 |
 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 2001, 21(13):4761-4771
Progressive Cerebellar, Auditory, and Esophageal Dysfunction
Caused by Targeted Disruption of the
frizzled-4 Gene
Yanshu
Wang1, 6,
David
Huso2,
Hugh
Cahill3, 4,
David
Ryugo3, 4, and
Jeremy
Nathans1, 4, 5, 6
1 Department of Molecular Biology and Genetics,
2 Division of Comparative Medicine,
3 Department of Otolaryngology-Head and Neck Surgery,
4 Department of Neuroscience, and
5 Department of Ophthalmology, 6 Howard Hughes
Medical Institute, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
 |
ABSTRACT |
Wnt signaling has been implicated in the control of cell
proliferation and in synapse formation during neural development, and
these actions are presumed to be mediated by
frizzled receptors. In this paper
we report the phenotype of mice carrying a targeted deletion of the
frizzled-4 (fz4) gene.
fz4( /) mice exhibit three distinct defects: (1)
progressive cerebellar degeneration associated with severe ataxia, (2)
absence of a skeletal muscle sheath around the lower esophagus
associated with progressive esophageal distension and dysfunction, and
(3) progressive deafness caused by a defect in the peripheral auditory
system unaccompanied by loss of hair cells or other auditory neurons.
As assayed using a lacZ knock-in reporter,
fz4 is widely expressed within the CNS. In particular, fz4 is expressed in cerebellar Purkinje cells,
esophageal skeletal muscle, and cochlear inner hair cells, and the
absence of Fz4 in these cells is presumed to account for the
fz4(/) phenotype. In contrast to the early cell
proliferation and patterning effects classically ascribed to Wnts, the
auditory and cerebellar phenotypes of fz4(/) mice
implicate Frizzled signaling in maintaining the viability and integrity
of the nervous system in later life.
Key words:
frizzled-4; cerebellar degeneration; Purkinje
cells; Wnt signaling; esophagus; progressive hearing loss
| |
INTRODUCTION |
Members of the Wnt family of
secreted signaling proteins are found throughout the animal kingdom and
play important roles in the control of tissue patterning, cell fate,
and cell proliferation (for review, see Cadigan and Nusse, 1997 ). Loss
of function mutations in Wnt genes have been extensively characterized
in Drosophila, Caenorhabditis elegans, and mice,
and these mutations demonstrate that a single Wnt can have multiple and
diverse functions in different developmental contexts. For example,
Drosophila Wingless (Wg), the most extensively studied Wnt
protein, plays an essential role in embryogenesis to maintain segment
polarity in the epidermis, define the identities of a subset of
developing neuroblasts, and pattern the midgut and heart; in imaginal
disc development, Wg acts to pattern the wings, legs, and eyes (for
review, see Klingensmith and Nusse, 1994). Gain of function mutations
that lead to Wnt overexpression have also been characterized in
Drosophila and mice. For example, overexpression of Wnt-1 or
Wnt-3a in mice, first identified as a consequence of retroviral
insertion, causes hyperproliferation and oncogenic transformation
(Nusse and Varmus, 1982; Roelink et al., 1990). Consistent with this
observation, recent work has implicated mutational activation of
downstream effectors of Wnt signaling in the pathogenesis of various
human cancers (Morin et al., 1997; Chan et al., 1999; Satoh et al., 2000). In general, the range of Wnt action is limited to the immediate vicinity of the site of synthesis and secretion, presumably as a
consequence of the tight binding of Wnts to extracellular matrix molecules.
A role for Wnt signaling in the development of the vertebrate CNS has
emerged from observations with knock-out mice: mutations in
Wnt-1 lead to a failure of midbrain development and a
partial or complete failure of cerebellar development (McMahon and
Bradley, 1990; Thomas and Capecchi, 1990), and mutations in
Wnt-3a (Lee et al., 2000) or Lef1 (Galceran et
al., 2000), a downstream effector of Wnt signaling, block the normal
expansion of hippocampal precursors. Moreover, the phenotype of
Wnt-1/Wnt-3a double knock-out mice shows that
these two Wnts act redundantly to expand the number of dorsal neural
crest precursors along the length of the spinal cord (Ikeya et al.,
1997). Consistent with these loss-of-function phenotypes, ectopic
expression of Wnt-1 in the developing spinal cord causes a
local increase in cell proliferation (Dickinson et al., 1994). These
observations indicate that one role of Wnts in CNS development involves
the stimulation of cell proliferation.
Wnts may also play a role in CNS synaptogenesis as indicated by the
recent demonstration that Wnt-7a stimulates axonal growth and
synaptogenesis by developing cerebellar mossy fibers as they find their
granule cell targets (Lucas and Salinas, 1997; Hall et al., 2000).
Interestingly, the defect in mossy fiber differentiation in
Wnt-7a knock-out mice is partial and transient, suggesting that other Wnts expressed in the cerebellum may largely compensate for
the loss of Wnt-7a. At present there are 18 known mammalian Wnt genes,
and many of them are expressed in the CNS (Salinas and Nusse, 1992;
Parr et al., 1993; Grove et al., 1998; Lako et al., 1998), raising the
possibility that additional aspects of CNS development may be
controlled by Wnt signaling.
Wnt signals are transduced by the Frizzled family of cell surface
receptors, the members of which are also found throughout the animal
kingdom. In mammals there are currently nine known Frizzled
genes, and for many of these there are highly conserved orthologs in
fish, birds, and amphibia. In vitro studies of
Wnt-Frizzled binding using soluble Wg or Xenopus Wnt-8
demonstrate that one Wnt can bind to any of a number of Frizzled
proteins, although the affinity of binding varies among different
ligand-receptor pairs (Hsieh et al., 1999; Rulifson et al., 2000).
Several experiments have begun to address the biological consequences
of this complex pattern of ligand-receptor specificity. Injection of
Wnt and Frizzled RNAs into Xenopus embryos has demonstrated
a specific interaction between Wnt-5A and Frizzled-5 but not
between Wnt-5A and any of six other Frizzled receptors tested,
indicating a high degree of ligand-receptor specificity in this
experimental system (He et al., 1997). In contrast, during
Drosophila embryogenesis, Frizzled and Frizzled-2 appear to
play largely redundant roles as Wg receptors, as judged by the
development of nearly normal embryos when either receptor is absent,
and the recapitulation of the full wg null phenotype
when both are absent (Bhat, 1998; Kennerdell and Carthew, 1998; Bhanot
et al., 1999; Chen and Struhl, 1999).
