.762.9 cells, n510 slices; Fig. 4g–i and Supplementary
Fig. 14). The coupling was inhibited by carbonoxelene (CBX), a
blocker for gap junctions (1.960.6 cells, n57 slices; Fig. 4g, h and
Supplementary Fig. 14), indicating that locally generated astrocytes
successfully integrated into existing glial networks. Another classical
function of astrocytes is to clear glutamate from the synaptic cleft27.
time course appeared in all the GFP1astrocytes recorded (20 of 20
GFP1cells, P12–19; Fig. 4j) and was sensitive to the glutamate
transporter blocker TBOA (Fig. 4j, k). The remaining currents, lasting
for more than 10s (decay time 13.360.4s, n54; Fig. 4j), correspond
to Kiractivation after neuronal excitation28. These observations thus
take up glutamate and K1ions after neuronal activity.
postnatal cortex provides a major glial source, at least in layers I–IV,
whereas astrocytes generated early in development are derived from
glioblasts)4–6,20. Once a subset of early astrocytes from those sources
colonize and differentiate in the cortex as ‘pioneers’, local division of
these differentiated astrocytes has a pivotal role in glial production
after birth in the cortex.
Astrocytic endfeet almost fully cover the blood vessels by postnatal
day20andarecrucialtothe regulation of cerebralbloodflow29andthe
transport of nutrients from blood to neurons30. It is not yet clear how
this large number of locally generated astrocytes can coordinate with
angiogenesis to form the complete gliovascular interface. Furthermore,
aberrant gene activity affecting glial proliferation is one potential cause
of gliomas, which comprise nearly 80% of primary malignant brain
tumours3. It will also be of interest to test whether gliomas could have
describedpreviously14. Forinvivo imaging,thepups with thinned skull(Fig.3h, l,
m) were immobilized with 4% agarose. The mouth of the pup was attached to a
1-ml pipette tip that was connected to a tube for inhalation. All data are given as
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 22 August 2011; accepted 14 February 2012.
Published online 28 March 2012.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank K. D. McCarthy and H. Zeng for providing us with the
hGFAP-CreER and Ai14 transgenic mice, respectively; A. Sakaue-Sawano for the
W. Zhou, F. Huang, and members of the Jan laboratory for discussion; G.-n. Li for help
on electroporation; Y. Li for advice on retroviral experiments; and E. Unger, C Guo,
Long-Term Fellowship of Human Frontier Science Program (HFSP) and National
Institute of Neurological Disorders and Stroke (NINDS) Pathway to Independence
Award. This work was supported by a NINDS K99/R00 award (1K99NS073735) to
W.-P.G., a National Institute of Mental Health R37 grant (4R37MH065334) to L.Y.J, a
National Institutes of Health (NIH) R01 grant (5R01MH084234) to Y.N.J., and grants
from the NIH/National Institute on Aging P01 AG010435, MH090258, Jeffry M. and
are Howard Hughes Medical Institute investigators.
Author Contributions W.-P.G. conceived the project, designed and performed the
design the experiments. W.-P.G. and L.Y.J. wrote the manuscript. F.H.G. and A.M.
provided MLV retrovirus and CAG-Fucci-Green transgenic mice, respectively. All
authors reviewed and edited the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article at
www.nature.com/nature. Correspondence and requests for materials should be
addressed to L.Y.J. (email@example.com).
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