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First published online 11 June 2008
doi: 10.1242/dev.018101
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Department of Biology, Duke University, Durham, NC 27708, USA
Author for correspondence (e-mail:
dmcclay{at}duke.edu)
Accepted 23 May 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: Numb, Notch, Delta, Sea urchin, Endomesoderm specification
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Baker, 2000).
Since that initial discovery, Notch signaling has been found to contribute to
development of many organisms. In the sea urchin, Notch signaling initiates
specification of the non-skeletogenic mesoderm (NSM) from the surrounding
endomesoderm (Sherwood and McClay,
1999). Initially, the endomesoderm cells express the Notch
receptor, and those closest to the vegetal pole receive a Delta signal from
micromeres (Sweet et al., 2002). Those cells in direct contact with the
Delta-expressing cells become NSM, and those cells not making direct contact
remain endomesoderm. This first signal initiates specification of NSM cells
that become pigment and blastocoelar cells, between the seventh to ninth
cleavage. Later, Notch signaling again contributes to the further subdivision
of the endomesoderm, this time initiating specification of muscle cells and
ceolomic pouch cells (McClay et al.,
2000; Sherwood and McClay,
1997; Sherwood and McClay,
1999; Sweet et al., 2002). Thus, the sea urchin embryo uses
Delta-Notch signaling to specify all of the NSM cell fates.
Both the Delta ligand and Notch receptor rely on a number of important
modifiers to influence the outcome of the signal. One of these modifiers is
Fringe, which is necessary for the Notch reception of the Delta signal
(Irvine and Wieschaus, 1994;
Panin et al., 1997;
Peterson and McClay, 2005).
Another modifier is Numb: a membrane-localized intracellular protein that
antagonizes Notch signaling in many contexts
(Cayouette and Raff, 2002). The
most detailed studies of Numb/Notch interactions examined their involvement in
asymmetric cell divisions in the Drosophila peripheral nervous
system, central nervous system and mesoderm, all of which rely on Delta-Notch
signals (Han and Bodmer, 2003;
Le Borgne and Schweisguth,
2003b; Ruiz Gomez and Bate,
1997; Skeath and Doe,
1998). A model developed from these studies suggests that Notch
and Delta segregate equally from a progenitor cell into both daughter cells,
whereas Numb segregates into only one of the daughter cells. The Delta ligand
signals to both cells, but activation of Notch target genes only occur in one
cell. In the other cell, Numb blocks the Notch signal by an unresolved
mechanism. Thus, Numb is modeled to be the molecular cue causing the asymmetry
between the daughter cells (Jan and Jan,
1998; Posakony,
1994). This antagonistic relationship between Numb and Notch also
occurs during vertebrate neurogenesis and myogenesis, and has been implicated
in breast cancer (Cayouette and Raff,
2002; Pece et al.,
2004).
Biochemical studies suggest that Numb has three protein-protein binding
domains that have the potential to influence its function. Numb binds to the
Notch intracellular domain via its N-terminal phosphotyrosine binding domain
(PTB domain). This domain is thought to act as a scaffolding domain that
targets proteins to the intracellular domain of the Notch receptor
(Guo et al., 1996;
Li et al., 1997;
Rice et al., 2001). Numb also
has a C-terminal proline rich region (PRR), which has an affinity for the SH3
domains of SRC-family tyrosine kinases, suggesting a link between Numb and
tyrosine-kinase-mediated signaling pathways
(Verdi et al., 1996). Finally,
an EH domain, which is located within the PRR domain, interacts with a network
of proteins involved in endocytosis, actin remodeling and intracellular
transduction of signals (Confalonieri and
Di Fiore, 2002; Santolini et
al., 2000). Thus, Numb may influence Notch signaling by bringing
multiple proteins to the Notch receptor.
Because Numb has been demonstrated to have important roles in the regulation of Notch signaling in many diverse contexts, we isolated the first echinoderm Numb homologue, LvNumb, and examined its role as a regulator of Notch signaling in the sea urchin. Surprisingly, our results indicate that LvNumb is not a negative regulator of Notch signal as expected; instead, it mediates specification of NSM in the sea urchin embryo, and is required for full NSM specification.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Phylogenetic analysis
Phylogenetic trees were created using the maximum likelihood method with
PhyML using the WAG substitution model
(http://atgc.lirmm.fr/phyml/)
(Guindon et al., 2005).
A consensus tree with a 50% cut-off value was derived from a 250 bootstrap analysis using Mega 3.1 (http://www.megasoftware.net/). Numbers above bootstraps represent posterior possibilities calculated from this consensus.
