First published online 29 March 2006
doi: 10.1242/dev.02347
Development 133, 1767-1778 (2006)
Published by The Company of Biologists 2006
Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation
Jianbo Wang1,
Natasha S. Hamblet1,*,
Sharayne Mark2,
Mary E. Dickinson3,
,
Brendan C. Brinkman4,
Neil Segil5,
Scott E. Fraser3,
Ping Chen2,
John B. Wallingford6 and
Anthony Wynshaw-Boris1,
1 Department of Pediatrics and Medicine, University of California, San Diego,
9500 Gilman Drive, MC 0627, La Jolla, CA 92093-0627, USA.
2 Department of Cell Biology and Otolaryngology, School of Medicine, Emory
University, 615 Michael Street, Atlanta, GA 30322, USA.
3 Divison of Biology and Beckman Institute, California Institute of Technology,
Pasadena, CA 91125, USA.
4 Department of Neuroscience, University of California, San Diego, 9500 Gilman
Drive, MC 0627, La Jolla, CA 92093-0627, USA.
5 Department of Cell and Molecular Biology, House Ear Institute, 2100 West Third
Street, Los Angeles, CA 90057.
6 Molecular Cell and Developmental Biology & Institute for Cellular and
Molecular Biology, 1 University Station C0930, University of Texas, Austin, TX
78712, USA.
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Fig. 1. Dvl1-/-; Dvl2-/- mutants
displayed reduced length-to-width ratio during neurulation. (A-D)
Five- to eight-somite stage Dvl1-/-; Dvl2+/- or
Dvl1-/-; Dvl2+/+ control embryos.
(A'-D') Dvl1-/-; Dvl2-/- mutants of
equivalent somite stages. Floor plates are expanded in Dvl1-/-;
Dvl2-/- mutants. (E) Length-to-width ratio (LWR)
changes of control and Dvl1-/-; Dvl2-/- mutant
embryos at different somite stage during neurulation. Each point represents
the average of at least three separate embryos. The error bars represent
standard deviation. *P<0.05;
**P<0.01.
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Fig. 2. In situ hybridization analysis of Brachyury (T) in
early headfold stage Dvl1-/-; Dvl2-/-
and Lp/Lp mutant embryos. (A) T, a known
downstream target gene of the canonical Wnt pathway, was expressed in the
notochord, node and primitive streak in control embryos
(Dvl1-/-; Dvl2+/-). (B) In
Dvl1-/-; Dvl2-/- mutants, T expression
in the node and primitive streak was maintained, suggesting normal activity of
the Wnt pathway. However, T expression in the notochord was
abnormally widened. T-positive cells were also observed outside of
the notochord (arrows), consistent with a disruption of cell movement towards
the central midline. Compared with wild-type littermates (C),
T expression was also widened in the notochord of Lp/Lp
embryos (D). T-positive cells were also observed outside of
the notochord (D, arrows).
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Fig. 3. Genetic interaction between Dvl2 and Lp and reduced
length-to-width ratio (LWR) in Lp/Lp and Dvl2-/-;
Lp/+ embryos during neurulation. (A) Similar to
Dvl1-/-; Dvl2-/- embryos, Lp/Lp
mutants displayed significant reduction of LWR during neurulation, while
Lp/+ embryos displayed intermediate reduction. At the eight-somite
stage, some of the neural folds were already apposed and started to fuse at
the dorsal midline in wild-type embryos (B). Parallel to the reduction
of LWR, however, the neural folds were still open in Lp/+ embryos
(C) and widely apart in Lp/Lp embryos (D) of the same
somite stage. Similar to both Dvl1-/-; Dvl2-/-
and Lp/Lp mutants, all Dvl2-/-; Lp/+ embryos also
displayed significant reduction of LWR during neurulation (E). From a
cross between Dvl1+/-; Dvl2+/-; Lp/+ and
Dvl2-/- mice, all progeny that were
Dvl2-/- or Dvl1+/-; Dvl2-/-
(F) displayed normal neural tube closure at E10.5. By contrast, all
Dvl1+/-; Dvl2-/-; Lp/+ and
Dvl2-/-; Lp/+ embryos displayed craniorachischisis
(G). The error bars represent s.d.
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Fig. 4. Domain requirement of Dvl2 for its involvement in convergent extension
and membrane distribution during neurulation. (A) Schematic diagram
of the genomic structure of the endogenous wild-type Dvl2 allele, the
targeted Dvl2 null allele (Dvl2 KO), the two wild-type
Dvl2 BAC transgenes, Dvl2-EGFP1 and Dvl2-EGFP2, and
three mutant Dvl2 BAC transgenes:  DIX-EGFP,
DEP-EGFP and dsh1-EGFP. Although Dvl2-EGFP1
had an EGFP cassette fused in-frame to the last codon of Dvl2 and a
LoxP site inserted into intron 2 for genotyping purpose (see Materials and
methods), Dvl2-EGFP2 contained an additional LoxP site inserted in
the 3' flanking gene Acadvl (acylcoenzyme A dehydrogenase, very
long chain). DIX-EGFP and DEP-EGFP deletion
mutant transgenes were generated by removing sequence encoding amino acids
67-159 and 442-736, respectively. dsh1-EGFP point mutation was
generated by replacing AAG with ATG to introduce a K M substitution at
amino acid 446. Lower panel showed sequence alignment of the wild-type
Dvl2-EGFP and the modified dsh1-EGFP BAC transgenes, as well
as endogenous mouse Dvl1, Dvl2, Dvl3, Xenopus XDsh,
Drosophila Dsh and the dsh1 mutation identified in fly.
