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The Journal of Neuroscience, October 1, 2002, 22(19):8563-8573
Frizzled-3 Is Required for the Development of
Major Fiber Tracts in the Rostral CNS
Yanshu
Wang1, 4,
Nupur
Thekdi1,
Philip M.
Smallwood1, 4,
Jennifer P.
Macke1, 4, and
Jeremy
Nathans1, 2, 3, 4
1 Departments of Molecular Biology and Genetics,
2 Neuroscience, and 3 Ophthalmology,
4 Howard Hughes Medical Institute, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
Many ligand/receptor families are known to contribute to axonal
growth and targeting. Thus far, there have been no reports implicating
Wnts and Frizzleds in this process, despite their large numbers and
widespread expression within the CNS. In this study, we show that
targeted deletion of the mouse fz3 gene leads to severe
defects in several major axon tracts within the forebrain. In
particular, fz3( /) mice show a complete loss of the
thalamocortical, corticothalamic, and nigrostriatal tracts and of the
anterior commissure, and they show a variable loss of the corpus
callosum. Peripheral nerve fibers and major axon tracts in the more
caudal regions of the CNS are mostly or completely unaffected. Cell
proliferation in the ventricular zone and cell migration to the
developing cortex proceed normally until at least embryonic day 14. Extensive cell death in the fz3(/) striatum occurs
late in gestation, perhaps secondary to the nearly complete absence of
long-range connections. In contrast, there is little cell death, as
assayed by terminal deoxynucleotidyl transferase-mediated biotinylated
UTP nick end labeling, in the cortex. These data provide the first link
between Frizzled signaling and axonal development.
Key words:
axonal growth; fiber tracts; frizzled; mouse brain
development; forebrain; Wnt signaling
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INTRODUCTION |
The founding member of the Frizzled
(Fz) family of transmembrane proteins was identified as the product of
a Drosophila gene required for the correct orientation of
cuticular hairs (trichomes) and bristles (Gubb and Garcia-Bellido,
1982 ; Vinson et al., 1989). In wild-type flies, these cuticular
structures have a definite and highly stereotyped orientation with
respect to the body axes, a property referred to as tissue or planar
cell polarity. Based on their finding that Fz is an integral membrane
protein, Adler and colleagues suggested that it might be the receptor
for an extracellular ligand that communicates directional information (Vinson et al., 1989; Park et al., 1994). Consistent with this general
hypothesis, experiments in cell culture and in Drosophila embryos have demonstrated that Fz and a related receptor, Fz2, function
as receptors for Wingless, a member of the Wnt family of extracellular
signaling proteins (Bhanot et al., 1996, 1999; Bhat, 1998; Kennerdell
and Carthew, 1998; Chen and Struhl, 1999). Ligand-receptor
relationships between Wnts and Fzs have also been inferred from
coinjection experiments in Xenopus embryos (Yang-Snyder et
al., 1996; He et al., 1997; Deardorff et al., 2001). However, intracellular signaling in tissue polarity appears to be distinct from
Wingless/Fz signaling in the embryo because the former uses a rho
GTPase pathway (Strutt et al., 1997; Winter et al., 2001), whereas the
latter depends on the modulation of gene expression by
 -catenin-lymphocyte enhancer factor (LEF)/T-cell factor (TCF) (van de Wetering et al., 1997).
Current models of Wnt/Fz signaling envision any of three downstream
signaling pathways, depending on the biological or experimental context: (1) -catenin-LEF/TCF mediated transcriptional control, as
seen in segment polarity during Drosophila embryogenesis
(van de Wetering et al., 1997) and in mammalian cell
division/tumorogenesis (Morin et al., 1997), (2) rho- and rac-dependent
control of cytoskeletal dynamics as seen in Drosophila
tissue polarity (Strutt et al., 1997; Winter et al., 2001) and in
Xenopus gastrulation (Habas et al., 2001), and (3)
G-protein-coupled calcium mobilization, as observed in mammalian cell
culture and early zebrafish development (Kuhl et al., 2000). An
additional complexity, and one that presents a major challenge in
dissecting the role of individual Wnt and Fz family members in
vivo, arises from the partial overlap of function within these
families. For example, in Drosophila, Fz and Fz2 are almost
completely redundant in mediating Wingless action in the embryonic
cuticle, gut, CNS, and heart (Bhanot et al., 1999; Chen and Struhl,
1999), and in mice, Wnt1 and Wnt3a have mostly redundant roles in
promoting cell division in the developing neural tube and crest (Ikeya
et al., 1997). Considering that typical mammalian genomes code for at
least nine Fzs and 18 Wnts
(http://www.stanford.edu/~rnusse/wntwindow.html), many of which have
partially overlapping patterns of expression, it is likely that at
least partial redundancy will be the rule rather than the exception.
Among developmental processes, two of the most complex are the
specification of neurons within the CNS and the correct targeting of
their axons. The latter process bears some formal resemblance to the
establishment of tissue polarity in the Drosophila cuticle because it involves the sensing of directional cues and a local polarized response. A wide variety of ligands, including netrins, ephrins, semaphorins, slits, and neurotrophins, together with their
corresponding receptors, are now known to contribute to axonal growth
and targeting. However, there have thus far been no reports implicating
Wnts and Fzs in this process, despite their large numbers and
widespread expression within the CNS (Wang et al., 1996; Grove et
al., 1998; Borello, 1999). Instead, Wnts and/or Fzs have been
implicated in CNS development in the context of cell fate
specification, cell proliferation, cell survival, and synaptogenesis.
