1 Department of Neurology, Graduate Programs in Neuroscience, Developmental
Biology and Biomedical Sciences, Room S-268, 513 Parnassus Avenue, University
of California, San Francisco, CA 94143, USA
2 Department of Pediatrics, University of California, San Francisco, CA 94143,
USA
3 Departments of Neurology, Psychology, Developmental Neuropsychobiology
Laboratory, Programs in Occupational Therapy, Neuroscience, Washington
University Medical School, St Louis, MO 63108, USA
Authors for correspondence (e-mail:
zhaocj{at}seu.edu.cn
or
samuelp{at}itsa.ucsf.edu)
Accepted 15 April 2005
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SUMMARY |
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Key words: Wnt, Dentate gyrus, Epilepsy, Mossy cell, Apoptosis
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Introduction |
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We have focused on the function of frizzled 9 because of its selective
expression pattern in the hippocampus throughout life
(Kim et al., 2001;
Zhao and Pleasure, 2004
).
Previous studies demonstrated that frizzled 9 may act as a Wnt receptor in the
canonical Wnt pathway, signaling via ß-catenin
(Karasawa et al., 2002
). A
further role for frizzled 9 in brain development is suggested by the fact that
frizzled 9 is within the Chr. 7q11 deletion interval for Williams syndrome, a
neurodevelopmental cognitive disorder in humans
(Wang et al., 1997
;
Wang et al., 1999
). Williams
syndrome patients have a characteristic cognitive profile that includes
sparing of language and social function but severe involvement of spatial
cognitive processing and memory (cognitive processes dependent on hippocampal
function), with sparing of language and social functions
(Bellugi et al., 1999
). Here we
present evidence that frizzled 9 null and heterozygous mutants have increased
apoptotic cell death and increased precursor proliferation during hippocampal
development, and that null mutants have severe defects in learning and memory
reflecting hippocampal functional deficits that may be reminiscent of Williams
syndrome.
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Materials and methods |
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Immunohistochemistry and semiquantitative cell counting in developing brains
Brains were either immersed (embryonic brains) into or perfused (older than
P0) by freshly prepared 4% PFA, cryoprotected in 30% sucrose, embedded in OCT
and sectioned at 14 µm (embryonic brains) or 40 µm (older than P8) on a
cryostat or sliding microtome. Sections were permeabilized by 0.1% Triton-X100
in PBS, blocked in 20% lamb serum and incubated with primary antibodies
overnight at 4°C, then with either biotinylated secondary antibodies that
were viewed by the ABC method with diaminobenzidine (DAB) or fluorescence
conjugated secondary antibodies. Antibodies and reagents used were: rabbit
anti-frizzled 9 antiserum produced using the COOH-terminal peptide sequence
CHYKAPTVVLHMTKTDPSLENPTHL; rabbit anti-prox1
(Bagri et al., 2002); rabbit
anti-phospho-Histone H3: Upstate, cat#06-570; rabbit anti-Calretinin
(Chemicon, cat#AB5054); rabbit anti-Calbindin (Chemicon); rabbit anti-GFAP
(Chemicon); AlexaFluor 594 goat anti-mouse IgG (Molecular Probes, A11005),
Alexafluor 488 goat anti-rabbit IgG (Molecular Probes, A11008), Biotinylated
goat anti-mouse IgG (Vector Laboratories, BA2000), Biotinylated goat
anti-rabbit IgG (Vector Laboratories, BA1000). VECTASTAIN ABC kit: Vector
Laboratories, pk-6100. 3, 3'-diaminobenzidine (DAB): Sigma, D-5637.
For counting anti-Phospho-Histone H3- or BrdU-labeled cells in developing brains, six sections at the same coronal level were chosen for each animal, labeled cells in the medial and lateral cortical wall at E14.5, in dentate gyrus at E18.5 and P8. Anti-Phospho-Histone H3-labeled cells in adult brains were counted using the same methodology as for the stereologic analysis (see below). For hilar mossy cell counts, brains were cut at 50 µm from anterior to posterior and one of every eight sections were chosen for counting. Three independent pairs of littermates at each age were quantified and the data was analyzed by Student's t-test.
To examine whether there was compensatory gliosis or mossy fiber sprouting due to the alterations in dentate structure, we used antibodies to GFAP (Chemicon) and to Calbindin (Chemicon) to qualitatively examine wild type, heterozygous and homozygous mutant mice. Immunohistochemistry was performed as described above.