Although Dvl2-EGFP1, Dvl2-EGFP2 (B) and
DIX-EGFP (F) can fully rescue the neural tube closure
defects in Dvl1-/-; Dvl2-/- mutants,
DEP-EGFP transgenes completely failed to rescue the neural
tube defects in Dvl1-/-; Dvl2-/- mutants
(C). Confocal analysis indicated that although DIX-EGFP was
primarily localized to the membrane (G,H), DEP-EGFP was
evenly distributed in the cytoplasm (D,E).
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Fig. 5. Dvl2-EGFP was primarily localized to the membrane in the neuroepithelium
during neurulation. Single optical sections from laser-scanning confocal
microscopy of neuroepithelium from neurulating embryos showed that, in the
wild-type background, Dvl2-EGFP was primarily localized to the plasma membrane
in the neuroepithelium (A,E,I). Consistent results were observed in wild-type
and Lp/Lp mutant two-somite stage embryos stained with an anti-EGFP antibody
(A,B) or through direct visualization of Dvl2-GFP signal from
briefly fixed five-somite stage embryo (E,F) or live
eight-somite stage embryos in culture (I,J). The apparently
diffuse cytoplasmic distribution of Dvl2-EGFP and actin in some cells in A,B
was due to scanning of the focal plane immediately underneath the apical
plasma membrane during confocal laser microscopy. Wild-type and mutant embryos
from the two- and five-somite stage were also stained with Phalloidin
(C,D,G,H) to label F-actin. When the entire series
of z stacks were examined (data not shown), it was clear that both
Dvl and actin are localized to the lateral plasma membrane as individual
optical sections are scanned more basally through the cell. Curiously,
Dvl2-EGFP and actin distribution also appeared to be more diffuse in the
somatic mesoderm at the five-somite stage (E-H). Overall Dvl2-EGFP
distribution in the neuroepithelium was not altered in the Lp/Lp
mutants (B,D,F,H,J,L; data not shown). (K,L) DIC images of live
wild-type (K) or Lp/Lp mutant (L) embryos in culture. NE, neuroepithelium; SM,
somatic mesoderm.
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Fig. 6. Localization of Dvl2-EGFP and dsh1-EGFP in the organ of Corti.
(A-E) Confocal scanning at the apical surface of hair cells in
whole-mount organ of Corti at E18.5, showing the native Dvl2-EGFP (A,C,E) or
dsh1-EGFP (B,D) signal. (A'-E') are the overlay of Dvl2-GFP (green,
A',C',E') or dsh1-EGFP (green, B',D') and hair cell membrane and stereociliary
bundles (red) outlined with phalloidin staining. At E18.5 in an otherwise
wild-type background, dsh1-EGFP localization along the distal membrane
appeared to be more punctate (asterisk in B and B') when compared with
wild-type Dvl2-EGFP (A,A'). By contrast, in Dvl1-/-;
Dvl2-/- background at E16.5, dsh1-EGFP localization on the
membrane was mostly lost and accumulation in the cytoplasm was observed
(arrowheads in D,D'). Dvl1-/-; Dvl2-/-;
dsh1-EGFP mutants also display mis-alignment of inner hair cells (arrows
in D'). Although wild-type Dvl2-EGFP could maintain an even distribution along
the distal membrane in Dvl1-/-; Dvl2-/-
background at E16.5 (C,C'), its membrane localization was reduced and no
longer restricted to the distal side (arrows in E,E') in Lp/Lp
mutants at this stage.
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Fig. 7. dsh1-EGFP failed to rescue the craniorachischisis, cochlear
elongation and stereociliary polarity phenotypes in
Dvl1-/-; Dvl2-/- embryos.
(A,D) Dvl1-/-; Dvl2-/-; dsh1-EGFP
embryos (right in A) displayed craniorachischisis at E9.5 and E16.5,
suggesting that dsh1-EGFP was not able to compensate for the convergent
extension defect in neurulation Dvl1-/-;
Dvl2-/- mutants. (B) Confocal analysis indicated that
dsh1-EGFP was primarily localized to the plasma membrane in the
neuroepithelium. (C) Confocal analysis of phalloidin staining.
(E,F) Cochlea recovered at p0 from Dvl1-/-;
Dvl2+/- and Dvl1-/-; Dvl2-/-;
dsh1-EGFP mice, indicating that dsh1-EGFP also failed to rescue
the cochlea elongation defect in Dvl1-/-;
Dvl2-/- mutants that was also thought to be due to disruption
of convergent extension. (G,I) Confocal scan at either the base
(G) or medial region (I) showing uniform stereociliary orientation in the
organ of Corti of a p0 Dvl1-/-; Dvl2+/+ mouse.
(H,J) However, in the organ of Corti of a
Dvl1-/-; Dvl2-/-; dsh1-EGFP mouse, the
stereociliary bundles were mis-oriented (arrows) in some hair cells.
Misalignment in the outer hair cells could also be observed.
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© The Company of Biologists Ltd 2006
