Dynamic Magnetic Resonance Imaging of Femoral Head Perfusion in Femoral Neck Fracture

Article (PDF Available)inBiochemical and Biophysical Research Communications 423(4):627-31 · May 2012with40 Reads
DOI: 10.1016/j.bbrc.2012.05.125 · Source: PubMed
Abstract
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)
Yasuko Isoe
a
, Teruhiro Okuyama
a
, Yoshihito Taniguchi
b
, Takeo Kubo
a
, Hideaki Takeuchi
a,
a
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
b
Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
article info
Article history:
Received 4 April 2012
Available online 31 May 2012
Keywords:
p53
Adult neurogenesis
Progenitor cell
Neuroblasts
abstract
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.
1. Introduction
In the adult brain of teleosts, most proliferating cells are ob-
served in well-defined zones of the brain (called proliferation
zones) [1]. The whole brain of teleosts, such as medaka (Oryzias lat-
ipes) [2], zebrafish (Danio rerio) [3], gymnotiform fish (Apteronotus
leptorhynchus) [4], and three-spined stickleback (Gasterosteus
aculeatus) [5], 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 [2]. Further, the distribution
of proliferation zones is largely conserved among some fish species
[2], suggesting that this distribution in the adult teleost brain is
important for the maintenance and development of the fundamen-
tal structure of fish brains [2].
To clarify the molecular basis underlying adult neurogenesis in
teleost fish, we focused on medaka p53 mutants [6]. 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
[7]. 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-
lates neurogenesis.
2. Materials and methods
2.1. Fish
Medaka fish (O. latipes), Cab strain and p53 mutants [6], 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-
tion studies.
2.2. Detection of mitotic cells in the young medaka brain
The detection of mitotic cells was performed as described pre-
viously [2]. 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-
l
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.
http://dx.doi.org/10.1016/j.bbrc.2012.05.125
Corresponding author. Fax: +81 3 5841 4448.
E-mail address: takeuchi@biol.s.u-tokyo.ac.jp (H. Takeuchi).
Biochemical and Biophysical Research Communications 423 (2012) 627–631
Contents lists available at SciVerse ScienceDirect
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journal homepage: www.elsevier.com/locate/ybbrc
ing was performed following standard procedures. Cell nuclei were
detected with DAPI staining (Invitrogen, Tokyo). BrdU-positive
cells were counted as described previously [2].
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
0
-TGTTACATTTTATAGCTGTGGAGCA-3
0
and
reverse primer 5
0
-TTGGGCTGAAAACAGCACAACCATAGTT-3
0
using
cDNA clone number orbr44c15 (Medaka National BioResource Pro-
ject [14]) 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-
l
m sections. Apoptotic cells were de-
tected using a DeadEnd™ Fluorometric TUNEL System (Promega,
Tokyo), according to the manufacturer’s protocol.
3. Results
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 [6]. p53 is expressed
in proliferating and newly formed neurons of the adult murine
brain [15]. 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
l
m.
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
[2]. 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 [16], 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 [2]. 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 [2], 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 [6]. The p53
E241X
allele has a G to T substitution that
changes Glu241 to a stop codon, and the p53
Y186X
allele has a T
to A substitution that changes Tyr186 to a stop codon [6]. 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 [6]. We found the 18 proliferation zones in the two mu-
tant strains, p53
Y186X/Y186X
(Supplemental Fig. 3) and p53
E241X/Y186X
(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
Y186X/Y186X
mutant (1849 ± 248; n = 4), or
p53
E241X/E241X
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
p53
Y186X/Y186X
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),
p53
E241X/E241X
, and p53
Y186X/Y186X
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
Y186X/Y186X
or p53
E241X/E241X
mu-
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
l
m.
Y. Isoe et al. / Biochemical and Biophysical Research Communications 423 (2012) 627–631
629
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 [17]
(Supplemental Fig. 4).
4. Discussion
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 [18]. 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 [11]. 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 [22]. 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
[23]. In the murine brain, there is some evidence for the prolifera-
tion of migrating neuroblasts [24], 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-
ka brain.
Positive regulation of p53 in adult medaka brain neurogenesis
appears to be the opposite of what is observed in murine p53 mu-
tants [11,20]
, 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
neurogenesis.
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
l
m) and the lower row
indicates the magnified view of the proliferation zone (Zone E; Scale bars, 40
l
m),
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
Acknowledgments
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.).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bbrc.2012.05.125.