Stereologic analysis of adult dentate granule and hippocampal pyramidal cell number and layer volume
An investigator blind to frizzled 9 genotype performed quantitative
stereologic analysis of cell number and layer volume in dentate and
hippocampus using nissl stain to identify all cells. Adult mice (all about 12
weeks old) from each genotype (wild type, heterozygous and homozygous,
n=5-7 per genotype) were sectioned at 40 µm (nissl) or 50 µm
(immunohistochemistry) in the coronal plane throughout the extent of
hippocampus. A series of every tenth section was randomly selected and
processed for nissl stain immunocytochemistry. Total cell number was estimated
using the optical fractionator method
(West et al., 1991) and Stereo
Investigator software (Microbrightfield, Inc., Williston, VT). Using accepted
anatomic boundaries (The Rat Brain, Paxinos and Watson), the relevant
structure was traced at low power (4x) using the live image generated
with a Nikon Eclipse 600 microscope and a digital video camera. Pilot studies
determined the dissector dimensions (Table
1) to count approximately one cell per sampling frame and allowing
for a guard zone above and below the sampling site. A 60 x immersion
oil, 1.4 numerical aperture objective was used to achieve optimal optical
sectioning during stereologic analysis. The Stereo Investigator software
placed dissector frames using a systematic-random sampling design within each
contour. Only `caps' were counted, defined as immunoreactive or nissl stained
somata that came into focus while focusing down through the dissector height.
Adequate sampling was confirmed by coefficients of error for cell number less
than 0.05 (Table 1). Dentate
granule and hippocampal pyramidal cell layer volume estimates were calculated
using the Cavalieri principle and contours traced at low power.
|
Brdu injection and detection
Mice were injected with 50 µg Brdu g1 body weight and
sacrificed 2 hours later. Cryostat sections were prepared as described above
and treated with 2N HCl for 30 minutes at 37°C, neutralized with 0.1 M
borate buffer pH 8.5 for 15 minutes, washed with PBS-0.1% Triton-X100. After
blocking in 20% lamb serum, sections were incubated with anti-Brdu antibody
(Roche, cat#1170376) overnight at 4°C, then detected by AlexaFluor 594
goat anti-mouse IgG (Molecular Probes, A11005) or with the ABC method.
RT-PCR
P0 mouse brains were dissected in cold DEPC treated PBS, flash-frozen in
dry ice, quickly weighed and immediately homogenized in lysis buffer provided
in the Absolutely RNA RT-PCR Miniprep Kit (Strategene, cat#400800), and
isolation of RNA was performed by following the instructions provided by the
manufacturer. cDNA was synthesized at 45°C for 20 minutes and
pre-denatured at 95°C for 2 minutes. PCR was performed by the following
primer sets: 5'-CCTGCCAGCACTCAAAACTATCG-3' and
5'-GCACTGTGTAAAGGATGGAAAAGACTCC-3' for amplification of frizzled
9; 5'-TAACCGTCACGAGCATCATCCTC-3' and
5'-CCAGGTAGCGAAAGCCATTTTTTG-3' for LacZ. The conditions
were 40 cycles of denaturation at 95°C for 30 seconds, annealing at
65°C for 30 seconds, extension at 72°C for 30 seconds, then followed
by a final extension at 72°C for 10 minutes using SuperScript One-Step
RT-PCR kit (Invitrogen, cat#10928-034). PCR products were analyzed in a 2%
agarose gel.
Seizure induction
Ten-month-old frizzled 9 mutants, heterozygotes and their wild-type
littermates were injected with pentylenetetrazole (Sigma, p-6500) i.p. at 40
µg g1 body weight. The mice were observed for the latency
to first twitch and to tonic-clonic (T-C) seizure after injection. The data
were analyzed by Student's t-test.
Analysis of visuospatial learning and memory
The mice were transferred from the University of California, San Francisco
to the Developmental Neuropsychobiology Laboratory (Director: C. Robert Almli)
at Washington University School of Medicine (St Louis, MO, USA) at 10 days
prior to the onset of behavioral testing. Subjects used for behavioral testing
were 29 adult (120 days of age) male frizzled 9 mice: /
(n=9), +/ (n=12), +/+ (n=8). Mice were
housed under a 12:12 hour light:dark cycle, with food and water freely
available throughout the study.
Spatial learning and memory was assessed in the Morris water maze
(Morris, 1984). The water maze
and the testing procedures were described in detail previously
(Almli et al., 2000
;
Altemus and Almli, 1997
).