For example, in chick and Xenopus embryos, Wnt/Fz signaling
is involved in early decisions regarding neural crest development and
specification (Deardorff et al., 2001; Kiecker and Niehrs 2001; Wilson
et al., 2001), and, in the mouse, Wnt1 plays a critical role in the
early stages of cerebellar development (McMahon and Bradley, 1990;
Thomas and Capecchi, 1990), Wnt3a and LEF/TEF are required for
cell proliferation in the developing hippocampus (Dickinson et al.,
1994; Galceran et al., 2000), Fz4 is required for cell survival in the
cerebellum (Wang et al., 2001), and Wnt7a plays a role in cerebellar
synaptogenesis (Lucas and Salinas, 1997; Hall et al., 2000).
In the present study, we report the phenotype of mice carrying a
targeted deletion of the fz3 gene. fz3(/)
mice exhibit massive defects in the development of fiber tracts within
the forebrain while leaving other aspects of CNS development mostly unaffected. These data provide the first link between Fz function and
axonal development.
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MATERIALS AND METHODS |
Generation of fz3(/) mice. The fz3
knock-in construct (see Fig. 1) was electroporated into R1 cells, and
colonies were grown in medium containing G418 and gancyclovir. Colonies
were picked 8 d after plating and screened by Southern blot
hybridization. Positive embryonic stem cell clones were injected
into C57BL/6 blastocysts.
Histochemistry. Throughout this study, the first day after
overnight mating is counted as embryonic day 0 (E0). Tissues for 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal),
NADPH diaphorase, and acetylcholine esterase staining were immersion fixed 2-6 hr at 4°C in PBS, 2.5% formalin, and 0.2%
gluteraldehyde; embedded in 3% agarose in PBS; and sectioned at 300 µm on a vibratome. Embryos younger than embryonic day E15 were fixed
intact; for later stage embryos, fixation proceeded with isolated heads
from which the skin had been removed. Before cutting vibratome
sections, later stage heads were incubated at 4°C for 5-7 d in 0.5×
PBS, 20 mM Na EDTA to decalcify the skull. For
X-gal staining of adult brains, perfusion with 2.5% formalin, 0.2%
gluteraldehyde, 2 mM MgCl2, and PBS
was followed by a 30 min room temperature incubation in the same
fixative before vibratome sectioning. Complete serial sections were
collected in the wells of 12-well tissue culture trays. X-gal staining
was performed for 24 hr at 37°C in standard X-gal staining solution
supplemented with 0.02% Tween 20. Acetylcholine esterase staining was
performed at room temperature with minor modifications of the method of
Karnovsky and Root (1964). The reaction mixture contained 0.1 M sodium phosphate, pH 6.0, 4 mM acetylcholine iodide, 5 mM sodium citrate, 3 mM
copper sulfate, 0.5 mM potassium ferricyanide,
and 100 µM tetraisopropylpyrophosphoramide. NADPH diaphorase staining was performed at 37°C for 1-3 hr in PBS
with 0.3% Triton X-100, 0.1 mM NADPH, and 0.25 mg/ml nitroblue-tetrazolium-chloride as described by Bancroft and
Stevens (1982).
Immunohistochemistry, bromodeoxyuridine, and terminal
deoxynucleotidyl transferase-mediated biotinylated UTP nick end
labeling. Immunostaining and terminal deoxynucleotidyl
transferase-mediated biotinylated UTP nick end labeling (TUNEL) were
performed on brains, whole heads, or various organs that were either
(1) fresh frozen, cryosectioned at 16 µm, and then postfixed in 4%
paraformaldehyde, PBS, or (2) immersion fixed in cold Carnoy's
fixative, dehydrated, embedded in paraffin, and cut at 8 µm. Reagents
were obtained from the following sources: anti-neurofilament
(anti-neurofilament M; Chemicon, Temecula, CA, or anti-neurofilament
200; Sigma, St. Louis, MO), anti-tyrosine hydroxylase (Chemicon),
anti-calretinin (Swant, Bellinzona, Switzerland), anti-dopamine
transporter (Chemicon), anti-GFAP and anti-MAP2 (Sigma),
anti-acetylated tubulin (TuJ1; a gift from Dr. Anthony Frankfurter,
University of Virginia, Charlottesville, VA),
anti-bromodeoxyuridine (BrdU) (Harlan Sera-lab, Loughborough, UK), and
fluorescein TUNEL reagents (Roche, Indianapolis, IN).
For BrdU labeling, pregnant females were given a single intraperitoneal
injection of 400 µl of 10 mM BrdU at the indicated gestational ages. Fetuses were recovered either 1 hr later (for pulse-labeling experiments) or at E18, immersion fixed in cold Carnoy's fixative for 6 hr at 4°C, dehydrated, and embedded in paraffin. Deparaffinized 8 µm sections were incubated in 4N HCl for
20 min, washed in PBS, blocked in 10% normal goat serum in 0.3%
Triton/PBS, and incubated in anti-BrdU antibody (1:10 in blocking
solution) overnight at 4°C. Sections were washed in PBS, incubated in
biotinylated goat anti-rat antibody (Vector Laboratories, Burlingame,
CA; 1:200) for 1 hr, followed by horseradish peroxidase-conjugated ExtrAvidin (Sigma, 1:100) for 30 min, and visualized with a standard diaminobenzidine/peroxidase reaction.
1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate tracing. For
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) tracing, intact E12-E15 embryos or embryonic day 18 heads in
which the dorsal aspect of the brain was exposed were fixed overnight
in PBS with fresh 4% paraformaldehyde. For E12-E15 embryos, a small
hole was made through the skull with a 30 gauge needle through which
DiI crystals were placed on and in the cortex. For E18 brains, DiI
crystals were placed on the exposed surface of the cortex or tectum
using a flame-drawn glass needle or on the cut surface of the striatum
after the most anterior aspect of the telencephalon had been cut away.
Tissues were incubated for 3-6 weeks in 0.5× PBS, 2%
paraformaldehyde, and 20 mM Na EDTA (for
decalcification), at either room temperature or 37°C, then embedded
in 3% agarose in PBS, and sectioned at 300 µm on a vibratome.
