RESEARCH ARTICLE
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Development 135, 197-206 (2009) doi:10.1242/dev.027938
Interaction with Notch determines endocytosis of specific
Delta ligands in zebrafish neural tissue
Miho Matsuda and Ajay B. Chitnis*
Mind bomb1 (Mib1)-mediated endocytosis of the Notch ligand DeltaD is essential for activation of Notch in a neighboring cell.
Although most DeltaD is localized in cytoplasmic puncta in zebrafish neural tissue, it is on the plasma membrane in mib1 mutants
because Mib1-mediated endocytosis determines the normal subcellular localization of DeltaD. Knockdown of Notch increases cell
surface DeltaA and DeltaD, but not DeltaC, suggesting that, like Mib1, Notch regulates endocytosis of specific ligands. Transplant
experiments show that the interaction with Notch, both in the same cell (in cis) and in neighboring cells (in trans), regulates DeltaD
endocytosis. Whereas DeltaD endocytosis following interaction in trans activates Notch in a neighboring cell, endocytosis of DeltaD
and Notch following an interaction in cis is likely to inhibit Notch signaling by making both unavailable at the cell surface. The
transplantation experiments reveal a heterogeneous population of progenitors: in some, cis interactions are more important; in
others, trans interactions are more important; and in others, both cis and trans interactions are likely to contribute to DeltaD
endocytosis. We suggest that this heterogeneity represents the process by which effective lateral inhibition leads to diversification
of progenitors into cells that become specialized to deliver or receive Delta signals, where trans and cis interactions with Notch play
differential roles in DeltaD endocytosis.
INTRODUCTION
Notch signaling has an evolutionarily conserved role in determining
distinct fates for adjacent cells in diverse contexts during metazoan
development. Activation of Notch prevents cells from differentiating
and plays a crucial role in maintaining progenitor populations during
growth and development of an animal (reviewed by ArtavanisTsakonas et al., 1999; Schweisguth, 2004).
The mature Notch receptor has an extracellular fragment,
NotchEC, bound to the extracellular stub of a membrane-spanning
fragment, NotchTM, which is generated by S1 cleavage of the fulllength receptor during synthesis. Cleavage in the extracellular
stub (S2) followed by cleavage in the NotchTM transmembrane
domain (S3) releases an intracellular fragment of Notch, NotchIC,
into the cell. NotchIC contains a trans-activation domain and
additional interaction domains that allow it to form a
transcriptional activator complex with Mastermind and a member
of the CSL family [CBF1/RBPjkappa, Su(H), Lag1]. Together,
they drive expression of target genes recognized by the DNAbinding CSL protein (reviewed by Mumm and Kopan, 2000;
Fiuza and Arias, 2007). The NotchEC fragment regulates release
of NotchIC into the cell because its association with the NotchTM
extracellular stub prevents access to the cleavage sites required
for release of NotchIC. Removal of NotchEC is a key step in the
‘activation’ of Notch as it leaves the S2 and S3 sites on NotchTM
accessible to sequential cleavage by an ADAM protease and a γsecretase complex (Gordon et al., 2007).
Laboratory of Molecular Genetics, Section on Neural Developmental Dynamics,
Eunice Kennedy Shriver National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, MD 20892, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 30 October 2008
Separation of the NotchEC and NotchTM fragments is determined
by interaction of Notch with DSL (Delta Serrate Lag2) ligands on
the surface of a neighboring cell. The NotchEC fragment contains
EGF repeats that bind DSL ligands, which are also trans-membrane
proteins that have EGF repeats in their extracellular domain. It is
thought that ubiquitin-dependent endocytosis of a Notch DSL
ligand, like Delta, bound to NotchEC on the surface of a neighboring
cell, promotes Notch activation because endocytosis of the DSL
ligand-NotchEC complex facilitates separation of NotchEC from the
extracellular stub of membrane-tethered NotchTM (Klueg and
Muskavitch, 1999; Parks et al., 2000; Nichols et al., 2007a; Nichols
et al., 2007b).
In zebrafish, the ubiquitin-mediated endocytosis of Delta
homologues is regulated by the RING ubiquitin ligase Mind
bomb (Mib). Previously, we have shown that a Mib homologue,
now called Mib1, ubiquitylates DeltaD, promotes its endocytosis,
and is essential for effective Notch activation in zebrafish during
early neurogenesis (Itoh et al., 2003). It remained unclear,
however, whether, in this context, Mib-mediated Delta
endocytosis is triggered by interaction of Delta with Notch in a
neighboring cell, or whether Mib-mediated endocytosis of Delta
is a process that occurs independently of an interaction of Delta
with Notch.
This study was initiated to examine the subcellular distribution
of endogenous Delta in the central nervous system and to
determine whether the previously described accumulation of
DeltaD on the cell surface of hair cells in otic vesicles in Mib
mutants (Itoh et al., 2003) is representative of changes in the rest
of the nervous system. We show that Mib-mediated endocytosis
is a crucial determinant of the subcellular distribution of DeltaD
throughout the nervous system. We also show that Notch is a
determinant of Delta endocytosis and that interaction with Notch,
both within the same cell (in cis) and in a neighboring cell (in
trans), is likely to regulate endocytosis of specific Delta
homologues. We discuss the potential significance of these
interactions during lateral inhibition.
DEVELOPMENT
KEY WORDS: Notch signaling, Endocytosis, DeltaD, DeltaA, DeltaC, Neurogenesis, Zebrafish, Lateral inhibition, Cis inhibition
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RESEARCH ARTICLE
MATERIALS AND METHODS
Fish maintenance and mutant strain
Zebrafish were maintained under standard conditions and staged according
to Kimmel et al. (Kimmel et al., 1995). The mind bomb, mibta52b and mibm178
(Itoh et al., 2003; Jiang et al., 1996), and deltaD, aeiag49 (Holley et al., 2000)
alleles have been described previously. Treatment with 50 μM DAPT
(Calbiochem) was started at 80% epiboly to block Notch signaling.
Plasmid construction and embryo injections
Membrane-tethered mRFP-myc gene [modified from Moriyoshi et al.
(Moriyoshi et al., 1996)] and HA-tagged zebrafish deltaD were cloned into
pCS2 vector. Capped mRNAs were synthesized using Ambion mMessage
mMachine kits. Morpholino and mRNA injections were performed at the
one- or two-cell stage. 1.5 ng of each morpholino was injected for double
morpholino experiments. Morpholinos were purchased from Gene Tools
(Philomath, OR). mRNAs were injected at the following concentrations: 50
pg for HA-deltaD, 25 pg for memb-myc and 100 pg for DN-Su(H).
Morpholinos used in this study are:
Notch1a-MO (5⬘-GAAACGGTTCATAACTCCGCCTCGG-3⬘) (Yeo et
al., 2007);
Notch3-MO (5⬘-ATATCCAAAGGCTGTAATTCCCCAT-3⬘) (Yeo et al.,
2007);
DeltaA-MO (5⬘-CTTCTCTTTTCGCCGACTGATTCAT-3⬘); and
DeltaD-MO (5⬘-AAACAGCTATCATTAGTCGTCCCAT-3⬘).