Briefly, a tub scaled specifically for mice (diameter=92 cm) was filled with
water (21±1°C) made opaque with nontoxic, white tempera paint. The
four walls of the maze room were differentially decorated with visually
distinct designs.
Each mouse was tested in each of three maze testing conditions (in the following order): Place Condition (spatial condition) the escape platform was hidden (submerged 1 cm below surface of the water) in a fixed location of a specific maze quadrant for each acquisition (learning) trial; Random Condition (unsolvable control condition) the escape platform was hidden (submerged 1 cm below surface of the water) in a randomly selected quadrant for each acquisition (learning) trial; Cue Condition (vision and motor ability control condition) the escape platform was visible (elevated 1 cm above the surface of the water) in a fixed location of a specific maze quadrant for each acquisition (learning) trial. The mice were `rested' for two weeks between testing on the place and random testing conditions, and between testing on the random and cue testing conditions, i.e. a 2 week `time-out' interval between testing conditions to reduce carry-over effects. This procedure was validated during extensive preliminary work with rats and mice.
During testing on the place, random and cue conditions, the mice were given six 1 minute acquisition (learning) trials per day for the consecutive 6 days. For each acquisition trial, the mouse was held against the wall of the maze in the center of a semi-randomly assigned quadrant not containing the escape platform. Time to escape to the platform (escape latency) was determined using Videomex-One Image Motion System and Water Maze Monitoring software (Columbus Instruments, Columbus, Ohio). After completion of the six acquisition trials each day, the platform was removed from the maze, and a single, 1 minute probe (memory) trial was performed. The total time that the mouse spent in quadrant that had previously contained the escape platform was measured as probe time (for testing under the random condition, a specific quadrant was arbitrarily designated as the `probe/platform' quadrant). In addition, the number of times that the mouse crossed the exact spot/position where the platform had be located during acquisition trials was measured as annulus crossings. Data analyses were performed with Statistica (Statsoft, Corp.), with alpha at P<0.05. Analysis of variance (ANOVA) with Tukey's HSD and Newman-Keuls post-tests were used.
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Results |
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Increased proliferation in the medial cortical wall and dentate gyrus
Previous studies have implicated Wnt signaling in regulating precursor
proliferation in the dorsal spinal cord and medial cortical wall
(Ikeya et al., 1997;
Alder et al., 1999
;
Lee et al., 2000
). These
studies predict that, if frizzled 9 is required for canonical
Wnt/ß-catenin signaling, then precursor proliferation should be decreased
in the medial cortical wall of mutant mice. Surprisingly, when we examined the
medial cortical wall of mutants we instead noted a small increase in precursor
proliferation at E14.5 (Fig.
2A,B). We quantified this data and found that there was a
statistically significant increase in the number of M-phase precursor cells in
the medial cortical wall of / mice and an intermediate change in
+/ mice (still statistically significant when compared to +/+ mice)
(Fig. 2B), while the number of
M-Phase precursors in the lateral cortical wall, where frizzled 9 is very
weakly expressed, showed no change according to genotype
(Fig. 2A,B).
By E18.5 proliferating dentate granule precursors have migrated to the
dentate hilus to form a displaced proliferative zone
(Bagri et al., 2002). At this
age, we also saw a statistically significant increase in M-Phase precursors in
the dentate gyrus (Fig. 2A,B).
By P8 and in adulthood we no longer saw any difference according to genotype
(Fig. 2A,B). Thus, frizzled 9
mutant mice had a small developmental increase in the number of dividing
precursor cells.
Although labeling of M-Phase cells with Phospho-Histone-H3 antibody has two advantages over BrdU labeling for counting acutely dividing precursors (these are the exclusion of potential teratogenic effects of BrdU and the decreased variability based on potential injection errors), acute BrdU labeling is the more established methodology. To determine if our approach of counting M-Phase cells was reliable to document the magnitude differences we observed, we also counted BrdU labeled after acute injection of BrdU at E18.5 and found the same magnitude difference based on genotype (Fig. 2C).
Since most granule cells are born postnatally
(Altman and Das, 1965a;
Altman and Das, 1965b
), it
seemed possible that the small increase in precursor cell numbers would lead
to a more dramatic change in granule cell number by adulthood despite the
overall normal organization of the dentate granule cell layer. To address this
question in 12-week old mice we used unbiased stereologic techniques to count
the total number of dentate granule cells and the total dentate volume in all
three genotypes. Unexpectedly this analysis showed that there was a
statistically significant approximately 20% decrease in the number of dentate
granule cells in mutant mice (Fig.
