First published online 27 April 2005
doi: 10.1242/dev.01844
Development 132, 2535-2546 (2005)
Published by The Company of Biologists 2005
Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex
Noriyuki Nishioka1,2,
Seiichi Nagano3,
Rika Nakayama4,
Hiroshi Kiyonari4,
Takashi Ijiri5,
Kenichiro Taniguchi6,
William Shawlot6,
Yoshihide Hayashizaki7,
Heiner Westphal8,
Richard R. Behringer9,
Yoichi Matsuda5,10,
Saburo Sakoda3,
Hisato Kondoh2 and
Hiroshi Sasaki1,*
1 Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
2 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka,
Suita, Osaka 565-0871, Japan
3 Department of Neurology, Graduate School of Medicine, Osaka University, D-4,
2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
4 Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN Center
for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, Hyogo
650-0047, Japan
5 Division of Bioscience, Graduate School of Environmental Earth Science,
Hokkaido University, North 10 West 8, Sapporo 060-0810, Japan
6 Department of Genetics, Cell Biology, and Development, University of
Minnesota, Minneapolis, MN 55455, USA
7 Laboratory for Genome Exploration Research Group, Genomic Sciences Center,
RIKEN, Yokohama, Kanagawa 230-0045, Japan
8 Laboratory of Mammalian Genes and Development, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda, MD
20892, USA
9 Department of Molecular Genetics, The University of Texas M. D. Anderson
Cancer Center, Huston, TX 77030, USA
10 Center for Advanced Science and Technology, Hokkaido University, North 10 West
8, Sapporo 060-0810, Japan
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Fig. 1. Gross phenotype of headshrinker mutants. (A-H) External views
(A,B) and skeletal specimens (C-H) of wild-type (A,C,E,G) and
headshrinker (hsk) mutant (B,D,F,H) P0 neonates. Higher
magnification views of the head region of C and D (C',D'), sternum
(E,F) and vertebral bones (G,H). Arrowheads and asterisks in E,F indicate
asymmetrical attachment of ribs to the sternum, and anteroposterior
dislocation of the sternum, respectively; arrowheads in H indicate bifurcation
and/or fusion of ribs. (I-P) The external morphologies of wild-type (I-L) and
hsk mutant embryos (M-P). The arrowhead in N indicates the
abnormality in the anterior neural folds. (Q) Phenotypic variability of
hsk mutants. Sections of wild-type (R) and hsk mutant (S)
embryos; asterisks indicate somites. c1, atlas; e, exoccipital bone; f,
frontal bone; i, interparietal bone; mn, mandible; mx, maxilla; n, nasal bone;
nt, neural tube; p, parietal bone; pm, premaxilla; s, supraoccipital bone.
Scale bar: 5 mm in C,D; 1 mm in C',D'; and 20 µm in R,S.
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Fig. 2. Identification of the transgene insertion site in headshrinker
mutants. (A,C) Chromosomal FISH showing the single transgene insertion site at
Chr4 C5-C6 (arrow). (B,D) Hoechst 33258 staining of the chromosomes shown in A
and C. (C,D) Close up of the transgene insertion site. (E) A physical map of
the area surrounding the transgene insertion site. The long horizontal bar at
the center represents Chr4, and the positions along the chromosome are given
in Mb above the bar. The short arrows below the bar and the numbers indicate
the positions of genes and their accession numbers, respectively. The upper
bar marked with thinner vertical lines shows the detailed structure of the
Ssdp1 gene. The vertical lines represent exons. Four copies of the
transgenes were inserted in the fourth intron of Ssdp1. P1, P2 and P3
indicate the positions of the PCR primers used for genotyping. (F,H,J,L)
Whole-mount in situ hybridization of Ssdp1. Left side view of ES
stage (F), LB stage (H), E9.0 (L) and frontal view of E8.0 (J) embryos.
(G,I,K) Transverse sections of embryos shown in F,H and J, respectively
(approximate position of sections are indicated by bars in F,H,J).
