Increasing p16INK4aexpression decreases forebrain
progenitors and neurogenesis during ageing
Anna V. Molofsky1*, Shalom G. Slutsky1*, Nancy M. Joseph1, Shenghui He1, Ricardo Pardal1†,
Janakiraman Krishnamurthy2, Norman E. Sharpless2& Sean J. Morrison1
Mammalian ageing is associated with reduced regenerative
capacity in tissues that contain stem cells1,2. It has been proposed
thatthisisatleast partiallycausedby the senescence of progenitors
with age3,4; however, it has not yet been tested whether genes
associated with senescence functionally contribute to physiological
declines in progenitor activity. Here we show that progenitor
proliferation in the subventricular zone and neurogenesis in the
olfactory bulb, as well as multipotent progenitor frequency and
self-renewal potential, all decline with age in the mouse forebrain.
These declines in progenitor frequency and function correlate with
kinase inhibitor linked to senescence5. Ageing p16INK4a-deficient
miceshowedasignificantly smaller decline insubventricular zone
proliferation, olfactory bulb neurogenesis, and the frequency and
self-renewal potential of multipotent progenitors. p16INK4a
deficiency did not detectably affect progenitor function in the
dentate gyrus or enteric nervous system, indicating regional
differences in the response of neural progenitors to increased
p16INK4aexpression during ageing. Decliningsubventricular zone
progenitor functionandolfactory bulb neurogenesisduring ageing
are thus caused partly by increasing p16INK4aexpression.
Stem cells must persist throughout adult life in numerous tissues,
including the central nervous system (CNS)6, in order to replace the
mature cells that are lost to turnover, injury, or disease. However, the
function of stem cells and other progenitors declines with age in
diverse tissues including the haematopoietic system7–9, muscle10,11
and brain6,12,13. Consistent with this, ageing tissues exhibit reduced
repair capacity and an increased incidence of degenerative disease1,4.
However, the mechanisms responsible for the age-related decline in
the function of stem cells and other progenitors remain uncertain.
p16INK4agene expression increases with age in a variety of
tissues14–16. Although induction of p16INK4aexpression can cause
the senescence of a variety of cell types in culture5,17and in vivo18,
some cells (including some neural progenitors) are unaffected by
increased p16INK4aexpression or p16INK4adeletion19–21. It is
thus unclear whether increased p16INK4aexpression causes
declines in progenitor function during ageing in vivo. We have
addressed this question by examining progenitor frequency,
proliferation and neurogenesis in the forebrain lateral ventricle
subventricular zone (SVZ) of ageing wild-type and p16INK4a-
deficient mice. The SVZ contains a mixed population of stem cells
and other progenitors that engage in neurogenesis throughout adult
life6,13,22. The rate of neurogenesis is known to decline in ageing
mammals13, but the physiological mechanisms responsible for this
decline have not been identified.
We compared various measures of progenitor function in the SVZ
of 60-day-old, 1-yr-old and 2-yr-old mice to determine the effects of
ageing. Consistent with a previous study6, we found a twofold
reduction with age in the frequency of SVZ cells that formed multi-
potent neurospheres in culture (Fig. 1a; asterisk, P , 0.05). This was
associated with an approximately twofold reduction in the self-
renewal potential of these multipotent neurospheres (Fig. 1b; asterisk,
P , 0.05). In vivo, we observed an approximately threefold reduction
in the rate of proliferation in the SVZ with age (Fig. 1c, d; asterisk,
P , 0.05). These data suggest that stem cell frequency and self-
renewal potential, as well as overall proliferation rate, decline with
age in the SVZ.
and self-renewal potential, as well as reduced proliferation in the
SVZ20. These effects are largely caused by increased p16INK4aand Arf
expression in the absence of Bmi1 (refs 19–21). We therefore
wondered whether the age-related changes observed in wild-type
mice (Fig. 1) were associated with increased p16INK4aor Arf
expression in neural progenitors. p16INK4aand Arf expression
increase with age in some tissues15,16, although these studies did
not examine neural progenitors. We did not detect any age-related
increase in Arf expression in uncultured SVZ cells at the RNA or
protein levels (data not shown). In contrast, p16INK4aexpression
increased substantially with age in uncultured SVZ cells (Fig. 1e).
p16INK4aexpressionwas not detectable by polymerase chain reaction
(PCR) in the SVZ of 60-day-old mice, but became detectable by 1yr
of age and further increased by 2yr of age. We were not able to
detect p16INK4aprotein in the SVZ by western blot, consistent with
previous studies that have also not been able to detect this protein in
most uncultured mouse tissues that express p16INK4amRNA15,16,
presumably due to its low expression level and the limited sensitivity
of available antibodies.