In the vertebrate CNS, several Frizzled genes are expressed during
development and in the adult (Wang et al., 1996; Shi et al., 1998;
Borello et al., 1999; Wheeler and Hoppler, 1999), but their roles in
vertebrate nervous system development and function are largely
unexplored. In this paper we report the phenotype of fz4
knock-out mice.
| |
MATERIALS AND METHODS |
Generation of knock-in mice. The fz4
knock-in construct 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, and
positive ES clones were injected into C57/Bl6 blastocysts.
Auditory brainstem response. Auditory brainstem responses
were recorded from adult fz4(/) and fz4(+/+)
littermates without revealing the genotype to the experimenter. Mice
were anesthetized with intraperitoneal injections of a 3-5 mg/kg
mixture of xylazine hydrochloride (2.5 mg/ml) and ketamine (25 mg/ml).
A subcutaneous injection of 1% lidocaine served to keep the animals
quiet and helped reduce muscle noise during signal averaging. The
absence of a withdrawal reflex to sharp paw pinches signaled adequate anesthesia. Electrodes were placed at the vertex of the skull, in the
dorsum of the neck, and in the postauricular region of the ear closest
to the speaker. Free field clicks (n = 1000) of 100 µsec duration were presented in 5 dB increments, starting at 5 dB and
progressing to 100 dB sound pressure level (SPL). At each intensity
level, recordings were collected for 20 msec and then averaged.
Immunohistochemistry and terminal deoxynucleotidyl
transferase-mediated biotinylated dUTP nick end labeling.
Immunostaining and terminal deoxynucleotidyl transferase-mediated
biotinylated dUTP nick end labeling (TUNEL) were performed on brain
sections that were fresh frozen, cryosectioned at 16 µm, and then
post-fixed in 4% paraformaldehyde in PBS, except for calbindin
immunostaining, which was performed with perfused tissues that were
cryosectioned at 16 µm. Immunostaining of the esophagus and lower
gastrointestinal (GI) tract was performed with tissue perfused
with 4% paraformaldehyde and cryosectioned at 10 µm. Reagents were
obtained from the following sources: anti-calbindin (Chemicon,
Temecula, CA), anti-type 1 IP3 receptor (Affinity BioReagents, Golden,
CO), anti-skeletal muscle myosin (Sigma, St. Louis, MO), anti-glial
fibrillary acidic protein (GFAP) (Dako, Carpinteria, CA),
anti-CGRP (Peninsula Laboratories, Belmont, CA), and fluorescein
TUNEL reagents (Roche, Indianapolis, IN).
Histochemistry. Unfixed leg muscles were snap-frozen in
isopentane and cryosectioned at 10 µm. Whole mounts of esophagus, stomach, and intestine were prepared by dissecting fresh tissues followed by fixation in fresh 4% paraformaldehyde in PBS.
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. NADH diaphorase staining was performed at room temperature for 30 min in 1 mM NADH, 0.2 M
Tris, pH 7.4, and 1 mg/ml nitro blue tetrazolium (NBT), as described in
Bancroft and Stevens (1982). 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 NBT, as described in
Bancroft and Stevens (1982). For semithin sections of the esophagus,
isolated esophagi were fixed in 2% paraformaldehyde, 2%
gluteraldehyde, in PBS with 1.5 mM
MgCl2 and then post-fixed in osmium tetroxide,
embedded in Epon, sectioned at 1 µm, and stained with toluidine blue.
For X-gal staining of temporal bones, esophagus, and embryos, or
vibratome sections of fresh brain, tissues were fixed for 5-10 min at
room temperature in 2.5% paraformaldehyde, 0.2% gluteraldehyde in PBS
with 2 mM MgCl2 before incubation in
X-gal overnight at 37°C. For inner ear staining, after the X-gal
reaction temporal bones were decalcified for 2 weeks in 0.25 M EDTA, 15% sucrose in PBS, equilibrated with OCT
over 2 d, and cryosectioned. Retinas were X-gal stained after
perfusion with 2.5% paraformaldehyde, 0.2% gluteraldehyde in PBS with
2 mM MgCl2, and cryosectioning of the eye.
The Sevier-Munger silver staining method, which highlights cerebellar
basket cells and nerve fibers, was performed on deparaffinized sections
of the CNS as described (Sevier and Munger, 1965; Luna, 1992). The
development step was timed by visual inspection to maximize specificity
and contrast before sections were rinsed, cleared, and mounted.
| |
RESULTS |
Expression pattern of a fz4-lacZ
knock-in allele
To investigate the function of fz4 in vivo,
we constructed a targeted deletion of all but the last 70 nucleotides
of the fz4 coding region (Fig.
1). The targeting construct also
incorporates a lacZ reporter beginning at the initiator
methionine of fz4. As an initial guide to the analysis of
the fz4(/) phenotype, we surveyed the pattern of
fz4 gene expression by assessing X-gal staining using the
fz4-lacZ knock-in allele. In the midgestation embryo, the fz4-lacZ reporter is expressed
widely (data not shown). In the adult, X-gal staining is observed in
many locations throughout the CNS, including the olfactory bulb,
cerebral cortex, midbrain, dorsal spinal cord, retina, and inner ear
(Fig. 2). Within the adult cerebellum,
X-gal staining is confined to Purkinje cells (Figs.
2A) and is seen at postnatal day (P) 15 but not at P8
(data not shown). In most locations within the CNS, X-gal staining
appears in a punctate pattern within the cell somata. This pattern
presumably reflects a heterogeneous distribution of active tetrameric
 -galactosidase, possibly as a consequence of low levels of
expression. However, retinal photoreceptors, inner hair cells in the
organ of Corti, and vestibular hair cells in the maculae and cristae
show a more intense and uniform pattern of X-gal staining (Fig.
2C-F), suggesting that the fz4
gene may be expressed at higher levels in these neurons. In all tissues
examined thus far, fz4(+/) and fz4(/) mice
exhibit identical or very nearly identical patterns of X-gal
staining.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1.
