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The Journal of Neuroscience, January 1, 1998, 18(1):284-293
Mouse Zic1 Is Involved in Cerebellar Development
Jun
Aruga1,
Osamu
Minowa2,
Hiroyuki
Yaginuma3,
Junko
Kuno2,
Takeharu
Nagai1,
Tetsuo
Noda2, and
Katsuhiko
Mikoshiba1, 4, 5
1 Molecular Neurobiology Laboratory, Tsukuba Life
Science Center and Brain Science Institute, Institute of Physical and
Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan,
2 Department of Cell Biology, Cancer Institute, Tokyo 170, Japan, 3 Institute of Basic Medical Sciences, University of
Tsukuba, Tsukuba, Ibaraki 305, Japan, 4 ; Department of
Molecular Neurobiology, Institute of Medical Science, University of
Tokyo, Tokyo 108, Japan, and 5 Calciosignal Net Project,
Exploratory Research for Advanced Technology, Tokyo 153, Japan
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ABSTRACT |
Zic genes encode zinc finger proteins, the
expression of which is highly restricted to cerebellar granule cells
and their precursors. These genes are homologs of the
Drosophila pair-rule gene odd-paired. To
clarify the role of the Zic1 gene, we have generated
mice deficient in Zic1. Homozygous mice showed
remarkable ataxia during postnatal development. Nearly all of the mice
died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the
external germinal layer (EGL), from 14 d postcoitum, and in
forward movement of the EGL. These findings suggest that
Zic1 may determine the cerebellar folial pattern
principally via regulation of cell proliferation in the EGL.
Key words:
Zic1; transcription factor; cerebellum; granule cell; cerebellar foliation; ataxia; gene targeting
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INTRODUCTION |
Determination of the molecular
mechanisms of cerebellar development remains one of the great
challenges of developmental neurobiology (Altman and Bayer, 1996 ).
Several genes have been shown to play a role in the patterning of this
region (for review, see Hatten and Heintz, 1995; Hatten et al., 1997;
Herrup and Kuemerle, 1997), and gene disruption studies in mice have
identified several genes that are involved in midbrain-hindbrain
patterning (McMahon and Bradley, 1990; Thomas and Capecchi, 1990;
Joyner et al., 1991; Wurst et al., 1994; Urbanek et al., 1997).
Zic1 was identified as a gene encoding a zinc finger protein
that was expressed in and highly restricted to the cerebellar granule
cell lineage (Aruga et al., 1994; Yokota et al., 1996). Genes that are
structurally related to Zic1 are also expressed in cerebella
(Aruga et al., 1996a,b). The amino acid sequence and genomic
organization of these genes suggest that they are the vertebrate
homologs of the Drosophila pair-rule gene
odd-paired (opa) (Benedyk et al., 1994; Cimbora
et al., 1995; Aruga et al., 1996a). The opa mutation causes severe pair-rule type segmentation abnormalities and impairment in the
timing of activation of the segment polarity genes wingless and engrailed (Benedyk et al., 1994). The vertebrate
homologs of these genes, En1, En2, and
Wnt-1, were shown to be involved in vertebrate neural
pattern formation (McMahon and Bradley, 1990; Thomas and Capecchi,
1990; Joyner et al., 1991; Wurst et al., 1994).
Furthermore, the zinc finger domain of the Zic protein is homologous to
those of Gli (Kinzler et al., 1988) and Opa and is capable of binding
to the Gli protein target sequence (Aruga et al., 1994). The Gli
proteins are vertebrate homologs of the Drosophila cubitus
interruptus (Orenic et al., 1990) and have been shown to play
essential roles in body pattern formation (Hui et al., 1994; Mo et al.,
1997).
In the mouse, three related genes, Zic1, Zic2,
and Zic3, are expressed in partially overlapping sites
(Nagai et al., 1997). Of these, the Zic1 transcript is the
most abundant throughout development, particularly in postnatal
cerebella (Aruga et al., 1996a). Zic1 was first detected in
the neuroectoderm during formation. Later, expression is restricted to
the dorsal neural tube and certain portions of somites and their
derivatives. Within the cerebella, expression was detected in the
entire cerebellar anlage as early as 12 d postcoitum [embryonic
day 12 (E12)], at which time the external germinal layer (EGL) had not
yet formed. Thereafter, expression became restricted to the EGL, which
contains granule cell precursors and granule cells.
These findings led us to hypothesize that Zic1 plays
significant roles in vertebrate development, particularly in cerebellar development. In the present study, we disrupted the Zic1
gene by homologous recombination in embryonic stem (ES) cells. Mice homozygous for the mutated Zic1 gene showed abnormal
behavior. Dysgenesis of the CNS, including the cerebellum, accounted
for these abnormalities. Our findings suggest that Zic1
plays essential roles in cerebellar development.
