The Notch signaling pathway controls embryonic cell-fate
decisions in a variety of cell lineages in flies, worms and
mammals (Artavanis-Tsakonas et al., 1999). Improper Notch
signaling by genetic alteration often leads to developmental
defects or cancer in humans and rodents (Harper et al., 2003;
Radtke et al., 2002). Thus, it is essential to identify and
understand the key components of the Notch signaling
The core components in Notch signaling include the ligands
Delta and Serrate, the receptor Notch, and the transcription
factor Suppressor of Hairless [Su(H)] in Drosophila. Notch
signaling is initiated by the interaction of the Notch receptor
with its ligands (Lai, 2004; Schweisguth, 2004). These
interactions induce proteolytic cleavage (S2) of the Notch
receptors, which results in membrane-bound Notch fragments
(Brou et al., 2000). After the S2 cleavage by metalloproteases,
the remaining receptor fragments are cleaved at a third site (S3)
within the membrane, by γ-secretase complexes containing
presenilin 1 and presenilin 2, nicastrin and Aph1 (De Strooper,
2003; Mumm et al., 2000). The released intracellular fragments
of Notch (Nicd) translocate to the nucleus to form
transcriptional activator complexes with Su(H)/CBF1/RBP-Jκ.
These complexes activate Notch target genes, such as
Hairy/E(spl)-related basic helix-loop-helix (bHLH) repressors
(Iso et al., 2003).
Although much is known about Notch signal transduction
afterthe receptor undergoes the ligand-dependent S2 cleavage,
the mechanism by which the Notch ligands engage Notch and
trigger itscleavage is less understood. It has been proposed that
the endocytosis of Notch ligands on signal-sending cells that
are bound to Notch on adjacent signal-receiving cells induces
the S2 and S3 cleavage of the receptor, thus activating signal
transduction (Parks et al., 2000). Delta-Notch interactions
result in the endocytosis of Delta in the signaling cell, which
carries along the bound Notch extracellular domain, and
endocytosis-defective Delta mutants have reduced signaling
capacity (Parks et al., 2000). These studies in Drosophila
suggested that the endocytosis of Notch ligands might be
important for effective Notch signaling.
To date, there are two candidate genes, neuralized (Neur;
Neurl in mouse) and mind bomb 1 (Mib1) that promote the
ubiquitination and the endocytosis of Notch ligands. The neur
and mib1 mutants have defects in Notch activation in
Drosophila and zebrafish, respectively (Boulianne et al., 1991;
Itoh et al., 2003). However, disruption of the Neur1 gene in
mice did not generate the characteristic Notch phenotypes
displayed by Drosophila neur mutants, suggesting that
unknown murine Neur1 homologues might compensate for the
loss of Neur1 expression in mammals (Ruan et al., 2001;
Vollrath et al., 2001). This discrepancy also raised the
possibility that Mib1 is the functional homologue of Neur1 in
The Delta-Notch signaling pathway is an evolutionarily
conserved intercellular signaling mechanism essential for
cell fate specification. Mind bomb 1 (Mib1) has been
identified as a ubiquitin ligase that promotes the
endocytosis of Delta. We now report that mice lacking Mib1
die prior to embryonic day 11.5, with pan-Notch defects in
cardiogenesis. The Mib1–/–
expression of Notch target genes Hes5, Hey1, Hey2 and
Heyl, with the loss of N1icd generation. Interestingly, in the
mutants, Dll1 accumulated in the plasma
membrane, while it was localized in the cytoplasm near the
embryos exhibit reduced
nucleus in the wild types, indicating that Mib1 is essential
for the endocytosis of Notch ligand. In accordance with the
pan-Notch defects in Mib1–/–embryos, Mib1 interacts with
and regulates all of the Notch ligands, jagged 1 and jagged
2, as well as Dll1, Dll3 and Dll4. Our results show that Mib1
is an essential regulator, but not a potentiator, for
generating functional Notch ligands to activate Notch
Key words: Notch signaling, Mind bomb, Endocytosis, Notch ligand,
Mind bomb 1 is essential for generating functional Notch ligands to
Bon-Kyoung Koo1,*, Hyoung-Soo Lim1,*, Ran Song1, Mi-Jeong Yoon1, Ki-Jun Yoon1, Jin-Sook Moon1,
Young-Woong Kim1, Min-chul Kwon1, Kyeong-Won Yoo3, Myung-Phil Kong1, Jinie Lee1, Ajay B. Chitnis2,
Cheol-Hee Kim3and Young-Yun Kong1,†
1Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, South Korea
2Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
3Department of Biology, Chungnam National University, Taejeon 305-764, South Korea
*These authors contributed equally to this work
†Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 31 May 2005
Development 132, 3459-3470
Published by The Company of Biologists 2005
mice, because both Neur and Mib1 interact with Delta and
promote its endocytosis through the ubiquitination (Le Borgne
and Schweisguth, 2003).
Zebrafish mib1 mutants exhibit not only a severe
neurogenic phenotype, but also a wide range of additional
defects in the development of somites, neural crest and
vasculature, all indicative of defective Notch signal
transduction (Jiang et al., 2000; Jiang et al., 1996; Lawson et
al., 2001). The phenotypes of zebrafish mib1 mutants are
much more severe than those of deltaA (dx2), deltaD (after
eight) and notch1 (deadly seven) (Bingham et al., 2003; Gray
et al., 2001; Riley et al., 1999). These remarkable phenotypes
have suggested that mib1 is likely to encode a core component
of the Notch pathway in zebrafish. However, the lack of other
zebrafish mutants with pan-Notch defects prevents a
comparative study between mutants. We reported that Mib1
promotes the ubiquitination of zebrafish DeltaD and DeltaB,
suggesting that Mib1 might regulate multiple ligands (Itoh et
al., 2003). However, there are four Delta homologues (deltaA,
deltaB, deltaC and deltaD) and three jagged (Jag) homologues
(jagged1, jagged2 and jagged3) in zebrafish. Thus, it is
necessary to determine whether Mib1 regulates other ligands,
such as jagged homologues and other Delta homologues, and
whether Mib1 is an essential core component in Notch
signaling from nematodes to mammals.