Strategy for targeted replacement of the
fz4 coding region by lacZ. A,
Top, Map of the murine fz4 locus. The
fz4 coding exons reside within a region of 3 kb,
indicated by a filled rectangle. B,
BamHI; E, EcoRI;
H, HindIII; N,
NcoI. Center, Structure of the
fz4 targeting construct. A 5' flanking segment of 8.5 kb
terminating at the initiator methionine of fz4 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
PGK-neo selectable marker. A thymidine kinase
selectable marker (MC1-TK) is located distal
to the 3' homology segment. Bottom, Structure of the
targeted allele. The targeting event precisely deletes all but the 3'
70 bp of the fz4 coding region. B,
Genotyping of fz4(+/+), fz4(+/), and
fz4(/) mice by EcoRI digestion and
Southern blotting with the 3' flanking probe indicated in
A. The wild-type and gene-targeted alleles generate
fragments of 10.4 and 12.0 kb, respectively.
|
|
View larger version (94K):
[in this window]
[in a new window]
|
Figure 2.
fz4 expression patterns as revealed
by X-gal staining of the lacZ reporter in
fz4(+/) and fz4(/) mice.
A, B, X-gal staining of cerebellum
(A) and a coronal section through the brain
(B) from adult fz4(+/) mice. In
the cerebellum, X-gal staining is detected only in Purkinje cells.
C, X-gal staining of the adult fz4(+/)
retina. The pigment epithelium is at the top, and the
ganglion cell layer is at the bottom. Uniform filling of
the photoreceptor soma by the X-gal reaction product contrasts with a
punctate pattern of X-gal staining in the inner retina.
D-F, X-gal staining pattern in the adult
fz4(/) inner ear. X-gal staining is found in inner
hair cells in the organ of Corti (D,
arrowhead) and at very low levels in outer hair cells
and in the primary sensory cells of the maculae
(E) and cristae (F). As
seen in the retina, the X-gal product exhibits uniform filling of
primary sensory cells. fz4(+/) and
fz4(/) mice exhibited indistinguishable patterns of
X-gal staining in each of these tissues. Scale bars: A,
200 µm; C-F, 40 µm.
|
|
Growth retardation, abnormal gait, and premature death in
fz4(/) mice
Despite the widespread expression of fz4 observed in
the embryo and documented previously in multiple adult organs by RNase protection (Wang et al., 1996), fz4(/) mice are born at
the expected Mendelian ratio in crosses between fz4(+/)
parents, and they grow at a normal rate during the first week of
postnatal life. However, beginning in the second postnatal week,
fz4(/) mice gain weight more slowly than their
sex-matched fz4(+/+) and fz4(+/) littermates,
and ~50% of fz4(/) mice die during the first several
months of postnatal life (Fig. 3). No
differences were observed between fz4(+/+) and
fz4(+/) mice with respect to weight gain, viability, or
any of the other phenotypes examined in this study.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 3.
Growth retardation and early lethality
in fz4(/) mice. A-C,
Weight gain during early life in a cohort of seven
fz4(+/+), six fz4(+/), and four
fz4(/) mice. No differences are observed between
fz4(+/+) and fz4(+/) mice. The four
fz4(/) mice in this cohort weighed less than their
fz4(+/+) and fz4(+/) littermates at
every time point examined and died between postnatal weeks 5 and 11. D, E, Weight gain over 35 weeks among 20 fz4(+/) and 13 fz4(/) mice.
Squares, circles, and
triangles represent different cohorts. Long-lived
fz4(/) mice remain underweight throughout life in
comparison to their fz4(+/) littermates.
F, A Kaplan-Meyer survival curve shows ~50%
lethality among fz4(/) mice between 3 and 15 weeks
of age. Many of the fz4(/) mice that survive beyond
this early period die during the ensuing year.
|
|
The earliest outward phenotypic difference between
fz4(/) mice and their fz4(+/+) and
fz4(+/) littermates is seen in coat color. In the
129/SVJ × C57BL/6J background of this line, fz4(+/+) and fz4(+/) mice are either black or agouti, whereas
fz4(/) mice are light black or silver, respectively.
This phenotype suggests that fz4 may play a role in the
expansion or migration of melanocyte precursors from the neural crest
or the differentiation and survival of melanocytes. Other outward
phenotypes that become increasingly apparent as fz4(/)
animals age are a characteristic hunchback posture and an abnormal gait
(Fig. 4). Older fz4(/)
mice walk with a shortened stride, minimal alternation between the left and right feet, and frequent failures to fully lift each foot between
steps. At rest, fz4(/) adults show a characteristically wide stance, as seen in the position of the hind feet in Figure 4C.
View larger version (39K):
[in this window]
[in a new window]
|
Figure 4.
Ataxia in fz4(/) mice.
A, B, Hind footprints from adult
fz4(+/) and fz4(/) mice.
fz4(/) mice exhibit a shortened stride, minimal
alternation between the left and right feet, and frequent failures to
fully lift each foot between steps. C,
fz4(/) adults shows a wide stance, as evident in the
position of the hind feet. As seen here, older animals also have a
characteristic hunchback posture.
|
|
Cerebellar degeneration in fz4(/) mice
The gait abnormalities noted above might be caused, at least in
part, by a peripheral neuropathy or by abnormalities intrinsic to
skeletal muscle. To test these possibilities, leg muscles from fz4(+/+) and fz4(/) adults were examined by
hematoxylin and eosin staining, the pattern of innervation was assessed
by acetylcholine esterase histochemistry, and the distribution of type
1 and type 2 muscle fibers was assessed by NADH diaphorase staining
(Fig. 5) [in this and all subsequent
figures, paired photographs show the fz4(+/+) or
fz4(+/) section on the left and the
fz4(/) section on the right]. This analysis
shows that the average diameter of the fz4(/) muscle
fibers is smaller than that of the fz4(+/+) fibers, a
difference that we attribute to myocyte atrophy associated with the
poor weight gain in fz4(/) mice. However, no other differences were observed between these muscle samples, and in particular, no evidence was found for denervation, loss, or
regeneration of myofibrils. Cresyl violet staining of serial cross
sections along the length of the fz4(+/+) and
fz4(/) spinal cord shows little or no difference between
the two (data not shown). Although we cannot rule out functional
defects in spinal motor or sensory mechanisms not accompanied by cell
loss, these data suggest that the gait abnormality is unlikely to arise
from lower motor neuron loss, peripheral neuropathy, or myopathy.
View larger version (79K):
[in this window]
[in a new window]
|
Figure 5.