To test the function of p16INK4ain ageing neural progenitors we
examined young (60-day-old) and old (2-yr-old) p16INK4a-deficient
affect the composition of the SVZ, with similar proportions of SVZ
cells staining positively for the glia marker GFAP or the neuroblast
marker doublecortin in wild-type and p16INK4a-deficient mice irre-
spective of age (data not shown). Similarly, apoptotic SVZ cells
(activated caspase-3þ) were rare in all treatments (data not shown).
Deletion of p16INK4ain young mice did not significantly affect the
frequency of SVZ cells that formed multipotent neurospheres in
culture (Fig. 2a), or the self-renewal potential of these cells upon
subcloning (Fig. 2b). This is consistent with our failure to detect
p16INK4aexpression in SVZ cells from young mice in vivo, as well as
with previous studies19,20. In contrast, p16INK4adeficiency in old
mice significantly increased the frequency of SVZ cells that formed
1Howard Hughes Medical Institute, Department of Internal Medicine, and Center for Stem Cell Biology, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.
2Departments of Medicine and Genetics, The Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina
27599-7295, USA. †Present address: Laboratorio de Investigaciones Biomedicas, Hospital Universitario Virgen del Rocio, Universidad de Sevilla, E-41013 Seville, Spain.
*These authors contributed equally to this work.
Vol 443|28 September 2006|doi:10.1038/nature05091
© 2006 Nature Publishing Group
multipotent neurospheres in culture (Fig. 2a; hash, P , 0.05 relative
to old wild-type mice), as well as the self-renewal potential of these
cells (Fig. 2b; hash, P , 0.05 relative to old wild-type mice).
Consistent with this finding, the rate of proliferation among SVZ
cells in vivo was also significantly increased by p16INK4adeficiency in
old but not young mice (Fig. 2c; hash, P , 0.05 relative to old wild-
type mice). Nonetheless, the percentage of proliferating cells in old
p16INK4a-deficient mice was still significantly less than observed in
normal or p16INK4a-deficient young mice (Fig. 2c; asterisk, P , 0.05
mice also exhibited enlarged lateral ventricles due to cortical
atrophy (Fig. 1c, data not shown). These observations indicate
that p16INK4adeficiency partially rescued the age-related declines in
progenitor activity in the SVZ but did not prevent cortical atrophy.
p16INK4adeficiency seemed to rescue completely the age-related
decline in cells that can form stem cell colonies in culture (Fig. 2a),
while only partially rescuing the overall decline in SVZ proliferation
(Fig. 2c). One possibility that would be consistent with previous
studies of p16INK4ain neural progenitors19–21is that p16INK4a
progenitors. A number of studies have argued that stem cells can be
identified within the SVZ based on their ability to retain the DNA
replication label 5-bromodeoxyuridine (BrdU; stem cells divide
infrequently andareretained within theSVZwhileother progenitors
we examined the effects of age and p16INK4adeficiency on the
frequency of BrdU label-retaining cells. We administered BrdU for
p16INK4adeficiency had no effect on the frequency of label-retaining
cells in the young adult SVZ (Fig. 2d). The frequency of label-
retaining cells declined significantly in old wild-type mice but not
in old p16INK4a-deficient mice (Fig. 2d). Thus, the frequencies of
label-retaining cells in vivo exhibited similar trends as observed for
the frequencies of cells that could form multi-lineage colonies in
the age-related decline in the frequency of early progenitors within
SVZ progenitors form neuroblasts throughout life that migrate
into the olfactory bulb and differentiate into neurons13,22. To test
whether the increase in p16INK4aexpression within the SVZ also
affects neurogenesis, we examined the effects of age and p16INK4a
administered BrdU to mice for 8days to mark dividing progenitors,
followed by a 4-week chase period with no BrdU (during which
neurons could migrate and differentiate), then killed the mice to
analyse sections through the olfactory bulb by confocal microscopy.