(G',I',K') Higher magnifications of G,I,K, respectively.
ADE, anterior definitive endoderm; AVE, anterior visceral endoderm; al,
allantois; de, definitive endoderm; ec, ectoderm; ep, epiblast (thickness
indicated by a bar); m, mesoderm; pp, prechordal plate; ps, primitive streak
(indicated by dotted lines in F,H); ve, visceral endoderm (thickness indicated
by arrowheads). Dashed lines in G',I',K' indicate positions
of AVE, ADE and pp, respectively. (M) In situ hybridization of Ssdp1
on a section of E9.0 embryo. The hybridization signals appeared brown. (N)
Whole-mount in situ hybridization showing the reduced expression of
Ssdp1 in hsk homozygous embryos. Embryos were grouped
according to their genotypes, and were subjected to whole-mount in situ
hybridization, performed at the same time in different wells. Similar results
were obtained by two independent experiments. Scale bars: 200 µm in L,N;
100 µm in F,H,I,J,K,M; 20 µm in G,G',I',K'. (O)
Northern blot analysis of E9.5 RNA showing expression of Ssdp1 RNA in
hsk homozygous embryos at a reduced level. (P) Relative expression
levels of the genes surrounding the transgene insertion site in hsk
mutants compared with wild type. Values shown represent the means and standard
errors of the relative expression levels. The number on each bar indicates the
number of samples analyzed. The expression level of Ssdp1 in
hsk heterozygotes (*P<0.01) and homozygotes
(**P<0.001) was reduced.
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Fig. 3. Rescue of the Ssdp1hsk/hsk mutant phenotype by the
Ssdp1 transgene. (A) Structure of the Ssdp1 transgene (TG).
(B-D) Lateral view of E9.5 embryos obtained by the cross of
TG#37/Ssdp1+/hsk or TG#141/Ssdp1+/hsk
with Ssdp1+/hsk mice. (B) Ssdp1hsk/hsk
embryo. (C) Completely rescued Ssdp1hsk/hsk embryo
carrying TG#37. (D) Partially rescued Ssdp1hsk/hsk embryo
carrying TG#141. Scale bar: 400 µm for B-D. (E) TG#37 completely rescues
the mutant phenotype. (F) The expression level of Ssdp1 in two TG
lines. Values shown represent the means and standard errors of the relative
expression levels of the sum of endogenous and transgenic Ssdp1 RNA,
as quantified by RT-PCR. The number on each bar indicates the number of
samples analyzed.
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Fig. 4. Expression of anterior patterning markers in
Ssdp1hsk/hsk mutants. In situ hybridization showing gene
expression in the anterior visceral endoderm (AVE) (A-D), anterior definitive
endoderm (ADE) (E-H) and the prechordal plate (I-P) in wild-type and
Ssdp1hsk/hsk mutants. Wild-type (A) and mutant (B,
n=5/5) expression of Lefty1; wild-type (C) and mutant (D,
n=7/7) expression of Lim1; wild-type (E) and mutant (F,
n=4/4) expression of Hhex; wild-type (G,I) and mutant (H,
n=4/4; J, n=2/9) expression of Cerl; wild-type (K)
and mutant (L, n=0/6) expression of Dkk1, wild type (M) and
mutant (N, n=0/6) expression of Foxa2, and wild-type (O) and
mutant (P, n=0/3) expression of Gsc. n, number of embryos
expressing the gene/number of embryos analyzed. Broken lines in A-D and E-H
indicate the AVE and ADE, respectively. Arrows in I-P indicate the prechordal
plate. (Q-T) Sagittal section of wild-type (Q,S) and mutant (R,T) embryos
stained with Hematoxylin and Eosin. Scale bars: in D, 100 µm for A-D; in H,
100 µm for E-H; in P, 200 µm for I-P; in R, 200 µm for Q,R; in T, 200
µm for S,T.