Electron microscopy. E18 brains were fixed overnight in PBS,
2% paraformaldehyde, 2% gluteraldehyde, and 1 mM MgCl2, embedded in 3% agarose in
PBS, and sectioned at 300 µm on a vibratome. Sections containing
cortex and striatum were osmicated, stained with uranyl acetate,
embedded in Unicryl resin (British Biotechnology, Inc., Cardiff,
UK), and cut with a glass knife.
Dissociated neuronal cultures. Freshly dissected E18
cortices or striata were digested in 0.5 mg/ml papain, 0.5 mM EDTA, and 0.13 mg/ml
L-cysteine in Earl's Balanced Salt Solution
(EBSS) at 37°C for 30 min, and then transferred to 1 mg/ml BSA, 1 mg/ml trypsin inhibitor, and 10 µg/ml DNaseI in EBSS, and gently
triturated. Cell suspensions were layered on a shelf of 10 mg/ml BSA
and 10 mg/ml trypsin inhibitor in EBSS and pelleted. Cell pellets were washed and resuspended in 10% fetal calf serum, 5% horse serum, 200 µM L-cysteine, and 2 mM glutamine in Minimum Essential Medium. Cells
were plated on poly-L-ornithine-coated coverslips
at 5 × 105 cells per well in 24-well
plates, and the plating media was replaced with neurobasal media
supplemented with B27 16 hr later. Half of the culture media was
replaced with fresh media every other day. Four days after plating,
uridine and 5-fluoro-2'-deoxyuridine were added to the culture to
inhibit glial cell division.
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RESULTS |
fz3 expression assayed with a
lacZ knock-in
To examine the role of Fz3 in vivo, a targeted
disruption of the fz3 gene was constructed in which the
first 129 codons, present within the first two coding exons, were
replaced by a lacZ reporter, followed by a
protamine-1 intron and polyadenylation site (Fig. 1). The deleted region codes for the
N-terminal signal sequence and the conserved extracellular
cysteine-rich domain; the latter has been shown to mediate Wnt binding
in a variety of Fz proteins (Bhanot et al., 1996; Hsieh et al., 1999;
Dann et al., 2001). The targeted allele is therefore presumed to be a
null.
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Figure 1.
Targeted replacement of the first coding exon of
fz3 by lacZ. Top, Partial
map of the murine fz3 locus. The first three
fz3 coding exons are indicated by filled
rectangles. B, BamHI;
Bg, BglII; E,
EcoRI; H, HindIII;
N, NcoI; X,
XbaI. Parenthesis indicates elimination
of that restriction enzyme site. Bottom, Structure of
the fz3 targeting construct and Southern blot
hybridization probe. A flanking segment of 8.3 kb located immediately
5' of the initiator methionine codon of fz3 was joined
to the initiator methionine codon of a -galactosidase expression
cassette. The -galactosidase coding region is followed by an intron
and poly(A) site from the mouse protamine-1 gene
(lacZ-mp1) (Peschon et al., 1987) and by a
phosphoglycerate kinase-neo selectable marker,
both with the same orientation. The 5 kb 3' homology segment
encompasses the 5' 454 bp of the third coding exon and adjacent
upstream intron sequences and is followed by a thymidine
kinase-negative selectable marker (MC1-TK).
Right, Genotyping of fz3(+/+),
fz3(+/), and fz3(/) mice by
BamHI digestion and Southern blotting with the 5'
flanking BamHI-XbaI probe indicated at
left. The wild-type and gene-targeted alleles generate
fragments of 18 and 12 kb, respectively.
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The pattern of lacZ expression in developing
fz3(+/) and fz3(/) embryos and in the adult
fz3(+/) brain is in good agreement with existing in
situ hybridization and RNase protection data showing transcripts
predominantly or exclusively in the nervous system (Fig.
2) (Wang et al., 1996; Borello et al.,
1999). At midgestation, X-gal staining is seen throughout the
developing CNS (Fig. 2A), and at E18, it is seen in
the cortex, diencephalon (including the major thalamic nuclei), and
brainstem, with the most intense staining in the striatum and
trigeminal ganglia (Fig. 2B-D). Figure
2B-D shows the pattern of X-gal staining in a
fz3(/) head at E18; a nearly identical pattern is seen
in fz3(+/) heads but with reduced intensity. In the adult
fz3(+/) brain, X-gal staining is most intense in the
cortex, the diencephalon, and the rostral brainstem, and little or
no staining is seen in the striatum or cerebellum (Fig.
2E-H).
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Figure 2.
Pattern of fz3 expression as
determined by X-gal staining of the knocked-in lacZ
reporter in fz3(+/) and fz3(/)
mice. A, fz3(+/) embryos at E12 show
widespread X-gal staining in the developing CNS. B-D,
fz3(/) embryos at E18; coronal series from rostral
(B) to caudal (D).
B, Centered on the striatum; C, centered
on the thalamus; D, centered on the tectum. Widespread
X-gal staining is seen in the cerebral cortex, diencephalon, and
brainstem, with the highest levels in the developing striatum
(arrow at 45° angle in B) and the
trigeminal ganglia (vertical arrow in B).
Staining is also seen in the inner ear (vertical arrow
in C), dorsal and ventral thalamic nuclei (45°
arrows in C), tectum
(arrow in D), and retina (data not
shown). fz3(+/) embryos at E18 show a nearly identical
pattern of X-gal staining but at lower intensity. E-H,
Adult fz3(+/) brains show X-gal staining in the
cerebral cortex and select midbrain structures with minimal staining in
the striatum (arrow in E), cerebellum
(arrow in H), and brainstem.
