1 Division of Molecular and Life Sciences, Pohang University of Science and
Technology, Pohang, Kyungbuk, 790-784, South Korea
2 Laboratory of Molecular Genetics, NICHD, NIH, Bethesda, MD 20892, USA
3 Department of Biology, Chungnam National University, Taejeon 305-764, South
Korea
Author for correspondence (e-mail:
ykong{at}postech.ac.kr)
Accepted 31 May 2005
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SUMMARY |
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Key words: Notch signaling, Mind bomb, Endocytosis, Notch ligand, Mouse
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Introduction |
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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
-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-J
. 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 after the receptor
undergoes the ligand-dependent S2 cleavage, the mechanism by which the Notch
ligands engage Notch and trigger its cleavage 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 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.
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Materials and methods |
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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 121, BD) and a PECAM antibody
(MEC13.3, BD), using the Vectastain Elite ABC kit (Vector Laboratories).
RT-PCR analysis
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 ß-actin standard, using the LightCycler software.
All amplicons (100200 bp) showed efficient amplification that allowed us
to equate one threshold cycle difference. Primer information can be provided
on request.
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 EN1 and Dll1 cDNAs were
cloned into the HpaI site of pMSCV. D. Hayward kindly provided the
8x wild-type and 8x 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,
2540 µg of protein containing supernatants were separated by size,
blotted with primary and secondary Abs and visualized with ECL plus (Amersham
Biosciences). 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 8x 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).
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Results |
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Transverse sections showed that the Mib1/ embryos had smaller and thinner hearts, with broaden pericardial cavity 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 phenotypes.
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.
|
At E9.09.5, 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 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
Hes1/5DKO mutant 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 (Fig. 2D,F,H).
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 lateral
inhibition in neurogenesis (de la Pompa et
al., 1997
; Ma et al.,
1998
).
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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 fate decisions.
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-J 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-J 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 Mib1 itself.
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 -secretase-mediated S3
cleavage.
To investigate whether the -secretase-mediated S3 cleavage and its
downstream signaling are intact in Mib1/
embryos, the Notch1 deleted extracellular domain (
EN1) was expressed in
embryonic fibroblasts (EF) from wild-type and
Mib1/ embryos.
EN1 is readily cleaved
by the
-secretase complex to release the active N1icd, independent of a
ligand/receptor interaction. In short,
EN1 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.
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 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 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.
|
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Discussion |
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The Mib1/ mice exhibit multiple
Notch-related 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-J (de la
Pompa et al., 1997
; Donoviel
et al., 1999
; Oka et al.,
1995
; Shi and Stanley,
2003
). In accordance with the pan-Notch defects in
Mib1/ embryos, expression of the Notch
target genes, such as Hes5, Hey1 and 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-J
. 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.
|
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 cells that 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 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 signaling.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3459/DC1
* These authors contributed equally to this work
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