Development 136 (2)
family, which in turn inhibit expression of Ngn1 (Takke et al., 1999).
So, by driving Delta expression, each Ngn1-expressing cell acquires
the ability to inhibit Ngn1 and Delta expression in neighboring cells.
As a consequence of this process of ‘lateral inhibition’, only a subset
of cells in the neurogenic domain acquire high enough levels of
Ngn1 expression to differentiate as neurons. In the absence of
effective Notch signaling, most cells within the neurogenic domain
express high levels of Ngn1 to become neurons (reviewed by
Chitnis, 1999; Louvi and Artavanis-Tsakonas, 2006).
We examined the distribution of endogenous Delta protein in
neurogenic domains using a monoclonal antibody for DeltaD, zdD2
(Itoh et al., 2003). Fixation of zebrafish embryos with trichloracetic
acid (TCA) improved the ability to visualize endogenous DeltaD
distribution. zdD2 staining correlates with the distribution of deltaD
transcripts both at the taibud stage, when deltaD is expressed in a
subset of cells within longitudinal neurogenic domains in the
prospective hindbrain (Fig. 1A,B) (Haddon et al., 1998; Appel and
Whole-mount in situ hybridization
DIG-labeled anti-sense riboprobes were synthesized using the DIG labeling
kit (Roche, Indianapolis, IN). Embryos were fixed in 4% paraformaldehyde
overnight at 4°C. Signals were detected with alkaline phosphatase-labeled
anti-DIG antibody and BM Purple substrate (Roche). Whole-mount
embryos were imaged with a ProgRes C14 camera mounted on a Leica
MZ12 stereomicroscope.
Immunohistochemistry
Embryos were fixed with ice-cold 10% TCA (Sigma) for 30 minutes and
permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. Primary
antibodies used were: mouse anti-zebrafish deltaD, zdD2, anti-zebrafish
deltaC, zdC1 (kindly provided by Dr Julian Lewis, Cancer Research UK
London Research Institute), rabbit anti-β-catenin (Sigma) (dilution 1:500)
and rabbit anti-myc (abCAM) (dilution 1:500), mouse anti-HA (Covance)
(dilution 1:400). Photos were taken with a confocal microscope (LSM
510META, Carl Zeiss).
Cell transplantation and analysis
RESULTS
Subcellular localization of DeltaD in neural
ectoderm
DeltaD is expressed in ‘neurogenic’ domains of the neural plate,
where cells express a bHLH transcription factor neurogenin 1
(Ngn1) (Blader et al., 1997; Kim et al., 1997). Ngn1 expression
gives cells the potential to become neurons and drives its own
expression. If cells acquire high enough levels of Ngn1, they initiate
expression of additional genes that allow them to differentiate as
neurons. However, Ngn1 also drives the expression of Delta, which
activates Notch in neighboring cells. Notch activation drives
expression of transcriptional repressors in the Hairy E(Spl) Related
Fig. 1. DeltaD protein is primarily localized in cytoplasmic puncta.
(A-D) deltaD transcript distribution (A,C) and anti-DeltaD mAb zdD2
staining (B,D) are similar. (A,B) Arrows indicate neural expression and
arrowheads indicate the mesodermal expression in tail bud stage
zebrafish embryos. (C,D) DeltaD expression in the hindbrain at 30 hpf.
zdD2 in green and β-catenin in red. (E-F’) Magnified images correspond
to squares in B and D. (E,F) zdD2 staining. (E’,F’) Merged images with
zdD2 (green) and β-catenin (red). DeltaD protein is mainly detected as
cytoplasmic puncta and does not colocalize with β-catenin.
DEVELOPMENT
EK fish embryos were injected with either standard MO provided by Gene
Tools or MOs against notch1a and notch3 with mRNA encoding membmRFP-myc or with mRNA encoding HA-DeltaD and memb-mRFP-myc,
MycPM. Twenty to 30 cells from donor embryos were transplanted into host
embryos at the 1000 cell stage, and embryos were allowed to develop until
the appropriate stage as described in the Results section. Transplanted cells
were visualized with rabbit anti-myc antibody, and endogenous DeltaD and
exogenous DeltaD-HA proteins were detected with zdD2 or anti-HA
antibodies, respectively. Z-series sections of the transplanted cells were
taken on an LSMmeta510 confocal microscope.
Eisen, 1998), and at 30 hours post fertilization (hpf), when deltaD is
expressed adjacent to rhombomere boundaries (Fig. 1C,D) (Cheng
et al., 2004; Riley et al., 2004; Amoyel et al., 2005). To examine the
subcellular distribution of DeltaD protein, embryos were doublestained with zdD2 mAb and anti-β-catenin pAb, an adherens
junction protein, which reveals the shape of individual cells. DeltaD
protein was mainly detected as cytoplasmic puncta without overlap
with β-catenin associated with the plasma membrane, both at the tail
bud stage (Fig. 1E,E⬘) and later in the hindbrain at 30 hpf (Fig.
1F,F⬘).
Mib1 requirement for DeltaD endocytosis
At the 10-somite stage, most of the DeltaD protein accumulated on
cell surface in mib1 mutants (Fig. 2D), whereas DeltaD was in
cytoplasmic puncta in wild-type siblings (Fig. 2C), suggesting that
Mib1-mediated endocytosis is a crucial determinant of endogenous
DeltaD distribution. However, at this stage, the deltaD transcript and
DeltaD protein in mib1 mutants is exaggerated (compare Fig. 2A,B
with Fig. 2C,D) as absence of Notch signaling allows cells in the
neurogenic domain to express much higher levels of ngn1 and
deltaD. The exaggerated deltaD expression in mib1 mutants raised
the possibility that surface accumulation of DeltaD could be due to
higher production of DeltaD rather than to reduced internalization
from the cell surface (Lai et al., 2005; Le Borgne et al., 2005;
Pitsouli and Delidakis, 2005; Wang and Struhl, 2005).
To distinguish between these possibilities, we compared
DeltaD protein distribution at the tail bud stage when the overall
level of deltaD transcript does not look as significantly
exaggerated (compare Fig. 2A,B with Fig. 2E,F). In wild-type
siblings, DeltaD was distributed in small puncta in a small subset
of cells within neurogenic domains. In mib1 mutants, failure of
Notch signaling allowed most of the cells in the neurogenic
domains to express DeltaD protein. Though DeltaD expression
was significantly lower at this stage, most of it was still on the cell
surface (Fig. 2G,H). Two different mib1 alleles, mibta52b and
mibm178, had similar changes in DeltaD distribution (Fig. 2H; Fig.
4C). These results suggest Mib1-mediated endocytosis has a
significant role in determining endogenous DeltaD distribution in
the neural tissue.