In this study, we examined whether Mib1 plays an essential
role in Notch signaling pathways by generating Mib1-gene
targeted mice. Mib1-deficient mice exhibited pan-Notch
defects, such as a lack of somitogenesis, impaired vascular
remodeling and accelerated neurogenesis. Consistent with
these findings, Mib1–/–embryos showed completely defective
Notch activation, in terms of Nicd generation and Notch-target
gene expression. Interestingly, Mib1 directly interacts with all
of the known canonical Notch ligands [Delta-like (Dll) 1, 3 and
4, Jag1 and Jag2]. These data show that Mib1 is an essential
core component of the mammalian Notch pathway that
controls the function of multiple Notch ligands.
Materials and methods
Generation of Mib1 knockout mice
The IRES-lacZ-puro cassette was fused to exon 6, with the deletion
of exon 7 encoding amino acid 303 to 364 of the murine Mib1 protein
(see Fig. S1A in the supplementary material). E14K ES cells were
screened, and six clones showed homologous recombination. Three
clones were used to generate chimeric mice after injection into
C57BL/6 blastocysts. Subsequent breeding was carried out with
C57BL/6 mice to generate congenic mice and with FVB/N to test the
effects of the genetic background.
In situ hybridization
Details of the RNA in situ hybridizations on whole-mount or
sectioned embryos were described (de la Pompa et al., 1997).
Antisense DIG-labeled (digoxigenin) riboprobes were generated from
pGEM-T vectors (Promega) containing amplified cDNA fragments
(about 700~800 bp). Staining patterns were confirmed by
comparisons with previously published data, except for Mib1. Probe
information can be provided on request.
Histology and immunohistochemistry
For histological analysis, embryos and tissues were fixed in 4%
paraformaldehyde overnight at 4°C and 4 μm sections were cut and
stained with Hematoxylin and Eosin. Sections were incubated with
antibodies (Abs) for Nestin (Chemicon) and huC/D (Molecular
Probes) and then with Alexa-546-conjugated anti-mouse IgG Ab
(Molecular Probes). For BrdU labeling, pregnant mice were injected
with BrdU (150 μg/g) 2 hours before they were sacrificed. BrdU-
incorporation was analyzed with an anti-BrdU FITC-conjugated Ab
(BD). Apoptotic cells were detected by an in situ Cell Death Detection
Kit (Roche). For Dll1 staining, 10 μm cryosections were stained with
anti-Dll1 (T-20; Santa Cruz Biotechnology) Ab, followed by an
Alexa-594-conjugated secondary Ab. Endothelial cell staining of
whole-mount preparations was performed with a Flk1 antibody (Avas
12α1, BD) and a PECAM antibody (MEC13.3, BD), using the
Vectastain Elite ABC kit (Vector Laboratories).
Total RNA was extracted from complete yolk sacs and embryos, using
an RNeasy Micro kit (Qiagen) according to the manufacturer’s
instructions. Aliquots of 1 or 2 μg RNA were used for reverse
transcription (Omniscript RT, Qiagen) with oligo-dT priming. Real-
time RT-PCR reactions with SybrGreen quantification were set up
with 1/25 of each cDNA preparation in a Roche LightCycler. Relative
expression levels and statistical significance were calculated based on
a β-actin standard, using the LightCycler software. All amplicons
(100~200 bp) showed efficient amplification that allowed us to equate
one threshold cycle difference. Primer information can be provided
cDNA cloning and plasmid construction
The mouse Mib1, Dll1, Dll3, Dll4, Jag1 and Jag2 cDNAs were cloned
into the pGFP-N3 (Clontech) or pCS-MT3 vectors. The ΔEN1 and
Dll1 cDNAs were cloned into the HpaI site of pMSCV. D. Hayward
kindly provided the 8? wild-type and 8? mutant CBF Luc. All of
the cDNAs amplified by PCR were sequenced and tested for
expression by Western blotting.
Western blot analysis and co-immunoprecipitation assay
Embryos were homogenized in lysis buffer [10 mM Tris (pH 7.5), 150
mM NaCl, 5 M EDTA] containing a protease inhibitor mixture
(Roche). Generally, 25~40 μg of protein containing supernatants were
separated by size, blotted with primary and secondary Abs and
visualized with ECL plus (AmershamBiosciences). The primary Abs
used were as follows: rabbit anti-mouse DIP-1/Mib1 (gift from Dr
Gallagher), rabbit anti-actin (Sigma), rabbit anti-mouse Hes5
(Chemicon), rabbit anti-N1icd (Cell Signaling) and mouse anti-
Notch1 (mN1A; Chemicon). Immunoprecipitation was performed
previously described (Koo et al., 2005).
Isolation of embryonic fibroblasts, MSCV infection, Luc
assay, and neurosphere forming assay
Embryonic fibroblasts were isolated from Trypsin/EDTA digested
E9.5 embryos. For MSCV virus infection, a high titer virus soup was
produced with gp2-293 cells transfected with pMSCV (Clontech)
and VSV-G vectors. Embryonic fibroblasts were infected for 24
hours and selected to eliminate the uninfected cells. For the CBF-
Luc assay, the 8? wild-type and mutant CBF luc cassettes were
transfected with pRL-TK, using Lipofectamine 2000 (Invitrogen).
Luciferase activities were measured with a Dual Luciferase kit
(Promega). Neurospheres were generated as described (Grandbarbe
et al., 2003).
Subcellular localization analysis and flow cytometry
COS7 cells were transiently transfected with various cDNAs.
Subcellular localization analysis was performed previously described
(Koo et al., 2005). To detect the internalization of XD, the cells were
detached with dissociation buffer (Sigma) and stained with anti-HA
Ab (Santa Cruz Biotechnology) followed by anti-mouse Ab
conjugated with PE (BD). All samples were analyzed by flow
cytometry using a FACScan (BD).
Development 132 (15)Research article
3461Mib1 is essential for Notch signaling
Generation of Mib1–/–mice
Mib1–/–mice were generated as described in the supplementary
material (see Figs S1, S2). At E9.5, homozygous Mib1–/–
embryos were severely growth retarded, but they were present
in the expected Mendelian ratio (see Table S1 in the
supplementary material). Mib1–/–embryos that approximately
resembled their littermates with respect to size and
developmental stage could be found at E8.5, but only dead and
resorbed embryos were found at E11.5-E12.5.
Pleiotropic Notch defects in embryos lacking Mib1
At E9.5, the Mib1–/–embryos always lacked blood circulation
and were posteriorly truncated. Heart looping did not occur,
and the Mib1–/–embryos displayed an enlarged balloon-like
pericardial sac (Fig. 1B). Somitogenesis had begun albeit
irregular and fused, and embryonic turning occurred in the
majority of the mutants. The optic vesicles, the otic vesicles
and the first branchial arches were also formed. However, the
second branchial arches were completely absent (Fig. 1B).