Histologic appearance of hindlimb skeletal muscle
in fz4(/) mice. A, B,
Hematoxylin and eosin staining. C, D,
Acetylcholine esterase histochemistry to visualize neuromuscular
junctions. E, F, NADH diaphorase
histochemistry to distinguish type 1 fibers (strongly stained) from
type 2 fibers (weakly stained). fz4(/) limb muscles
show modest atrophy consistent with weight loss but no evidence of
inflammation, fibrosis indicative of myopathy, or clustering of fiber
types indicative of peripheral neuropathy. Scale bars, 40 µm.
|
|
To investigate the possibility of a central neurological basis for the
gait abnormalities in fz4(/) mice, sections of adult brain were surveyed for evidence of developmental anomalies or cell
loss using cresyl violet, hematoxylin and eosin, and the Sevier-Munger
silver stain (Sevier and Munger, 1965), which highlights neuronal
processes, and anti-calbindin immunostaining, which highlights cerebellar Purkinje cells (Fig. 6). This
survey revealed a hypocellular cerebellum in the context of grossly
normal cerebellar architecture (Fig.
6A,B). Closer examination revealed
a dramatic loss of cerebellar granule and Purkinje cells in a laminar
pattern that followed the crests and sulci of the cerebellar folia with
accompanying vacuolization that extended into the subjacent myelinated
tracts (Fig.
6C,D,G-N).
Adult fz4(/) cerebella also exhibited astroglial activation and altered astroglial architecture as assayed by GFAP immunostaining (Fig. 6E,F).
Other brain regions in adult fz4(/) mice appear to be
unaltered. The disorganization of cerebellar structure in
fz4(/) mice suggests that the gait abnormalities in
fz4(/) mice reflect a progressive cerebellar ataxia.
View larger version (115K):
[in this window]
[in a new window]
|
Figure 6.
Cerebellar degeneration in adult
fz4(/) mice. A, B,
Cresyl violet staining shows hypocellularity in the
fz4(/) cerebellum in the context of normal
cerebellar architecture. C, D,
I, J, Silver staining by the method of
Sevier and Munger (1965), which visualizes neuronal processes
(black), and hematoxylin and eosin staining
(K, L) show vacuolation, loss of Purkinje
and granule cells, and disorganization of the molecular and granule
cell layers in adult fz4(/) cerebella.
E, F, Increased activation of glia and
disorganization of activated glial morphology in the adult
fz4(/) cerebellum. G,
H, M, N, Purkinje cell
loss in the adult fz4(/) cerebellum as revealed by
calbindin immunostaining. Scale bars: A,
B, 1 mm;
C-F, K-N,
40 µm; G, H, 0.5 mm;
I, J, 400 µm.
|
|
Analysis of neuronal cell death in the cerebellum at different
developmental times was performed using cresyl violet staining and
in situ detection of DNA fragmentation by TUNEL labeling
(Gavrieli et al., 1992). By cresyl violet staining the cerebella of
fz4(/) mice appear to develop normally up to P19 (Fig.
7A,B).
However, TUNEL analysis at P8, P14, P19, and P30 shows massive death of granule cells in the fz4(/) cerebellum beginning between
P14 and P19 (Fig.
7C-F,M). At P8
and P14, both fz4(/) and fz4(+/+) cerebella
show small numbers of TUNEL+ cells in the external granule cell layer
and in the developing inner granule cell layer. At P19, at least
50-fold more TUNEL+ cells are present in the fz4(/) cerebellum compared with the fz4(+/) cerebellum, and > 95% of these are in the granule cell layer. By P30, the number of
TUNEL+ cells in the fz4(/) cerebellum is reduced
greatly. Elsewhere within the CNS, fz4(+/+), fz4(+/), and
fz4(/) littermates show differences of less than twofold
in the number of TUNEL+ cells during postnatal development and in
adulthood.
View larger version (112K):
[in this window]
[in a new window]
|
Figure 7.
Extensive granule cell death between weeks 2 and 5 in the fz4(/) cerebellum.
A-J, Cerebella from
fz4(+/) and fz4(/) mice at P19.
A, B, Cresyl violet staining reveals a
grossly normal appearance including roughly normal numbers of Purkinje
and granule cells. DAPI staining (C, D)
and TUNEL labeling of DNA fragments (E,
F) in the same sections reveal extensive granule
cell death in the fz4(/) cerebellum
(F) and minimal cell death in the
fz4(+/) control (E).
G, H, Type I IP3 receptor
immunostaining at P19 shows increased separation of Purkinje cell
bodies and processes in the fz4(/) cerebellum.
I, J, Activated glia, as revealed by GFAP
immunostaining, show subtle disorganization in the
fz4(/) granule cell layer. K,
L, Type I IP3 receptor immunostaining at P8
shows that developing Purkinje cells of fz4(+/) and
fz4(/) mice have indistinguishable morphology.
M, Histogram showing, at different ages, the number of
TUNEL-positive nuclei per square millimeter of surface area in a series
of randomly selected 16-µM-thick sections of cerebellum.
Because cell sizes are smaller and cell densities are higher in younger
animals, the total number of cells per square millimeter is higher in
younger animals. Scale bars, 100 µm.
|
|
In fz4(/) mice, TUNEL labeling of cerebellar Purkinje
cells was rarely seen, but this simply may reflect their paucity
relative to granule cells. However, a progressive loss of Purkinje
cells proceeds throughout adulthood in the fz4(/)
cerebellum as judged by immunostaining for type-1
IP3 receptor and calbindin, both of which are
specifically expressed in Purkinje cells (Figs.
6M,N, 7G,H,K,L).
Interestingly, the peak of granule cell death occurs at a time (P19)
when the vast majority of Purkinje cells are still present. At this
time, the only Purkinje cell abnormality evident in
fz4(/) mice is a small but distinct separation of the
Purkinje cell bodies from the network of processes in the molecular
layer (Fig. 7G,H). The organization of
activated astroglial cells is also subtly altered in the granule cell
layer at P19 but not at earlier times, as determined by immunostaining
with anti-GFAP (Fig. 7I,J).
If the cerebellar phenotype results from a lack of fz4
activity in Purkinje cells, this temporal pattern implies that granule
cell death in the fz4(/) cerebellum is not a consequence of Purkinje cell loss but is secondary to some alteration in the properties of the Purkinje cells.