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MATERIALS AND METHODS |
Construction of targeting vector. The  phage
clones containing the mouse Zic1 gene were obtained from a
129/Sv mouse genomic library (Stratagene, La Jolla, CA). The genomic
structure was described previously (Aruga et al., 1996a). The 5
flanking region and first exon comprising an 8 kb
EcoRI-SalI fragment and a 2.4 kb
BglII-XhoI fragment containing the first intron
to the third exon were subcloned into Bluescript vectors. An
XhoI-SalI fragment containing a neo
gene driven by the phosphoglycerate kinase (PGK) promoter (Rudnicki et
al., 1992) or an XhoI-SalI fragment containing the diphtheria toxin A gene driven by the MC1 promoter (Yagi et al.,
1993) was inserted into the XhoI site of a plasmid. The
SalI-NotI fragments from this plasmid were
inserted into a NotI-SalI-digested plasmid. The
resultant targeting vector was linearized at a NotI site at
the 5 end of the 5 homologous fragment before electroporation into ES
cells.
Electroporation and generation of germ line-transmitting
mice. J1 ES cells (Li et al., 1992) were electroporated with 20 µg of linearized targeting vector DNA using a Bio-Rad (Hercules, CA)
Gene Pulser. The electroporated cells were then cultured on feeder
cells, which had been prepared from G418-resistant primary embryonic
fibroblasts as described previously (Rudnicki et al., 1992) and
selected for resistance to G418 (175 µg/ml) over a 6-11 d period.
Selected clones were cultured for an additional 7 d; the clones
were then frozen, and their genomic DNA was isolated for Southern blot
analysis.
Homologous recombinants, identified by Southern blot analysis
using the 3 external probe, were expanded for injection and DNA
isolation. Southern blot analyses of 126 ES cell clones correctly identified one targeted clone. The correctly targeted clone was confirmed by Southern blot, using several restriction enzymes, and then
expanded. Mutant ES cells were trypsinized, centrifuged, and
resuspended in ES medium. ES cells were injected into blastocoel cavities of E3.5 blastocysts from C57BL/6J mice. Injected blastocysts were surgically transferred into the uteri of pseudopregnant ICR recipients at postcoitum day 2.5. Male chimeras with extensive ES cell
contributions to their coats were bred with C57BL/6J female mice, and
germ line transmission of the dominant agouti coat color marker was
observed. Germ line transmissions of the Zic1
allele were screened by applying Southern blot analysis, and heterozygous F1 animals were intercrossed. Genotyping of embryos and
postnatal animals was performed by Southern blot or PCR with the
Zic1com primer (5-TCGAACAGAAAGGACTCAAGAAAGTCCCTG-3), the PGK1
primer (5-GCTAAAGCGCATGCTCCAGACTGCCTTG-3), and the Zic1w primer
(5-CGCGTTCAGAGAACCTCAAGATCCACAA-3), which correspond specifically to
the mutated allele (Zic1com and PGK1) and wild-type (Zic1com and Zic1w)
Zic1 allele. PCR consisted of 30 cycles at 94°C for 1 min
and 70°C for 2 min. The initial heterozygotes were mated with
C57BL/B6 or C3H/HeN, and their heterozygous offspring (C57BL/B6, N2;
C3H/HeN, N1) were subsequently back-crossed with C57BL/B6 or C3H/HeN
until the N5 or N4 generation, respectively. The homozygotes
(Zic1 /) were obtained by double heterozygous breeding of
mice within both genetic backgrounds. There was no change in phenotype
among different generations until C57BL/6J N5 or C3H/HeN N4 with regard
to abnormalities in the cerebella and axial skeleton. The majority of
results presented herein were obtained from C3H/HeN N1 and N2 except
for the pictures in Figure 1A-H (C57BL/6J N2). The
mice were maintained by the Division of Experimental Animal Research at
the Institute of Physical and Chemical Research (RIKEN).
In situ hybridization. In situ hybridizations
were performed essentially as described by Aruga et al. (1994).
Briefly, the postnatal animals were perfused transcardially with 4%
paraformaldehyde. The dissected tissue was immersed in the same
fixative for 2-12 hr. The tissues were then immersed in a 20% sucrose
solution and embedded in OCT compound. Sections of 10 µm in thickness
were prepared using a cryostat. Immunohistochemistry was performed as
described elsewhere (Aruga et al., 1994).