As we observed for progenitor function, p16INK4adeficiency had
no effect on neurogenesis in young mice, but neurogenesis signifi-
cantly decreased with age, and p16INK4adeficiency significantly
increased the frequency of newly generated olfactory bulb neurons
in 15–19-month-old mice(Fig. 3i). Notably, p16INK4adeficiency had
no effect on the frequency of non-neuronal cells within the olfactory
Figure 2 | p16INK4acauses age-related declines in stem and progenitor cell
neurospheres in culture significantly declined in 2-yr-old wild-type mice as
compared with 60-day-old mice (asterisk, P , 0.01) but significantly
increasedinoldmicewithp16INK4adeficiency(hash,P , 0.01relativetoold
wild-type mice). b, Self-renewal potential (the number of secondary
neurospheres generated per subcloned primary neurosphere) significantly
declined in old wild-type mice as compared to young mice (asterisk,
P , 0.05). p16INK4adeficiency significantly increased the self-renewal of
neurospheres from old but not young mice (hash, P , 0.05 relative to old
of BrdU significantly declined in old as compared to young mice (asterisk,
P , 0.01), but significantly increased in old mice with p16INK4adeficiency
(hash, P , 0.01 relative to old wild-type mice). d, The frequency of BrdU
compared with young wild-type mice (asterisk, P , 0.05), but significantly
increasedinoldmicewithp16INK4adeficiency(hash,P , 0.05relativetoold
wild-type mice). All values are mean ^ s.d. for at least three independent
experiments. All mice were histologically negative for intracranial
Figure 1 | Neural progenitor function declines with age. a, The percentage
of SVZ cells that formed multipotent neurospheres in culture declined with
age (asterisk, P , 0.05 relative to 60-day-old mice; three independent
experiments; 5–6 mice per age; error bars for all panels are ^s.d.). b, The
self-renewal potential of these primary neurospheres also declined with age
(asterisk, P , 0.05; three independent experiments; 5–6 mice per age).
c, d, Proliferation in the SVZ (percentage of BrdUþcells after a 2-h pulse)
also declined significantly with age (three mice per age; 5–7 sections per
mouse). The SVZ thinned in old mice (c, arrows), and the lateral ventricle
expanded (asterisk) due to cortical atrophy (c, lateral ventricle is not visible
at this magnification in young mice; scale bar, 200mm). e, p16INK4amRNA
expression increased with age as detected by quantitative (real-time) PCR
in uncultured SVZ cells (three independent samples per age). Note that
1-yr-old samples were set to 1.0 as the reference sample. ND, not detected.
NATURE|Vol 443|28 September 2006
© 2006 Nature Publishing Group
bulb (Fig. 3j). This may at least partially reflect our previous obser-
restricted progenitors20. p16INK4adeficiency thus partially rescued the
age-related decline in neurogenesis in the olfactory bulb in addition
to rescuing partially the decline in progenitor function in the SVZ.
To test whether these effects of p16INK4aoccurred throughout
the CNS, we also examined the effect of p16INK4adeficiency on
progenitor activity andneurogenesis inthedentategyrus26.Therates
of progenitor proliferation and neurogenesis in the dentate gyrus
decline markedly with age12. To test whether this is affected by
p16INK4a, we administered BrdU for 8days to mark proliferation in
the subgranular layer, followed by a 4-week chase period without
BrdU to examine neurogenesis in the granular layer. p16INK4a
deficiency did not significantly affect the rate of proliferation
among progenitors in the subgranular layer, or the frequency of
BrdUþNeuNþnewly generated neurons or BrdUþNeuN2non-
neuronal cells in the granular layerof the dentate gyrus (Supplemen-
tary Fig. 1). Thus, although p16INK4adeficiency consistently
increased all measures of progenitor activity and neurogenesis in
the ageing subventricular zone/olfactory bulb, it did not detectably
affect proliferation or neurogenesis in the dentate gyrus.
We also examined the effect of age and p16INK4adeficiency on
the neural crest stem cells that persist throughout adult life in the
enteric nervous system (in the gut wall)19,20,27. The frequency
of these neural crest stem cells declined with age, and p16INK4a
expression increased with age in p75þ(neurotrophin-receptor
expressing) gutcellsenrichedfor neural creststem cells(Supplemen-
tary Fig. 2). However, p16INK4adeficiency had no effect on neural
crest stem cell frequency in young or old mice. These results indicate
decline in SVZ progenitor function and neurogenesis, other mech-
in the hippocampus and peripheral nervous system.
