Dynamic Magnetic Resonance Imaging of Femoral Head Perfusion in Femoral Neck Fracture
Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zones in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.
p53 Mutation suppresses adult neurogenesis in medaka ﬁsh (Oryzias latipes)
, Teruhiro Okuyama
, Yoshihito Taniguchi
, Takeo Kubo
, Hideaki Takeuchi
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
Received 4 April 2012
Available online 31 May 2012
Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain.
Here, we report that the p53 null mutation in medaka ﬁsh (Oryzias latipes) suppressed neurogenesis in
the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohisto-
chemistry, we identiﬁed 18 proliferation zones in the brains of young medaka ﬁsh; in situ hybridization
showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the num-
ber of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant ﬁsh.
Immediately after BrdU exposure, the number of BrdU-positive cells did not differ signiﬁcantly between
them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which
was accompanied by an increased number in the WT brain. In contrast, no signiﬁcant increase was
observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling
revealed that there was no signiﬁcant difference in the number of apoptotic cells in the telencephalon
of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the
mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death.
These results suggest that p53 positively regulates neurogenesis via cell proliferation.
Ó 2012 Elsevier Inc. All rights reserved.
In the adult brain of teleosts, most proliferating cells are ob-
served in well-deﬁned zones of the brain (called proliferation
zones) . The whole brain of teleosts, such as medaka (Oryzias lat-
ipes) , zebraﬁsh (Danio rerio) , gymnotiform ﬁsh (Apteronotus
leptorhynchus) , and three-spined stickleback (Gasterosteus
aculeatus) , contains a large number of proliferation zones. Pre-
viously, we identiﬁed 17 proliferation zones (Zones A–Q) in the
adult medaka brain using sexually mature ﬁsh (age, more than
3 months) and demonstrated that there is persistent cell prolifera-
tion in these brain regions in the adult brain, irrespective of sex,
body color, or growth environment . Further, the distribution
of proliferation zones is largely conserved among some ﬁsh species
, suggesting that this distribution in the adult teleost brain is
important for the maintenance and development of the fundamen-
tal structure of ﬁsh brains .
To clarify the molecular basis underlying adult neurogenesis in
teleost ﬁsh, we focused on medaka p53 mutants . p53 is a se-
quence-speciﬁc DNA-binding transcription factor that induces
apoptosis or cell cycle arrest in response to genotoxic stress, thus
preventing DNA mutations from transmitting to progeny cells
. In murine brains, the p53 null mutation enhanced cell prolifer-
ation in the adult subventricular zone (SVZ) and, in association
with their rapid differentiation, resulted in an increased number
of newborn neurons and oligodendrocytes [8–11]. Here, we show
the distribution of proliferating zones largely overlapped that of
p53-expressing cells in the medaka brain. Furthermore, the meda-
ka p53 null mutant phenotype suggested that p53 positively regu-
2. Materials and methods
Medaka ﬁsh (O. latipes), Cab strain and p53 mutants , were
maintained in groups in plastic aquariums (12 13 19 cm).
Sexually immature medaka ﬁsh (approximately 1 month after
hatching; body length, 15 mm) without secondary sexual character-
istics were used for immunohistochemistry and in situ hybridiza-
2.2. Detection of mitotic cells in the young medaka brain
The detection of mitotic cells was performed as described pre-
viously . Dividing cells were labeled with 5-bromo-29-deoxyur-
idine (BrdU), by exposure to water containing 1 g/L BrdU (Sigma
Aldrich, Tokyo) for 4 h. BrdU-positive cells were detected by anti-
BrdU immunohistochemistry. Parafﬁn sections (10-
m thick) were
cut with a microtome (LR-85, Yamato Kohki, Tokyo). Immunostain-
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
Corresponding author. Fax: +81 3 5841 4448.
E-mail address: firstname.lastname@example.org (H. Takeuchi).
Biochemical and Biophysical Research Communications 423 (2012) 627–631
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ing was performed following standard procedures. Cell nuclei were
detected with DAPI staining (Invitrogen, Tokyo). BrdU-positive
cells were counted as described previously .
2.3. In situ hybridization
In situ hybridization of tissue sections was performed as de-
scribed previously [12,13]. The p53 cDNA fragment was ampliﬁed
with forward primer 5
reverse primer 5
cDNA clone number orbr44c15 (Medaka National BioResource Pro-
ject ) as a template. The digoxigenin (DIG)-labeled riboprobes
were synthesized by T7 or SP6 polymerase with a DIG labeling
mix (Roche, Tokyo) from a template containing the p53 cDNA frag-
ment. Micrographs were obtained with a BX50 optical microscope
(Olympus, Tokyo). The micrographs were processed with Photo-
shop software (Adobe, San Jose, CA).