Histology and bromodeoxyuridine labeling. The fixation,
paraffin embedding, sectioning, and hematoxylin-eosin staining were performed as described previously (Aruga et al., 1994). The folial patterns were examined by preparing sagittal or coronal serial sections
through entire cerebella. Bromodeoxyuridine (BrdU) labeling was
performed as described by Yuasa et al. (1991). Briefly, the pregnant
mice were injected with BrdU (150 mg/kg, i.p.). The mice were killed 5 hr after injection by decapitation. The brains of embryos were immersed
in 95% ethanol and 5% acetic acid for 12 hr at room temperature. The
samples were dehydrated and embedded in paraffin. Paraffin sections (8 µm) were deparaffinized and immersed in 0.1N HCl for 15 min at 4°C
and then in a solution consisting of 50% formamide and 0.01× PBS for
5 min at 80°C. The sections were then reacted with anti-BrdU antibody
(Beckton Dickinson, Mountain View, CA; 1:50) overnight at 4°C. The
bound antibody was detected histochemically as described (Aruga et al.,
1994). At a minimum, three pairs of Zic1+/+ and
Zic1/ mice derived from two independent litters were
analyzed at each developmental stage.
Morphometric analyses. The cell number counting and the
perimeter measurements were performed on a Macintosh computer using an
NIH Image program (developed at the National Institutes of Health,
Bethesda, MD; available on the Internet at
http://rsb.info.nih.gov/nih-image/). Four sets of Zic1+/+,
Zic1+/, and Zic1/ mice derived from four independent litters were used. The midsagittal sections and sections through the cerebellar hemisphere, which were prepared from P21-P27 cerebella (5 µm), were stained with hematoxylin-eosin. Four sections per mouse were analyzed.
Electron microscopy. Three pairs of Zic1+/+ and
Zic1/ mice (P17) derived from three independent litters
were perfused under deep sodium pentobarbital anesthesia with Ringer's
solution, followed by a mixture of 2% paraformaldehyde and 2%
glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. The
cerebella were removed and cut into parasagittal sections with razor
blades. Sections through the cerebellar midline were chosen and
immersed in the same fixative at 4°C overnight. The sections were
osmificated, dehydrated through a graded ethanol series, and embedded
in Epon 812 (TAAB). Thin sections containing the cerebellar cortex
facing the primary fissure were cut on an ultramicrotome, stained with
uranyl acetate and lead citrate, and examined on a Hitachi H-7000
electron microscope.
Twenty electron micrographs were taken randomly from the middle layer
of the cerebellar molecular layer in each mouse at an original
magnification of 6000×. The number of synaptic profiles was counted
for each electron micrograph printed at a final magnification of
16,500×. The neuropil area, which contains no blood vessels, cell
bodies, or dendritic shafts thicker than 2 µm in caliber, was
measured following a method described previously by Kano et al. (1997).
The mean synaptic number/100 µm2 of neuropil area
was calculated for each mouse.
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RESULTS |
Targeted disruption of Zic1 and generation of
mutant mice
A targeting vector was constructed to replace a portion of the
first exon of the Zic1 gene with a neomycin resistance gene, resulting in homologous recombination and deletion of an initiator methionine and three of five zinc finger motifs (Fig.
1A). Electroporated ES
cells (J1 cell) were selected for G418 resistance, and DNAs extracted
from the drug-resistant colonies were analyzed by Southern blotting.
Correctly targeted ES cells were injected into C57BL/6J host
blastocysts to generate chimeric mice. Heterozygotes were obtained by
cross-breeding of the chimeras with C57BL/6J mice. Heterozygotes
(Zic1+/) were fertile and had no apparent
abnormalities.
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Figure 1.
Mouse Zic1 gene, targeting
construct, mutated Zic1 gene, and verification of the
targeted insertion. A, The wild-type mouse Zic1 gene (middle) consists of three
exons (Aruga et al., 1996a). The first contains the initiator
methionine and three of five C2H2 type zinc finger motifs. The
targeting vector (top) contains 8 and 2.4 kb regions
homologous to the Zic1 gene and a neomycin resistance
gene, respectively, driven by the PGK promoter (NEO). The diphtheria toxin A fragment gene driven by the MC1 promoter (DT) (Yagi et al., 1993) was inserted in the 3
end of the Zic1 gene to eliminate nonspecific
integration. In a properly mutated allele (bottom), the
protein coding region and splicing donor site of the mouse
Zic1 gene exon 1 has been replaced by the neomycin resistance gene expression unit. B,
EcoRI-digested genomic DNA samples extracted from a
litter were analyzed by Southern blotting using three probes
(5, 3, NEO). The band size and the corresponding fragments are indicated A and B,
respectively. B, BglII; R, EcoRI; S,
SalI; X, XhoI. C,
Viability of Zic1/ mice. Survival rates of 13 Zic1/ mice with the C57BL N2 background and 13 with
the C3H N2 background were determined up to postnatal day
(PND) 21. Pups in each group were derived from at least
five litters. There were no deaths among Zic1+/+ and
Zic1+/ littermates during the experimental
period.