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DeltaD distribution in DAPT and DN-Su(H)
embryos
To further evaluate how increased deltaD production following loss
of Notch signaling contributes to surface expression of DeltaD, we
examined the changes in DeltaD distribution following other
manipulations that inhibit Notch signaling: DAPT treatment, which
inhibits the γ-secretase dependent S3 cleavage of Notch (Geling et
al., 2002); injection of mRNA encoding a dominant negative form
of Su(H), DN-Su(H), which lacks its DNA binding domain
(Wettstein et al., 1997); and injection of morpholinos (MO) against
two homologues of Notch, notch1a and notch3 (previously called
notch5) expressed in the neural plate during early neurogenesis
(Hsiao et al., 2007; Yeo et al., 2007).
As in mib1 mutants, these manipulations induced a neurogenic
phenotype with more cells in the neurogenic domain expressing
DeltaD. However, at the tail bud stage, when cells did not show
significant exaggeration in deltaD transcript (see Fig. S1 in the
supplementary material), DAPT-treated or DN-Su(H)-expressing
embryos had zdD2 in cytoplasmic puncta (Fig. 3B,B⬘ and Fig.
3C,C⬘, respectively), not at the cell surface as seen in mib1 mutants
(Fig. 2H). Later, at the six-somite stage, when there was exaggerated
expression of DeltaD (compare Fig. 3D,D⬘ and Fig. 3E,E⬘), zdD2
did accumulate at the cell surface in DAPT-treated (Fig. 3E,E⬘) and
DN-Su(H)-injected (data not shown) embryos. However, unlike the
situation in mib1 mutants, the surface accumulation was now
accompanied by intense cytoplasmic puncta of DeltaD. These
observations suggest that surface accumulation seen in mib1 mutants
at an early stage is specifically due to failure of Mib1-mediated
endocytosis. Later, as failure of Notch signaling results in
exaggerated deltaD expression, some of the DeltaD does accumulate
at the cell surface because it exceeds the cells capacity for DeltaD
endocytosis.
Interactions with Notch are required for DeltaD
endocytosis
In contrast to changes seen following DAPT treatment or injection
of DN-Su(H), embryos with notch1a and notch3 simultaneously
knocked down had an increase in surface DeltaD similar to that
seen in mib mutants: DeltaD was at the cell surface at the tail bud
Fig. 2. Accumulation of DeltaD protein on the plasma
membrane in the mib mutant. (A,B) Hindbrain expression of
deltaD transcript in wild-type (WT) and mibta52b 10-somite stage
(10 ss) zebrafish embryos. (C,D) DeltaD protein in areas
corresponding to the rectangles in A and B. Embryos are stained
with zdD2. The patches of DeltaD expression in D are in
rhombomeres 3, 4 and 5. (E,F) deltaD transcript in wild type and
mibta52b at the tail bud stage (tb) in hindbrain neurogenic
domains. Area shown here corresponds approximately to
rectangular areas in Fig. S1A,B (see supplementary material).
mibta52b embryos do not have significantly exaggerated deltaD
expression. (G,H) zdD2 staining in corresponding wild-type and
mibta52b neurogenic domains. In mibta52b mutants, most DeltaD is
at the cell surface, even when expression is not so exaggerated.
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Notch regulates Delta endocytosis
RESEARCH ARTICLE
Development 136 (2)
Fig. 3. The effect of loss of Notch signaling on surface
accumulation of DeltaD. DeltaD distribution in DMSO-treated (A,A’),
DAPT-treated (B,B’) or DN-Su(H)-expressing zebrafish embryos (C,C’) at
the tail bud stage, and in DMSO-treated (D,D’) and DAPT-treated
(E,E’)embryos at the six-somite stage (6 ss). Areas shown in A-C’
correspond approximately to rectangular areas in Fig. S1E,F (see
supplementary material). Embryos were stained with zdD2 (A-E) and
double-stained (A’-E’) with β-catenin (zdD2 in green, β-catenin in red).
DAPT and DN-Su(H) treatment increased the numbers of cells
expressing DeltaD but did not result in DeltaD accumulation at the cell
surface at the tail bud stage. Some cell surface DeltaD is detected in
DAPT-treated embryos by the six-somite stage; however, it is
accompanied by intense cytoplasmic puncta.
Fig. 4. The effect of loss of Notch receptors on surface
accumulation of DeltaD. (A-C’) A comparison of DeltaD distribution
in wild type, notch1a and notch3 morphants, and mibm178 mutant
zebrafish embryos at the tail bud stage. Areas shown here
approximately correspond to rectangular areas in Fig. S1A-D (see
supplementary material). Embryos are double-stained with zdD2 (grey
in A-C, green in A’-C’) and β-catenin (red in A’-C’). DeltaD-expressing
cells are increased and DeltaD is mainly localized at cell surface in both
Notch morphants (B,B’) and mibm178 mutants (C,C’). (D-F’) DeltaD
distribution in Notch morphants with (F,F’) or without (E,E’) co-injection
of deltaA and deltaD (deltaA&D) MOs. Though co-injection of
deltaA&D MO dramatically reduces zdD2 staining (compare E with F),
DeltaD is still localized at the cell surface.
stage (Fig. 4B,B⬘) before there was a significant increase in the
level of deltaD (supplementary material Fig. S1C-E⬘) and the
surface accumulation was not accompanied by prominent
cytoplasmic distribution of DeltaD. In fact, the surface
accumulation was more prominent than in mib1m178 mutants
(compare Fig. 4B,B⬘ with Fig. 4C,C⬘). To demonstrate that excess
DeltaD production is not responsible for its accumulation at the
cell surface, DeltaD protein was reduced in Notch MO embryos
by co-injection of deltaA and deltaD MO (compare Fig. 4E with
Fig. 4F). The resulting reduction of DeltaD protein was still
accompanied by surface accumulation of DeltaD (Fig. 4F,F⬘).
This suggested that DeltaD interactions with Notch are essential
for effective DeltaD endocytosis.
Interactions with Notch in trans and cis are
required for DeltaD endocytosis
Cell transplantation experiments were performed to determine
whether interaction of DeltaD with Notch in the same cell (in cis) or
in a neighboring cell (in trans) is required for triggering endocytosis.
Donor embryos were injected with mRNA encoding a plasma
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membrane-tethered mRFP and Myc-tag (MycPM), so that donor cells
could be identified in host embryos by surface expression of RFP or
a red fluorescent secondary antibody to visualize MycPM. DeltaD
was labeled with zdD2 and visualized with a green fluorescent
secondary antibody. Embryos were co-injected with notch1a and
notch3 MO (Notch MO) or control MO (WT) to evaluate how the
reduction of Notch affects Delta distribution. When WT cells were
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transplanted into WT embryos, DeltaD protein was localized in
cytoplasmic puncta both in the donor cells and host embryos (28/28
of the transplanted cells) (Fig. 5G,G⬘). When Notch MO cells were
transplanted into Notch MO embryos, DeltaD protein remained at
the cell surface (24/24 cells) (Fig. 5I,I⬘). This showed that the
transplantation process does not significantly alter the distribution
of DeltaD.