Progressive hearing loss in fz4(/) mice
As part of a general screen for behavioral deficits, we observed
that fz4(/) mice show a grossly normal auditory startle response at 1-2 months of age, whereas older fz4(/)
mice show a diminished or absent startle response. Vestibular function
in fz4(/) mice appears grossly intact as judged by their
ability to remain balanced on a slowly rotating drum. Auditory
brainstem responses recorded from adult fz4(/) mice and
from age-matched fz4(+/+) or fz4(+/) controls
confirmed the presence of elevated auditory thresholds in
fz4(/) adults. In the example shown in Figure
8, the auditory brainstem response
threshold is at least 40 dB higher than the average control value of
~60 dB. Some adult fz4(/) mice with detectable startle
responses have auditory brainstem thresholds that are elevated but
still below the 100 dB ceiling of the stimulus used here. The failure
to observe the earliest component of the auditory brainstem response in
fz4(/) mice localizes the hearing defect to the
peripheral auditory system, although this does not rule out the
possibility of a coexisting central auditory defect.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 8.
Auditory brainstem responses in adult
fz4(/) mice reveal a peripheral hearing defect.
Representative ABRs in 11-month-old fz4(+/+) and
fz4(/) mice are shown. The click stimulus at the
indicated sound pressure levels (dB SPL) triggers the
line sweep and occurs at the 0 msec position. Each trace
is an average of 1000 responses. The fz4(+/+) mouse
shows a threshold sensitivity of ~60 dB SPL, whereas the
fz4(/) littermate shows no response at any of the
stimulus intensities tested. This fz4(/) mouse also
shows no startle reflex when tested with a stimulus generated by
striking two metal rods.
|
|
Strong expression of the fz4-lacZ knock-in
reporter is evident in inner hair cells in the organ of Corti in
both fz(+/) and fz4(/) mice (Fig.
2D). These results suggest that the peripheral auditory defect might be referable to defects in these cells. At the
light microscope level, however, there is no morphological evidence of
inner or outer hair cell loss in fz4(/) mice. Thus, in
this instance, primary sensory cell loss is not responsible for the
hearing loss. Similar analyses of vestibular sensory cells show strong
expression of the fz4-lacZ knock-in reporter in
both fz(+/) and fz4(/) mice (Fig.
2E,F) but no evidence of
sensory cell loss. Thus, the mechanism of progressive hearing loss is presently unknown, and the question of whether there may also be a
defect in the vestibular system remains open.
Esophageal dysfunction and distension in
fz4(/) mice
The only gross internal anatomic defect present in
fz4(/) mice is progressive enlargement of the esophagus
(Fig.
9A,B,E,F), an abnormality that is apparent at P8 but not at P0. A concomitant defect in esophageal peristalsis and gastric sphincter function is
suggested by the observations that when fed ad libitum
before sacrifice, adult fz4(/) mice typically have large
quantities of food within the esophagus, whereas the esophagi from
fz4(+/+) and fz4(+/) mice have little or no
food. Moreover, in fz4(/) mice, large numbers of
bacteria are found adhering to the esophageal epithelium, which also
shows extensive desquamation (Fig. 9C,D). These
observations suggest that the growth retardation in
fz4(/) mice may arise from a feeding defect caused by
esophageal dysfunction.
View larger version (92K):
[in this window]
[in a new window]
|
Figure 9.
Esophageal distension and absence of skeletal
muscle in the lower esophagus in adult fz4(/) mice.
A, B, Cross sections of the esophagus
from fz4(+/) and fz4(/) mice
stained with hematoxylin and eosin. B shows one of the
more extreme examples of esophageal distension seen in
fz4(/) mice. C, D, The
esophageal wall in fz4(+/) and
fz4(/) mice; the lumenal face is at the
top. Epon sections (1 µm) were stained with toluidine
blue. The outer layer of the fz4(+/+) esophagus is
composed of skeletal muscle, whereas the outer layer of the
fz4(/) esophagus is composed of smooth muscle. The
lumenal face of the fz4(/) esophagus shows extensive
desquamation of the epithelium and large numbers of bacteria, seen as
clusters of darkly stained dots in the higher
magnification inset. E, F,
Cross sections of the esophagus from fz4(+/) and
fz4(/) mice immunostained with anti-skeletal muscle
myosin (red). Cell nuclei are counterstained with DAPI
(blue). Only the fz4(+/) esophagus
shows a ring of skeletal muscle. G, H,
Acetylcholine esterase histochemistry of lower esophagus flat mounts
from fz4(+/+) and fz4(/) mice. Large
acetylcholine esterase-positive motor endplates characteristic of
skeletal muscle are seen throughout the fz4(+/+)
esophagus and in the upper esophagus of fz4(/) mice
(data not shown). In the lower esophagus of fz4(/)
mice, a fine network of acetylcholine esterase-stained fibers is seen
instead of motor endplates. I, Whole-mount X-gal
staining of an isolated fz4(/) esophagus at the
junction between the rostral two-thirds (left), which is
ensheathed in skeletal muscle, and the caudal one-third
(right), which is ensheathed in smooth muscle. The
lacZ reporter is strongly expressed in the esophageal
skeletal muscle but not in the smooth muscle. The location of this
transition zone varies among fz4(/) mice and in
extreme cases can be found within 1-2 mm of the rostral end of the
esophagus. The fz4(+/) esophagus shows a pattern of
X-gal staining along its entire length, which is less intense but
otherwise resembles that seen in the skeletal muscle of the
fz4(/) esophagus. J,
K, Absence of significant differences in the lower
gastrointestinal tract of fz4(+/+) and
fz4(/) mice as seen in the indistinguishable
patterns of NADPH diaphorase-stained enteric neurons in flat mounts of
duodenum. Scale bars: A, B, 400 µm;
C, D, G, H,
J, K, 40 µm; E,
F, 200 µm.
|
|
A potential mechanism for defective esophageal peristalsis and
sphincter function is suggested by the observation that, among fz4(/) mice, a variable length of the lower esophagus,
ranging from the lower one-fourth to nearly the entire length of the
esophagus, is devoid of the normal sheath of skeletal muscle.
Toluidine blue staining of 1 µm plastic sections and
immunostaining with anti-skeletal muscle myosin show the expected ring
of skeletal muscle encircling the fz4(+/) esophagus (Fig.
9C,E). By contrast, a ring of smooth muscle
encircles the fz4(/) esophagus (Fig.
9D,F). Acetylcholine esterase staining of whole mounts of a fz4(+/+) esophagus
reveals the expected pattern of large motor endplates terminating on
skeletal muscle fibers along the entire length of the esophagus (Fig.