2.4. TUNEL (TdT-mediated dUTP-biotin nick-end labeling) staining
Medaka brains were ﬁxed in 4% paraformaldehyde (prepared in
phosphate buffer saline) overnight and embedded in parafﬁn. Each
brain was sliced into 10-
m sections. Apoptotic cells were de-
tected using a DeadEnd™ Fluorometric TUNEL System (Promega,
Tokyo), according to the manufacturer’s protocol.
3.1. Distribution of proliferation zones and p53-expressing cells in
brains of young medaka ﬁsh
To elucidate the molecular basis underlying cell proliferation in
the medaka brain, we focused on medaka p53 . p53 is expressed
in proliferating and newly formed neurons of the adult murine
brain . To examine whether medaka p53-expressing cells were
Fig. 1. Mapping proliferation zones in the brain of young medaka. (a) Schematic drawing of the lateral view of the medaka brain. The positions of sections I–IX are indicated
by the lines. Te: telencephalon, OT: optic tectum, Cb: cerebellum. (b) Schematic representation of the distribution of the 18 proliferation zones. Red dots indicate proliferating
cells. Zone A: marginal zones of the anterior part of the telencephalon, Zone B: marginal zones of the dorsolateral part of the telencephalon, Zone C: medial zones of the
telencephalon, Zone D: dorsolateral part of the posterior part of the telencephalon, Zones E and F: preoptic area, Zone G: pineal body, Zone H: habenular nucleus, Zone I:
ventromedial nucleus, Zones J and K: optic tectum, Zone L: anterior part of marginal zones of third ventricular zone, Zone N: hypothalamus, Zones O–Q: cerebellum, Zone R:
periventricular grey zone (layer 3), and Zone S: Ependyme. Roman numerals in the panels correspond to section numbers shown in (a). Proliferation zones were determined
according to the medaka ﬁsh brain atlas (Supplemental Fig. 1). (c) Distribution of BrdU-positive cells in the different proliferation zones. A magniﬁed photo for zones P and Q
(cerebellum) in panel XI is shown in Supplemental Fig. 2. Scale bars indicate 100
628 Y. Isoe et al. / Biochemical and Biophysical Research Communications 423 (2012) 627–631
present in the proliferation zones, we mapped the proliferation
zones and performed in situ hybridization for detecting p53 tran-
scripts. In the present study, we used young medaka before they
developed secondary sexual characteristics, because the smaller
brain of the young medaka makes it easier to quantify newborn
cells within a speciﬁc brain structure such as the telencephalon
. As a detailed description of the cell proliferation zones in the
whole brain of sexually immature medaka is not available, we
mapped the proliferation zones by identiﬁcation of mitotic cells
as determined by BrdU uptake. Based upon the distribution of DAPI
staining and the medaka brain atlas , we identiﬁed the loca-
tions of the parafﬁn sections in the whole brain. We then mapped
the BrdU-positive cells and identiﬁed 18 proliferation zones, A–L
and N–S (Fig. 1, Supplemental Fig. 1). Sixteen zones (A–L and N–
Q), were identical to those previously identiﬁed in sexually mature
medaka . In the present study, we could not conﬁrm that there is
a proliferation zone in the pituitary gland (zone M) previously
identiﬁed in mature ﬁsh, as the pituitary gland is likely to be sep-
arate from the whole brain in the young ﬁsh. The 16 zones (A–L
and N–Q) were mapped to the telencephalon (zones A–D), preoptic
area (zones E and F), pineal body (zone G), habenular nucleus (zone
H), ventromedial nucleus (zone I), optic tectum (zones J and K),
marginal zone of the third ventricular zone (zone L), hypothalamus
(zone N), and cerebellum (zones O–Q) (Supplemental Fig. 2). The
two additional zones (R and S) were identiﬁed in the periventricu-
lar grey zone (layer 3) and ependyme, respectively, which were not
previously found in the mature ﬁsh , suggesting that these two
proliferation zones might disappear or integrate into the surround-
ing proliferation zones during the sexual maturation (Fig. 1). Next,
to examine whether p53 is expressed in proliferating zones in the
medaka brains, we performed in situ hybridization. We demon-
strated that medaka p53 expressed selectively in at least 12 zones
(zones A–E, H–K, N, P, and Q) (Fig. 2).
