Current Biology, Vol. 13, 1129–1133, July 1, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S 09 60 - 98 22 ( 03 )0 0 37 4- 9
Mutation of Celsr1 Disrupts Planar Polarity
of Inner Ear Hair Cells and Causes
Severe Neural Tube Defects in the Mouse
John A. Curtin,1,8 Elizabeth Quint,2,8 Vicky Tsipouri,1
Ruth M. Arkell,3 Bruce Cattanach,3
Andrew J. Copp,4 Deborah J. Henderson,5
Nigel Spurr,1 Philip Stanier,6 Elizabeth M. Fisher,7
Patrick M. Nolan,3 Karen P. Steel,2
Steve D.M. Brown,3 Ian C. Gray,1
and Jennifer N. Murdoch3,*
1
GlaxoSmithKline Pharmaceuticals
New Frontiers Science Park
Harlow
Essex, CM19 5AW
2
MRC Institute of Hearing Research
University Park
Nottingham, NG7 2RD
3
MRC Mammalian Genetics Unit
Harwell
Oxon, OX11 0RD
4
Neural Development Unit
Institute of Child Health
University College London
30 Guilford Street
London, WC1N 1EH
5
Institute of Human Genetics
University of Newcastle upon Tyne
International Centre for Life
Central Parkway
Newcastle upon Tyne, NE1 3BZ
6
Department of Obstetrics and Gynaecology
Institute of Reproductive and Developmental
Biology
Imperial College School of Medicine
Hammersmith Campus
London, W12 0NN
7
Department of Neurodegenerative Disease
Institute of Neurology
Queen Square
London, WC1N 3BG
United Kingdom
Summary
We identified two novel mouse mutants with abnormal
head-shaking behavior and neural tube defects during
the course of independent ENU mutagenesis experiments. The heterozygous and homozygous mutants
exhibit defects in the orientation of sensory hair cells
in the organ of Corti, indicating a defect in planar cell
polarity. The homozygous mutants exhibit severe neural tube defects as a result of failure to initiate neural
tube closure. We show that these mutants, spin cycle
and crash, carry independent missense mutations within
the coding region of Celsr1, encoding a large protocadherin molecule [1]. Celsr1 is one of three mammalian homologs of Drosophila flamingo/starry night,
which is essential for the planar cell polarity pathway in
*Correspondence: j.murdoch@har.mrc.ac.uk
8
These authors contributed equally to this work.
Drosophila together with frizzled, dishevelled, prickle,
strabismus/van gogh, and rhoA [2, 3]. The identification of mouse mutants of Celsr1 provides the first
evidence for the function of the Celsr family in planar
cell polarity in mammals and further supports the
involvement of a planar cell polarity pathway in vertebrate neurulation.
Results and Discussion
Two mutants, spin cycle (Scy) and crash (Crsh), were
identified from independent ENU mutagenesis experiments ([4] and B.C., unpublished data) by their headshaking behavior and subsequently were shown to exhibit belly curling and spinning during tail suspension.
These heterozygous phenotypes suggest some vestibular dysfunction, although we have found no clear defect
in the peripheral vestibular system of adult Scy or Crsh
mutants (data not shown). Both mutants exhibit a positive Preyer reflex in response to a 20 kHz, 90 dB SPL tone
burst; this finding indicates that they are not profoundly
deaf. However, examination of the adult cochlea revealed that both Scy and Crsh heterozygotes contain an
excess of misoriented outer hair cell (OHC) stereociliary
bundles (Figures 1A and 1B), particularly in the apical
turn of the cochlea (in Scy/⫹, apex 3.60%, base 1.68%
rotated; in ⫹/⫹, apex 0.66%, base 0.18% rotated). Inner
hair cell stereociliary bundles appeared to be unaffected
(Figures 1A and 1B). Crsh heterozygotes showed a
slightly milder phenotype than Scy, with fewer hair cells
affected (data not shown). The general organization into
three rows of outer and one row of inner hair cells appears to be normal in Scy and Crsh heterozygotes, although there was a small amount of OHC degeneration
in the basal turn (see the Supplemental Data available
with this article online). Furthermore, the upper surfaces
of supporting cells immediately surrounding the affected hair cells showed abnormal shapes (Figures 1A
and 1B). We examined cochleae from embryonic day
(E) 16.5 and 18.5 (Figures 1C–1F) and found that, even
at the earliest stages of OHC differentiation (E16.5 in
the apex), bundle misorientation was observed in the
mutants, while controls showed normal orientation. The
phenotype is particularly pronounced in homozygotes,
with apparently random orientation of OHC bundles,
including some bundles with a rotation of almost 180⬚
compared with the normal plane of orientation (Figures
1E and 1F). This OHC phenotype is consistent with a
defect in the establishment of planar cell polarity (PCP)
of cells within the sensory epithelium.