9G), and a similar pattern is seen for variable distances in
the upper esophagi of fz4(/) mice (data not shown).
However, acetylcholine esterase staining of the lower esophagus of
fz4(/) mice shows no motor endplates and reveals instead
a fine network of neuronal processes (Fig. 9H). In
both fz4(+/) and fz4(/) mice, the
fz4-lacZ reporter is expressed in the skeletal
muscle fibers that ensheath the upper esophagus, but the reporter is
not expressed in the smooth muscle that lines the lower esophagus of
fz4(/) mice (Fig. 9I).
Gross and microscopic examination of the stomach and lower
gastrointestinal tract by hematoxylin and eosin staining and
microscopic examination of the associated enteric neurons by NADPH
diaphorase and acetylcholine esterase histochemistry and by anti-CGRP
immunostaining reveal few if any differences between
fz4(/) and fz4(+/+) or fz4(+/)
mice (Fig. 9J,K).
| |
DISCUSSION |
The experiments reported here establish a role for Fz4 in
maintaining the structure and function of the cerebellum, inner ear,
and esophagus during postnatal life. Although the discussion that
follows assumes that the involvement of Fz4 in these processes reflects
its inferred role as a Wnt receptor, we note that for Frizzled-mediated
tissue polarity in Drosophila there is no evidence at
present for Wnt involvement, suggesting the possibility that in some
contexts Frizzled proteins may function independently of Wnts.
Earlier studies of Wnt and lef-1 mutant mice revealed a role for Wnts
and Wnt signaling in the proliferation of neural precursors, the
patterned expression of cell adhesion molecules (Shimamura et al.,
1994), and synaptogenesis. In each of these cases, Wnt activity was
manifest during prenatal or immediately postnatal life. By contrast,
the progressive cerebellar degeneration and auditory dysfunction seen
in older fz4(/) mice suggest that defects in Wnt
signaling may also play a role in progressive neurodegenerative processes in later life. In keeping with this general hypothesis, recent work has implicated Wnt signaling in the suppression of apoptosis in cultured fibroblasts (Chen et al., 2001) and has suggested
-catenin destabilization by mutant presenilin-1 as a mechanism that
enhances apoptosis in the brain in patients with Alzheimer's disease
(Zhang et al., 1998). Whether the progressive esophageal enlargement in
older fz4(/) mice simply represents the late sequela of
an early defect in esophageal skeletal muscle development remains to be
determined. In the paragraphs that follow we consider each of the
fz4(/) phenotypes in the context of previous work with
genetically modified mice and related human diseases.
Esophageal enlargement and dysfunction
Under normal circumstances, the esophagus propels food by
peristalsis in a rostrocaudal direction, selectively relaxes the lower
esophageal sphincter to allow passage of food into the stomach, and
maintains lower esophageal sphincter tone to block reflux of gastric
contents (Pelot, 1995). In humans, esophageal enlargement is found in
the context of achalasia, a disorder of esophageal motility caused by
diminished peristalsis of the esophageal body and impaired relaxation
of the lower esophageal sphincter (Ouyang and Cohen, 1995). Primary
achalasia is thought to arise from defects in esophageal innervation,
involving either the enteric neurons within the myenteric plexus or
extrinsic innervation from the dorsal motor nucleus via the vagus
nerve. A similar etiology has been described for achalasia secondary to
Chagas's disease, in which loss of enteric neurons follows infection
by Trypanosoma cruzi. Although the expression of the
fz4-lacZ reporter in skeletal muscle within the
esophagus suggests that the progressive esophageal dilatation seen in
fz4(/) mice may be of myogenic origin, the data do not
rule out a neurogenic contribution.
The finding of smooth rather than skeletal muscle in the lower
esophagus of fz4(/) mice is of interest because in
normal development the mouse esophagus is initially ensheathed in
smooth muscle. During late fetal and early postnatal life the
esophageal smooth muscle transdifferentiates into skeletal muscle, a
process that proceeds in a rostrocaudal direction until the entire
esophagus is ensheathed in skeletal muscle (Patapoutian et al., 1995).
It is possible that loss of Fz4 directly or indirectly causes a
developmental arrest in the process of esophageal transdifferentiation.
Interestingly, in humans the lower half of the esophagus is ensheathed
exclusively in smooth muscle, whereas the upper half of the esophagus
contains a mixture of smooth and striated muscle (Pelot, 1995). Thus,
the absence of skeletal muscle per se in the lower esophagus of
fz4(/) mice is not necessarily the cause of esophageal
enlargement and dysfunction.
It is of interest to compare the esophageal phenotype in
fz4(/) mice with that seen in MASH1(/)
mice (Guillemot et al., 1993; Sang et al., 1999). Neonatal
MASH1(/) mice do not show evidence of milk in their
stomachs, indicating that either they fail to nurse or having nursed
fail to ingest the milk. In the neonatal MASH1(/)
esophagus, enteric neurons are missing almost completely, but
transdifferentiation of esophageal muscle and vagal innervation appear
to be undisturbed. Sang et al. (1999) have hypothesized that the
failure to find milk in the stomachs of MASH1(/) pups
reflects a failure to relax the lower esophageal sphincter secondary to
the nearly complete absence of nitric oxide synthase-containing enteric
neurons. The esophageal dilatation in fz4(/) mice could
also reflect a failure of lower esophageal relaxation. Because
fz4(/) mice have no difficulty nursing, the severity of
any esophageal sphincter defect is presumably less than that
hypothesized for MASH1(/) mice.
Progressive hearing loss
In the human population, ~0.1% of individuals suffer from some
form of early onset hereditary deafness (Keats and Berlin, 1999). The
incidence of late onset deafness, whether hereditary or acquired,
increases with age, such that 30% of individuals over 65 years of age
suffer from hearing impairment (Weinstein, 2000). Numerous mouse models
of early onset Mendelian hearing disorders have been identified as
naturally occurring mutants or have been constructed by targeted gene
disruption (Holme and Steele, 1999). Most of these mouse models
exhibit severe early onset defects in auditory or vestibular function,
or both, and their phenotypes closely resemble the corresponding
Mendelian disorders in humans.