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Viability and behavioral abnormalities of Zic1
mutant mice
Examination of the embryonic genotypes generated by double
heterozygous breeding showed no distortion of Mendelian inheritance (Zic1+/+, 26%; Zic1+/, 51%; and
Zic/, 24%; n = 95 at E18.5), indicating
that Zic1/ mice did not die during embryonic
development. However, ~50% of the Zic1/ animals
generated by C57BL/F2 Zic1+/ mating died within 1 d
after birth, and most homozygous mutants died within 3 weeks (Fig.
1C). In contrast, the survival of Zic1/ mice
with the C3H genetic background was better than that of mice with the
C57BL background. The C3H Zic1/ mice occasionally
survived as long as 8 weeks. Therefore, most of the analyses on
cerebella were done on C3H N1 and N2 (See Materials and Methods). The
neonatal death potentially may not be associated with the cerebellar
abnormality described below, and the cause of death is currently under
investigation.
The Zic1/ mice were significantly smaller than
littermates after the P2, and their weights were one-third to one-half
of those of littermates. The stomachs of homozygotes nearly always contained less milk than those of littermates, based on observations made through their thin body walls. Therefore, the growth retardation appeared to be attributable, in part, to the poor suckling. Further examinations are required to elucidate the mechanism of this growth retardation.
The behavioral abnormality manifests in Zic1/ mice after
P2. They often lie on their backs, and their forelimbs are extended anteriorly with irregular motions (Fig.
2A). At P10,
Zic1+/+ and Zic1+/ pups were able to turn over
easily within a few seconds when they were placed on their backs. In
contrast, it took the Zic1/ mice more than 1 min to turn
over, and, in some cases, mice were completely unable to recover their
posture.
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Figure 2.
Abnormal behavior was observed in the
Zic1/ mice. A,
Zic1/ mice (P10) (right) often lay on
their backs in infancy, whereas littermates (left) did
not. The mouse distorted its body in an attempt to turn over but was
unable to do so. When the mice were suspended by their tails, the
Zic1/ mice (P14) often crossed or clasped their
hindlimbs (C), whereas the normal mice usually extended and shook their hindlimbs (B).
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The gait of 2-week-old Zic1/ mice was also severely
disturbed, as they often dragged their hindlimbs behind
them.The Zic1/ mice were generally
unable to walk in a straight line, and most fell to one side. When the
mice were lifted by the tail, typically, their hindlimbs were clasped,
whereas the wild-type mice extended their legs (Fig.
2B,C). Thus, the loss of coordinated movements involving all four limbs was peculiar to Zic/ mice
during their development. Two weeks after birth, the ataxia became more
severe, and some mice showed tonic convulsions.
In addition to the behavioral phenotypes, the thoracic regions of the
Zic1/ mice were variably kyphotic, concomitant with marked cervical flexion. These findings may be attributable to abnormal
patterning of the developing sclerotome in which Zic1 is
also strongly expressed. The phenotypes will be described in detail
elsewhere.
Cerebellar abnormalities
In addition to the highly restricted Zic1 expression in
the CNS, the abnormal behaviors of Zic1/ mice suggested
alteration in the CNS. We therefore examined the morphological features
of the CNS in homozygous mice. Macroscopically, the most remarkable change found in the CNS was hypoplasia of the cerebellum (100% in 53 Zic1/ pups after birth) (Fig.
3A,B). In addition to
reduction of the cerebella, the cerebellar fissures and folia were also deformed in Zic1/ mice. In almost all cases, a folia in
the anterior lobe was completely missing, and the crus and
paraflocculus lobule were hypoplastic (Fig. 3C-F).
However, foliation pattern abnormalities were not confined to specific
portions of the cerebella, because all of the fissures appeared to be
shallow. We found no overt abnormalities in the Zic1+/
cerebella by macroscopic observation. In contrast to the abnormal
foliation, the layer organization of Zic1/ cerebella was
grossly normal (Fig. 3G,H). The Purkinje cell and
granule cell layers were equally clearly observed in the cerebella of
Zic1/, Zic1+/+, and Zic1+/
mice.