Crosses between head-shaking heterozygotes from
either mutant stock generated litters containing fetuses
that exhibit severe neural tube defects (NTD; Figures
2A–2E). This craniorachischisis is characterized by the
failure to close almost the entire neural tube, from the
midbrain/hindbrain boundary throughout the spine, and
leads to perinatal lethality. Homozygous fetuses often
also exhibit a delay or failure of eyelid closure (Figures
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Figure 1. Scy Heterozygotes and Crsh Homozygotes Exhibit Defects in Planar Cell Polarity of the Inner Ear Sensory Epithelium
(A–F) Scanning electron micrographs of the
organ of Corti from (A and C) wild-type (⫹/⫹),
(B and D) heterozygous (Scy/⫹), or (E and F)
homozygous (Crsh/Crsh) mice. At (A and B)
3–5 months and (C and D) E18.5, heterozygotes exhibit misoriented outer hair cell bundles, as indicated by arrows; the orientation
of each arrow depicts the altered plane of
polarity of the bundle. (B) Adjacent supporting cell apices are also distorted (asterisk),
but the inner hair cells (arrowheads in [A] and
[B]) appear to be unaffected. (E) In homozygotes, the OHCs are extensively rotated, and
the image shown is typical of the entire length
of the cochlear duct. At E16.5, toward the
apex, normal hair cells show the first sign of
PCP, whereby the thicker kinocilium migrates
away from the center to the lateral edge of
the cell. Even at this early stage, the defect
in PCP can be seen in homozygotes as the
kinocilium moves to abnormal positions (arrowheads in [F]; the arrow shows a central
kinocilium). The scale bars represent 2.5 ␮m.
2J and 2K). We analyzed embryonic litters earlier in gestation and found that the NTD are preceded by failure
to initiate neural tube closure in the future cervical region
at E8.5, so-called Closure 1 (Figures 2F and 2G), although neural tube closure in the forebrain and rostral
midbrain occurs normally. Intercrosses between Scy
and Crsh heterozygotes generated fetuses with severe
NTD (Figures 2H and 2I); this phenotype is indistinguishable from the phenotype of homozygous mutants and
consistent with the possibility of allelism.
We initially localized Scy on Chromosome 15 [4] and
confirmed this by genotyping with additional markers in
a total of 231 mutant mice. This placed Scy within a 1.7
cM (ⵑ2.6 Mb) interval between D15Gsk1-D15Gsk2 and
D15Mit241 (Figure 3A). Haplotype analysis of Crsh homozygotes (n ⫽ 29) established linkage of Crsh to an
overlapping region of Chromosome 15 (Figure 3B). The
Scy critical interval contains 12 known genes, including
3 plausible candidates for Scy: Wnt7b, Sca10, and the
seven-pass transmembrane protein-coding gene, Celsr1.