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Fig. 5. Head defects in Ssdp1hsk/hsk mutants. In situ
hybridization showing the expression of various regional markers in wild-type
and Ssdp1hsk/hsk embryos. Wild-type (A,E) and mutant
Six3 expression (B, n=2/5; F, n=0/8); wild-type
(C,G) and mutant (D, n=7/7; H, n=1/5) Otx2
expression; wild-type (I) and mutant (J, n=0/2) Pax6
expression; and wild-type (K) and mutant (L, n=2/3) En2
expression. (M,N) Fgf8 was expressed in the commissural plate
(arrowhead) and MHB (black arrow) in wild-type embryos (M), while MHB
expression in Ssdp1hsk/hsk mutants was observed at the
anterior tip (black arrow in N, n=6/10). Expression of
Krox20 was unaffected (M,N, white arrows). (O,P) Wnt1
expression in the MHB (O; arrow) was observed at the anterior tip in some
Ssdp1hsk/hsk mutants (arrow in P, n=3/5). Scale
bars: in D, 200 µm for A-D; in P, 400 µm for E-P.
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Fig. 6. Apoptosis and cell proliferation in Ssdp1hsk/hsk
mutants. Sections showing the distribution of apoptotic (A,B) and
proliferating (E,F) cells in the head region of wild-type (A,E) and
Ssdp1hsk/hsk mutant (B,F) embryos. (D) Scale bar in F, 400
µm for A,B,E,F. (C,D,G,H) Quantitation of apoptotic and proliferating cells
in various tissues of wild type and Ssdp1hsk/hsk mutants
at E8.0 and E8.5. Values shown represent the means and standard errors of the
percentages of the apoptotic (C,D) or proliferating (G,H) cells to total
cells. Black and white bars represent wild-type and
Ssdp1hsk/hsk mutant embryos, respectively. The number
above each bar indicates the number of samples analyzed. The tissues with
significant differences between wild-type and mutant are indicated
(*P<0.05, **P<0.01). mesen,
mesenchyme.
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Fig. 7. Ssdp1 acts as a coactivator component of a Lim1-Ldb1-Ssdp1 complex. (A)
E8.0 Lim1-lacZ knock-in embryo stained for ß-galactosidase
activity. (B) Cross-section of the embryo shown in A; approximate sectioning
position is indicated in A. (C) Whole-mount in situ hybridization of Ldb1 in
E8.0 embryo. (D) Cross-section of embryo shown in C. Scale bars in C,D: 200
µm. (E) Schematic representation of the effector and the reporter plasmid
used in the transfection assay described in F. (F) The fusion protein
comprised of the GAL4 DNA-binding domain and Ssdp1 activated reporter gene
expression in a dose-dependent manner. (G) Schematic representation of the
effectors and a reporter used in the transfection assays described in H and I.
(H) Effects of Lim1, Ldb1 and Ssdp1 on the Gsc promoter. (I) Effects
of varying Ssdp1 concentration on the activity of Lim1- and Ldb1-mediated
reporter gene expression. Values are the means and standard errors of
duplicate experiments. (J) A model for the action of Ssdp1. Ssdp1, Ldb1 and
Lim1 constitute a ternary complex and regulate genes expressed in the
prechordal plate.
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Fig. 8 Genetic interactions of Ssdp1 with Lim1 and
Ldb1. (A-D) E9.5 embryos obtained by crossing
Ssdp1+/hsk and Lim1+/- mice. (A)
Wild-type embryo. (B-D) Double heterozygotes, which can be classified into
three types: I, normal (B, n=6); II, microcephaly (C, n=5);
III, dwarfism (D, n=4). (E-G) E9.5 embryos obtained by crossing
Ssdp1+/hsk and Ldb1+/- mice. (E)
Wild-type embryo. (F,G) Double heterozygotes showing no apparent abnormalities
(F, n=7) or varying degrees of abnormality (G, n=6). Scale
bar: 400 µm.
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© The Company of Biologists Ltd 2005