To define the effect of the Zic1 mutation on gross
cerebellar organizations more clearly, morphometric analyses were
performed on both midsagittal sections and parasagittal sections
through cerebellar hemispheres of P21-P27 cerebella, in which the
inward migration of the granule cells is nearly complete. When the
areas of the granule cell and molecular layers and the length of the Purkinje cell layer were compared, the abnormalities in
Zic1+/ and Zic1/ cerebella became apparent.
Although the Zic1+/ cerebella were significantly smaller
than Zic1+/+ cerebella with respect to all three parameters,
the extent of reduction was not as severe as that in
Zic1/ cerebella, in which the values were approximately half those in Zic1+/+ cerebella (Fig.
4A,B).
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Figure 4.
Morphometric analyses of midsagittal sections of
mature cerebella. A, Areas of the granular cell layer
(GCL) and molecular layer (ML).
B, Perimeter of the Purkinje cell layer in both
midsagittal (mid) and cerebellar hemisphere
(lat) regions. C, Ratios of anterior GCL
area (rostral to primary fissure) to posterior GCL area (caudal to
primary fissure). Granule cell density (D) and
Purkinje cell number per millimeter (E) in
midsagittal regions were measured in Zic1+/+
(open bar), Zic1+/ (hatched
bar), and Zic1/ (shaded bar)
cerebella. Error bars indicate SEM. *p < 0.01;
**p < 0.001.
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Because a folium rostral to the primary fissure was absent in the
Zic1/ cerebella, we compared the granule cell layer
(GCL) areas anterior and posterior to the primary fissure (Fig.
4C). As expected, the ratio (anterior GCL/posterior GCL) was
reduced, markedly in homozygotes and mildly in heterozygotes. In
contrast to the severe reduction in granule cell layer area, granule
cell density was not changed (Fig. 4D), suggesting
that the total number of granule cells was decreased because of a
reduction in the size of the granule cell layer. On the other hand,
Purkinje cell density was greater in the Zic1/ cerebella
(Fig. 4E). This may suggest that the total Purkinje
cell number is not as severely reduced as that of granule cells.
We next assessed whether cells in the mature Zic1/
cerebella had undergone normal differentiation. Hematoxylin-eosin (HE) sections were almost indistinguishable from those of a normal specimen,
the exception being the more slender Purkinje cell body in the
Zic1/ compared with Zic1+/+ cerebella. Then,
we performed in situ hybridization and immunohistochemical
staining to examine the expression of cell type-specific markers.
Molecular markers for granule cells Zic2 (Aruga et al.,
1996a; Nagai et al., 1997) and En2 (Davis and Joyner, 1988)
(Fig. 5), which encode transcription factors located in the granule cells and markers for Purkinje cells,
type 1 inositol trisphosphate receptor (Furuichi et al., 1989), and
cGMP-dependent protein kinase (Schlichter et al., 1980) were normally
expressed in the Zic1/ cerebella (data not shown).
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Figure 5.
In situ hybridization of
tissues from Zic1+/+ (A, C) or
Zic1/ (B, D) cerebella. In
situ hybridization using En2 (A,
B) and Zic2 (C, D) probes showed
the expression of these genes to be essentially unchanged in the
Zic1/ cerebella. The cerebella were taken from C3H
N1 mice. Scale bar, 1 mm.
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When the mutant cerebella were examined electron microscopically (Fig.
3I,J), it became clear that normal synapses were
present, that the number of synapses between the parallel fibers of
granule and Purkinje cells was not changed (Zic1+/+,
23.12 ± 3.38; Zic1/, 22.58 ± 3.81 synapses/100 µm2) and that granule cells and
Purkinje cells were morphologically indistinguishable in
Zic1+/+ and Zic1/ mice. Thus, taken together with the normal expression of several cerebellar markers, we assumed that differentiation and neural circuit formation were fundamentally normal in Zic1/ cerebella.
Abnormalities of developing cerebella
To understand the basis of abnormalities found in
Zic1/ cerebella, we next examined the process of
cerebellar development. Before E17.5, the Zic1/ brains
were apparently indistinguishable from those of Zic1+/ or
Zic1+/+ animals. Abnormalities were first recognized in the
cerebella of E17.5 mice (Fig.
6A,D). At this stage,
there was a lucent area in the immature Purkinje cell layer, which may
correspond to the most recently localized Purkinje cells on the caudal
end of the prospective lobule VI (Altman and Bayer, 1996) (Fig.
6A,D, closed arrowheads). This site was more anterior in the Zic1/ cerebella than in the Zic1+/+
cerebella. The migrating granule cells and the site of the primary
fissure were more anteriorly located in E18.5 cerebella (Fig.