The mechanisms that account for the increase in p16INK4a
could be developmentally programmed to increase with age to
counter the increasing incidence of cancer that is observed in the
accumulates from oxidative metabolism or other stresses. Although
Bmi1 is important for p16INK4arepression19,20,28, we did not detect
any change in Bmi1 expression with age at the RNA or protein levels
in SVZ cells (Supplementary Fig. 3). Nonetheless, subtle reductions
in Bmi1 expression or activity cannot be excluded and could be
functionally important, as even the twofold reduction in Bmi1
expression in Bmi12/þmice leads to increased p16INK4aexpression
(data not shown). Additional work will be required to resolve the
potentially multifactorial mechanisms that regulate age-related
changes in p16INK4aexpression.
The simplest interpretation of our data is that p16INK4aacts cell
autonomously within neural stem cells, because p16INK4adeficiency
increased the self-renewal of single neural stem cells in culture.
However, we cannot exclude the possibility of non-cell-autonomous
effects by p16INK4ain vivo. In addition to investigating this issue
further, it will also be of interest to determine whether circulating
humoral factors11influence p16INK4aexpression during ageing.
and p19ARFin transgenic mice does not detectably accelerate gross
measures of ageing or change lifespan, despite reducing cancer
incidence29. However, that study did not examine neural progenitor
function or neurogenesis. We also examined p16INK4abacterial
artificial chromosome (BAC) transgenic mice that exhibited a
moderate increase in p16INK4aexpression and found no effect of
p16INK4aoverexpression on stem cell frequency or self-renewal
potential, although old transgenic mice did exhibit a modest
reduction in SVZ proliferation (data not shown). It seems that the
levels of p16INK4athat are required to deplete stem cells are quali-
tatively higher than the levels that are required to inhibit carcino-
genesis. Together, the data suggest that although physiological
increases in p16INK4aexpression during ageing reduce neural pro-
overexpression of p16INK4adoes not substantially amplify these
Figure 3 | p16INK4acauses age-related declines in olfactory bulb
neurogenesis. a–d, Low-magnification images (scale bar, 20mm) from
sagittal sections of the olfactory bulb of old wild-type (a, b; same field of
view) and p16INK4a-deficient (c, d; same field of view) mice.Arrows point to
new neurons in the granular layer (BrdUþNeuNþ) whereas arrowheads
images (scale bar, 10mm) from one field of view from an old p16INK4a-
deficient mouse. Arrow indicates BrdUþNeuNþneuron; arrowhead
indicates BrdUþNeuN2non-neuronal cell. Panel h is a three-dimensional,
reconstructed side view (808 turn in the z-axis) of panel g. i, Neurogenesis
significantly (asterisk, P , 0.05) declined with age (BrdUþNeuNþneurons
level of neurogenesis in young mice, but significantly (hash, P ¼ 0.02
relative to old wild-type mice) increased neurogenesis in old mice. j, The
frequency of BrdUþNeuN2non-neuronal cells was not significantly
affected by p16INK4adeficiency (also as a percentage of NeuNþneurons).
The same trends were observed when the counts were expressed per unit
area (not shown). Values are mean ^ s.d. from 25 to 30 fields of view per
mouse, three mice per treatment.
NATURE|Vol 443|28 September 2006
© 2006 Nature Publishing Group
effects or accelerate gross measures of ageing.
Although physiological p16INK4aexpression reduces stem/pro-
genitor cell function and neurogenesis with age, how this relates to
overall ageing or lifespan remains unclear. It is difficult to examine
the effect of p16INK4adeficiency on mouse lifespan because p16INK4a
deficiency increases cancer incidence30in addition to affecting age-
related changes in stem/progenitor cell function. Declining stem/
progenitor cell function may be a major cause of the decline in
regenerative capacity and the increase in degenerative disease that is
observed in ageing tissues. On the other hand, increases in the death
related morbidity. It is similarly unknown whether physiological
differences in the rate at which stem/progenitor cell activity declines
with age has a detectable impact on longevity. The fact that p16INK4a
did not affect progenitors in certain regions of the nervous system
suggests that p16INK4ais not likely to promote generically the ageing
of all cells. Indeed, it is likely that there will be important differences
between tissues, and perhaps between progenitor cells and differ-
entiated cells, in terms of the genes that regulate the ageing process.