Coronal series from rostral (E) to caudal
(H). E, Centered on the
striatum; F, centered on the thalamus; G,
centered on the colliculus; H, centered on the
cerebellum.
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Functional and anatomic defects in
fz3(/) mice
fz3(+/) mice are indistinguishable from their
fz3(+/+) littermates in viability, growth, appearance,
fertility, and the gross anatomic and histologic appearance of the
brain. Therefore, in the remainder of this study, we will present both
fz3(+/+) and fz3(+/) mice as interchangeable
representatives of the wild-type phenotype. fz3(/)
neonates have a curly tail [a sensitive indicator of neural tube
defects (Peeters et al., 1998)] and flexed lower limbs (Fig.
3A,B),
and they breathe irregularly and typically die within 30 min of birth.
fz3(/) neonates show a normal withdrawal reflex when
pinched, but unlike their fz3(+/) and fz3(+/+)
littermates, they do not vocalize in response to the stimulus.
fz3(/) mice do not appear to be lost during gestation,
because a survey of 122 E18 embryos from 15 consecutive litters derived
from fz3(+/) parents revealed
fz3(+/+):fz3(+/):fz3(/) progeny
in a ratio of 37:54:31, close to the expected Mendelian ratio of 1:2:1.
This analysis also revealed a perfect correlation between the
fz3(/) genotype and the curled tail phenotype and
between the fz3(+/+) and fz3(+/) genotypes and
the straight tail phenotype.
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Figure 3.
Gross anatomic anomalies in
fz3(/) mice. A, B,
Newborn fz3(/) mice have a curled tail and flexed
hindlimbs. C-H, E18 fz3(/) mice have
enlarged lateral ventricles, a thinned cerebral cortex, and a smaller
striatum but essentially normal patterns of NADPH diaphorase
(C-F) and acetylcholine esterase
(G, H) activity in the CNS.
Arrows in C, D,
G, and H point to the higher density of
stained cells in the lateral striatum. I,
J, fz3(+/) and
fz3(/) littermates at E18 from a litter in which 2/2
fz3(/) fetuses have an open cephalic neural
tube.
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At present, the pathogenic mechanism of the fz3(/)
breathing defect is unknown. fz3(/) neonates appear
cyanotic in room air but become as well oxygenated as their wild-type
littermates when placed in 100% oxygen. When the lungs of oxygenated
fz3(/) animals are examined several hours after birth,
they are typically found to be partially inflated, and in those regions
which are inflated, the fz3(/) alveoli and bronchi look
microscopically normal. Whole-mount acetylcholine esterase staining
shows no abnormalities of the intercostal muscles or their innervation.
The characteristically irregular breathing together with the finding of
gross neuroanatomic defects in the cortex and striatum (described
below) suggest that there may be a defect in the brainstem control of
respiration (Richter and Spyer, 2001), although we have not observed
histologic abnormalities in this region of the CNS.
In the mixed 129/SVJ × C57BL/6 background of the
fz3(+/) line, there is a failure of cephalic neural tube
closure in a small percentage of fz3(/) embryos but not
in fz3(+/) or fz3(+/+) littermates (Fig.
3I,J). This phenotype most
likely arises from the effect of genetic modifiers, because it occurs
at higher frequency in some crosses, as seen, for example, in the two
affected fz3(/) littermates in Figure 3J. In
one experiment aimed at examining possible interactions between
fz3 and Wnt-1, 3 of 44 E18 progeny obtained from
crossing fz3(+/);Wnt-1(+/) double
heterozygous parents exhibited a failure of cephalic neural tube
closure. Two of these three animals were
fz3(/);Wnt-1(+/+), and the third was
fz3(/);Wnt-1(+/) [out of a total of 11 fz3(/) progeny]. This small series supports a model in
which the cephalic closure phenotype requires both loss of
fz3 and one or more genetic modifiers, and it shows no
indication that this phenotype is enhanced by loss of
Wnt-1.
Defects in major fiber tracts in the
fz3(/) CNS
At E18, fz3(/) mice exhibit a single gross
neuroanatomic defect: a marked enlargement of the lateral ventricles
secondary to shrinkage of the striatum and thinning of the cortex
(Figs. 3C-H). A complete survey of acetylcholine
esterase and NADPH diaphorase histochemistry throughout the
fz3(/) brain at E18 revealed minimal differences from
the wild type (Figs. 3C-H). A developmental survey of brain structure using cresyl violet staining revealed little or no
difference between the wild-type and fz3(/) striatum and cortex at E13 (Fig.
4A,B,E,F).
By E15, the lateral ventricles of the fz3(/) brain are
enlarged, but the cortex still appears nearly normal (Fig.
4C,D,G,H).
However, by E18, the fz3(/) cortex is significantly
thinner than the wild-type cortex, mostly because of changes within the
ventricular, subventricular, and intermediate zones (Fig.
4I,J), and the
fz3(/) striatum is more compact and lacks the internal
capsule.
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Figure 4.
Morphology of the fz3(/)
cerebral cortex between E13 and E18. Cresyl violet-stained paraffin
sections at E13 (A, B, E,
F), E15 (C, D,
G, H), and E18 (I,
J). The fz3(/) cortex has a
normal or nearly normal thickness and lamination at E13
(E, F) and E15 (G,
H); by E18, the intermediate, subventricular, and
ventricular zones are reduced in thickness (I,
J). The fz3(/) lateral
ganglionic eminence (LGE) and medial ganglionic eminence
(MGE) are of normal size at E13 (A,
B) but are reduced in size by E15 (C,
D). CP, Cortical plate;
ML, marginal layer; PP, preplate;
SP, subplate; IZ, intermediate
zone; SV, subventricular zone; VZ,
ventricular zone.