Vasculogenesis of the yolk sac was impaired in the mutants
(Fig. 1D). Although an initial vascular plexus and primitive red
blood cells formed, the organization into a discrete network of
vitelline vessels did not occur. Furthermore, the yolk sacs had
a blistered appearance. Toluidine Blue staining of semi-thin
sections from wild-type and mutant yolk sacs revealed that the
mutants had only small capillaries, and lacked large vitelline
collecting vessels (Fig. 1F).
Transverse sections showed that the Mib1–/–embryos had
smaller and thinner hearts, with broaden pericardial cavity
Fig. 1. Pleiotropic Notch defects in
Mib1–/–embryos. (A,B) Wild-type (A)
and Mib1–/–(B) embryos at E9.0. The
wild-type embryo has the first and
second branchial arches (A, arrows),
while the Mib1–/–embryo only has the
first branchial arch (B, arrow) with a
distended pericardial sac (asterisk) and
small, irregular somites (arrowheads).
(C-F) Vascular defects in the yolk sac of
E9.5 Mib1–/–embryos (D,F), as
compared with the wild type (C,E). The
blood vessels are indicated (asterisks).
(G,H) Transverse sections of wild-type
(G) and Mib1–/–(H) embryos at E9.5.
The Mib1–/–embryo has a smaller dorsal
aorta (da; inset i), a thinner neural tube
(nt), loss of mesenchymal cells
(asterisk), an enlarged pericardial cavity
(#) and a fused notochord (inset ii).
(I,J) Transverse section of wild-type (I)
and coronal section of Mib1–/–(J)
embryos at E9.5. A kinked neural tube
(asterisk) and irregular somites (arrows)
are evident in the Mib1–/–embryo (K)
Myogenin expression (bracket) in E9.5
wild-type (left) and Mib1–/–(right)
embryos. (L-P) Expression of Uncx4.1
(L), Dll1 (M), Hes7 (N), lunatic fringe
(Lfng) (O) and Heyl (P) in E8.5 wild-
type (left) and Mib1–/–(right) embryos.
The Mib1–/–embryos lack the
characteristic expression of Uncx4.1 (L,
arrows), Dll1 (M, arrows and inset),
Hes7 (M, arrow and bracket), Lfng (O,
arrow and bracket) and Heyl (P, arrow
when compared with the wild-type embryos (Fig. 1H). These
sections also revealed that the mutants had a smaller dorsal
aorta, and displayed loss of mesenchyme cells and fusion of
the notochord to the neural tube (Fig. 1H). These data suggest
that Mib1 might be essential for somitogenesis, vasculogenesis
and cardiogenesis, which are reminiscent of the Notch-related
Impaired somitogenesis in Mib1–/–embryos
At E8.5, the Mib1–/–embryos were of normal size and
appearance, but failed to show normal somite segmentation
(not shown). Transverse sections of wild-type embryos and
coronal sections of Mib1–/–embryos at E9.5 also revealed
unevenly divided somites, with kinked neural tubes in Mib1–/–
embryos (Fig. 1I,J), while six or seven irregular somites were
present in anterior region of the E9.0 wild-type and Mib1–/–
embryos (Fig. 1K). To characterize the somitogenesis defects
in the Mib1–/–embryos, we analyzed the expression of
Uncx4.1, Dll1, Hes7, lunatic fringe (Lfng) and Heyl. The
expression of Uncx4.1, a homeobox gene expressed in the
posterior half of each somite (Leitges et al., 2000), was
undetectable in the segmental plate of E8.5 Mib1–/–embryos
(Fig. 1L). Consistently, the expression of Dll1 in the posterior
half of each somite was also absent (Fig. 1M). Hes7 and Lfng
expression normally oscillates in the presomitic mesoderm
(PSM) of E8.5 wild-type embryos, but the Hes7 expression was
disturbed and Lfng expression was lost in the Mib1–/–embryos
(Fig. 1N,O). Interestingly, the expression of Heyl, another
Notch target gene, was completely absent (Fig. 1P). Taken
together, the Mib1–/–embryos show a lack of somatic polarity
and oscillation, whereas their somitic myogenesis is not altered
in very early embryogenesis.
Premature neurogenesis in Mib1–/–embryos
As in zebrafish, murine Mib1 mutants also exhibit a severe
neurogenic phenotype. A histological analysis of Mib1–/–
embryos isolated at E9.5 revealed localized areas of cell death,
which did not seem to be random, but instead appeared
preferentially in regions of the brain tissue. Picnotic cells were
found in the neuroepithelium of the central nervous system,
particularly in the optic lobe and the hindbrain (Fig. 2B,D,H).
At E9.0~9.5, the forebrain and hindbrain of wild-type
embryos mostly consisted of nestin-positive neural precursor
cells, and only a small population of cells became huC/D
Development 132 (15) Research article
Fig. 2. Premature neurogenesis in
Mib1–/–embryos. (A,B) Sagittal
sections of wild-type (A) and Mib1–/–
(B) embryos at E9.5. The Mib1–/–
embryo has a smaller head, with
picnotic cells in the brain and inside
the ventricles. Enlarged views of the
forebrain are shown in insets.
(C-H) Sagittal and transverse sections
of E9.0-E9.5 brain regions from wild-
type (C,E,G) and Mib1–/–(D,F,H)
embryos. Sections were stained for
nestin (C,D; in red) and huC/D (E-H;
in red). BrdU incorporation (E,F; in
green) and TUNEL staining
(C,D,G,H; in green) were used for the
detection of proliferative and
apoptotic cells, respectively. Nuclear
DNA was stained with Hoechst (in
blue). (I) Impaired neurosphere
formation in Mib1–/–forebrains.
Neural stem cells are absent in E9.5
(n=3) and E9.75 (n=5) Mib1–/–
forebrains. (J) Expression of
neurogenin 1 (Ngn1), Dll1, Hes5 and
Lfng in the neural tubes of E9.5 wild-
type and Mib1–/–embryos.