Sequence analysis of these three genes found no mutations in either Wnt7b or Sca10. A single point mutation,
which results in an asparagine to lysine substitution at
codon 1110, was identified in the second exon of Celsr1
(T3337A of GenBank sequence AF031572) (Figures 3C
and 3D). Sequence analysis of Celsr1 cDNA from Crsh
mutants identified a single point mutation (A3126G) that
results in an aspartate to glycine substitution at codon
1040 (Figures 3E and 3F). Residue 1040 lies within the
eighth cadherin repeat of the extracellular domain of
Celsr1, while residue 1110 forms part of the linker region
connecting adjacent cadherin domains. Both residues
are highly conserved within the Celsr family and within
other cadherins in diverse species (see the Supplemental Data). Indeed, X-ray crystallography of E-cadherin
suggests that asparagine 1110 is likely to be involved
in coordinating Ca2⫹ binding [5], and mutation of this
residue may reduce the extracellular structural integrity
of Celsr1.
Celsr1 is expressed during neurulation and in the developing inner ear and eyelids [1, 6–8]; this finding is
consistent with the mutant phenotypes seen in Crsh and
Scy. Celsr1 is first detectable at E6.5, within the primitive
streak [1, 7, 8], and is intensely expressed in the neuroepithelium immediately prior to and during neurulation
[1, 7]. Examination of Crsh mutants detects no abnormalities in Celsr1 mRNA expression at the time of
neurulation (data not shown). We have also investigated
further the expression of Celsr1 in the inner ear. At E14.5,
Celsr1 is expressed in a broad stripe including the developing organ of Corti in the cochlea, as well as in the
vestibular sensory patches (Figures 4A and 4B); this
finding is consistent with an early role in sensory patch
development. At E16.5, the stage at which the first signs
of hair cell PCP appear (Figure 1F), and later at P0,
Celsr1 is expressed in the organ of Corti and in adjacent
greater epithelial ridge cells (Figures 4C–4E). At P0, comparison with expression of lunatic fringe (Lfng), a known
Celsr1 Acts in Planar Polarity and Neurulation
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Figure 2. Crsh and Scy Homozygotes and Compound Heterozygotes Exhibit Severe Neural Tube Defects
Figure 3. Positional Cloning of the Mutations in Scy and Crsh
(A–E) Homozygous mutant fetuses for (B and C) Crsh ([B], E13.5;
[C], E14.5) or (E) Scy (E12.5) alleles exhibit the severe neural tube
defect of craniorachischisis, whereby the neural tube is open from
the midbrain/hindbrain boundary throughout the spine. (A and D)
Wild-type littermates exhibit a completely closed neural tube.
(F–G) Embryos collected at E8.5 (ⵑ10 somite stage) reveal initiation
of neural tube closure in the (F) wild-type (between arrows) but
reveal failure of Closure 1 in the (G) Crsh/Crsh mutant.
(H and I) (I) Compound heterozygous fetuses (Scy/⫹, Crsh/⫹) also
demonstrate the severe neural tube defect (E13.5), whereas (H) wildtype littermates exhibit complete closure of the neural tube.
(J and K) (J) Eyelids have completely fused in the wild-type fetus
at E17.5, while a (K) homozygous Crsh littermate reveals the absence
of eyelids.
The scale bar represents 2 mm in (A)–(E), (H), and (I), 0.6 mm in (F)
and (G), and 2.4 mm in (J) and (K).
(A and B) Genetic mapping of (A) Scy and (B) Crsh mutations on
Chromosome 15. The figures to the left of the chromosome indicate
the distances between markers (in cM). The numbers following the
marker name (in parentheses) indicate the Mb position in the last
release of the mouse genomic sequence (nd ⫽ not determined).
The mapping data indicate that Scy and Crsh are localized in region
85.4–88.0 Mb and 74.7–88.3 Mb along Chromosome 15, respectively. Celsr1 is positioned at 86.7 Mb.
(C–F) Sequence electropherograms showing nucleotide substitutions at base 3337 of Celsr1 in (C) Scy heterozygotes compared to
(D) wild-type, and at base 3126 in (E) Crsh homozygotes compared
to (F) wild-type.
