First published online 21 June 2006
doi: 10.1242/dev.02457
Development 133, 3005-3013 (2006)
Published by The Company of Biologists 2006
The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development
Masayo Sakaki-Yumoto1,*,
Chiyoko Kobayashi1,*,
Akira Sato2,
Sayoko Fujimura1,
Yuko Matsumoto2,
Minoru Takasato3,
Tatsuhiko Kodama4,
Hiroyuki Aburatani4,
Makoto Asashima3,
Nobuaki Yoshida5 and
Ryuichi Nishinakamura1,2,6,*,
1 Division of Integrative Cell Biology, Institute of Molecular Embryology and
Genetics, Kumamoto University, Kumamoto 860-0811, Japan.
2 Division of Stem Cell Regulation, The Institute of Medical Science, The
University of Tokyo, Tokyo 108-8639, Japan.
3 Graduate School of Sciences, The University of Tokyo, Tokyo 113-8654,
Japan.
4 Research Center for Advanced Science and Technology, The University of Tokyo,
Tokyo 153-8904, Japan.
5 Laboratory of Gene Expression and Regulation, The Institute of Medical
Science, The University of Tokyo, Tokyo 108-8639, Japan.
6 PRESTO, JST, Saitama 332-0012, Japan.
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Fig. 1. Embryonic lethality of Sall4-deficient mice. (A)
Targeting strategy of Sall4. Ovals represent the zinc-finger domains.
E, EcoRI. (B) Southern blot analysis using the probes
described in Fig. 1A. (C) Hematoxylin and Eosin staining of wild-type
(+/+) and Sall4-deficient (-/-) embryos at E6.5. Arrow indicates
epiblast. (D) In situ hybridization of Sall4, epiblast markers
(Fgf4, Nodal and Oct3/4), and a marker for trophectoderm and
extra-embryonic ectoderm (H19) in wild-type (+/+) and
Sall4-deficient (-/-) embryos at E5.8. H19 signal is absent in the
epiblast of both +/+ and -/- embryos. Black arrowhead, epiblast; white
arrowhead, extra-embryonic ectoderm. Serial sections from two wild-type and
two Sall4-deficient embryos are shown. Left three columns and right
two comulns are from different embryos.
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Fig. 2. Requirement of Sall4 for inner cell mass proliferation in
blastocysts in vitro. (A) Normal development of wild-type (+/+),
heterozygous (+/-) and Sall4-null (-/-) blastocysts (E 3.5). Sall4 is
expressed in the inner cell mass and trophectoderm (left column). Oct3/4 (red)
and Cdx2 (green) staining shows that commitment to the inner cell mass and
trophectoderm occurs normally in the Sall4 homozygotes (compare
middle and right columns). (B) Reduction of inner cell mass in
Sall4-null blastocysts cultured in vitro. Uppermost row shows
phase-contrast photo at 5 day of culture. Second row shows phase contrast at 3
day of culture. Lower two rows show immunostaining of Oct3/4 and Sall4. E,
primitive endoderm; I, inner cell mass; T, trophectoderm. (C) RT-PCR
analysis of markers in blastocysts cultured for 3 days. All the lineage
markers are expressed. (D) Reduced proliferation of the inner cell mass
of Sall4-null blastocysts cultured for 3 days. BrdU incorporation of
the inner cell mass is reduced in Sall4-null blastocysts (arrow),
compared with wild type (arrowhead). (E) Failure of growth of
Sall4-null inner cell mass free from the trophectoderm.
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Fig. 3. Reduced proliferation of Sall4-null ES cells. (A)
Northern blot analysis showing the reduction of Sall4 upon
Sall4-siRNA treatment. KD (knockdown), treated with Sall4-siRNA; NC (negative
control), treated with control siRNA. (B) Transiently reduced
proliferation of ES cells upon Sall4-siRNA treatment. Cells were counted in
triplicate. (C) Conditional disruption of Sall4 in ES cells.
When a Sall4-IRES-Hyg vector was introduced into cells heterozygous
for a floxed allele of Sall4 (flox/+), both alleles were targeted
with a similar frequency, resulting in two types of cells: flox/- and +/-.
Upon infection with adenovirus expressing Cre, flox/-cells became almost
Sall4-null by day 3, determined by western blot (lowest panels).