3A,B). At the same time, we found no changes in the pyramidal cell
number of volume of the pyramidal cell fields using stereologic methods
(Fig. 3B).
Cell death is elevated in the dentate gyrus of frizzled 9 mutant mice
How can increased numbers of precursors lead to a decreased number of
dentate granule neurons? If there is a consistent increase in precursor number
throughout development this should translate into a substantial increase in
neurons by adulthood. This suggested the possibility that some other cellular
process might be perturbed in the mutant mice. To explain this conundrum we
examined programmed cell death, using TUNEL staining, in dentate development.
We reasoned that the small increase in precursor number might be partial
compensation for a more substantial alteration in cell death.
By E14.5 the dentate anlage was a site of fairly abundant TUNEL+ nuclei in
wild-type mice. At low power, these cells were consistently present in 1-2
clusters each consisting of 1-4 cells when examined at higher power
(Fig. 4A,B). In mutant
/ mice there was dramatic (more than five-fold) increase both in
the number of clusters and the number of cells per cluster
(Fig. 4A,B). Frizzled
9+/ mice were intermediate in both the number of clusters and number of
TUNEL+ cells (Fig. 4A,B). The
differences between genotypes both in clusters of dying cells and the total
number of dying cells were all statistically significant. We also examined the
medial cortical wall before the dentate gyrus begins to form (at E12.5, when
frizzled 9 protein is very weakly expressed) and found no change in dying
cells in the region of the future dentate (the neuroepithelium immediately
dorsal to the cortical hem) and cortical hem
(Fig. 4A). This region was
previously noted to be a hotspot for developmental apoptosis under the control
of BMP signaling (Furuta et al.,
1997; Hebert et al.,
2002
; Panchision et al.,
2001
) but was not altered in frizzled 9 mutants.
Later in gestation both the dentate hilus, containing the bulk of the
ongoing proliferating dentate precursors, and the fimbria continued to be
areas with substantial numbers TUNEL+ cells in wild-type mice. At this age the
fimbria marked the boundary of the still prominent migratory route for granule
neurons and precursors (Bagri et al.,
2002). Again there was an almost three-fold increase in TUNEL+
cells in the dentate and fimbria of frizzled 9 null animals and an
intermediate increase in heterozygotes
(Fig. 4A,B). At P8, when the
displaced dentate proliferative zone was still active, there was a smaller but
still statistically significant increase in TUNEL+ cells in nulls but not
heterozygotes (Fig. 4A,B). In
adults we detected no alteration dependent on genotype, however the very low
level of apoptosis at these later ages may have made differences difficult to
discern (data not shown).
Mutants had increased numbers of hilar mossy cells
One of the other important hilar neuronal populations are the mossy cells.
These excitatory neurons are one of the chief synaptic targets of the granule
cells and their participation in the hippocampal circuit has been postulated
to be crucial in the etiology of temporal lobe epilepsy in humans
(Ratzliff et al., 2002;
Sloviter et al., 2003
). We
also noted quite prominent expression of frizzled 9 in this other population
of dentate neurons (Zhao and Pleasure,
2004
; Zhao and Pleasure,
2005
), so we examined their numbers as well. Interestingly we
found a dramatic increase in mossy cell number leading to substantially
increased neuronal density in the hilus
(Fig. 5). Previous anatomic
studies showed that these neurons originate from the germinative zone
immediately adjacent to the primordial dentate granule ventricular zone
(Nowakowski and Rakic, 1981
;
Nowakowski and Rakic, 1979
),
thus it seems likely that the increase in precursor proliferation in the
ventricular zone at mid-gestation included the cells generating mossy cells.
Thus, the loss of frizzled 9 led to reduction in the total numbers of granule
cells but increased mossy cells. This raised the possibility that there might
be functional, behavioral defects in the frizzled 9 mutant mice.
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We focused on spatial `memory' behaviors measured during probe (memory) trials for testing under the place (hidden platform in fixed location) condition in the maze. These measures are: probe times (time spent in the maze quadrant that previously contained the platform) and number of annulus crossings (number of times crossing the exact same position/spot where the platform had been previously located) (Fig. 7).