Fig. 5. Delta-Notch interactions in trans and cis contribute to DeltaD endocytosis in zebrafish. (A-F) Schematic of changes in DeltaD
distribution expected following transplantation if endocytosis was determined primarily by interactions in trans (C,D) or in cis (E,F). If Notch
interactions in ‘trans’ were essential for Delta endocytosis: (C) DeltaD would be in cytoplasmic puncta in Notch MO donor cells in a WT host, (D)
DeltaD would accumulate on the cell surface of WT donor cells in Notch MO host, and some Delta would now also be internalized in host Notch
MO cells following interaction with Notch in adjacent WT donor cells. If ‘cis’ interactions with Notch were essential for Delta endocytosis: (E) DeltaD
would remain on the surface of donor Notch MO cells in a WT host and (F) DeltaD would remain in cytoplasmic puncta in a donor WT cell in Notch
MO hosts. (G-J’) Transplantation results. (G,G’) In WT to WT transplants, DeltaD remains in intracellular puncta in WT donor and host cells. (H,H’) In
Notch MO to WT transplants, some DeltaD in Notch MO donor cells becomes localized in cytoplasmic puncta (arrows in H’), suggesting that
interactions with in trans promote endocytosis. However, some DeltaD remains on the cell surface (arrowheads in H) in Notch MO donor cells,
suggesting an additional requirement for interactions in cis. (I,I’) In Notch MO to Notch MO transplants, DeltaD remains on the cell surface in Notch
MO donor cells in Notch MO hosts. (J,J’) In WT to Notch MO transplants, some adjacent Notch MO host cells now show DeltaD in cytoplasmic
puncta (arrows in J), suggesting DeltaD endocytosis following interactions in trans. Observation of cytoplasmic DeltaD puncta in WT donor cells in
Notch MO hosts (black arrowheads in J’) suggest Notch interactions in cis contribute to DeltaD endocytosis. (K,L) Quantitative analysis. Cumulative
pixel intensity within the cell or at the cell surface, representing the fluorescence of a secondary antibody recognizing zdD2, was measured in
individual cells to quantify the distribution of DeltaD protein. Each spot represents an individual cell. (K) Scatter plot of relative intracellular (x-axis)
versus cell surface (y-axis) distribution in transplanted cells from WT to WT (blue), Notch MO to Notch MO (green) and Notch MO to WT (red). The
broken line indicates 10 K units and the area below 5 K units is shadowed. (L) Cumulative intracellular intensity in Notch MO host cells adjacent to
WT transplants and Notch MO host adjacent to Notch MO transplants.
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Notch regulates Delta endocytosis
RESEARCH ARTICLE
Transplantation of Notch MO cells into WT embryos, and WT
cells into a Notch MO embryo was expected to have distinct effects
on the distribution of DeltaD if interactions with Notch either in cis
or trans were primarily responsible for triggering endocytosis
(schematized in Fig. 5A-F). If interactions in trans were essential for
endocytosis, DeltaD would be effectively internalized in a Notch
MO donor cell transplanted to a WT embryo, as DeltaD in the Notch
MO donor cell would interact with Notch in surrounding WT host
cells (Fig. 5C). Similarly, Notch MO host cells adjacent to a
transplanted WT cell would show some internalized DeltaD
following successful interaction with Notch on the cell surface of
transplanted WT cells (Fig. 5D). At the same time, the transplanted
WT cell itself would now be expected to accumulate DeltaD on the
cell surface due to absence of Notch in neighboring Notch MO host
cells (Fig. 5D). However, if interactions with Notch in cis were
essential to trigger DeltaD endocytosis, DeltaD would remain on the
surface of a Notch MO donor cell when placed adjacent to WT host
cells (Fig. 5E), and DeltaD would continue to be internalized in a
WT donor cell when placed in a Notch MO host embryo (Fig. 5F).
Our results suggest that neither cis nor trans interactions are
exclusively responsible for DeltaD endocytosis, but rather both
types of interactions are likely to contribute. When Notch MO cells
were transplanted into WT host embryos (Fig. 5H,H⬘), some Notch
MO donor cells still had some DeltaD on the cell surface (Fig. 5H,
arrowheads), suggesting that interactions with Notch in cis are
required for normal levels of DeltaD endocytosis. However, some
of the Notch MO cells (Fig. 5H⬘, arrows) now had significant
intracellular DeltaD puncta, suggesting that interactions in trans with
surrounding Notch-expressing WT cells had triggered DeltaD
endocytosis. When WT donor cells were transplanted into a Notch
MO host (Fig. 5J,J⬘), some donor WT cells had prominent
intracellular DeltaD puncta (Fig. 5J⬘, black arrowheads), suggesting
that cis interactions with Notch within the transplanted cell were
adequate for some internalization of DeltaD. In addition, some host
Notch MO cells adjacent to donor WT cells now had intracellular
DeltaD (Fig. 5J, arrows), suggesting that trans interactions with
Notch in transplanted WT cells had promoted DeltaD endocytosis
in Notch MO host cells.
To quantify how much interactions in cis versus trans contribute
to endocytosis of DeltaD, we compared surface DeltaD to
cytoplasmic DeltaD in individual transplanted cells, where DeltaD
had the ability to interact with Notch both in trans or in cis (WT cells
into WT hosts), neither in trans nor in cis (Notch MO cells into
Notch MO hosts), and only in trans (Notch MO cells into WT hosts).
We did not evaluate WT cells in Notch MO hosts, where interactions
only take place in cis, because DeltaD on the surface of the
transplanted cells could not be distinguished from DeltaD on the
surface of surrounding host cells. The cell surface or cytoplasmic
boundary was outlined in a representative confocal image of each
cell and surface versus intracellular DeltaD was quantified by
measuring the cumulative pixel intensity within the defined
boundaries using Image J software. The relative distribution of
DeltaD for each cell, within each experimental group, was
represented in a scatter plot with cumulative intracellular pixel
intensity on the x-axis and cumulative cell surface pixel intensity on
the y-axis (Fig. 5K).
The scatter plot revealed that, although WT cells transplanted into
WT host embryos (Fig. 5K, blue spots) had variable amounts of
intracellular DeltaD, none had a surface intensity higher than 10,000
(10 K) units (Fig. 5K, broken line). By contrast, all but one of the
Notch MO cells transplanted into Notch MO host embryos (Fig. 5K,
green spots) had surface intensity higher than 10 K and intracellular
Development 136 (2)
intensity less than 10 K units. These changes in distribution illustrate
ineffective DeltaD endocytosis in the absence of both trans and cis
interactions. The scatter diagram also illustrates that although all the
WT cells transplanted into WT hosts have low cell surface DeltaD,
10/28 (36%) of the cells had lower than 5 K units of both surface and
intracellular intensity (Fig. 5K, shadowed area). By contrast, 23/24
(96%) of the Notch MO cells transplanted into Notch MO embryos
had cumulative intensities greater than 5 K. This difference is likely
to be related to the role of lateral inhibition in restricting Delta
expression to a subset of WT cells and failure of lateral inhibition to
allow a larger fraction of transplanted Notch MO cells to express
DeltaD.