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A survey of major fiber tracts in the E18 fz3(/) brain,
visualized by anti-neurofilament immunostaining and DiI tracing, revealed a complete absence of the anterior commissure, a nearly complete absence of major corticofugal and thalamocortical fibers, a
variable decrease in the size of the corpus callosum,
and a marked thinning or in some cases a
complete loss of the intermediate zone of
the developing cortex (Figs. 5, 6; Table
1); none of these defects were observed
in fz3(+/+) or fz3(+/) littermates. In
fz3(/) brains at E18, the hippocampus appears minimally
affected (Fig. 5I,J). DiI
placed in the cortex of fz3(+/+) and fz3(+/) brains at E18 efficiently diffuses along fibers in the intermediate zone of the cortex (Fig. 6I-L) and through the
internal capsule toward the thalamus (Fig.
6D-H,I-L), but DiI similarly placed in the
fz3(/) cortex shows mostly local diffusion (Fig.
6M-P). A similar result is obtained when DiI
crystals are placed in the caudal region of the striatum: in wild-type
brains, the internal capsule and adjacent intermediate zone of the
cortex are labeled on the ipsilateral side, and the anterior commissure
is labeled on the contralateral side (Fig. 6A),
whereas in fz3(/) brains, DiI labels only local
structures (Fig. 6B). An examination of well
separated cortical cells filled by application of microcrystals of DiI
to the cortical surface revealed a range of approximately normal
morphologies among cortical pyramidal cells (Fig. 6C).
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Figure 5.
Absence of major fiber tracts in the cortex and
striatum in fz3(/) brains. Anti-NF200 immunostaining
of horizontal sections of the forebrain at E15
(A, D) and at E18 (B,
E, dorsal region; C, F,
ventral region) and of coronal sections of the forebrain
(G, H) and midbrain
(I, J) regions. Corticothalamic
and thalamocortical fibers passing through the striatum are missing at
E15 (arrows in A and D). A
complete or nearly complete absence of callosal
(leftward arrows in B and
E), corticofugal and thalamocortical (rightward
arrows in B and E;
arrows in G and H),
anterior commissural (arrows in C and
F), and cortical (arrows in
I and J) fibers is apparent in
fz3(/) embryos at E18. The loss of fibers is most
prominent in the rostral cortex and striatum (G,
H) and is minimal in the hippocampus
(I, J).
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Figure 6.
DiI labeling of cortical, commissural, and tectal
fiber tracts at E18. A, DiI crystals placed in the
caudal region of the striatum of a fz3(+/) brain label
the external capsule and the genu of the corpus callosum in the
ipsilateral cortex and the anterior commissure in the contralateral
hemisphere (vertical arrows). B, In the
fz3(/) brain, there is little diffusion of the DiI
beyond the site of crystal placement. C, Individual
fz3(/) cortical neurons labeled with microcrystals
of DiI from the cortical surface (top) show normal
morphologies. D-L, Serial coronal sections through two
fz3(+/) brains in which DiI crystals were placed in
the dorsolateral (D) or lateral
(I) cortex. Prominent labeling is seen in
cortical fibers traversing the internal capsule (vertical
arrows in E-H and horizontal
arrows in J-L) and in the genu of the corpus
callosum (arrows at 45° angle in J-L).
M-P, Serial coronal sections through a
fz3(/) brain in which DiI crystals were placed in
the lateral cortex. The DiI has spread locally but has labeled only a
small number of fibers traversing the internal capsule
(arrow in N). Q-U,
Serial sagittal sections through an fz3(/) brainstem
in which DiI crystals were placed in the lateral tectum.
R-U, Efficient labeling of the tectospinal tract
(arrows in S-U) with midline
crossing in T, a labeling pattern indistinguishable from
that seen in fz3(+/+) or fz3(+/)
brains. Sections are 300 µm in thickness.
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The defects in major fiber tracts appear to be mostly confined to the
cortex and diencephalon, because neurofilament immunostaining within
the spinal cord and brainstem appears normal in fz3(/) mice at E18 (data not shown). DiI tracing of tectospinal fibers at E18
shows the normal decussation of fibers at the isthmus of the brain stem
with no apparent differences between fz3(/) mice and
their fz3(+/+) or fz3(+/) littermates (Fig.
6Q-U). Similarly, a normal pattern of cranial and
spinal nerves was observed in whole-mount E11 fz3(/)
embryos stained for neuron-specific tubulin with monoclonal antibody
(mAb) TuJ1 (Lee et al., 1990). At E12, fz3(/) and
wild-type spinal cords show indistinguishable patterns of
immunostaining with antibodies to Nkx2.2, HNF3-, Lim2, Isl1, and
neurofilament. Whether the loss of fz3 produces some subtle alteration in spinal cord development that is not evident from this
analysis, as seen for example in the Wnt1/Wnt3a double mutant (Muroyama
et al., 2002), remains to be determined. In the enteric nervous system,
E18 fz3(/) mice showed normal densities and morphologies
of enteric neurons, as visualized with NADPH diaphorase and
acetylcholine esterase staining.
To examine a single immunochemically defined fiber tract within the
affected region of the CNS, we immunostained the striatum for tyrosine
hydroxylase (TH) and the dopamine transporter, two well characterized
presynaptic markers for the dopaminergic nigrostriatal pathway. At E18,
the wild-type striatum shows robust immunostaining for both markers,
whereas the fz3(/) striatum is devoid of
immunoreactivity (Fig. 7A-D).
Importantly, dopaminergic neurons appear to be present in normal
numbers within the fz3(/) substantia nigra as revealed by TH immunostaining (Fig.
7E,F).
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Figure 7.
Dopaminergic neurons from the substantia nigra
fail to innervate the striatum in fz3(/) brains.