(K,L) Neurogenin 1 (Ngn1) (K) and
Neurod1 (L) expression in E9.5 wild-
type (left) and Mib1–/–(right)
embryos. Note that the expression of
both Ngn1 and Neurod1 in the
trigeminal ganglia is increased in the
Mib1–/– embryos (arrows), and Ngn1
expression is also increased in the
neural tube of the Mib1–/– embryos
3463Mib1 is essential for Notch signaling
positive committed early neurons of the pial surface (Fig.
2C,E,G). By contrast, most of the brain cells in mutant
embryos became huC/D positive (Fig. 2F,H). Interestingly, in
spite of the massive neurogenesis, most of the cells still
remained nestin positive in the mutant brains (Fig. 2D), which
is similar to their phenotype in the Hes1/5DKOmutant brain
(Ohtsuka et al., 1999). In the Mib1–/–mutant brains, most of
the huC/D and nestin double-positive neuronal cells might be
postmitotic differentiating neurons. Consistent with the
premature differentiation into immature neurons in Mib1–/–
mice, the mutant brains had virtually no BrdU-positive cells,
whereas the wild-type brains had proliferative zones along with
the ventricle (Fig. 2E,F), indicating that the nestin-positive
cells in the mutant brain were not proliferative. Moreover, the
E9.5 and E9.75 Mib1–/–embryos lacked neurosphere-forming
cells, suggesting the absence of a neuronal stem cell population
in these stages (Fig. 2I). The mutant embryos had many
TUNEL-positive cells, and large numbers of detached
apoptotic cells were often visible as a mass in the ventricle
To further examine neurogenesis in Mib1–/–embryos, we
used in situ hybridization to analyze the expression of
neurogenin 1 and Neurod1, which are bHLH transcription
factors that are expressed during neuronal determination and
neuronal differentiation, respectively (Ross et al., 2003). In
E9.0 wild-type embryos, both neurogenin 1 and Neurod1 were
expressed mainly in the developing trigeminal ganglia (Fig.
2K,L). In E9.0 Mib1–/–embryos, neurogenin 1 was ectopically
overexpressed in the neural tube, and both markers were highly
induced in the trigeminal ganglia (Fig. 2K,L). To test whether
the massive neurogenesis in Mib1–/–embryos is caused by a
lack of Notch signaling, we examined the Hes5 and Lfng
expression in the neural tube. As expected, the Mib1–/–
embryos lacked Hes5 and Lfng expression, whereas Dll1
expression was upregulated (Fig. 2J). Thus, Mib1 is a critical
component of Notch-mediated
neurogenesis (de la Pompa et al., 1997; Ma et al., 1998).
lateral inhibition in
Vascular defects with the loss of arterial fate in
Dll4–/–, Notch1–/–and Hey1/2DKO mice comprise the molecular
cascades for suitable
(Dll4rNotch1rHey1/2) (Duarte et al., 2004; Fischer et al.,
2004; Gale et al., 2004; Krebs et al., 2000; Krebs et al., 2004).
We examined whether Mib1–/–embryos also have
similar vascular defects, with the loss of arterial
fate decision. For a detailed analysis of the
vasculature, the E9.5 wild-type and Mib1–/–
embryos were immunostained with an anti-Flk1
antibody to detect endothelial cells and
endothelial precursors (Fig. 3A-D). The Flk1
expression revealed that vasculogenesis had
occurred in both the wild-type and Mib1–/–
embryos. In the Mib1–/–embryos, however, the
development of the vasculature was impaired. In
the head region, the vessels were thin and
arterial fate decision
Fig. 3. Vascular defects in Mib1–/–embryos.
(A-D) Whole-mount Flk1 antibody staining of E9.0
wild-type (A,C) and Mib1–/–(B,D) embryos. Mib1–/–
embryos have relatively thin and disorganized blood
vessels. (A,B) Lateral view of the head; (C,D) Dorsal
view of the trunk. (E-N) Transverse sections of E9.0
wild-type (E,G,I,K,M) and Mib1–/–(F,H,J,L,N)
embryos, stained with an anti-PECAM antibody (E,F)
or labeled by in situ hybridization with specific probes
for ephrin B2 (G,H), Sm22 (I,J), Dll4 (K,L) and Hey1
(M,N). The PECAM-stained sections revealed the
marked reduction or loss of the dorsal aorta (da) in the
Mib1–/–embryos. The lack of vascular ephrin B2
expression (H; inset), smooth muscle cell recruitment
(J; inset), and Hey1 expression (N; inset) in the Mib1–/–
embryos is evident, whereas the Dll4 expression is
normal (L; inset). (O,P) Expression of vascular Notch
target genes (Hey1 and Hey2) and genes for
vasculogenesis (ephrin B2, EphB4, Vegf and Shh).
Total RNA from E9.0 wild-type and Mib1–/–yolk sacs
was analyzed by semi-quantitative RT-PCR (O). The
expression of Hey1 and Hey2 was analyzed by real-
time quantitative RT-PCR (P). The numbers on each
bar indicate the mean fold of induction, and the error
bars indicate the standard deviation. β-actin was used
for normalization. The results are representative of
three independent experiments. ***P<0.0001,
truncated, and did not form a finely branched tree (Fig. 3B).
The intersomitic vessels that form through angiogenic
sprouting were not present (Fig. 3D). This might be caused by
the lack of somitic structures. However, the anterior part of the
intersomitic vessels also showed an irregular ladder shape,
where the somitic defects were rather mild.
To examine the defects in arterial fate decision, we analyzed
the expression of PECAM, ephrin B2 (mRNA) and sm22
(mRNA) in transverse sections of E9.0 wild-type and Mib1–/–
embryos. PECAM and ephrin B2 were used as a pan-
endothelial cell marker and an arterial endothelial marker,
respectively (Fischer et al., 2004). sm22 is a marker for smooth
muscle cells that are recruited to the arterial endothelium
(Fischer et al., 2004). All of the mutant embryos had markedly
smaller dorsal aorta compared with the anterior cardinal veins
(Fig. 3F). Similar to the Dll4–/–, Notch1–/–and Hey1/2DKO
mutants, the Mib1–/–embryos had no or significantly reduced
expression of ephrin B2 in the endothelium of the dorsal aorta
(Fig. 3H) (Fischer et al., 2004). Smooth muscle cell
recruitment to the dorsal aorta was also dramatically reduced,
which might be caused by the loss of arterial identity (Fig. 3J).