DeltaD in Notch MO cells transplanted into WT hosts (Fig. 5K,
red spots) was neither primarily restricted to the cell surface as in
Notch MO to Notch MO transplants, nor primarily intracellular as
in WT to WT transplants. In 11/32 (34%) of the cells, more than 10
K units intensity remained on the cell surface and fewer than 10 K
units was intracellular, suggesting that in these cells trans
interactions with Notch in surrounding WT cells were not capable
of restoring high intracellular DeltaD distribution as seen in WT
cells. In another 5/32 (16%) of the cells, however, fewer than 10 K
units pixel intensity was on the cell surface and more than 10 K units
was intracellular, suggesting that interactions in trans had restored
sufficient endocytosis to promote significant intracellular
distribution in these cells. In another 5/32 (16%) of cells, there was
greater than 10 K units of intensity both on the cell surface and in an
intracellular compartment, suggesting that although interactions in
trans had permitted significant amounts of Delta endocytosis, the
absence of interactions in cis allowed significant amounts of DeltaD
to remain on the cell surface. These observations suggest that
interactions with Notch in cis and trans have differential roles in the
endocytosis of DeltaD in different cells.
As seen in Notch MO cells transplanted into Notch MO embryos,
a large fraction of Notch MO cells transplanted into WT embryos
(21/32 or 66%) had levels of DeltaD intensity higher than 10 K units
and only a small fraction had lower than 5K units of pixel intensity.
As discussed earlier, it is likely that reduced Notch signaling in
Notch MO cells allowed a large fraction of these cells to express
high levels of DeltaD when transplanted into WT embryos.
Intracellular DeltaD levels in Notch MO host cells adjacent to
either transplanted Notch MO or transplanted WT cells were also
quantified (Fig. 5L). Most host Notch MO cells (21/22 or 95%) had
lower than 10 K units intracellular intensity when they were adjacent
to transplanted Notch MO cells. However, when the host Notch MO
cells were adjacent to transplanted WT cells, 10/39 (26%) of the
cells had higher than 10 K units intensity, suggesting that interaction
of DeltaD in trans with Notch in adjacent transplanted WT cells
triggered endocytosis of DeltaD in host Notch MO cells.
Stabilization of DeltaD protein in Notch morphant
Next, we asked whether Notch-dependent DeltaD endocytosis
determines DeltaD degradation. As loss of Notch signaling allows
increased deltaD transcription, it is hard to evaluate if the change in
the amount of endogenous DeltaD protein is determined by
increased synthesis or decreased degradation. To avoid complexities
introduced by endogenous transcriptional regulation, mRNAs for
deltaD-HA and mycPM were co-injected into one-cell stage embryos
and expression levels of DeltaD-HA and MycPM were compared in
the presence and absence of Notch (Fig. 6). It should be noted that
for reasons that remain unclear, significantly higher amounts of
exogenously provided DeltaD-HA protein localize at the cell surface
compared with endogenous DeltaD (Fig. 6A,A⬘). Nevertheless,
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Notch regulates Delta endocytosis
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Fig. 6. Notch-mediated DeltaD endocytosis results in DeltaD degradation. (A-B’) Distribution of DeltaD-HA in WT and Notch MO zebrafish
embryos. In vitro synthesized mRNA encoding DeltaD-HA and MycPM was co-injected with control (WT in A,A’) or notch1a and notch3 MOs (Notch
MO in B,B’). Embryos are labeled with anti-HA mAb (green) and anti-myc pAb (red) at 80% epiboly stage. More DeltaD-HA is localized in
cytoplasmic puncta in WT than in Notch MO cells. (C-H’) Comparison of DeltaD-HA (green) and MycPM stability with (F-H) and without Notch
morpholinos (C-E). DeltaD-HA expression reduces from 80% epiboly (C) to the two-somite stage (2 ss) (D) and is barely detectable at the fivesomite stage (5 ss) (E) in WT. By comparison, MycPM expression levels are progressively increased from 80% epiboly to 5 ss (C’-E’). In Notch MO
embryos, Delta-HA (F-H) behaves like MycPM (F’-H’) and its expression is not decreased.
that Notch knockdown reduces DeltaD-HA protein degradation. At
this stage, we have not been able evaluate whether interactions with
Notch in cis or in trans are more effective promoting degradation of
DeltaD-HA.
Interactions with Notch are also required for
DeltaA endocytosis
Although we used zdD2 as a specific antibody for zebrafish DeltaD
protein, we noticed that deltaD/aeiAG49 mutants, with an early
termination signal in the deltaD coding sequence (Holley et al.,
2000), have faint zdD2 labeling when the sensitivity of fluorescence
detection is increased (see Fig. S2C,E in the supplementary
material). As the distribution of the faint signal resembled the deltaA
expression pattern, we surmised that the staining was due to cross
reactivity of zdD2 with DeltaA. Injection of deltaA MO into
deltaD/aeiAG49 mutant eliminated this faint signal (data not shown),
confirming that zdD2 faintly labels DeltaA in deltaD/aeiAG49
mutants. We took advantage of this cross-reactivity and examined
the role of Notch interactions in DeltaA endocytosis. We found that
whereas zdD2 is primarily in intracellular puncta (see Fig. S2E,E⬘
in the supplementary material), its distribution becomes restricted to
DEVELOPMENT
intracellular DeltaD-HA puncta seen in WT embryos are no longer
seen in Notch MO embryos (compare Fig. 6A,A⬘ with Fig. 6B,B⬘),
confirming that endocytosis of DeltaD-HA is also regulated by
Notch.
Following co-injection of deltaD-HA and mycPM mRNA, MycPM
expression levels progressively increased when examined at 80%
epiboly the two-somite and the five-somite stage (compare Fig. 6C⬘E⬘). This suggested that MycPM is relatively stable and it
progressively accumulates on the cell surface as more is translated.
By contrast, progressively lower levels of DeltaD-HA were detected
at the cell surface between 80% epiboly (Fig. 6C) (n=6 embryos)
and the two-somite stage (Fig. 6D) (n=10), and it was hard to see by
the five-somite stage (Fig. 6E) (n=9). As Delta-HA and MycPM were
transcribed from identical vectors with the same 5⬘- and 3⬘-UTR,
this difference suggests that DeltaD-HA is degraded more rapidly
than it is translated during this period in control embryos. When
notch1a and notch3 MOs were co-injected, there was no longer a
progressive reduction in DeltaD-HA expression. Instead, similar
levels of DeltaD-HA were seen at 80% epiboly (Fig. 6F) and at the
two-somite stage (Fig. 6G), while slightly increased DeltaD-HA was
seen at the five-somite stage (Fig. 6H). These observations suggest
RESEARCH ARTICLE
the plasma membrane when Notch MOs are injected into
deltaD/aeiAG49 mutants (see Fig. S2F,F⬘ in the supplementary
material). As zdD2 labels DeltaA in deltaD/aeiAG49 mutants, this
suggests that interactions with Notch are also crucial for DeltaA
endocytosis.
