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First published online 4 December 2008
doi: 10.1242/dev.027938
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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: chitnisa{at}mail.nih.gov)
Accepted 30 October 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Notch signaling, Endocytosis, DeltaD, DeltaA, DeltaC, Neurogenesis, Zebrafish, Lateral inhibition, Cis inhibition
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; 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 full-length
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 DNA-binding
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 
-secretase
complex (Gordon et al.,
2007).
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.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). 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.
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)] 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').
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
EK fish embryos were injected with either standard MO provided by Gene
Tools or MOs against notch1a and notch3 with mRNA encoding
memb-mRFP-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.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
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 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 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 double-stained 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').
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;
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.
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 -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 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.
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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 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, 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.
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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 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.
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), 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.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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 Mib1-mediated
endocytosis. Nevertheless, it is likely that in vivo, under 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 wild-type 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
Delta-Notch complexes following interactions in cis may help to tune the
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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/197/DC1
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Footnotes |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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.[Abstract]
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.[Abstract]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
Chitnis, A. B. (1999). Control of
neurogenesis-lessons from frogs, fish and flies. Curr. Opin.
Neurobiol. 9,18
-25.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[Abstract]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
Jacobsen, T. L., Brennan, K., Arias, A. M. and Muskavitch, M.
A. (1998). Cis-interactions between Delta and Notch modulate
neurogenic signalling in Drosophila. Development
125,4531
-4540.[Abstract]
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.[Abstract]
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.[CrossRef][Medline]
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.
203,253
-310.[Medline]
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.[Abstract]
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
.[CrossRef][Medline]
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.[CrossRef][Medline]
Louvi, A. and Artavanis-Tsakonas, S. (2006).
Notch signalling in vertebrate neural development. Nat. Rev.
Neurosci. 7,93
-102.[CrossRef][Medline]
Moriyoshi, K., Richards, L. J., Akazawa, C., O'Leary, D. D. and
Nakanishi, S. (1996). Labeling neural cells using adenoviral
gene transfer of membrane-targeted GFP. Neuron
16,255
-260.[CrossRef][Medline]
Mumm, J. S. and Kopan, R. (2000). Notch
signaling: from the outside in. Dev. Biol.
228,151
-165.[CrossRef][Medline]
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 signaling-constantly on the move.
Traffic 8,959
-969.[CrossRef][Medline]
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.[Abstract]
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.[CrossRef][Medline]
Schweisguth, F. (2004). Regulation of notch
signaling activity. Curr. Biol.
14,R129
-R138.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[Abstract]
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.[Abstract]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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.[CrossRef][Medline]
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