To test whether the defect in artery formation is caused by
the lack of Notch activation, we examined the expression of
Dll4 and Hey1, a Notch ligand and a downstream target gene
for the arterial fate decision, respectively. Interestingly, Dll4
expression was unaffected in Mib1–/–dorsal aorta, but Hey1
expression was undetectable (Fig. 3L,N; see Fig. S3 in the
supplementary material), indicating that Dll4 expression itself
is not sufficient for the activation of the Notch target gene in
the absence of Mib1. To further test the notion that Hey1 and
Hey2 are downstream of Mib1-regulated Notch activation,
RNA from the embryonic yolk sacs of E9.0 wild-type and
mutant embryos was analyzed by RT-PCR and real-time
quantitative RT-PCR. Both Hey1 and Hey2 were expressed in
the wild-type yolk sacs, but the amounts of these transcripts
from the Mib1–/–yolk sacs were strongly reduced, by factors
of 3.7 and 1.6, respectively (Fig. 3O,P). We also detected
downregulation of the arterial-specific marker ephrin B2 in the
yolk sacs of Mib1–/–embryos, while the transcript level of its
receptor, Ephb4, which is highly expressed in veins, was
upregulated (Fig. 3O). Taken together, these results strongly
suggest that Mib1 is an essential component involved in arterial
Notch signaling defects in Mib1–/–mice
Based on the multiple Notch-related phenotypes observed in
Mib1–/–embryos, we tested the expression patterns of Notch-
related genes. Previous studies of mutants lacking presenilin
1/2, RBP-Jκ and POFUT1 revealed the marked upregulation of
Dll1 in the neural tube and brain, with the combined loss of
Hes5 expression in the neural tube (de la Pompa et al., 1997;
Donoviel et al., 1999; Shi and Stanley, 2003). In E9.0 Mib1–/–
embryos, Dll1 expression was strongly upregulated in the
neural tube (Fig. 4A, part a′) with the loss of Notch target
genes, such as Hes5 and Hey1 (Fig. 4A, parts g′,h′). Similar
up- and downregulation of these genes were also detected using
RT-PCR and quantitative real-time RT-PCR of E8.5 wild-type
and mutant embryos (Fig. 4B,C). Dll1 transcripts in mutant
embryos were upregulated about sevenfold and Hes5
transcripts were reduced about fivefold, when compared with
their wild-type counterparts. Hes1 was downregulated in the
first branchial arches (Fig. 4A, part f′). Hey1 was also
downregulated in the branchial arches and the forming somites
(Fig. 4A, part h′). In situ hybridization of Jag1 and Lfng in E9.0
embryos showed their reduced expression levels in the
branchial arch and the PSM, respectively, of mutant embryos
(Fig. 4A, parts b′,e′), while Notch1 and Notch2 showed
comparable expression levels and patterns except in the PSM
and somites (Fig. 4A, parts c′,d′). All of these results are
indicative of Notch signaling defects.
To investigate the possibility that the Notch components
(Notch1, Notch2, Dll1, Jag1, presenilins, mastermind 1, RBP-
Jκ and neur) are defective in Mib1–/–embryos, RT-PCR
analyses were performed. In short, all of these molecules were
normally expressed or upregulated in E8.5 Mib1–/–embryos
(Fig. 4B,C). Thus, we excluded the possibility that the Mib1–/–
embryos lack essential components for Notch signaling, except
To evaluate directly whether these remarkable changes in
gene expression are caused by the lack of Notch activation, we
examined the generation of the Notch1 intracellular domain
(N1icd) and its target gene product, Hes5. In E9.0 wild-type
whole-embryo lysates, N1icd was readily detected by western
blotting. By contrast, N1icd was not observed in E9.0 Mib1–/–
whole-embryo lysates (Fig. 5A). In accordance with the
defective generation of N1icd, Hes5 expression was markedly
reduced in Mib1–/–embryos (Fig. 5A). These defects in N1icd
generation and Hes5 expression in Mib1–/–embryos were not
due to the lack of Notch1 expression, as the Notch1 expression
in the Mib1–/–embryos was comparable with that in the wild-
type embryos (Fig. 5A). These results indicate that the Mib1–/–
embryos have defects in Notch activation, especially upstream
of the γ-secretase-mediated S3 cleavage.
To investigate whether the γ-secretase-mediated S3 cleavage
and its downstream signaling are intact in Mib1–/–embryos, the
Notch1 deleted extracellular domain (ΔEN1) was expressed in
embryonic fibroblasts (EF) from wild-type and Mib1–/–
embryos. ΔEN1 is readily cleaved by the γ-secretase complex
to release the active N1icd, independent of a ligand/receptor
interaction. In short, ΔEN1 was cleaved in both the wild-type
and Mib1–/–EFs, and the cleaved N1icd was readily translocated
to the nucleus to activate the transcriptional activity of
downstream target genes (Fig. 5B,C,D). These results clearly
show that the Mib1–/–embryos have no defect in the
downstream signaling of S3 cleavage or in S3 cleavage itself.
To evaluate directly the ligand function of the Mib1–/–embryos,
Xdelta1-Myc (XD-Myc) was expressed in the EFs from wild-
type and Mib1–/–embryos. XD-Myc-expressing EFs were co-
cultured with C2C12-Notch1 cells containing CBF-Luciferase
reporter gene (CBF-Luc). Notch activation was readily
observed in the co-culture with XD-Myc-expressing wild-type
EFs, but not in the co-culture with XD-Myc-expressing Mib1–/–
cells (Fig. 5F). When the mutant CBF-Luc reporter was used,
both co-cultures did not induce luciferase activity. Furthermore,
when Dll1-Myc and Jag1-Myc were used instead of XD-Myc,
the Notch activation was observed only in the co-culture with
wild-type EFs (Fig. 5F). To test whether murine Mib1 directly
induces the internalization of ligand, HA-tagged Xdelta1 (HA-
XD-Myc) was co-expressed with Mib1-GFP in COS7 cells. As
expected, Mib1-GFP induced internalization of Xdelta1 (Fig.
5E). Thus, the Notch signaling defects in the Mib1–/–embryos
might be due to the defective endocytosis of Notch ligands.