The means (plus standard errors) for two measures of spatial memory during place condition probe trials are presented in Fig. 7 (A=probe time in seconds, B=number of annulus crossings). Inferential statistical analyses of these measures of spatial memory revealed that: (1) the / mice displayed significantly shorter probe times during probe/memory trials under the place/spatial condition than the +/+ (P<0.04) or +/ (P<0.03) mice (Fig. 7A); and (2) the / mice displayed significantly fewer annulus crossings during probe/memory trials under the place/spatial condition than the +/+ (P<0.004) and +/ (P<0.009) mice (Fig. 7B). For both probe time and annulus crossing measures during the probe (memory) trials under the place/spatial condition, the +/+ mice and +/ mice did not statistically differ from one another (P>0.05). The three groups of mice did not statistically differ (P>0.05) for either probe times or annulus crossings during the probe/memory trials when tested under either the cue (visible platform in fixed location) or random (hidden platform in random location) testing conditions.
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Discussion |
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Wnts and the control of developmental apoptosis
Our analysis strongly suggests that Wnt signaling may control survival of
immature cells in the developing hippocampus along with its well-described
role in regulating cellular proliferation. Recent studies have shown similar
findings for other morphogenic signaling molecules in the developing CNS. The
roles of Sonic Hedgehog as both a developmental morphogen and regulator of
proliferation have been widely reported
(Ruiz et al., 2002;
Marti and Bovolenta, 2002
),
but recently Patched, the receptor for Sonic Hedgehog, was shown to regulate
developmental apoptosis of neural tube precursors
(Thibert et al., 2003
).
Interestingly this effect was blocked by ligand binding to Patched. In this
case a morphogenic signal is also required to block programmed cell death in
cells expressing the receptor for this molecule. The role of BMP signaling in
regulating developmental apoptosis has long been known
(Furuta et al., 1997
), but
recent studies have shown a particular role for BMP signaling in controlling
apoptosis required to shape the early development of the medial cortical wall
(Hebert et al., 2002
;
Panchision et al., 2001
). In
the case of BMPs, the ligands are positive regulators of apoptosis in the
medial cortical wall. Our study demonstrates that the loss of a Wnt receptor,
selectively expressed in the medial cortical wall, leads to an increase in
apoptosis. This implies that a Wnt signal is normally required to inhibit
apoptosis in the developing dentate gyrus, perhaps in opposition to BMP
effects. At this time we do not know which ligand would be operative in this
pathway since there are so many candidate ligands expressed in this region
developmentally.
Previous studies have failed to show any evidence of alterations in cell
death in the hippocampus of mutants with defects in the canonical Wnt
signaling pathway (Lee et al.,
2000; Galceran et al.,
2000
; Zhou et al.,
2004
). In fact, in these studies loss of Wnt signaling was shown
to cause a decrease in precursor proliferation in the hippocampus and dentate
gyrus, an effect quite inconsistent with the results of this study. This
raises the very real likelihood that frizzled 9 is active in a non-canonical
Wnt signaling pathway (Kuhl et al.,
2000
; Wallingford et al.,
2000
). It is still quite possible that frizzled 9 is also active
normally in the canonical Wnt pathway but that this function is largely
redundant with other frizzled proteins, several of which are expressed in
domains overlapping with frizzled 9
(Rattner et al., 1997
;
Kim et al., 2001
). Previous
studies have shown that several members of the frizzled family may be active
in a variety of Wnt signaling pathways in vertebrates and that this may depend
on the specific Wnt ligand they interact with in any given situation
(Kuhl et al., 2000
).
|
|
The hippocampal anatomic phenotypes of the frizzled 9 mutants (both
+/ and / genotypes) are likely to be quite significant
for understanding Williams syndrome on a number of levels. As we have shown,
frizzled 9 +/ and / mutants have reduced latency to onset
of seizures in response to a chemoconvulsant. This is likely due to a network
imbalance in two important classes of excitatory neurons in the hippocampus
that are generally synaptic partners (granule neurons and mossy cells) and is
consistent with the fact that about half of patients with Williams syndrome
are epileptic (Trauner et al.,
1989). Also predicted from our finding that there is a decrease in
granule cell number in adult frizzled 9 mutants is the failure of the null
mice to perform appropriately on Morris water maze testing. Since this is a
test of hippocampal visuospatial learning it is likely to be significant given
the selective visuospatial processing defects in Williams syndrome
patients.
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ACKNOWLEDGMENTS |
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Footnotes |
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