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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
indicate 80
l
m (Left) and 20
l
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
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    • "leptorhynchus: (Zupanc and Horschke, 1995; Zupanc et al., 1996; Zupanc, 2001; Hinsch and Zupanc, 2006); Eigenmannia sp: (Zupanc and Zupanc, 1992); and Brachyhypopomus gauderio: (Dunlap et al., 2011)]. The spatial distribution of brain proliferation zones in adult wave type weakly electric gymnotids roughly resembles that of other teleosts [Astatotilapia burtoni (Maruska et al., 2012); Austrolebias (Fernández et al., 2011); Carassius auratus (Raymond and Easter, 1983; Delgado and Schmachtenberg, 2011); Danio rerio (Maeyama and Nakayasu, 2000; Zupanc et al., 2005; Adolf et al., 2006; Grandel et al., 2006; Ampatzis and Dermon, 2007; Kaslin et al., 2009; Ito et al., 2010; März et al., 2010; Zupanc, 2011); Gasterosteus aculeatus (Ekström et al., 2001); Nothobranchius furzeri (Terzibasi et al., 2012); Odontesthes bonariensis (Strobl-Mazzulla et al., 2010); Oreochromis mossambicus (Teles et al., 2012); Oryzias latipes (Nguyen et al., 1999; Candal et al., 2005a; Alunni et al., 2010; Kuroyanagi et al., 2010; Isoe et al., 2012) and Salmo trutta fario (Candal et al., 2005b)], despite the phylogenetic distance to most of those species. However, differences have been observed that were attributed to the functional specialization of weakly electric fish (Zupanc and Horschke, 1995; Grandel et al., 2006; Grandel and Brand, 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Proliferation of stem/progenitor cells during development provides for the generation of mature cell types in the CNS. While adult brain proliferation is highly restricted in the mammals, it is widespread in teleosts. The extent of adult neural proliferation in the weakly electric fish, Gymnotus omarorum has not yet been described. To address this, we used double thymidine analog pulse-chase labeling of proliferating cells to identify brain proliferation zones, characterize their cellular composition, and analyze the fate of newborn cells in adult G. omarorum. Short thymidine analog chase periods revealed the ubiquitous distribution of adult brain proliferation, similar to other teleosts, particularly Apteronotus leptorhynchus. Proliferating cells were abundant at the ventricular-subventricular lining of the ventricular-cisternal system, adjacent to the telencephalic subpallium, the diencephalic preoptic region and hypothalamus, and the mesencephalic tectum opticum and torus semicircularis. Extraventricular proliferation zones, located distant from the ventricular-cisternal system surface, were found in all divisions of the rombencephalic cerebellum. We also report a new adult proliferation zone at the caudal-lateral border of the electrosensory lateral line lobe. All proliferation zones showed a heterogeneous cellular composition. The use of short (24h) and long (30day) chase periods revealed abundant fast cycling cells (potentially intermediate amplifiers), sparse slow cycling (potentially stem) cells, cells that appear to have entered a quiescent state, and cells that might correspond to migrating newborn neural cells. Their abundance and migration distance differed among proliferation zones: greater numbers and longer range and/or pace of migrating cells were associated with subpallial and cerebellar proliferation zones.
    Full-text · Article · Aug 2014
    • "Thus, heat-inducible Cre/loxP site-specific recombination is also applicable for investigating adult neurogenesis in medaka fish. In contrast to mammals with limited neurogenesis in the adult brains, teleost fish such as medaka fish and zebrafish constitutively generate newborn neurons in numerous proliferating zones across the whole brain throughout life [21], [26], [39]–[41]. As the distribution of proliferation zones in the adult brain is mostly conserved among teleost fish, adult neurogenesis is believed to be important for the maintenance and development of the fundamental structure of the fish brain throughout life [21], [41]. "
    [Show abstract] [Hide abstract] ABSTRACT: Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg) medaka lines, Tg (HSP:Cre) and Tg (HuC:loxP-DsRed-loxP-GFP). We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP) embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP) embryos (0-1 day post fertilization [dpf]) in a thermalcycler (39°C) led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO) system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system in both juvenile and adult stages.
    Full-text · Article · Jun 2013
  • [Show abstract] [Hide abstract] ABSTRACT: The Japanese medaka fish, Oryzias latipes, has become a powerful vertebrate model organism in developmental biology and genetics. The present study explores the dielectric properties of medaka embryos during pre-hatching development by means of the electrorotation (ROT) technique. Due to their layered structure, medaka eggs exhibited up to three ROT peaks in the kHz-MHz frequency range. During development from blastula to early somite stage, ROT spectra varied only slightly. But as the embryo progressed to the late-somite stage, the ROT peaks underwent significant changes in frequency and amplitude. Using morphological data obtained by light and electron microscopy, we analyzed the ROT spectra with a three-shell dielectric model that accounted for the major embryonic compartments. The analysis yielded a very high value for the ionic conductivity of the egg shell (chorion), which was confirmed by independent osmotic experiments. A relatively low capacitance of the yolk envelope was consistent with its double-membrane structure revealed by transmission electron microscopy. Yolk-free dead eggs exhibited only one co-field ROT peak, shifted markedly to lower frequencies with respect to the corresponding peak of live embryos. The dielectric data may be useful for monitoring the development and changes in fish embryos' viability/conditions in basic research and industrial aquaculture.
    Article · Oct 2012
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