3.2. The p53 mutation had no effect on either the distribution of the
proliferating zones or the number of proliferating cells
To examine whether p53 is involved in cell proliferation in the
medaka brain, we mapped proliferation zones using two p53 mu-
tant strains . The p53
allele has a G to T substitution that
changes Glu241 to a stop codon, and the p53
allele has a T
to A substitution that changes Tyr186 to a stop codon . The
two mutated p53 genes encode truncated proteins that terminate
within a DNA-binding domain. These proteins lack the nuclear
localization signal and tetramerization domain required for full
activity. Thus, these nonsense mutations probably lead to a null
phenotype . We found the 18 proliferation zones in the two mu-
tant strains, p53
(Supplemental Fig. 3) and p53
(data not shown), indicating that loss of p53 has no effect on the
distribution of proliferation zones. To examine whether the num-
ber of proliferating cells was affected by the p53 null mutation,
we counted the number of BrdU-positive cells in the entire telen-
cephalon (zones A–D). There was no signiﬁcant difference in
BrdU-positive cells between the wild-type (WT) (average ± SE,
2316 ± 598; n = 4), p53
mutant (1849 ± 248; n = 4), or
mutant (1728 ± 366; n =3)(Fig. 3D and F).
3.3. The p53 mutation led to decreased numbers of differentiated
progenitors 1 week after BrdU exposure
To determine whether p53 mutation affects survival and/or pro-
liferation of progeny cells, we compared the distribution pattern of
differentiated newborn cells in the brains of WT (Cab strain) and
mutant medaka. One week after BrdU exposure,
BrdU-positive cells migrated from the proliferation zones
(Fig. 3E) in the telencephalon of both WT and mutant strains, sug-
gesting that there is no substantial difference in the migration pat-
tern between the two strains. However, in some brain regions, such
as the telencephalon (zone C) (Fig. 3E) and hypothalamus (zone N)
(Supplemental Fig. 4B), the number of BrdU-positive neurons
seemed to reduce in the mutant strain compared to the WT. Next,
we quantiﬁed the number of BrdU-positive cells in WT (Cab strain),
, and p53
in the telencephalon (zones A–D).
In the WT, the number of BrdU-positive cells 1 week after BrdU
exposure (6300 ± 535, average ± S.E, n = 4) increased over twofold
(Fig. 3F), suggesting proliferation of the migrated progenitors. In
contrast, there was no signiﬁcant increase in BrdU-positive cells
1 week after BrdU exposure in p53
tants (3596 ± 572 and 2378 ± 560, respectively). These results
raised two possibilities: (1) the p53 mutation enhanced cell death
Fig. 2. Distribution of medaka p53-expressing cells in the brain of young medaka. Zones A–D: telencephalon, zone E: preoptic area, zone H: habenular nucleus, zone I:
ventromedial nucleus, zones J and K: optic tectum, zone N: hypothalamus, zones P and Q: cerebellum. Scale bars indicate 100
Y. Isoe et al. / Biochemical and Biophysical Research Communications 423 (2012) 627–631
of differentiated progenitors (neuroblasts) or (2) the p53 mutation
repressed neuroblast proliferation and/or repressed differentiation
of stem cells to an active, proliferating, neuroblast subpopulation.
To examine whether cell death was enhanced in the p53 mutant
strains, we compared TUNEL-positive cells in the telencephalon
of WT and p53 mutants. The number of TUNEL-positive cells was
far less than the number of BrdU-positive cells in both WT and
p53 mutants, with no difference between the WT and p53 mutants
(Fig. 4A and B). We conﬁrmed that TUNEL-positive signals were
localized in nuclei stained with DAPI (Fig. 4A), and numerous TUN-
EL-positive cells were detected when using medaka pancreas sec-
tions, which are known to be susceptible to apoptosis 
(Supplemental Fig. 4).
In the present study, we demonstrated that the p53 mutation
did not affect the number of BrdU-positive cells immediately after
BrdU exposure. In the SGZ of murine brains, adult neurogenesis
originates from radial glia-like stem cells (Type 1 cells) through a
proliferating stage (Type 2 cells) generating neuroblasts (Type 3
cells) and dentate granule interneurons . Our ﬁnding strongly
suggests that loss of medaka p53 did not affect highly proliferating
progenitors, which correspond to Type 1 and 2 cells. This seems
inconsistent with a previous study indicating that genetic ablation
of p53 enhanced proliferation of stem cells in the adult murine
brain . There was no defect in stem cells in the p53 mutant me-
daka brain. Most mice, zebraﬁsh, and medaka with p53 function
defects develop without any obvious morphological defects
[6,18,19–23], as p53 family proteins are redundant and can com-
pensate for each other in various organs. Our results imply that
other p53 family members may compensate for a p53 deﬁciency
in medaka brain stem cells.