Tyrosine hydroxylase (A, B) and the
dopamine transporter (C, D), markers for
presynaptic nigrostriatal processes, are absent from the
fz3(/) striatum at embryonic day 18. E, F, Tyrosine hydroxylase staining of
cell bodies in the substantia nigra demonstrates normal numbers of
dopaminergic neurons in the fz3(/) brain at
embryonic day 18.
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To distinguish between an early failure of axonal outgrowth or
pathfinding versus a subsequent loss of axons from their targets, corticofugal and thalamocortical axons were visualized by DiI tracing
at E12, E13, and E15 (Sheth et al., 1998; Auladell et al., 2000) (Table
1), and axon tracts throughout the forebrain were visualized by
anti-neurofilament immunostaining at E13, E14, E15, and E16. DiI
tracing at E13 and E15 revealed a clear failure of thalamocortical and
corticothalamic tract formation in the fz3(/) brain
(Fig. 8 and data not shown). In contrast,
robust and indistinguishable DiI labeling of the developing trigeminal ganglia was observed in fz3(+/+), fz3(+/), and
fz3(/) preparations at E12, E13, and E15, presumably via
DiI labeling of fibers that innervate the scalp (Fig.
8F,L,R,X).
Anti-neurofilament staining at these early gestational ages similarly
reveals a massive failure in the development of the major forebrain
fiber tracts (Fig. 5A,D).
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Figure 8.
DiI tracing from the cortex at E13. Paired
bright-field (A-F) and fluorescent
(G-L) images of 300-µm-thick serial horizontal
sections of an E13 fz3(+/) head. M-X,
The analogous paired series for an E13 fz3(/) head.
Local spreading of DiI within the cortex and adjacent striatum and
labeling of the ipsilateral trigeminal ganglion (arrows
in F, L, R, and
X), presumably from DiI in the scalp, are seen in
both samples. However, DiI labeling of the thalamus is only seen in the
fz3(+/) brain (arrows in
C-E and I-K).
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The ultrastructural consequences of the forebrain fiber defects are
seen in Figure 9. In the wild-type
striatum at E18, densely packed clusters of axons course between groups
of striatal neurons, whereas in the fz3(/) striatum, few
if any axons are seen, and instead, numerous spaces separate the
resident cells (Fig. 9A,B). Similarly, in the intermediate zone of the wild-type cortex at E18,
large numbers of axons are separated by sparsely distributed cell
bodies, whereas in the fz3(/) intermediate zone, the
intercellular space is mostly devoid of axons or other cellular
material (Fig. 9C,D). The presence of lacunae
rather than fiber tracts within the fz3(/) striatum and
cortex suggests either that some axonal outgrowth occurred followed by
a subsequent loss or that there are pre-existing extracellular spaces
through which axons normally course.
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Figure 9.
Ultrastructural defects in the
fz3(/) striatum and cortex at E18. A,
B, A high density of synapses, dendritic processes, and
axons is seen in the fz3(+/+) striatum. These
structures are mostly missing in the fz3(/)
striatum, which show numerous spaces between neurons. C,
D, The subventricular region of the
fz3(+/+) cortex shows a dense packing of fibers and cell
bodies. In the corresponding region of the fz3(/)
cortex, fibers are mostly missing, and numerous spaces separate the
remaining cell bodies. Scale bars, 10 µm.
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Generation, differentiation, and survival of neurons in the
fz3(/) cortex and striatum
The diminution of striatal volume and thinning of the cortex in
fz3(/) mice at E18 could simply reflect a decrease in
tissue mass referable to the missing axons. It could also be caused in part by a decrease in the number of cortical or striatal cells as a
result of decreased proliferation and/or increased cell death. To
examine these possibilities, we analyzed cell proliferation by BrdU
pulse labeling for 1 hr at E12, E14, and E15, during the peak of
cortical and striatal neurogenesis (Bayer and Altmann, 1995), and also
at E18 (Fig. 10A-D).
In 8 µm sections, the number of BrdU-labeled cells in the
fz3(/) forebrain ventricular zone is larger than that
from the corresponding region from wild-type littermates by 0, 6, 12, and 18% at E12, E14, E15, and E18, respectively. These differences are
likely to be within the limits of experimental variability of the BrdU
method. Consistent with this interpretation, wild-type and
fz3(/) brains show virtually identical densities of
proliferating cells within the ventricular zone at E15 and E18 as
determined by immunostaining for proliferating cell nuclear antigen.
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Figure 10.
Normal proliferation and differentiation of
cortical neurons in fz3(/) brains.
A-D, One hour BrdU pulse labeling at E12
(A, B) or E14 (C,
D) shows nearly identical numbers of proliferating cells
in the ventricular zone of wild-type and fz3(/)
forebrains. E, F, Calretinin
immunoreactivity in the dorsal cortex at E15 reveals Cajal-Retzius
cells at the marginal zone (top) together with a subset
of neurons in the cortical plate. G, H,
Calretinin immunoreactivity in the medial forebrain at E14 reveals a
subset of neurons in the most superficial cortical layers (the lateral
ventricle is at the top left; the midline is at the
right). I, J, TuJ1
immunostaining at E14 shows widespread expression of brain-specific
tubulin in postmitotic neurons. K, L,
MAP2 immunostaining shows the differentiation and lamination of
cortical neurons at E14.
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Differentiation of cortical neurons was assessed by immunostaining for
MAP2, calretinin, and neuron-specific tubulin (with mAb TuJ1). At E13
and E14, anti-MAP2 shows indistinguishable patterns of immunostaining
in the wild-type and fz3(/) forebrain, revealing the
early lamination of the cortex (Fig. 10K-L). At E14,
uniform and intense TuJ1 immunostaining is seen in all regions outside of the ventricular zone (Fig.