6B,E, open arrowheads). These results indicate that
the anterior cerebellar lobe was more severely reduced than the
posterior lobes, an observation consistent with the morphometric
analyses of mature cerebella. The sites where primary fissures appeared
in Zic1/ cerebella were always rostral to those of
Zic1+/+. These abnormalities in the developing cerebella were consistently observed irrespective of the genetic background (C57BL or C3H).
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Figure 6.
Phenotypes of the developing cerebella. A series
of sagittal sections through the midsagittal planes of the
Zic1+/+ (A-C, G) or
Zic1/ (D-F, H) cerebella were
stained with hematoxylin-eosin. E17.5 (A, D), E18.5
(B, E), and P5 (C, F) cerebella
were examined. CP, Choroid plexus; T,
tectum; IV, fourth ventricle; e, external germinal layer; closed arrowheads, lucent portions
indicating the cessation of the immature Purkinje cell layer
(asterisk); open arrowheads, primary
fissures. G, H, Higher magnifications of
C and F, respectively. e,
External germinal layer; g, granule cell layer;
m, molecular layer; p, Purkinje cell
layer. Note that the Purkinje cells of Zic1/
cerebella are morphologically immature at this time point. All panels
indicate the C3H N1 cerebella. Scale bars: A-C, 200 µm; G, 100 µm.
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During postnatal development, folial pattern abnormalities and reduced
cerebellar mass became increasingly obvious. Poor development of all
folia was observed in the Zic1/ P5 cerebella (Fig.
6C,F). Purkinje cells were not aligned in a
monolayer, and the apical cytoplasms were hypertrophic and rectangular
(Fig. 6G,H). These features are consistent with those
of immature Purkinje cells (Altman, 1972). There may thus be a delay in
the maturation of cerebella in these animals.
The driving force behind cerebellar foliation has been thought to be
the existence of an expanding germinal cell layer on the surface of the
cerebellum. We initially performed terminal deoxynucleotidyl
transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining to
compare the frequency of apoptotic cell death from E14 to E17 and at P7
when cell death peaked (Wood et al., 1993). However, we found no
changes (data not shown). In addition, there were very few differences
in the number of pyknotic cells at all stages examined. Because the
cerebellar abnormality manifests as early as E18.5, cell death probably
contributes less to the Zic1/ cerebellar phenotype.
We therefore considered whether the proliferative capacity of the EGL
is altered in the Zic1/ cerebella. To answer this question, the proliferating cell number in the cerebellar anlage was
examined by BrdU labeling (Fig. 7). We
counted the BrdU-labeled cells in an entire EGL. As expected, there was
a significant reduction of BrdU-labeled cell number in the EGL region.
This reduction was apparent from the beginning of EGL
forma- tion (E14.5) to birth (Fig. 7A). We also measured the
extent of the EGL in the anterior to posterior direction during the
same period (Fig. 7B). The Zic1+/+ EGL was longer
than that of Zic1/, starting from E15.5, with the
difference gradually becoming larger. When we divided the BrdU-labeled
cell number by the EGL length, it became clear that there was still a
consistent reduction of BrdU-labeled cell number in
Zic1/ cerebella (p < 0.001, paired t test) (Fig. 7C). These results indicate
that cell proliferation activity is lower in the Zic1/
EGL than in the Zic1+/+ EGL from E14.5 through E18.5.
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Figure 7.
BrdU labeling study of developing cerebella.
A, BrdU-labeled cells in the EGL, on sections of the
cerebellar sections anlage, were counted. B, Change in
EGL length in the developing cerebella. Rostral to caudal lengths of
the EGL were measured at each developmental stage. In A
and B, all data for Zic1/ are
significantly different from those for Zic1+/+ cerebella
(p < 0.001, t test).
C, BrdU-labeled cells are compared in a lengthwise
portion. Open and closed bars (circles) indicate the Zic1+/+ and
Zic1/ cerebella, respectively. From E14 through E18,
the data for Zic1/ cerebella are significantly different from those for Zic1+/+ cerebella
(p < 0.001, paired t test).
The data are presented as means. Error bars indicate SEM.
*p < 0.01; **p < 0.001.
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The reduction in BrdU-labeled cells in the E14.5 cerebellar anlage was
the earliest sign of the cerebellar abnormality in Zic1/
mice, although Zic1+/+ and Zic1/ cerebella
were indistinguishable at this time point in HE sections. These
findings suggest that Zic1 may promote cell proliferation in
the EGL in wild-type cerebella. However, the difference might be traced
back to an earlier stage. For example, there may be different numbers
of initial progenitors or different proliferation start times. Further
studies will address these points.