Furthermore, we showed that the number of newborn cells that
migrate from the proliferation zones increased during the 1-week
period after BrdU exposure in a p53-dependent manner. These data
suggested that p53 positively regulated the number of migrating
progenitors, which may correspond to Type 3 cells (neuroblasts).
Dividing neuroblasts are also found in the cerebellum (zone Q) of
the zebraﬁsh adult brain . The shift in the distribution of
BrdU-positive cells from the proliferation zone into the granule cell
layers is accompanied by an increase in the number of labeled cells
. In the murine brain, there is some evidence for the prolifera-
tion of migrating neuroblasts , which originate from stem cells
located in the SVZ of the lateral ventricles, moving along the rostral
migratory stream. To determine which subpopulation of progeni-
tor cells is regulated by p53, it will be crucial to characterize the
subtype and maturation sequence of progenitor cells in the meda-
Positive regulation of p53 in adult medaka brain neurogenesis
appears to be the opposite of what is observed in murine p53 mu-
, where p53 negatively regulates neurogenesis. One
possible explanation is that the p53 N-truncated isoform, which
has the opposite effect, may function in the medaka brain. In
mice and zebraﬁsh, the p53 family genes (including p63 and
p73) have 2 isoforms—full length and N-truncated—with an alter-
native transcriptional start site [10,20,25]. Because the latter iso-
form lacks a transactivation domain, it is thought to function in a
dominant-negative fashion to inhibit the transcriptional activity
of full-length p53 family members. In the murine brain, p53 fam-
ily proteins interact with each other in a cell-type/stage-speciﬁc
manner and coordinated expression of the two isoforms is re-
quired for stem cell maintenance in adult neurogenesis
[10,20,25,26]. As positive regulation of p53 in neurogenesis has
not been indicated in the murine brain, a p53 study using medaka
may shed a light on a novel mechanism underlying adult
Fig. 3. Comparison of the distribution and number of newborn cells in WT and p53
mutants. (A) Schematic drawing of the medaka brain and position of the
telencephalon in the brain. (B) Schematic drawing of the transverse section of the
medaka head. The section of images in (D) and (E), are indicated by a line and a
square in (A) and (B), respectively. The pink area represents the medaka
telencephalon. (C) The time schedule of this experiment. (D and E) Anti-BrdU
immunohistochemistry of parafﬁn sections from wild-type (WT) medaka (Cab
strain) and p53 mutants (Magenta). Nuclei were stained with DAPI (Blue). The
upper row indicates the transverse sections (Scale bars, 200
m) and the lower row
indicates the magniﬁed view of the proliferation zone (Zone E; Scale bars, 40
represented by the white rectangles in the upper row. (D) Immunohistochemistry
was performed immediately after BrdU exposure. (E) Immunohistochemistry was
performed 1 week after BrdU exposure. (F) Number of BrdU-positive cells in the
telencephalon of WT and p53 mutants medaka brains. Signiﬁcant differences were
observed between a and b, and b and c (p < 0.01 and p < 0.05, respectively; ANOVA
with Bonferroni–Dunn post hoc test; n = 3–4 per group).
630 Y. Isoe et al. / Biochemical and Biophysical Research Communications 423 (2012) 627–631
We thank the National BioResource Project Medaka, which is
supported by the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT) of Japan for supplying the medaka p53 cDNA
and mutants. We thank Dr. S. Kanda for technical assistance. This
work was supported by National Institute for Basic Biology Priority
Collaborative Research Project (10-104), MEXT, Scientiﬁc Research
on Grant-in-Aid for Scientiﬁc Research (B), and Grant-in-Aid for JSPS
fellows (to T.O.).
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Fig. 4. Cell death in the telencephalon of young medaka. (A) Confocal images show double-labeling of TUNEL (Magenta) and DAPI (Green) in Zone D. Red arrow head indicate
TUNEL-positive cells. For each strain, images in the right column are the magniﬁed images of the region outlined by the white rectangle in the left column images. Scale bars
m (Left) and 20
m (Right) (B) quantiﬁcation of TUNEL-positive cells. No signiﬁcant difference was detected (ANOVA with a Bonferroni–Dunn post hoc test;
n = 3–4 per group).
Y. Isoe et al. / Biochemical and Biophysical Research Communications 423 (2012) 627–631