DeltaC endocytosis is regulated differently from
DeltaD and DeltaA
Previous studies have suggested that whereas DeltaD is a substrate
for Mib1, DeltaC, expressed in a small subset of neuronal
progenitors (Smithers et al., 2000), is a substrate for both Mib1 and
its paralog, Mib2 (Zhang et al., 2007a; Zhang et al., 2007b).
Consistent with the specific requirement of Mib1 for DeltaD
ubiquitylation, DeltaD is on the cell surface in both mib1ta52b and
mib1m178 embryos (Fig. 2H; Fig. 4C,C⬘). By contrast, DeltaC
cellular distribution is not dramatically affected in mib1m178 embryos
(compare Fig. 7A,A⬘ with Fig. 7B,B⬘), even though its expression
is exaggerated as a consequence of reduced Notch signaling. This is
consistent with Mib2 function being sufficient to mediate DeltaC
endocytosis in mib1m178 embryos. However, DeltaC is primarily on
the cell surface in mib1ta52b mutants (Fig. 7C,C⬘). This is consistent
with the reported dominant inhibitory effect of the Mib1ta52b protein
reducing both Mib1 and Mib2 function (Fig. 7C,C⬘) and therefore,
DeltaC endocytosis (Zhang et al., 2007a).
We also examined the requirement for Notch interactions in
DeltaC endocytosis. Surprisingly, DeltaC endocytosis was not
dramatically affected by notch1a and notch3 knockdown (Fig.
7D,D⬘), though DeltaC expression itself was exaggerated as a
consequence of failed Notch signaling (compare Fig. 7A,D). These
results suggest that DeltaC and DeltaD not only have distinct
requirements for Mib1 and Mib2, their endocytosis is differentially
regulated by interactions with Notch.
DISCUSSION
In this study, we examined the subcellular localization of
endogenous Delta protein in zebrafish neural tissue and we showed
that DeltaA, DeltaC and DeltaD is primarily located in cytoplasmic
puncta. Our analysis indicates that whereas Mib1 is crucial for
endocytosis of DeltaA and DeltaD, Mib1 and Mib2 have redundant
roles in mediating DeltaC endocytosis. Furthermore, while Mib1mediated endocytosis is likely to have a crucial role in determining
the cytoplasmic distribution of DeltaD, overproduction of deltaD in
Development 136 (2)
the neural tissue following loss of Notch signaling can also
eventually contribute to some DeltaD accumulation on the cell
surface.
Surface accumulation of DeltaD induced by dominant-negative
Su(H) expression or DAPT treatment is accompanied by
accumulation of intense cytoplasmic puncta of DeltaD. By contrast,
DeltaA and DeltaD accumulation on the cell surface is not
accompanied by cytoplasmic puncta when notch1a and notch3
function is knocked down. This observation revealed that
interactions with Notch1a and Notch3 have a crucial role in
regulating DeltaA and DeltaD endocytosis. Loss of Notch1a and
Notch3 does not, however, affect the cellular distribution of DeltaC.
This suggests that although interactions with Notch1a and Notch3
are likely to regulate endocytosis of DeltaA and DeltaD, they are not
crucial for endocytosis of DeltaC.
Surface localization of DeltaA and DeltaD in mib1m178 mutant
embryos revealed a crucial role for Mib1 in determining cytoplasmic
distribution of DeltaA and DeltaD. The mib1m178 allele encodes a
truncated Mib1m178 protein that lacks the third RING domain
essential for its ubiquitin ligase function (Itoh et al., 2003).
Nonsense-mediated degradation of mib1m178 transcript significantly
reduces the amounts of the mutant protein and its phenotype is like
that of a null mutant (Zhang et al., 2007b). By contrast, the mib1ta52b
transcript, which is relatively stable, encodes a Met to Arg
substitution in the third RING domain. This interferes with its
ubiquitin ligase function and Mib1ta52b has a dominant-negative
effect on both Mib1 and Mib2. Consistent with these distinctions
and with the reported ability of both Mib1 and Mib2 to promote
DeltaC endocytosis in cultured cells, DeltaC endocytosis does not
appear to be significantly reduced in mib1m178 mutants where only
Mib1 function is lost, whereas endocytosis is compromised in
mib1ta52b mutants where the function of both Mib1 and Mib2 is
compromised.
Though our study suggests that interactions with Notch1a and
Notch3 regulate DeltaA and DeltaD endocytosis in zebrafish
embryos, it remains unclear whether Delta interactions with Notch
are specifically required for Mib-mediated endocytosis. Previously,
we have shown that Mib1 promotes DeltaD ubiquitylation and its
endocytosis in Cos-7 cells (Itoh et al., 2003). In that context,
however, where both DeltaD and Mib1 were provided in excess, an
interaction with Notch did not appear to be essential for Mib1mediated endocytosis. Nevertheless, it is likely that in vivo, under
Fig. 7. Mib1, Notch1a and Notch3 are not essential for
DeltaC endocytosis. (A-C’) Distribution of DeltaC in wild-type
zebrafish embryos (A,A’) and in two mib alleles, mibm178 (B,B’)
and mibta5ab (C,C’). (D,D’) Distribution of DeltaC in Notch MO
embryos. Eight-somite stage embryos were double stained with
zebrafish DeltaC antibody zdC1 (grey in A-D, green in A’-D’)
and β-catenin (red in A’-D’). A dorsal view of a lateral
neurogenic domain in the caudal neural plate is shown. In WT
embryos, only subset of cells express DeltaC in cytoplasmic
puncta (A,A’). A neurogenic phenotype allows more cells to
express DeltaC in both mib mutants and Notch MO embryos as
Notch signaling is reduced (B-D). However, DeltaC distribution
remains cytoplasmic in mibm178 (B,B’) and Notch MO embryos
(D,D’), and it only accumulates on the cell surface in mibta52b
mutant embryos (C,C’).
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204
physiological conditions, interaction with Notch in trans triggers
Mib1-mediated DeltaD endocytosis as Mib-mediated Delta
endocytosis is required for Notch activation. However, it is not clear
whether Mib is required for Delta endocytosis when Delta and
Notch interact in cis. Determining when and how Delta-Notch
interactions trigger Mib1-mediated Delta endocytosis remains a
question for future studies.
Interactions with Notch both in cis and trans
regulate DeltaD endocytosis
One particularly interesting role for Notch revealed in this study is
that Delta-Notch interactions both ‘in trans’ and ‘in cis’ regulate
Delta endocytosis. Endocytosis of Delta following its interaction
with Notch in trans is expected to ‘activate’ Notch, as it helps to
separate the NotchEC domain from the NotchTM domain, and this
in turn facilitates sequential S2 and S3 cleavage of NotchTM and
release of the NotchIC fragment into the cytoplasm. By contrast,
Delta endocytosis following Delta-Notch interactions in cis, on the
surface of the same cell, is not expected to activate Notch. Instead,
if Delta and Notch remain bound in this context, endocytosis is
likely to help to clear Delta and Notch from the cell surface,
making both unavailable for interactions with partners in a
neighboring cell.