White triangle, Frt; black triangle, loxP. (D) Reduced proliferation of
Sall4-null ES cells. Cell expansion rate over 16 days is shown, using
Sall4-null (-/-) versus flox/-cells obtained upon the same Cre
treatment. Analysis was carried out in triplicate. (E) Reduced S phase
and increased G1 phase in the Sall4-null ES cells. Consistent data
were obtained from two independent experiments using three Sall4-null
cells, and the representative data is shown. (F) Normal morphology and
positive staining of Oct3/4 of a Sall4-null ES colony. (G)
Northern blot analysis of Sall4-deficient ES cells. Two heterozygous
(flox/-), and two Sall4-null ES (-/-) clones are shown. (H)
Chimeric embryo formation from Sall4-null ES cells transfected with
GFP. (left) High contribution of GFP-expressing cells in the E7.5 embryo.
(Right) A section of the chimera was stained by an anti-GFP antibody and
detected by DAB.
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Fig. 4. Phenotypes caused by Sall4 haploinsufficiency. (A)
Anal stenosis (above) and megacolon (below) of 5-week-old
Sall4-heterozygous mice. Black arrowhead, anus in wild type; open
arrowhead, imperforate anus in the heterozygotes. (B) Ventricular
septum defect in Sall4 heterozygotes at E18.5. Black arrowhead,
ventricular septum in wild-type; open arrowhead, ventricular septum defect in
the heterozygotes. Hematoxylin and Eosin staining. (C) Normal formation
of digits, metacarpus and metatarsus in new born Sall4 heterozygotes.
Stained with Alcian Blue and Alizarin Red S. (D) Normal formation of
abducens nuclei (arrowhead) and facial nerve (arrow) in 8-week-old
Sall4 heterozygotes. Serial sections were examined by
Klüver-Barrera staining. (E) Normal development of inner ear
structure in Sall4 heterozygotes at E17.5. Hematoxylin and Eosin
staining. (F) Exencephaly of Sall4-heterozygous mice at
E14.5.
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Fig. 5. Genetic interactions of Sall4 and Sall1.
(A) Bilateral renal agenesis in Sall1/4 heterozygotes. Six out
of the 38 compound heterozygotes analyzed had this phenotype, while 10 had
unilateral agenesis. a, adrenal glands; b, urinary bladder; c, colon; k,
kidney. (B) Anal stenosis in Sall1/4 heterozygotes at E17.5
(right two panels). Black arrowhead shows complete stenosis of the rectoanal
junction; white arrowhead shows absence of the rectum. a, anus; b, urinary
bladder; r, rectum. (C) In situ hybridization of Sall4 and
Sall1 at E8.5. The upper side is the anterior region of the embryo
(transverse section). Black and white arrowheads indicate the mesenchyme and
neuroepithelium, respectively. (D) Sall4 and Sall1
expression in the anorectal region at E11.5 (arrowheads). Heterozygotes of
Sall4-ßgeo and Sall1-lacZ
(Nishinakamura et al., 2001 )
were stained using X-gal. (E) Overlap of Sall4 and
Sall1 in the developing heart at E11.5. Sall4 is expressed
in myocardium (arrowhead), while Sall1 is expressed in myocardium
(arrowhead) and endocardium (arrow). Sall4-ßgeo and
Sall1-lacZ mice were stained using X-gal. (F)
Immunocytochemistry of Sall4 and Sall1, and counterstaining with DAPI in ES
cells. (G) Binding of Sall4 and Sall1 shown by immunoprecipitation
using ES cell lysates.
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Fig. 6. Mislocalization of Sall4 from the heterochromatin by a truncated Sall1
that functions in a dominant-negative manner. (A) Requirement of
the C-terminal zinc finger (Zn4) of Sall4 for heterochromatin localization
(rectangle). Zn4 is also sufficient for heterochromatin localization (broken
outline). Mutants of Sall4-GFP fusion were expressed in NIH 3T3 cells.
Zinc-finger clusters are categorized as Zn1, Zn2, Zn3 and Zn4, as shown.
(B) Requirement of the C-terminal zinc fingers (Zn4 and Zn5) of Sall1
for heterochromatin localization (rectangle). Zn4 and Zn5 are also sufficient
for heterochromatin localization (broken outline). Mutations in Zn2 and Zn5
also show a defect in heterochromatin localization, though these two clusters
are not sufficient for proper localization in the heterochromatin. (C)
Co-transfection of truncated Sall1
(Sall11-435-DsRed) and Sall4-GFP into NIH 3T3
cells. Heterochromatin localization of Sall4 is disrupted by C-terminally
truncated Sall1.
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© The Company of Biologists Ltd 2006