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DISCUSSION |
Role of the Zic1 gene in cerebellar granule
cell development
The present study showed that one role of the Zic1 gene
is regulation of cerebellar development. Zic1 may regulate
granule cell proliferation throughout periods of germinal cell
expansion in the EGL.
EGL cells only give rise to cerebellar granule cells (Gao and Hatten,
1994; Zhang and Goldman, 1996). Embryonic precursor cells from the
rhombic lip are specific to cerebellar granule neurons (Alder et al.,
1996). Therefore, cells in the EGL may represent the single source of
granule cells. As a consequence, the number of granule cells is
determined by the extent of cellular proliferation in the EGL as well
as by cell death in later stages (Wood et al., 1993). The involvement
of cell death in reducing the cell number in Zic1/ mice
is probably minimal, because the abnormal pattern was already defined
at birth, and TUNEL staining showed that apoptotic cell death occurred
equally in Zic1/ and Zic1+/+ mice at E17 and
P7. The role of Zic1 in cerebellar development therefore
probably involves promotion of cellular proliferation of granule cell
precursors.
In our previous study, we showed that Zic1 is persistently
expressed during granule cell development (Aruga et al., 1994). Zic1 expression in the rhombic lip, EGL, and mature granule
cells is abundant and highly restricted. Although other members of the Zic family are also expressed in the adult cerebellum, their
levels of expression are considerably lower than that of
Zic1 (Aruga et al., 1996a,b). The phenotypes observed in
this study appear to correspond closely with the persistent expression
of Zic1 in the granule cell precursor, and with
Zic1 being the predominantly expressed member of the
Zic family. However, the role of the Zic1 gene in
mature cerebella is not clear as yet, given that we found no evidence
of abnormalities in granule cell differentiation during synapse
formation or in marker expression. Further study, including physiological characterization, is necessary to resolve this issue.
Human Zic1 is also expressed in the cerebellar granule cell
lineage in a highly restricted manner (Yokota et al., 1996). In addition, among various human tumors, the gene is predominantly expressed in medulloblastoma. Therefore, the expression of
Zic1 is compatible with cells in a proliferative state,
supporting the observations made in this study. It would be interesting
to examine whether Zic1 is structurally altered in
medulloblastoma.
Role of the Zic1 gene in cerebellar patterning
Zic1/ cerebella lacked a lobule rostral to the
primary fissure. This abnormality may be relevant to the behavioral
abnormalities, because afferents from the hindlimbs, trunk, and
forelimbs have abundant terminations in the anterior lobe (Oscarsson,
1973), in which mass reduction was most severe in the
Zic1/ mice. Abnormal foliation was consistently observed
in the Zic1/ cerebella, suggesting that Zic1
has an essential role in determining the cerebellar foliation pattern.
Therefore, we speculated on how Zic1 may be involved in
cerebellar patterning.
First, the phenotypes may be produced by the reduction of forward
movement of granule cell precursors. The forward movement of the
granule cell precursors may in part be driven by cell proliferation in
the EGL. The majority of forward-moving cells have a rounded or
irregular shape, rather than the elongated spindle shape of streaming
cells, with the long axes of the cells oriented in the direction of
dispersal, as is typical of actively migrating cells (Altman and Bayer,
1996). Therefore, insufficient cell proliferation in the EGL might
account, in part, for the abnormal foliation pattern. Consistent with
this, we showed the forward extension of the EGL and that cell
proliferation is reduced in Zic1/ cerebella.
Second, foliation of the cerebella may result from the postnatal
expansion of the cells in the EGL and differences in germinative activities between areas where fissures will form and areas destined to
be folial convexities (Mares and Lodin, 1970). Destruction of
proliferating cells in the rat EGL by x-ray irradiation from birth
disturbs the formation of folial patterns (Altman and Anderson, 1972).
In addition, EGL agenesis leads to the loss of foliation in
Math1 mutant mice (N. Ben-Arie and H. Zoghbi, personal
communication). These findings indicate that the germinative activity
of the EGL is essential in cerebellar foliation.
However, the expression of Zic1 in the EGL is uniform, based
on in situ hybridization results. We must therefore consider a third possibility; that Zic1 may cooperate with other
spatially restricted factors. The present studies, as well as analyses
of development of En2/ hemisphere foliation (Millen et
al., 1994), indicate that the basic plan of foliation is determined
before birth, as early as E17.5. Similarly, Millen et al. (1995)
reported that homologs of the Drosophila segment polarity
genes En2, En1, Wnt-7B, Pax-2, and Gli are
expressed in perinatal cerebella in spatially restricted manners. In
addition, several genes are differentially expressed along the anterior
to posterior axis in developing cerebella (Herrup and Kuemerle, 1997).