Development 132 (15)Research article
3465Mib1 is essential for Notch signaling
Interactions between Mib1 and all
known Notch ligands
Based on molecular interactions between
Mib1 and Delta, we speculated that the pan-
Notch phenotypes of Mib1–/–embryos might
be caused by the lack of multiple Notch
ligand-mediated signaling. To test this
possibility, we examined the interaction of
each murine Notch ligand (three Dll and two
Jag homologues) with Mib1. HA-tagged Mib1 (HA-Mib1)
protein was co-immunoprecipitated with all of the Myc-tagged
Delta-related Notch ligands (Dll1, Dll3 and Dll4) in HEK-
293A cells (Fig. 6A). Surprisingly, Jag1 and Jag2, the Serrate-
related Notch ligands, also co-immunoprecipitated with HA-
Mib1 under the same conditions (Fig. 6A).
To characterize the consequences of the interactions between
Notch ligands and Mib1, we tested whether murine Mib1
promotes the endocytosis of Notch ligands. Overexpression of
Myc-tagged Xenopus Delta (XD-Myc), murine Dll1 (Dll1-
Myc) and murine Jag1 (Jag1-Myc) in COS7 cells resulted in
the characteristic plasma membrane expression or cytoplasmic
expression with mesh-like patterns (Fig. 6B, parts b,c). When
GFP-tagged Mib1 (Mib1-GFP) alone was expressed in COS7
cells, it was localized in the cytoplasm as punctate structures
(Fig. 6B, part a). However, when both XD-Myc and Mib1-GFP
were co-expressed, the XD-Myc expression on the cell surface
was decreased, and the XD-Myc accumulated in the cytoplasm
as vesicular structures where it was co-localized with Mib1-
GFP, as previously described (Fig. 6B, part f) (Itoh et al.,
2003). Likewise, when Dll1-Myc and Mib1-GFP (Fig. 6B, part
d) or Jag1-Myc and Mib1-GFP (Fig. 6B, part e) were co-
expressed in COS7 cells, the expression patterns of Dll1-Myc
and Jag1-Myc were also changed, as in XD-Myc. In addition,
we found the similar localization of other Notch ligands, Dll3,
Dll4 and Jag2, when those ligands were co-expressed with
Mib1 (data not shown).
To directly evaluate whether Mib1 regulates the endocytosis
of Notch ligand in vivo, sections from E9.0 wild-type and
Mib1–/–embryos were stained with anti-Dll1 antibody. The
Fig. 4. Notch signaling defects in Mib1–/–
embryos. (A) Lateral view of E8.75~9.0 wild-type
(wt; a-h) and Mib1–/–(mt; a′-h′) embryos probed
for Dll1 (a,a′), Jag1 (b,b′), Notch1 (c,c′), Notch2
(d,d′) Lfng (e,e′), Hes1 (f,f′), Hes5 (g,g′) and Hey1
(h,h′) expression. There is ectopic overexpression
of Dll1 in the neural tube (a′; asterisk) and the
downregulation of Dll1 in the somite and PSM (a;
arrows, a′; arrowhead); loss of Jag1 expression in
the head, branchial arches and presomitic region
(b; arrows, b′; arrowhead); loss of Notch1 and
Notch2 expression in somites (c,d; arrows, c′,d′;
arrowheads); and ectopic overexpression of
Notch1 in the neural tube and trigeminal ganglia
(c′; asterisk); the loss of Lfng expression in the
trigeminal ganglia, newly forming somites and
PSM (e′; arrowheads); and the loss of Hes1, Hes5
and Hey1 expression in the first branchial arches
and PSM (Hes1, f′; arrowheads), the forebrain and
neural tube (Hes5, g′; arrowheads), and the first
branchial arches and newly forming somites
(Hey1, h′; arrowheads) of the Mib1–/–embryos.
(B) Expression of Notch target genes (Hes1, Hes5,
Hes7, Hey1, Hey2, Heyl) and Notch pathway
genes [Notch1, Notch2, Neur, RBP-jκ, Pres1
(presenilin 1), Pres2 (presenilin 2), Maml1
(mastermind-like1), Dll1, Jag1 and Mib1]. Total
RNA from E8.5 wild-type (wt) and Mib1–/–(mt)
embryos was analyzed by RT-PCR. β-actin was
used for normalization. The results are
representative of three independent experiments.
(C) Real-time quantitative RT-PCR for Notch1,
Dll1, Jag1, Hes5, Hey1 and Hey2, using RNA
from E8.5 wild-type (white bars) and Mib1–/–
(black bars) embryos. Numbers in each bar
indicate the mean fold of induction, and error bars
indicate the standard deviation. ***P<0.0001,
Dll1 in wild-type embryos was localized in the cytoplasm near
the nucleus (Fig. 6C). Surprisingly, in Mib1–/–embryos, Dll1
exclusively accumulated in the plasma membrane (Fig. 6C).
All of these observations indicate that murine Mib1 is essential
for the endocytosis of Notch ligand.
In large-scale mutagenesis screens, zebrafish Mib1 mutants
were initially identified by their neurogenic phenotype, which
is a hallmark for the disruption of Notch signaling (Jiang et al.,
1996; Schier et al., 1996). Recently, Mib1 has been identified
as an E3 ubiquitin ligase that interacts with Delta to promote
its ubiquitination and internalization, and to potentiate its
signaling activity in the signal-sending cells (Itoh et al., 2003).
As the overexpression of Xdelta in zebrafish Mib1 mutants
rescues the neurogenic defect, it has been suggested to be a
potentiator in generating functional Delta ligands to activate
Notch (Itoh et al., 2003). However, by generating Mib1–/–mice,
we have clearly demonstrated that Mib1 is an essential
regulator of Notch ligand activation, but not a potentiator, and
this ligand activation is absolutely required for Notch
phenotypes, such as defects in somitogenesis, neurogenesis,
vasculogenesis and cardiogenesis. In addition, the Mib1–/–
embryos showed an enlarged balloon-like pericardial sac,
fusion of the notochord to the neural tube, disorganization of
the trunk ventral neural tube, loss of mesenchyme cells, and
abnormal heart and second branchial arch development. The
phenotypes of the Mib1–/–embryos most closely resemble
those of embryos that lack core Notch signaling components,
such as Pofut, presenilins 1/2 and RBP-Jκ (de la Pompa et al.,
1997; Donoviel et al., 1999; Oka et al., 1995; Shi and Stanley,
Development 132 (15)Research article
Fig. 5. Defective Notch activation in Mib1–/–
embryos. (A) Notch1 intracellular domain
(N1icd) generation and Hes5 expression.
Lysates from E9.0 wild-type (wt) and Mib1–/–
(mt) embryos were immunoblotted with
rabbit anti-N1icd, mouse anti-Notch1, rabbit
anti-mouse Hes5 and rabbit anti-Mib1
antibodies. The results are representative of
three independent experiments. (B-D) S3
cleavage of Notch1 by γ-secretase.