(G) Comparison of nucleotide at bases 3126 and 3337 in Scy, Crsh,
and inbred mouse strains. The Celsr1 mutations are unique to Scy
and Crsh and are not seen in their parental strains (C3H/HeH and
BALB/c for Scy, and C3H and 101 for Crsh) or in six other strains
tested, including Mus musculus castaneus and Mus spretus.
marker for supporting cells in the sensory patch, suggests that Celsr1 and Lfng are expressed in a complementary pattern, with Celsr1 clearly expressed in hair
cells and Lfng expressed only in supporting cells (data
not shown).
Celsr1 is a mammalian homolog of Drosophila flamingo/starry night, which acts in the PCP signaling pathway together with frizzled, dishevelled, strabismus/van
gogh, and prickle [3]. Our analysis of the mouse Celsr1
mutants demonstrates that Celsr1 plays a role in PCP
in the mammalian inner ear, and that Celsr1 is also required for vertebrate neurulation. Although it remains
to be demonstrated whether Celsr1 acts in neurulation
through the PCP pathway or by some other mechanism,
it is intriguing that two other mouse mutants that demonstrate this severe NTD phenotype are also implicated in
the vertebrate PCP pathway. The loop-tail mutant has
a mutation in Vangl2 (formerly Lpp1/Ltap), which is the
homolog of Drosophila strabismus/van gogh [9, 10],
while a similar severe NTD phenotype is exhibited by
the double knockout of dishevelled 1 & 2 [11]. It has been
postulated that a vertebrate equivalent of the Drosophila
PCP signaling cascade is required for convergent extension movements during gastrulation in Xenopus and
zebrafish embryos [3, 12, 13]. Normal convergent extension is essential for neural tube closure in Xenopus and
zebrafish, suggesting a possible mechanism by which
PCP may regulate neurulation in mammalian embryos.
The coordinated orientation of stereocilia bundles reveals the planar cell polarity of the sensory hair cells in
the vertebrate inner ear [14, 15]. The misorientation of
hair cells seen in Scy and Crsh mutants from the earliest
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Figure 4. Celsr1 Expression in the Developing Inner Ear
(A–E) In situ hybridization of Celsr1 in the (A
and B) developing inner ear at E14.5 and in
the (C–E) cochlea at P0. Celsr1 is expressed
in patches corresponding to the maculae and
cristae of the inner ear and in an intense band
along the length of the cochlea that includes
the developing organ of Corti (square
bracket). At P0, supporting cells do not appear to be labeled by the Celsr1 antisense
probe, as shown by the pale regions between
outer hair cells (blue arrows). OC, organ of
Corti; OHC, outer hair cell rows 1–3; IHC, inner
hair cell; PC, posterior crista; AC, anterior
crista; LC, lateral crista; UM, utricular macula;
SM, saccular macula; GER, greater epithelial
ridge. The scale bars represent 250 ␮m in
(A)–(C), 20 ␮m in (D), and 10 ␮m in (E).
stage that hair bundle orientation is detectable indicates
a defect in the establishment of this polarity and is a
phenotype unique to the Celsr1 mutants. The distorted
shapes of supporting cell surfaces suggest that it is
polarity of the cell and not just the hair bundle that is
abnormal. Although many other mouse mutants have
been characterized with hair cell defects [16–22], none
appear to affect the initial establishment of planar polarity. For example, in waltzer (Cdh23) and shaker1 (Myo7a)
mouse mutants, hair bundles show normal polarity at
first, and progressive disorganization follows, indicating
that these molecules are required for maintenance but
not for the establishment of polarity of the bundle [16,
19, 22]. Some mutants with abnormal patterning of the
organ of Corti (e.g., Jag1, Jag2) [17, 18, 20, 21] may
show slightly disorganized polarity of outer hair cells,
but nothing like the extent of polarity disruption we see in
homozygous Scy or Crsh mutants. Rotated hair bundles
have been reported in OHCs of golden hamsters and
guinea pigs [23–26]. However, the phenotype was unlike
the pattern we see in the Celsr1 heterozygotes in that
a larger proportion of cells showed rotation, the rotation
was largely confined to the first row of outer hair cells,
the surrounding supporting cells looked normal, and it
was not clear if the cause was genetic.