Internalization of Delta-Notch complexes following interactions
in cis would leave either a net excess of Delta or Notch at the cell
surface, making cells specialized to deliver or receive Notch
activation, respectively. As a cell with an excess of Delta becomes
specialized in activating Notch in its neighbor, endocytosis of Delta
might become progressively more dependent on interactions with
Notch in trans. By contrast, Delta endocytosis in a cell with an
excess of Notch might become progressively less dependent on
interactions in trans.
We found that when Notch MO cells were transplanted into wildtype embryos, three types of cells were identified: (1) cells with
relatively low surface DeltaD and high intracellular DeltaD where
interactions in trans might have been adequate for effective
endocytosis; (2) a population with high surface Delta and low
intracellular DeltaD where interactions in cis might have been more
important; and (3) a third population with high levels of both surface
and intracellular DeltaD where both interactions in cis and in trans
were likely to have been contributing to DeltaD endocytosis. The
distinctions in the requirement for interactions in trans or cis with
Notch for DeltaD endocytosis seen in transplanted cells may reflect
progressive differentiation into distinct populations specialized to
deliver or receive Delta signals. It remains to be seen whether
interactions in trans predominantly determine Delta endocytosis in
cells specialized to deliver Delta signals, whether Delta interactions
with Notch in cis determine endocytosis in cells that are not as yet
specialized or whether they dominate in cells with an excess of
Notch specialized in receiving activation.
Interactions in cis may help tune a system for
more effective lateral inhibition
Lateral inhibition is expected to amplify differences in neurogenic
potential between adjacent cells and allow the progenitors with
greatest neurogenic potential to differentiate, while they inhibit their
neighbors from doing the same. However, when cells have high
levels of both Delta and Notch on their surface, relative differences
in the amounts of Delta and Notch may not be enough to prevent
significant cross activation of Notch by neighbors and mutual
inhibition of neurogenesis. In this context, internalization of DeltaNotch complexes following interactions in cis may help to tune the
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205
system for more effective lateral inhibition as it leaves a net excess
of Delta or Notch on the surface of cells. In this context, cells with
an excess of Delta would activate Notch in neighbors left with a net
excess of Notch, without receiving effective activation from their
neighbors. However, this model also suggests that if all the cells
expressed an excess of Delta, internalization of Delta-Notch
complexes following interactions in cis could deplete surface Notch
and lead to failure of Notch signaling. But we know that ectopic
expression of delta mRNA results in inhibition of neurogenesis
(Chitnis et al., 1995; Dornseifer et al., 1997; Haddon et al., 1998;
Appel and Eisen, 1998). This suggests that when all cells express an
excess of Delta, Notch signaling does not fail. Instead, interactions
with Notch in trans dominate and mutual Notch activation inhibits
neurogenesis. This implies that when cells express both Delta and
Notch, and in principle both interactions in cis and trans are possible,
interactions in trans are likely to dominate and are more likely to be
productive compared with cis interactions. Future studies will
determine whether this is true.
The models described above assume that both Delta and Notch
are internalized following an interaction in cis. Our data at present
show only that Delta endocytosis is regulated by Notch. We do not
have evidence for Delta regulating Notch trafficking in zebrafish.
Tests of these models await future experiments using tools for
visualizing the trafficking of Notch receptors in zebrafish. In
Drosophila, it has been shown that cis interactions of another DSL
ligand, Serrate, with Notch inhibit Notch signaling (Jacobsen et al.,
1998; Li and Baker, 2004) by promoting endocytosis of Notch
(Glittenberg et al., 2006). Similarly in C. elegans it has been
suggested that interactions of a Notch homologue, Lin12, with a
DSL ligand in cis inhibit the ability of a prospective vulval cell to
deliver a signal and activate Lin12 in its neighboring cells (Chen and
Greenwald, 2004).
In conclusion, our study confirms the role of Mib proteins in
regulating Delta endocytosis and it reveals a crucial role for
interactions with Notch in regulating endocytosis of specific Notch
ligands. Future studies will reveal how differential trafficking of
different Notch ligands contributes in unique ways to regulation of
cell fate and behavior in distinct developmental contexts.
We thank Julian Lewis (Cancer Research UK, London) for providing zdC1 and
zdD2 antibodies; Motoyuki Itoh (Nagoya University, Japan) for valuable
discussions and preliminary exploration of these questions; and all the
members of Chitnis laboratory for their comments. This work was supported
by the Intramural Research Program of the NIH/NICHD. This work was also
supported by a JSPS research fellowship (M.M.). Deposited in PMC for release
after 12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/197/DC1
References
Amoyel, M., Cheng, Y. C., Jiang, Y. J. and Wilkinson, D. G. (2005). Wnt1
regulates neurogenesis and mediates lateral inhibition of boundary cell
specification in the zebrafish hindbrain. Development 132, 775-785.
Appel, B. and Eisen, J. S. (1998). Regulation of neuronal specification in the
zebrafish spinal cord by Delta function. Development 125, 371-380.
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling:
cell fate control and signal integration in development. Science 284, 770-776.
Blader, P., Fischer, N., Gradwohl, G., Guillemot, F. and Strahle, U. (1997). The
activity of neurogenin1 is controlled by local cues in the zebrafish embryo.
Development 124, 4557-4569.
Chen, N. and Greenwald, I. (2004). The lateral signal for LIN-12/Notch in C.
elegans vulval development comprises redundant secreted and transmembrane
DSL proteins. Dev. Cell 6, 183-192.
Cheng, Y. C., Amoyel, M., Qiu, X., Jiang, Y. J., Xu, Q. and Wilkinson, D. G.
(2004). Notch activation regulates the segregation and differentiation of
rhombomere boundary cells in the zebrafish hindbrain. Dev. Cell 6, 539-550.
DEVELOPMENT
Notch regulates Delta endocytosis
RESEARCH ARTICLE
Chitnis, A. B. (1999). Control of neurogenesis-lessons from frogs, fish and flies.
Curr. Opin. Neurobiol. 9, 18-25.
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D. and Kintner, C. (1995).
Primary neurogenesis in Xenopus embryos regulated by a homologue of the
Drosophila neurogenic gene Delta. Nature 375, 761-766.
Dornseifer, P., Takke, C. and Campos-Ortega, J. A. (1997). Overexpression of a
zebrafish homologue of the Drosophila neurogenic gene Delta perturbs
differentiation of primary neurons and somite development. Mech. Dev. 63,
159-171.
Fiuza, U. M. and Arias, A. M. (2007). Cell and molecular biology of Notch. J.
Endocrinol. 194, 459-474.