Embryonic fibroblasts (EFs) isolated from
E9.5 wild-type (wt) and Mib1–/–(mt) embryos
were infected with the MSCV vector driving
the expression of Myc-tagged extracellular
domain-deleted Notch1 (ΔEN1). (B) Western
blot analysis of Nicd generation. Cell lysates
were immunoblotted with anti-Notch1, anti-
N1icd and anti-Mib1 antibodies. HEK293A
cells (HEK) were used as a positive control
for infection and N1icd generation. There is
intact N1icd generation in Mib1–/–EFs.
(C) Nuclear transport of Nicd. Infected cells
were fixed and immunostained with an anti-
Myc antibody, followed by anti-mouse Alexa-
488 (in green). Nuclear DNA was stained
with Hoechst (in blue). N1icd are transported
intra-nuclearly in Mib1–/–EFs. (D) CBF-
luciferase (Luc) activation by Nicd. The 8?
wild-type and mutant CBF-Luc vectors were
transfected to measure the activity of N1icd in
MSCV_ΔEN1-infected wild-type and Mib1–/–
EFs. The 8? mutant CBF-Luc lacks the CBF-
binding sites and was used as a control. The
relative Luc activities of wild-type and
Mib1–/–EFs are comparable.
(E) Internalization of Delta by murine Mib1.
COS7 cells were transfected with either mock
GFP or HA and Myc-tagged Xenopus Delta
(HA-XD-Myc) (black) or Mib1-GFP and
HA-XD-Myc (green). Twenty-four hours
after transfection, the cells were stained with
anti-HA Ab followed by PE-conjugated anti-
mouse Ab and analyzed by flow cytometry. (F) Defective Notch signaling in the Mib1–/–EFs. The wild-type (white bar) and Mib1–/–(black bar)
EFs infected with MSCV vector driving the expression of Myc-tagged Xenopus Delta (XD), Myc-tagged mouse Delta1 (Dll1) or Myc-tagged
mouse jagged 1 (Jag1) were co-cultured with C2C12-Notch1 cells transfected with the 8? wild-type and mutant CBF-Luc vectors. Twenty-four
hours after co-culture, luciferase activity was measured. The 8? mutant CBF Luc was used as a control for Notch activation.
3467Mib1 is essential for Notch signaling
Fig. 6. Interactions between Mib1 and all known Notch ligands. (A) Co-immunoprecipitation (Co-IP) of murine Mib1 with murine Notch
ligands (Dll1, Dll3, Dll4, Jag1 and Jag2). HA-tagged Mib1 (HA-Mib1) or control vectors were co-expressed with Myc-tagged Notch ligands in
HEK293A cells. The top panels show IP of Notch ligands by HA-Mib1, and the middle and bottom panels show the expression of HA-Mib1
and Notch ligand-Myc, respectively, in total cell lysates. (B) Subcellular localization of Mib1 (in green) and Notch ligands (Dll1, Jag1 and XD;
in red). Mib1-GFP and/or Myc-tagged Notch ligand constructs were co-expressed in COS7 cells. Myc epitopes were detected with an anti-Myc
antibody followed by a TRITC-labeled antibody. Nuclear DNA was stained with Hoechst (in blue). (a-c) Expression of Mib1-GFP (a), Dll1-
Myc (b) and Jag1-Myc (c). (d-f) Co-transfection of Mib1-GFP with either Dll1-Myc (d), Jag1-Myc (e) or XD-Myc (f). Overlapping expression
is yellow. (C) Transverse sections of E9.0 wild-type (wt) and Mib1–/–(mt) embryos stained with anti-Dll1 antibody. Dll1 is localized in the
cytoplasm in wild-type neural tube, and accumulates in the plasma membrane in Mib1–/–neural tube (in red). Nuclear DNA was stained with
Hoechst (in blue). The overall images are shown in insets. (D) Notch signal transduction. The pan-Notch phenotype in Mib1–/–embryos and the
molecular interactions between Mib1 and multiple Notch ligands suggest a new core-Notch component that regulates the endocytosis of Dll and
Jag ligands. The endocytosis of the Notch ligands by Mib1 stimulates the S2 and S3 cleavages of Notch receptors and the released Nicd
translocates to the nucleus to express the Notch target genes, such as Hes5, Hey1 and Heyl.
2003). In accordance with the pan-Notch defects in Mib1–/–
embryos, expression of the Notch target genes, such as Hes5,
Heyl, was dramatically downregulated. In
neurogenesis, the Mib1–/–embryos exhibited the characteristic
loss of Hes5 expression in the neural tube and premature
neuronal differentiation accompanied by the depletion of
neural stem cells (de la Pompa et al., 1997; Donoviel et al.,
1999). In somitogenesis, the Mib1–/–embryos showed the loss
of Uncx4.1 expression and defects in Hes7 and Lfng oscillation
(Barrantes et al., 1999). Moreover, the Mib1–/–embryos
displayed the loss of ephrin B2 and Hey1 expression in the
dorsal aorta (Krebs et al., 2004). These results are all indicative
of a lack of Notch activation and are characteristic pan-Notch
phenotypes in mutants lacking core Notch signaling
components, such as Pofut, presenilins 1/2 and RBP-Jκ. In
addition to the downregulation of Notch target gene
expression, the Mib1–/–embryos also showed a complete loss
of N1icd generation, despite the normal expression of core
components for Notch signaling, such as presenilins, Notch
proteins and Notch ligands.
There are five canonical Notch ligands (Dll1, Dll3, Dll4,
Jag1 and Jag2) in mammals (Lai, 2004). A previous study
revealed that Mib1 directly regulates the endocytosis of
zebrafish DeltaB and DeltaD (Itoh et al., 2003). However, the
endocytosis of Serrate-related ligands by E3 ubiquitin ligases
has not been investigated, although it has been shown that
Serrate, like Delta, is a transmembrane ligand that can
participate in the trans-endocytosis of Notch to the ligand
expressing cells (Klueg and Muskavitch, 1999). Each Notch
ligand has redundant and non-redundant roles in the Notch
activation pathway at various cell fate decisions. Dll1 and Dll3
are required for the proper anteroposterior polarity of each
somite (Barrantes et al., 1999; Hrabe de Angelis et al., 1997;
Kusumi et al., 1998). Dll4 is crucial in the arterial fate decision
(Duarte et al., 2004; Gale et al., 2004; Krebs et al., 2004). Jag1
and Jag2 are essential for remodeling of the embryonic
vasculature, and for limb and craniofacial development,
respectively (Jiang et al., 1998; Xue et al., 1999). The pan-
Notch phenotypes in Mib1–/–embryos could be explained by
the combined loss of multiple ligands. The defects in
somitogenesis, the loss of Uncx4.1 and Heyl expression, and
the impaired oscillations of Hes7 and Lfng can be explained by
the combined loss of Dll1 and Dll3. The defects in
vasculogenesis and the loss of Hey1 and ephrin B2 expression
closely resemble the vascular phenotypes in Dll4–/–mice.