Although these questions remain unanswered, it may be possible to
gain important new insights into the ageing process at a cellular level
by studying individual cell types that have known physiological
functions in vivo, such as haematopoietic or neural stem cells.
Our data suggest that stem cell function is regulated by a balance
between proto-oncogenes, like Bmi1 (that promote stem cell main-
tenance and regenerative capacity but can also contribute to neo-
plastic proliferation), and tumour suppressors, like p16INK4a(that
reduce regenerative capacity and promote ageing but also reduce
cancer incidence). This balance changes with age and is influenced
byother proto-oncogenes and tumour suppressors that also regulate
p16INK4aexpression. The networks of proto-oncogenes and tumour
suppressors that regulate stem cell self-renewal, cancer cell prolifera-
tion and stem cell ageing may have evolved to balance the need
neoplasms with age. p16INK4athus reduces cancer incidence5,30but
also contributes to ageing by reducing progenitor function and
neurogenesis in at least certain regions of the nervous system.
Mice. p16INK4aþ/2mice were backcrossed at least six times onto a C57BL
background. p16INK4agenotyping was performed by PCR as described30.
Isolation of CNS progenitors. Adult SVZ was obtained by microdissecting the
lateral walls of the lateral ventricles, then dissociating for 20min at 378C in
0.025% trypsin/0.5mM EDTA (Calbiochem) plus 0.001% DNase1 (Roche).
containing 1mgml21BSA (Sigma A-3912), 10mM HEPES (pH7.4) and 1%
penicillin/streptomycin (BioWhittaker)) containing 0.014% soybean trypsin
inhibitor (Sigma) and 0.001% DNase1, the cells were washed and re-suspended
in staining medium, triturated, filtered through nylon screen (45mm, Sefar
America), counted by haemocytometer, and plated.
Cell culture and self-renewal assay. Cells were plated at clonal density
(1.3cellsper ml) on ultra-low-binding plates (Corning) to grow neurospheres.
Culture mediumwasbasedon a 5:3 mixture of DMEM-lowglucoseas described
previously19: neurobasal medium (Gibco) supplemented with 20ngml21
human bFGF (R&D Systems), 20ngml21EGF (R&D Systems), 1% N2 sup-
plement (Gibco), 2% B27 supplement (Gibco), 50mM 2-mercaptoethanol, 1%
were maintained at 378C in 6% CO2/balance air. To measure self-renewal,
individual neurospheres were dissociated by trituration then replated at clonal
density as above. Secondary neurospheres were counted 5–10 days later to
determine the number of secondary neurospheres formed per primary neuro-
sphere. CNS neurospheres were tested for multipotency by replating one
neurosphere per well into 48-well plates and then culturing adherently for
3–5days before triple staining for oligodendrocytes (O4), neurons (TuJ1) and
astrocytes (GFAP). See Supplementary Methods for details.
BrdU incorporation/proliferation assays. To quantify SVZ proliferation, mice
were injected intraperitoneally with 50mgkg21of BrdU (Sigma), and killed 2h
after BrdU injection. To quantify proliferation in the dentate gyrus, mice
received a single injection of 50mgkg21BrdU, followed by 1mgml21BrdU
in their drinking water for 8days before being killed and analysed. To quantify
neurogenesis in the dentate gyrus and the olfactory bulb and BrdU retention in
taken off of BrdU for 4weeks before the animals were killed. The brains were
dissected, fixed in 4% paraformaldehyde overnight, then cryo-protected in 15%
sucrose, embedded in 7.5% gelatin/15% sucrose, and flash frozen. Twelve-
micrometre sections were cut on a Leica cryostat.
For detection of BrdU in the tissue sections, DNAwas denatured in 2M HCl
for 30min at room temperature and neutralized with 0.1M sodium borate.
Sections were pre-blocked for 1h at room temperature in goat serum solution
(PBS containing 5% goat serum, 1% BSA and 0.3% Triton X-100 (Sigma)).
Primary rat anti-BrdU (1:500, Accurate Chemical) diluted in goat serum
solutionwas incubated overnight at 48C, followed by fluorescein isothiocyanate
(FITC)-conjugated anti-rat Fc fragment (Jackson Labs) for 3–4h at room
temperature. Slides were counter stained in 2.5mg ml21DAPI for 10min at
room temperature, then mounted using ProLong antifade solution (Molecular
To ensure that BrdU incorporation results were not skewed by dividing
endothelial or haematopoietic cells, we double-labelled SVZ sections with
antibodies against BrdU and either anti-CD45 (to identify blood cells) or anti-
VE-cadherin (to identify endothelial cells). We did not detect any BrdUþ
haematopoietic or endothelial cells.