10I,J). In the developing
rodent cortex, calretinin immunoreactivity marks Cajal-Retzius cells in
the marginal zone and a subset of neurons in the subplate (Fonseca et
al., 1995). At E14 and E15, wild-type and fz3(/)
forebrains show indistinguishable patterns of calretinin
immunoreactivity in both of these classes of cells, as well as in a
broad band of cortical cells adjacent to the midline (Fig.
10E-H). The indistinguishable patterns of
calretinin staining in the marginal zone persist through at least E17.
At E15 and E17, calretinin staining is also seen in axons in the wild
type but not the fz3(/) cortex.
Migration and layering of cortical neurons were examined by pulse
labeling with BrdU at E13 or E14, followed by histologic analysis at
E18. As seen in Figure
11A-H, wild-type and
fz3(/) cortex and striata at E18 exhibit similar numbers
and arrangements of BrdU-labeled cells. The principal differences
between the two appear to be a relative thinning of the intermediate
and subventricular zones of the fz3(/) cortex, as noted
earlier, and a greater packing density of labeled cells in the
fz3(/) striatum. In summary, cell proliferation and
differentiation in the forebrain are remarkably normal in
fz3(/) mice during a time window when axonogenesis is
severely disrupted.
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Figure 11.
Cortical migration and striatal cell death in
fz3(/) brains at E18. A-H, BrdU
labeling at E13 (A-D) or E14
(E-H); animals were killed at E18. The
density of striatal neurons and the density and layering pattern of
cortical neurons are similar between fz3(+/) and
fz3(/) brains. The fz3(/) cortex
shows a thinning of the genu of the corpus callosum and the
subventricular zone. I-L, TUNEL-labeled cells in 16 µm sections of striatum and cortex at E18. Red dots,
The border between the striatum (str) and cortex
(ctx). I, J, Typical
appearance of fz3(/) and fz3(+/)
sections; K, a fz3(/) section with an
unusually high density of TUNEL-positive cells. L, Mean
and SDs of TUNEL-positive cells per 16 µm section of striatum at E18,
counted from fz3(+/) (n = 24 sections) or fz3(/) (n = 28 sections) brains.
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Cell death in the developing brain was analyzed at E16 and E18 by
quantitating the number of TUNEL-positive nuclei (Gavrieli et al.,
1992). Although minimal labeling was observed in wild-type and
fz3(/) brains at E16, at E18 fz3(/)
brains showed a dramatic increase in the number of TUNEL-positive
nuclei in the striatum (Fig. 11I-L). In contrast,
TUNEL-positive cells were exceedingly rare in other brain regions,
including the cortex. It will be of interest to determine whether the
absence of striatal afferants or efferants secondarily triggers the
death of striatal neurons or whether their death results from some
other fz3-dependent process.
If the absence of major fiber tracts in vivo reflects an
intrinsic defect in the production, growth, or maintenance of axons, such a defect might be manifest by individual dissociated cells in
culture. To test this possibility, we harvested and dissociated cells
from either the cortex alone or from the striatum and pyriform cortex
at E18 and examined their survival and morphology over the ensuing
25 d in culture. Both wild-type and fz3(/) cultures show similar high levels of cell viability and similar ratios of
neurons to glia (identified by immunostaining for MAP2 and GFAP,
respectively) (Fig.
12A-D,K-N).
Moreover, no differences between wild-type and fz3(/)
cultures were observed in glial or neuronal morphologies or in the
density of either dendrites or axons, as determined by immunostaining
for MAP2 or neurofilaments (Fig.
12E,F), and by transfecting
green fluorescent protein (GFP) cDNA and subsequent
visualization of isolated GFP-expressing cells by immunostaining (data
not shown). Synapse formation in vitro also appears to be
unaffected by loss of fz3 as assessed by immunostaining for
synaptophysin (Fig. 12G,H) or bassoon, a
presynaptic cytoplasmic matrix protein found at both excitatory and
inhibitory synapses (Fig.
12I,J) (Dieck et al., 1998).
Thus, the axon tract defects in the fz3(/) brain appear
to reflect a defect in the interaction between axons and their
microenvironment in vivo rather than an intrinsic defect in
axonal outgrowth per se.
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Figure 12.
Normal numbers and morphologies of dissociated
neurons and glia cultured from E18 neocortex or combined E18 striatum
and pyriform cortex. Cultures were prepared from pooled
fz3(+/) and fz3(+/+) brains (designated
"+/") or from pooled fz3(/)
brains. A-J, Neocortex; K-N, combined
striatum and pyriform cortex. Immunostaining was performed after 3-25
d in culture. A, B, K,
L, Dendrites visualized by anti-MAP2 immunostaining.
C, D, M, N,
Glia visualized by anti-GFAP immunostaining. E,
F, Axons visualized with anti-neurofilament
immunostaining. G-J, Synapses visualized with
anti-synaptophysin (G, H) or
anti-bassoon (I, J)
antibodies.
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DISCUSSION |
In this study, we show that targeted deletion of the mouse
fz3 gene leads to severe defects in several major axon
tracts within the forebrain. In particular, fz3(/) mice
show a complete loss of the thalamocortical, corticothalamic, and
nigrostriatal tracts and of the anterior commissure, and they show a
variable loss of the corpus callosum. Axonal defects are apparent
beginning at E13. In contrast, peripheral nerve fibers and major axon
tracts in the more caudal regions of the CNS are mostly or completely unaffected. Cell proliferation in the ventricular zone and cell migration to the developing cortex proceed normally. Extensive cell
death in the fz3(/) striatum occurs late in gestation, perhaps secondary to the nearly complete absence of long-range connections. Curiously, the cortex exhibits little or no cell death, as
determined by histochemical detection of DNA fragmentation, despite a
nearly complete absence of connections to subcortical structures. The
fz3(/) axonal phenotype is in marked contrast to the
phenotypes reported for mutations that affect axon guidance, in which
cases axonal outgrowth is readily observed, but the axons follow
erroneous trajectories (Bagri et al., 2002). As discussed more fully
below, it is also in marked contrast to the phenotypes described thus
far for Wnt mutants.