We have shown that Celsr1 plays a role in planar cell
polarity mechanisms in mammals. However, a number
of other molecules have been shown to be involved in
establishing PCP in Drosophila [2], and it is likely that
similar components are involved in mammalian systems.
There are, in addition, at least three Celsr genes in mammals, Celsr1-3, all of which are expressed in the developing organ of Corti [8], and they may act in a combinatorial
manner to influence planar cell polarity. It will therefore
be important to examine mutants in other members of
this gene family and explore their interactions with other
genes of the vertebrate PCP pathway.
Experimental Procedures
Linkage Analysis and Mutation Screening
Genetic mapping of Scy was performed on mutant (n ⫽ 231)
[Scy/⫹ ⫻ C3H/HeH] ⫻ C3H/HeH backcross mice [27], while linkage
analysis of Crsh was performed on fetuses with the severe neural
tube defect (n ⫽ 29), derived from intercrosses of [BALB/c ⫻ Crsh/⫹]
F1 mice. Candidate genes were evaluated by comparative sequence
analysis of coding exons in mutant and wild-type mice.
In Situ Hybridization and Scanning Electron Microscopy
Cochleae were dissected to remove the outer shell and to expose
the organ of Corti for hybridization, and segments of the organ of
Corti were removed and mounted on slides to view as a surface
preparation after processing (E14.5, n ⫽ 5 antisense, 1 sense; E16.5,
n ⫽ 8 antisense, 2 sense; P0, n ⫽ 8 antisense, 3 sense). For SEM,
cochleae from Scy (aged 3–5 months, n ⫽ 4 Scy/⫹, 4 ⫹/⫹; aged
E18.5, n ⫽ 1 Scy/Scy, 13 Scy/⫹, 14 ⫹/⫹) and Crsh (aged 2–6 months,
n ⫽ 11 Crsh/⫹, 7 ⫹/⫹; aged E18.5, n ⫽ 8 Crsh/Crsh, 12 Crsh/⫹, 6
⫹/⫹; aged E16.5, n ⫽ 6 Crsh/Crsh, 18 Crsh/⫹, 5 ⫹/⫹) mutants were
prepared by the osmium tetroxide-thiocarbohydrazide (OTOTO)
method as described previously [16]. All comparisons were carried
out at equivalent locations along the length of the cochlear duct.
The utricular macula and all three cristae were examined by SEM
by using the same OTOTO protocol in Scy/⫹ (n ⫽ 12) and ⫹/⫹ (n ⫽
Celsr1 Acts in Planar Polarity and Neurulation
1133
12) adults. For quantification of rotation, cells were counted with
angles of rotation beyond the usual range (20⬚ toward base or apex
with respect to the length of the organ of Corti).
Supplemental Data
Supplemental Data including more detailed Experimental Procedures are available at http://www.current-biology.com/content/
supplemental.
Acknowledgments
We thank Nicholas Greene, Carles Gaston-Massuet, Caroline
Formstone, and Ivor Mason for many helpful discussions and Sarah
Breeds, Colin Clapham, Kit Doudney, Diana Hoult, Caroline Paternotte, Charlotte Rhodes, Lesley Rooke, Matthew Sims, Sarah
Smythe, and Lucie Vizor for technical assistance. This work was
supported by grants from the Medical Research Council, the Wellcome Trust, SPARKS, the British Heart Foundation, the Welton
Foundation, Defeating Deafness, the European Union, EC contract
QLG2-CT-1999-00988, and the Birth Defects Foundation.
Received: January 24, 2003
Revised: April 8, 2003
Accepted: May 8, 2003
Published online: May 14, 2003
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