The complex etiology of late onset hearing loss has made it more
difficult to study. Environmental factors that confound genetic analysis include chronic exposure to loud sounds and ototoxic compounds. In a few cases, a genetic etiology for late onset hearing loss in humans has been identified as arising from partial defects in
genes that are essential for inner ear function in the mouse. For
example, heterozygosity for an 8 bp deletion in the human POU3F4/Brn-3c/Brn3.0 transcription factor gene produces late onset hearing loss (Vahava et al., 1998), whereas homozygous deletion of the
orthologous gene in mice results in a complete loss of auditory and
vestibular hair cells early in development (Erkman et al., 1996; Xiang
et al., 1997, 1998). The expression of fz4 in auditory and
vestibular hair cells together with the late onset hearing loss
exhibited by fz4(/) mice in the absence of auditory or
vestibular hair cell death suggests that Fz4 plays a role in maintaining hair cell function, but that it is not essential for the
initial development and functioning of the inner ear. These observations suggest the general possibility that derangements in Wnt
signaling might play a role in human hearing loss.
Cerebellar degeneration
The size, cellularity, and overall structure of the
fz4(/) cerebellum appear normal before the third week of
postnatal life. Thus, cell proliferation, migration, and arborization
occur with little or no disruption. Cerebellar degeneration in
fz4(/) mice begins in the third postnatal week with
extensive granule cell death, and over the ensuing months Purkinje
cells also die. The time window for the initial wave of granule cell
death overlaps with the peak of synaptogenesis during cerebellar
development, roughly P10-P20, suggesting that granule cell death could
arise from a defect in some aspect of synaptogenesis.
Among the various mouse cerebellar mutants, the
leaner mutant most closely approximates the
fz4(/) mice with respect to the time course and pattern
of cell loss (Herrup and Wilczynski, 1982; Fletcher et al.,
1996). In leaner mice, granule cell death begins at P10,
occurs mostly over the ensuing several months, and is characterized by
large numbers of TUNEL-positive granule cells. Purkinje cell death
begins several weeks later and continues for at least 6 months.
Leaner, and its alleles tottering and
rolling mouse Nagoya, are caused by mutations in the
gene encoding the  1A calcium channel (Fletcher et al., 1996).
Despite the widespread expression of this gene in the CNS, the cell
death phenotype is largely confined to the cerebellum. This similarity
in phenotype suggests that cell death in the fz4(/)
cerebellum could involve misregulation of intracellular calcium or of a
common effector that is regulated by both calcium and Wnt signaling.
On the basis of the expression of the
fz4-lacZ reporter in Purkinje cells and our
inability to detect reporter expression in cerebellar granule cells,
granule cell death in fz4(/) mice may arise as a
secondary consequence of Purkinje cell dysfunction. However, we note
that fz4 is widely expressed in the CNS, including in
brainstem nuclei that project to the cerebellum (e.g., the dorsal
cochlear nuclei). Thus, granule cell loss could also arise from a
defect in some fz4-dependent property of cells that project to the cerebellum.
Clear precedents exist for developmental signaling from Purkinje cells
to granule cells in the context of granule cell proliferation in the
external granule layer and granule cell survival in the immediate
postmitotic period (Goldowitz and Hamre, 1999; Heintz and Zoghbi,
2000). In staggerer mice, a deletion in the gene
encoding ROR-, a nuclear hormone receptor expressed
specifically in Purkinje cells (Hamilton et al., 1996), leads to a
block in Purkinje cell development and a secondary decrease in granule
cell proliferation and survival (Herrup, 1983; Sonmez and Herrup,
1984). A similar loss of granule cells secondary to Purkinje cell loss
is observed in heterozygous Lurcher mice (Caddy and
Biscoe, 1979) and after targeted ablation of cerebellar Purkinje cells
using transgenic diphtheria toxin (Smeyne et al., 1995). In
Lurcher heterozygotes a point mutation in the
Grid2 ionotropic glutamate receptor gene produces a
constitutively active channel that causes excitotoxic Purkinje cell
death beginning in the second postnatal week (Wetts and Herrup, 1982;
Zuo et al., 1997). By contrast, in the Purkinje cell
degeneration mouse, Purkinje cell death begins in the third postnatal week and is associated with minimal granule cell death (Mullen et al., 1976).
Signaling between Purkinje and granule cell precursors appears to be
mediated, at least in part, by Sonic hedgehog (Shh). Purkinje cells
produce Shh, granule cells express the Shh receptor Patched, and Shh
acts to promote proliferation and block differentiation of granule cell
precursors (Dahmane and Ruiz-i-Altaba, 1999; Wechsler-Reya and Scott,
1999). The late granule cell death in fz4(/) mice suggests that communication between Purkinje and granule cells continues beyond the proliferative phase of cerebellar development and
that this communication remains essential for granule cell viability.
It will be of interest to determine whether cell-cell communication is
similarly implicated in any of the human cerebellar degenerations.
Diversity and redundancy in frizzled function
The complex and widespread patterns of expression exhibited by
many of the mammalian frizzled genes suggest that each
Frizzled receptor is likely to play multiple roles in different tissues and at different developmental times. Moreover, the significant overlap
among different frizzled family members in the time and place of expression suggests that partial redundancy of Frizzled function may be the rule rather than the exception. The present work
reveals a role for fz4 in three unrelated processes. As
noted in the introductory remarks, earlier work on Frizzled and
Frizzled-2 in Drosophila revealed their nearly complete
functional redundancy as Wingless receptors in the embryo (Bhat, 1998;
Kennerdell and Carthew, 1998; Bhanot et al., 1999; Chen and Struhl,
1999). Therefore it will be of great interest to determine whether the
pattern of frizzled redundancy seen in Drosophila
also holds in mammals. If so, then we can anticipate that mice
harboring mutations in fz4 together with mutations in other
frizzled genes will reveal a range of phenotypes
significantly richer than those revealed by the corresponding single
gene mutations.
| |
FOOTNOTES |
Received Feb. 15, 2001; revised April 17, 2001; accepted April 19, 2001.
This work was supported by the Howard Hughes Medical Institute (Y.W.,
J.N.) and National Institutes of Health Grant DC00232 (H.C., D.R.). We
thank Se-jin Lee for the 129/SVJ genomic library; Andras Nagy and Janet
Rossant for ES cells; Richard Behringer for the knock-out vector;
Jen-Chih Hsieh for assistance with mapping of genomic clones; Phil
Smallwood for help with animal husbandry; Mitra Cowan, Diane Blesh, and
Chip Hawkins for blastocyst injection; Anthony Wynshaw-Boris and Amy
Chen for advice on ES cell methodology; David Linden, Mark Molliver,
Elizabeth O'Hearn, Randy Reed, and Amir Rattner for helpful
discussions; and Tudor Badea 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.
| |
REFERENCES |
-
Bancroft JD,
Stevens A
(1982)
In: Theory and practice of histological techniques. Edinburgh: Churchill Livingstone.