Models for Fz3 function
How might the absence of Fz3 cause defects in axon growth? One
clue comes from the observation that fz3(/) neurons
grown in culture exhibit grossly normal axonal and dendritic growth and
synaptogenesis. This observation suggests that the axonal growth
defects in the fz3(/) brain may involve inhibitory
interactions within the developing CNS that are relieved or bypassed
when neurons are grown at relatively low density in culture. For
example, axonal growth in the forebrain might require Fz3 to overcome
inhibitory or adhesive interactions with ECM components. If such a
mechanism were to apply generally within the CNS, then we would presume that in more caudal regions, it is mediated by other Fz family members
either alone or redundantly with Fz3.
Alternatively, Fz3 may function within developing neurons to coordinate
cell and cytoskeletal polarity in a manner analogous to the action of
Drosophila Fz in tissue polarity. In the developing wing
epithelium, Fz function is required to determine the location from
which each cell's single cuticular hair will emerge (Adler and Lee,
2001). In the wild type, the hair emerges from the tip of the cell that
is furthest from the thorax, and it grows away from the thorax (Wong
and Adler, 1993). The initial outgrowth of axons within the CNS
presents a conceptually similar, albeit vastly more complex, cell
biological challenge. If Fz3 functions at this point in neuronal
development, then the data presented here would imply that a failure to
correctly polarize the budding axon leads to abortive axonal outgrowth.
In the developing Drosophila wing epithelium, Fz appears to
localize within each cell to the point from which the single hair emerges (Strutt, 2001). [We note that this observation was made with
an overexpressed Fz-GFP fusion protein; the results are presumed to
hold for the endogenous Fz protein, the low abundance of which has thus
far hindered its immunolocalization (Park et al., 1994).] This
localization, together with genetic evidence that rac and rho act downstream of fz in the tissue polarity
pathway (Strutt et al., 1997; Winter et al., 2001), suggest that in
this developmental context, Fz acts locally to organize the
cytoskeleton. Therefore, determining the subcellular localization of
Fz3 within developing neurons and, in particular, determining whether
it is uniformly distributed over the plasma membrane or clustered
within a subdomain, although technically challenging given the low
abundance of the Fz3 protein, might provide a significant clue to its
action in the CNS.
The best characterized role for Wnts in the CNS, and, by inference, a
role for Fzs, is in cell proliferation. Loss of Wnt3a or LEF/TCF
function produces a massive failure to generate cells within the
developing hippocampus (Galceran et al., 2000; Lee et al., 2000). The
partial or complete absence of the cerebellum seen in
Wnt1(/) mice (McMahon and Bradley, 1990; Thomas and Capecchi, 1990) may represent an analogous failure of cell
proliferation within the midbrain. In support of this general idea,
Wnt1/Wnt3a double mutant embryos show a defect in cell proliferation
among dorsal neural crest precursors (Ikeya et al., 1997), and ectopic expression of Wnt-1 in the developing spinal cord causes a local increase in cell proliferation (Dickinson et al., 1994). In
contrast, the normal or nearly normal pattern of cell proliferation
seen in the fz3(/) CNS argues against mechanisms common
to those implicated for the Lef-1, Wnt-1, and
Wnt-3a mutants. Similarly, the late demise of postmitotic
cerebellar granule and Purkinje cells reported in fz4(/)
mice does not fit into the paradigm of a cell proliferation defect
(Wang et al., 2001).
As the above discussion indicates, it is unclear which one or more of
the three characterized Fz signaling pathways is used by Fz3. Indeed,
this remains an open question for all of the mammalian Fz proteins. As
noted in the introductory remarks, the precedent from studies of
Drosophila Fz action in the embryo and wing would suggest
that the answer is likely to be specific to the biological context.
Diversity and redundancy in Fz3 function
Studies in Xenopus using expression of wild-type and
dominant negative Xfz3 mutants suggest that, in the
Xenopus embryo, XFz3 functions in the specification of the
developing eye fields and, in conjunction with XWnt1, in the
specification of neural crest (Shi et al., 1998; Deardorff et al.,
2001; Rasmussen et al., 2001). The absence of effects on either of
these processes in fz3(/) mice suggests that the
phenotypes we report here may represent only a small fraction of the
functions normally mediated by Fz3, the others being covered in the
mouse by the redundant action of one or more of the remaining eight Fz
proteins encoded in the genome. This level of redundancy among
mammalian Fzs is certainly plausible given the nearly complete
redundancy shown by Drosophila Fz and Fz2 in mediating a
variety of Wingless actions in embryonic development (Bhat, 1998;
Kennerdell and Carthew, 1998; Bhanot et al., 1999; Chen and Struhl,
1999). Thus, one challenge for the future will be to dissect the
multiple layers of redundancy to fully define the role of each Fz in
CNS development.
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FOOTNOTES |
Received March 13, 2002; revised July 5, 2002; accepted July 22, 2002.
This work was supported by the Howard Hughes Medical Institute (Y.W.,
P.M.S., J.N.) and the Medical Scientist Training Program (N.T.). We
thank the following: Drs. Pradeep Bhide, Anirvan Ghosh, Lori Redmond,
Hengye Man, and Lin Ding for advice; the Johns Hopkins Transgenic Core for blastocyst injections; Carol Cooke for assistance with electron microscopy; and Drs. Anirvan Ghosh, Alex Kolodkin, Tudor
Badea, and Hui Sun for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Jeremy Nathans, 805 PCTB, 725 North Wolfe Street, Johns Hopkins University School of Medicine,
Baltimore, MD 21205. E-mail: jnathans{at}jhmi.edu.
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TOP
ABSTRACT
INTRODUCTION