Western blots and quantitative RT–PCR were performed as described in
analysed by analysis of variance. Statistically significant results were followed up
with Student’s t-tests.
Confocal analysis of neurogenesis in the olfactory bulb. A Zeiss LSM 510
confocal laser-scanning microscope was used to obtain 25–30 random fields of
view throughout all regions of one entire olfactory bulb of each mouse with a
£40 or £63 objective lens. For each image, 1-mm-thick optical sections were
scanned with three different lasers through a 12-mm sagittal tissue section
creating a Z-series stack with three distinct channels of fluorescence. An
ultravioletenterpriselaser wasusedtodetect theDAPIsignal labellingall nuclei.
An argon laser detected FITC (BrdU), and a HeNe laser detected Cy3 (NeuN).
Channels were merged together to determine whether DAPI, BrdU and NeuN
signals co-labelled at every 1-mm slice of the Z-series. Three-dimensional
projections were made using LSM 510 software.
Received 10 April; accepted 25 July 2006.
Published online 6 September 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements This work was supported by the National Institute on Aging
(grants to S.J.M. and N.E.S.) and the National Institute of Neurological Disorders
and Stroke (to S.J.M.). S.J.M. is an Investigator of the Howard Hughes Medical
Institute. N.E.S. is supported by the Sidney Kimmel Foundation for Cancer
Research, the Paul Beeson Physician Scholars program, and the Ellison Medical
Foundation. A.V.M. and N.M.J. were supported by National Research Service
Awards from the National Institutes of Health. We thank K. Yeager for tissue
sectioning and C. Mountford for mouse colony management.
Author Contributions A.V.M. studied the effect of age on forebrain progenitors,
p16INK4aexpression and function during ageing in the subventricular zone (Figs 1
and 2), and Bmi1 expression during ageing (Supplementary Fig. 3). S.G.S.
contributed to studies of p16INK4aexpression during ageing, and the effect of
p16INK4aon proliferation and neurogenesis in the subventricular zone and
hippocampus (Figs 2 and 3 and Supplementary Fig. 1). N.M.J. studied
neurogenesis in the olfactory bulb and hippocampus (Fig. 3 and Supplementary
Fig. 1). S.H. and R.P. examined p16INK4aexpression in the ageing enteric nervous
system and its effect on neural crest stem cells (Supplementary Fig. 2). J.K. and
N.E.S. provided ageing p16INK4a-deficient and control mice for some of the
experiments and discussed results throughout the project. S.J.M. helped to
design and interpret experiments and wrote the manuscript with help from
A.V.M., S.G.S. and N.M.J.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to S.J.M.
NATURE|Vol 443|28 September 2006
© 2006 Nature Publishing Group
Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter
Page 1 of 3
Supplementary figure 1: Ink4a does not significantly affect proliferation or neurogenesis in the dentate gyrus
of old mice. Panels a, c, and e show one field of view from the dentate gyrus of an old wild type mouse while panels
b, d, and f show one field of view from the dentate gyrus of an old Ink4a-deficient mouse. NeuN (red, a,b,e,f) labels
neurons in the dentate gyrus while BrdU (green, c,d,e,f) labels cells that were born during the BrdU pulse (8 days for
the analysis of proliferation in the subgranular layer (g) or 8 days followed by a 4 week chase without BrdU for the
analysis of neurogenesis in the granular layer (a-f; h)). A BrdU+NeuN+ neuron is shown in panels a,c,e (arrow). A
BrdU+NeuN- non-neuronal cell is shown in panels b,d,f (arrowhead). In the proliferation analysis, the number of
BrdU+ cells per mm of subgranular layer (SGL) in old mice was not affected by Ink4a-deficiency (g). The numbers
of BrdU+NeuN- non-neuronal cells and BrdU+NeuN+ neurons per mm of dentate gyrus (DG) in old mice were also
not significantly affected by Ink4a deficiency (h). Note that control mice appeared to have more BrdU+NeuN+
neurons per mm of dentate gyrus (h) due to a single control mouse that had an unusually high frequency of newborn
neurons for unknown reasons. All data are based on three independent experiments and error bars represent standard
Supplementary figure 2: Neural crest stem cell frequency decreases, and p16Ink4a expression increases with
age in the gut but Ink4a deficiency does not rescue stem cell frequency. The frequency of neural crest stem cells
within the myenteric plexus of the enteric nervous system decreases with age, whether measured by the frequency of
cells that could form multilineage colonies in culture (a; data are from four independent experiments) or by flow-
cytometric analysis of the frequency of p75+ cells, which are highly enriched for neural crest stem cells in the adult
gut19,27 (b; data are from four independent experiments; *, p<0.05 relative to 30 day old mice; #, p<0.05 relative to 1
year old mice). p16Ink4a expression increased with age in p75+ gut cells by quantitative PCR (c), and by Western blot
of gut wall cells (myenteric plexus plus muscle layer) (d). Ink4a deficiency did not affect the frequency of cells from
the gut wall that formed multilineage neural crest stem cell colonies in culture (e; *, p<0.05 relative to young wild
type mice; data are from four independent experiments). All values are mean±s.d.
Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter
Page 2 of 3
Supplementary figure 3: Bmi-1 mRNA and protein levels did not detectably change with age in the
subventricular zone of 60 day old, 1 year old, or 2 year old mice. Comparisons of Bmi-1 mRNA levels were
performed by quantitative (real-time) PCR (a; values are mean±s.d. from 3 independent samples per age), and Bmi-1
protein levels were assessed by Western blot (b) as in prior studies 19,20. ß-actin was used as a protein loading
Publisher: NPG; Journal: Nature: Nature; Article Type: Biology letter
Page 3 of 3
To stain neural progenitor colonies for makers of neurons (TuJ1), astrocytes (GFAP), and oligodendrocytes
(O4), plates were incubated first in anti-O4 antibody, and then in donkey anti-mouse IgM secondary antibody
conjugated to horse radish peroxidase (Jackson Immunoresearch) followed by nickel diaminobenzidine staining 27.
The cultures were then fixed in acid ethanol (5% glacial acetic acid in 100% ethanol) for 20 min at –20°C. After
blocking in PBS containing 4% horse serum, 0.4% BSA, 0.05% sodium azide, and 0.1% Igepal (Sigma), the cultures
were stained with anti-Tuj1 (1:500, Covance, Princeton, NJ) and anti-GFAP (1:200, Sigma G-3893) primary
antibodies followed by FITC-conjugated goat anti mouse IgG1, and PE-conjugated goat anti mouse IgG2a
antibodies (Southern Biotech, Birmingham, AL). Cells were stained for 10 min at room temperature with 10 µg/ml
DAPI (Sigma D-8417) to visualize nuclei.
Western blots and quantitative RT-PCR
Cells or tissues were resuspended in ice-cold cell lysis buffer (Cell Signaling Technology) with protease
inhibitor cocktail (complete mini tablet, Roche), and incubated for 15-30 minutes on ice. Tissues were briefly
homogenized, then all samples were sonicated for 1 minute at 20-30% power in a microson ultrasonic cell disruptor,
spun down for 5 minutes at 16,000g and the supernatant quantified colorimetrically (BioRad protein assay). 50-100
ug of protein per lane were separated in 12% denaturing SDS-PAGE gels and transferred overnight at 4°C to PVDF
membranes (Bio-Rad). The membranes were blocked in Tris buffered saline with 0.1% tween-20 and 5% milk,
incubated with primary and secondary antibodies, and washed following standard procedures. Horseradish
peroxidase conjugated secondary antibodies were detected by chemiluminescence (ECL Plus; Amersham-
Pharmacia). Primary antibodies were mouse monoclonal anti-ß-actin (Ab-1; Oncogene), mouse monoclonal anti-
Bmi-1 (Upstate biotechnologies), and rabbit polyclonal anti-p16Ink4a (M-156; Santa Cruz Biotechnology).
Quantitative RT-PCR was performed as described previously 20. Primers used to amplify Ink4a were
5’CGAACTCTTTCGGTCGTACCC-3’ (sense) and 5’-CGAATCTGCACCGTAGTTGAGC-3’ (antisense). These
primers amplified an 89 nucleotide product that spanned an intron and that was identical to Ink4a upon sequencing.