Geling, A., Steiner, H., Willem, M., Bally-Cuif, L. and Haass, C. (2002). A
gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe
neurogenic phenotype in zebrafish. EMBO Rep. 3, 688-694.
Glittenberg, M., Pitsouli, C., Garvey, C., Delidakis, C. and Bray, S. (2006). Role
of conserved intracellular motifs in Serrate signalling, cis-inhibition and
endocytosis. EMBO J. 25, 4697-4706.
Gordon, W. R., Vardar-Ulu, D., Histen, G., Sanchez-Irizarry, C., Aster, J. C.
and Blacklow, S. C. (2007). Structural basis for autoinhibition of Notch. Nat.
Struct. Mol. Biol. 14, 295-300.
Haddon, C., Smithers, L., Schneider-Maunoury, S., Coche, T., Henrique, D.
and Lewis, J. (1998). Multiple delta genes and lateral inhibition in zebrafish
primary neurogenesis. Development 125, 359-370.
Holley, S. A., Geisler, R. and Nusslein-Volhard, C. (2000). Control of her1
expression during zebrafish somitogenesis by a delta-dependent oscillator and
an independent wave-front activity. Genes Dev. 14, 1678-1690.
Hsiao, C. D., You, M. S., Guh, Y. J., Ma, M., Jiang, Y. J. and Hwang, P. P.
(2007). A positive regulatory loop between foxi3a and foxi3b is essential for
specification and differentiation of zebrafish epidermal ionocytes. PLoS ONE 2,
e302.
Itoh, M., Kim, C. H., Palardy, G., Oda, T., Jiang, Y. J., Maust, D., Yeo, S. Y.,
Lorick, K., Wright, G. J., Ariza-McNaughton, L. et al. (2003). Mind bomb is a
ubiquitin ligase that is essential for efficient activation of Notch signaling by
Delta. Dev. Cell 4, 67-82.
Jacobsen, T. L., Brennan, K., Arias, A. M. and Muskavitch, M. A. (1998). Cisinteractions between Delta and Notch modulate neurogenic signalling in
Drosophila. Development 125, 4531-4540.
Jiang, Y. J., Brand, M., Heisenberg, C. P., Beuchle, D., Furutani-Seiki, M.,
Kelsh, R. N., Warga, R. M., Granato, M., Haffter, P., Hammerschmidt, M. et
al. (1996). Mutations affecting neurogenesis and brain morphology in the
zebrafish, Danio rerio. Development 123, 205-216.
Kim, C. H., Bae, Y. K., Yamanaka, Y., Yamashita, S., Shimizu, T., Fujii, R., Park,
H. C., Yeo, S. Y., Huh, T. L., Hibi, M. et al. (1997). Overexpression of
neurogenin induces ectopic expression of HuC in zebrafish. Neuroscience Lett.
239, 113-116.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F.
(1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253310.
Klueg, K. M. and Muskavitch, M. A. (1999). Ligand-receptor interactions and
trans-endocytosis of Delta, Serrate and Notch: members of the Notch signalling
pathway in Drosophila. J. Cell Sci. 112, 3289-3297.
Development 136 (2)
Lai, E. C., Roegiers, F., Qin, X., Jan, Y. N. and Rubin, G. M. (2005). The
ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating
the localization and activity of Serrate and Delta. Development 132, 2319-2332.
Le Borgne, R., Remaud, S., Hamel, S. and Schweisguth, F. (2005). Two distinct
E3 ubiquitin ligases have complementary functions in the regulation of delta and
serrate signaling in Drosophila. PLoS Biol. 3, e96.
Li, Y. and Baker, N. E. (2004). The roles of cis-inactivation by Notch ligands and of
neuralized during eye and bristle patterning in Drosophila. BMC Dev. Biol. 4, 5.
Louvi, A. and Artavanis-Tsakonas, S. (2006). Notch signalling in vertebrate
neural development. Nat. Rev. Neurosci. 7, 93-102.
Moriyoshi, K., Richards, L. J., Akazawa, C., O’Leary, D. D. and Nakanishi, S.
(1996). Labeling neural cells using adenoviral gene transfer of membranetargeted GFP. Neuron 16, 255-260.
Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev.
Biol. 228, 151-165.
Nichols, J. T., Miyamoto, A., Olsen, S. L., D’Souza, B., Yao, C. and
Weinmaster, G. (2007a). DSL ligand endocytosis physically dissociates Notch1
heterodimers before activating proteolysis can occur. J. Cell Biol. 176, 445-458.
Nichols, J. T., Miyamoto, A. and Weinmaster, G. (2007b). Notch signalingconstantly on the move. Traffic 8, 959-969.
Parks, A. L., Klueg, K. M., Stout, J. R. and Muskavitch, M. A. (2000). Ligand
endocytosis drives receptor dissociation and activation in the Notch pathway.
Development 127, 1373-1385.
Pitsouli, C. and Delidakis, C. (2005). The interplay between DSL proteins and
ubiquitin ligases in Notch signaling. Development 132, 4041-4050.
Riley, B. B., Chiang, M. Y., Storch, E. M., Heck, R., Buckles, G. R. and Lekven,
A. C. (2004). Rhombomere boundaries are Wnt signaling centers that regulate
metameric patterning in the zebrafish hindbrain. Dev. Dyn. 231, 278-291.
Schweisguth, F. (2004). Regulation of notch signaling activity. Curr. Biol. 14,
R129-R138.
Smithers, L., Haddon, C., Jiang, Y. J. and Lewis, J. (2000). Sequence and
embryonic expression of deltaC in the zebrafish. Mech. Dev. 90, 119-123.
Takke, C., Dornseifer, P. v., Weizsacker, E. and Campos-Ortega, J. A. (1999).
her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a
target of NOTCH signalling. Development 126, 1811-1821.
Wang, W. and Struhl, G. (2005). Distinct roles for Mind bomb, Neuralized and
Epsin in mediating DSL endocytosis and signaling in Drosophila. Development
132, 2883-2894.
Wettstein, D. A., Turner, D. L. and Kintner, C. (1997). The Xenopus homolog of
Drosophila Suppressor of Hairless mediates Notch signaling during primary
neurogenesis. Development 124, 693-702.
Yeo, S. Y., Kim, M., Kim, H. S., Huh, T. L. and Chitnis, A. B. (2007). Fluorescent
protein expression driven by her4 regulatory elements reveals the spatiotemporal
pattern of Notch signaling in the nervous system of zebrafish embryos. Dev. Biol.
301, 555-567.
Zhang, C., Li, Q. and Jiang, Y. J. (2007a). Zebrafish Mib and Mib2 are mutual E3
ubiquitin ligases with common and specific delta substrates. J. Mol. Biol. 366,
1115-1128.
Zhang, C., Li, Q., Lim, C. H., Qiu, X. and Jiang, Y. J. (2007b). The
characterization of zebrafish antimorphic mib alleles reveals that Mib and Mind
bomb-2 (Mib2) function redundantly. Dev. Biol. 305, 14-27.
DEVELOPMENT
206