It is possible that Zic1 interacts with a spatially
restricted gene to define a uniform folial pattern.
A combination of these processes is also possible. First, a spatially
restricted factor may define the sites of major fissures, possibly from
the caudal end of the cerebellar anlage. Then, Zic1 defines
the anterior extent of the EGL. Finally, differential germinative
activity in the EGL results in the formation of folia.
Comparisons with other genetic cerebellar folial abnormalities
The abnormal foliation observed in this study is clearly different
from other genetically defined abnormal cerebellar foliation patterns,
such as Wnt1, Swaying, meander tail, leaner, and
cerebellar foliation pattern. (Herrup and Wilczynski, 1982;
McMahon and Bradley, 1990; Neumann et al., 1990; Thomas and Capecchi,
1990; Fletcher et al., 1991; Thomas et al., 1991). However, the
abnormal foliation patterns in En2 mutant mice (Joyner et
al., 1991) are somewhat similar to that of Zic1/ mice.
In En2/ cerebella, reductions in the extents of crus I
and crus II folia and the paraflocculus lobule were observed, as in
Zic1 homozygotes (Joyner et al., 1991; Millen et al., 1994).
En2 is also expressed in granule cells and their precursors
(Davis and Joyner, 1988). These findings suggest a possible genetic
interaction between Zic1 and En2 as is the case
in their Drosophila homologs odd-paired and
engrailed (Benedyk et al., 1994). Although in
situ hybridization showed that the expression of En2 in
the adult was not changed in this manner, it would be interesting to
analyze further the pattern of En2 gene expression in
Zic1/ embryos as well as the cerebellar phenotypes of
Zic1 and En2 compound mutants.
Possible interactions with other Zic genes
The abnormalities found in the Zic1/ mice
are grossly restricted in later development of the CNS and somite
derivatives. Both tissues express Zic1 (Aruga et al., 1994;
Nagai et al., 1997). However, even in cerebella, Zic2 and
Zic3 are expressed in addition to the Zic1 gene
(Aruga et al., 1996a). As shown in this study, Zic2, which
is the most similar Zic family gene to Zic1, is
still expressed in mature granule cells as well as in the developing granule cell lineage of Zic1/ mice (J. Aruga,
unpublished data), indicating that the expression of Zic2 is
independent of that of Zic1. The role of the Zic
family in cerebellar development may be more clearly defined by
analyses of compound Zic family mutants.
Although Zic1 expression begins at an earlier stage of
development, i.e., almost simultaneous with neuroectoderm formation (Nagai et al., 1997), no abnormalities were observed in the
Zic1 mutants at this stage. We found that a
Xenopus homolog of the Zic gene has a role in
neuroectoderm formation (Nakata et al., 1997). The absence of overt
abnormalities in early stage Zic1/ embryos may be
explained by functional redundancy among members of the Zic
family. The expression patterns of Zic1, Zic2,
and Zic3 overlap considerably during gestation as well
(Nagai et al., 1997). Furthermore, the amino acid sequence is highly
conserved among Zic family members (Aruga et al., 1996a). Therefore, it is possible that, to some extent, Zic genes have similar
roles.
Besides the interaction with other Zic genes, the
relationships between Zic1 and other genes, such as those of
the Gli, Pax, and En families, await
further clarification. The production of compound mutants involving
mutations of these genes might further clarify the role of the
Zic1 gene in mammalian development.
| |
FOOTNOTES |
Received July 25, 1997; revised Oct. 10, 1997; accepted Oct. 15, 1997.
This work was supported by special coordination funds for promoting
science and technology, Grants from the Japanese Ministry of Education,
Science, and Culture to J.A. and K.M., a Core Research for Evolutional
Science and Technology grant from the of Japan Science and Technology
Cooperation to J.A., and Japanese Brain Science Foundation funds
awarded to J.A. We thank Drs. Hidehiro Mizusawa, Shigeki Yuasa, Toshio
Terashima, Katsunori Nakata, Toshiyuki Nakagawa, Hitoshi Niwa, Ken-ichi
Yamamura, and Masahiko Watanabe for their helpful advice, Drs. Nissim
Ben-Arie and Huda Zoghbi for sharing their results before publication,
Dr. Paul Greengard for providing one of the antibodies, Dr. Takeshi
Yagi for plasmids, and Ms. Qing Nie, Naoko Fujimoto, Yoko Nishi, Noriko
Sugae, and Megumi Yakuwa for technical assistance.
Correspondence should be addressed to Jun Aruga, Molecular Neurobiology
Laboratory, Tsukuba Life Science Center, Institute of Physical and
Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan.
| |
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Top
Abstract
Introduction