-
Bhanot P,
Fish M,
Jemison J,
Nusse R,
Nathans J,
Cadigan KM
(1999)
Frizzled and frizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.
Development
126:4175-4186.
-
Bhat KM
(1998)
Frizzled and frizzled2 play a partially redundant role in wingless signaling and have similar requirements to wingless in neurogenesis.
Cell
95:1027-1036.
-
Borello U,
Buffa V,
Sonnino C,
Melchionna R,
Vivarelli E,
Cossu G
(1999)
Differential expression of the Wnt putative receptors Frizzled during mouse somitogenesis.
Mech Dev
89:173-177.
-
Caddy KW,
Biscoe TJ
(1979)
Structural and quantitative studies on the normal C3H and Lurcher mutant mouse.
Philos Trans R Soc Lond B Biol Sci
287:167-201.
-
Cadigan KM,
Nusse R
(1997)
Wnt signaling: a common theme in animal development.
Genes Dev
11:3286-3305.
-
Chan EF,
Gat U,
McNiff JM,
Fuchs E
(1999)
A common human skin tumour is caused by activating mutations in beta-catenin.
Nat Genet
21:410-413.
-
Chen C,
Struhl G
(1999)
Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila.
Development
126:5441-5452.
-
Chen S,
Guttridge DC,
You Z,
Zhang Z,
Fribley A,
Mayo MW,
Kitajewski J,
Wang CY
(2001)
Wnt-1 signaling inhibits apoptosis by activating beta-catenin/T cell factor-mediated transcription.
J Cell Biol
152:87-96.
-
Dahmane N,
Ruiz-i-Altaba A
(1999)
Sonic hedgehog regulates the growth and patterning of the cerebellum.
Development
14:3089-3100.
-
Dickinson ME,
Krumlauf R,
McMahon AP
(1994)
Evidence for a mitogenic effect of Wnt-1 in the developing central nervous system.
Development
120:1453-1471.
-
Erkman L,
McEvilly RJ,
Luo L,
Ryan AK,
Hooshmand F,
O'Connell SM,
Keithley EM,
Rapaport DH,
Ryan AF,
Rosenfeld MG
(1996)
Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.
Nature
381:603-606.
-
Fletcher CF,
Lutz CM,
O'Sullivan TN,
Shaughnessy Jr JD,
Hawkes R,
Frankel WN,
Copeland NG,
Jenkins NA
(1996)
Absence epilepsy in tottering mutant mice is associated with calcium channel defects.
Cell
87:607-617.
-
Galceran J,
Miyashita-Lin EM,
Devaney E,
Rubenstein JL,
Grosschedl R
(2000)
Hippocampus development and generation of dentate gyrus granule cells is regulated by LEF1.
Development
3:469-482.
-
Gavrieli Y,
Sherman Y,
Ben-Sasson SA
(1992)
Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J Cell Biol
119:493-501.
-
Goldowitz D,
Hamre K
(1999)
The cells and molecules that make a cerebellum.
Trends Neurosci
21:375-382.
-
Guillemot F,
Lo LC,
Johnson JE,
Auerbach A,
Anderson DJ,
Joyner AL
(1993)
Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons.
Cell
75:463-476.
-
Grove EA,
Tole S,
Limon J,
Yip Y,
Ragsdale CW
(1998)
The hem of the embryonic cerebral cortex is defined by the expression of multiple Wnt genes and is compromised in Gli3-deficient mice.
Development
125:2315-2325.
-
Hall AC,
Lucas FR,
Salinas PC
(2000)
Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling.
Cell
100:525-535.
-
Hamilton BA,
Frankel WN,
Kerrebrock AW,
Hawkins TL,
FitzHugh W,
Kusumi K,
Russell LB,
Mueller KL,
van Berkel V,
Birren BW,
Kruglyak L,
Lander ES
(1996)
Disruption of the nuclear hormone receptor RORalpha in staggerer mice.
Nature
379:736-739.
-
He X,
Saint-Jeannet J-P,
Wang Y,
Nathans J,
Dawid I,
Varmus R
(1997)
A member of the Frizzled protein family mediating axis induction by Wnt-5A.
Science
275:1652-1654.
-
Heintz N,
Zoghbi H
(2000)
Insights from mouse models into the molecular basis of neurodegeneration.
Annu Rev Physiol
62:779-802.
-
Herrup K
(1983)
Role of staggerer gene in determining cell number in cerebellar cortex I granule cell death is an indirect consequence of staggerer gene action.
Dev Brain Res
11:267-274.
-
Herrup K,
Wilczynski SL
(1982)
Cerebellar cell degeneration in the leaner mutant mouse.
Neuroscience
7:2185-2196.
-
Hsieh J-C,
Rattner A,
Smallwood PM,
Nathans J
(1999)
Biochemical characterization of Wnt-Frizzled interactions using a soluble, biologically active vertebrate Wnt protein.
Proc Natl Acad Sci USA
96:3546-3551.
-
Holme RH,
Steele KP
(1999)
Genes involved in deafness.
Curr Opin Genet Dev
9:309-314.
-
Ikeya M,
Lee SMK,
Johnson JE,
McMahon AP,
Takada S
(1997)
Wnt signaling required for expansion of neural crest and CNS progenitors.
Nature
389:966-970.
-
Karnovsky MJ,
Root L
(1964)
A "direct coloring" thiocholine method for choline esterases.
J Histochem Cytochem
12:219-221.
-
Keats BJB,
Berlin CI
(1999)
Genomics and hearing impairment.
Genome Res
9:7-16.
-
Kennerdell JR,
Carthew RW
(1998)
Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.
Cell
95:1017-1026.
-
Klingensmith J,
Nusse R
(1994)
Signaling by wingless in Drosophila.
Dev Biol
166:396-414.
-
Lako M,
Lindsay S,
Bullen P,
Wilson DI,
Robson SC,
Strachen T
(1998)
A novel mammalian wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain.
Hum Mol Genet
7:813-822.
-
|
TOP
ABSTRACT
INTRODUCTION