Although the Mib1–/–embryos do not clearly explain the
phenotypes in Jag1–/–and Jag2–/–mice, the thin vascular
network and the defect in the second branchial arch
development appear to represent the loss of Jag1 and Jag2
activity, respectively. In accordance with the pan-Notch
phenotypes in Mib1–/–embryos, Mib1 interacts with all of the
Notch ligands, Jag1 and Jag2 as well as Dll1, Dll3 and Dll4,
and induces their endocytosis, suggesting that all of these
defects might be due to the inactivation of most, if not all, of
the Notch ligand functions.
A crucial step for efficient Notch signaling by Delta was
revealed by an analysis of Drosophila neur and zebrafish Mib1
mutants (Deblandre et al., 2001; Itoh et al., 2003; Lai et al.,
2001; Pavlopoulos et al., 2001). These two genes promote the
endocytosis of Delta in a ubiquitination-dependent manner. It
has been proposed that the endocytosis of Notch ligands, on
signal-sending cellsthat are bound to Notch on adjacent signal-
receiving cells, induces the S2 cleavage of the receptor, thus
activating signal transduction (Parks et al., 2000). Surprisingly,
in Mib1–/–mutants, the expression of Dll1 was accumulated in
the plasma membrane, while it was localized in the cytoplasm
near the nucleus in the wild type, indicating that Mib1 is
essential for the endocytosis of Dll1. Consistent with the
endocytic defects of Notch ligand, Notch activation, such as
the generation of Nicd and the activation of Notch-target genes,
was abolished. Thus, our data clearly show that Mib1 is an
essential regulator of Notch ligand endocytosis and activation
of Notch signaling.
Although both Neur and Mib1 interact with Delta and
regulate its endocytosis, it is not clear whether they have
redundant or unique, non-redundant functions in Notch
signaling. Consistent with previous studies in zebrafish Mib1
mutants, our Mib1–/–mice exhibited pan-Notch defects,
indicating that Mib1 has non-redundant roles in Notch
signaling in both zebrafish and mammals. Most recently, two
groups have reported the serrate-related function of Drosophila
Mib1 and they also showed replaceable function of Drosophila
Mib1 and Drosophila Neur (Lai et al., 2005; Le Borgne et al.,
2005). In contrast to our Mib1–/–mice exhibiting pan-Notch
defects, Drosophila Mib1 mutants display part of the Notch
phenotypes, suggesting functional redundancy between
Drosophila Mib1 and Drosophila Neur. In our recent study, we
identified a Mib1 paralogue, Mib2, which has similar activities,
but different expression patterns compared with those of Mib1
(Koo et al., 2005). Mib2 is mainly expressed in the adult
tissues, but not in early embryonic stages, whereas Mib1 is
abundantly expressed in both embryos and adult tissues,
suggesting Mib1 might have a dominant role during early
embryogenesis. However, it is not tested whether Drosophila
mib2 (CG17492) has an essential or redundant role in the
Drosophila Notch signaling pathway. neur mutants in
Drosophila are characterized by neurogenic phenotypes and
have defects in Notch activation (Boulianne et al., 1991; Price
et al., 1993). However, the disruption of the Neur gene in mice
did not generate the characteristic Notch phenotypes as in
Drosophila neur mutants (Ruan et al., 2001; Vollrath et al.,
2001). This discrepancy suggests that unknown murine neur
homologues and/or murine Mib1 can compensate for the loss
of Neur in mammals. There are two murine neuralized genes,
Neur1 and Neur2, which have similar phylogenic distances
from Drosophila neur (R.S. and Y.-Y.K., unpublished),
suggesting that double mutants of murine Neur1 and Neur2
might have defects equivalent to those observed in Drosophila
neur mutants. Considering the evolutionary conservation of the
Notch signaling pathway, it will be interesting to examine
whether these two regulators, Neur and Mib1, play cooperative
but non-redundant roles in mammals.
Mib1 has been suggested to be a potentiator in generating
functional ligands because of the residual Notch activity in
zebrafish Mib1 mutants (Cheng et al., 2004; Itoh et al., 2003).
The ectopic overexpression of Xdelta rescues the neurogenic
defect in zebrafish Mib1 mutants and Notch activation was
further suppressed by expression of dominant-negative Su(H).
By contrast, we have identified Mib1 as an essential regulator
in generating functional Notch ligands (Fig. 6D). This
discrepancy might be due to the effects of maternal genes as
Mib1 transcripts are expressed maternally in zebrafish
Development 132 (15)Research article
3469Mib1 is essential for Notch signaling
unfertilized eggs (Itoh et al., 2003). Furthermore, Mib1
interacts with all of the murine Notch ligands, and genetic
inactivation of Mib1 results in multiple developmental defects
that are characteristic of impaired Notch signaling. Thus, our
data provide the first evidence in mammals that the E3
ubiquitin ligase Mib1 is an essential core component of Notch
Supplementary material for this article is available at
We thank D. S. Lim and J. K. Han for helpful comments, P. J.
Gallagher for the DIP-1 antibody, D. Hayward for the 8? wild-type
and mutant CBF-Luc, G. Weinmaster for the C2C12-Notch1 cell line,
and J. M. Penninger for critical reading of the manuscript. This work
was supported by grants from the Vascular System Research Center
from KOSEF, Basic Research Program of the Korea Science and
Engineering Foundation (R02-2003-000-10057-0), the 21C Frontier
Functional Human Genome Project from Ministry of Science and
Technology of Korea (FG04-22-05), and the Molecular and Cellular
BioDiscovery Research Program grant from the Ministry of Science
and Technology, South Korea (M1-0106-01-0001).
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Development 132 (15)Research article