Cell isolation and transplantation
Micromeres were removed at the 16-cell stage by hand using a small glass
pipette as previously described (McClay,
2000). Embryos were halved by incubation of 60-120 cell stage
embryos in Ca2+-free seawater and separation of the halves through
the animal and vegetal poles. A half from a control embryo injected with the
green fluorescent stain CFDA-SE (carboxyfluorescein diacetate succinimidyl
ester) (Invitrogen) was combined with a half from a morpholino-injected embryo
of the same stage.
Cloning a LvNumb fragment
LvNumb was isolated during a search of the sea urchin genome for
components of the Notch signaling pathway. Exact primers were designed against
a small region of S. purpuratus DNA corresponding to Numb and used to
amplify SpNumb from midgastrula cDNA via PCR. The amplified, 105 bp
product was cloned into the pGEMT vector (Promega) and sequenced
bi-directionally (Duke Sequencing Core). Clones were identified as PCR
products of SpNumb by BLAST search. SpNumb was used to probe
a L. variegatus cDNA library macroarrayed on filters.
Northern analysis
Total RNA was isolated from embryos with Trizol. RNA (10 µg) from each
developmental stage was loaded onto a 1% agarose formaldehyde gel, fractioned
by electrophoresis and blotted onto Nylon membrane using Turboblot (Schleicher
and Schuell) and hybridized with a LvNumb fragment lacking the PTB
domain. Blots were given two 5 minute washes with 6xSSPE, 0.5% SDS at
room temperature, one 45-minute wash with 1xSSPE, 0.1% SDS at 37°C,
and one 45 minute wash with 1xSSPE, 0.1% SDS at 50°C. The blot was
placed on film for 72 hours at -80°C with an intensity screen.
Generation of LvNumb constructs
A LvNumb clone was generated by splicing partial clones into a
pCS2 expression vector. The 5'UTR and first 1300 bp of the open reading
frame were cloned from the macroarray screens. The remaining sequence was
cloned by 3'RACE. The pCS2 vector has a 5'UTR that provides an
excellent translation start site for mRNA constructs in the sea urchin (Sweet
et al., 2002). pCS-2 constructs containing the sequence of LvNumb, the PTB
domain of LvNumb and the PRR domain of Numb were generated by standard
molecular biology techniques. All clones were verified by sequence
analysis.
mRNA preparation and injection
LvnNumb-pCS2, PTB domain-pCS2 and PRR domain-pCS2
constructs were linearized with NotI and used as template to generate
in vitro transcribed 5' capped mRNA using the SP6 mMessage Machine kit
(Ambion). mRNA concentrations were determined by spectrophotometry. Injections
were carried out as described (Sherwood
and McClay, 1999).
QPCR analysis
RNA from 25 L. variegatus embryos was isolated with Trizol
(Invitrogen). The samples were treated with Dnase I (Ambion) and then reverse
transcription reactions were performed using a Taqman Gold RT-PCR kit (Applied
Biosystems). A LightCycler instrument and Fast Start SYBR Green PCR kit
(Roche) were used for QPCR analysis based on manufacturers instructions.
Primers used were ubiquitin (Ub) (Davidson
et al., 2002a), and two sets of primers designed to LvNumb. The
primer sets generated similar results. A pCS2 plasmid containing the
LvNumb clone was used to determine the specificity and efficiency of
each primer set. The data from each cDNA sample was normalized against
ubiquitin mRNA. QPCR analysis was performed on three separate samples at least
two times, and each reaction product was confirmed by gel electrophoresis.
Ubiquitin was used as a standard to determine LvNumb transcript
numbers.
Counts of SMC types
The number of SMC-derived cell types was examined in 50-55 hour pluteus
larvae, as previously described (Sherwood
and McClay, 1999).
Immunolocalization and image analysis
Embryos were fixed either in 2% paraformaldehyde for 10 minutes and then
washed through methanol for 1 minute, or they were fixed in methanol for 2
minutes. The embryos were returned to SW plus 4% normal goat serum and
immunochemistry was performed as described previously
(Sherwood and McClay,
1999).
Morpholino injection
The LvNumb sequence was used to design two morpholino antisense
oligonucleotides, which GeneTools produced. Sequences of morpholino
oligonucleotides are: Numb 1, 5'-GTATAATACATGAGAAGAAGACCAC-3';
Numb 2: 5'-GAGAAGAAGACCACTGTTTATATCC-3'.
mRNA encoding a GFP reporter construct fused to the 5'UTR of LvNumb was co-injected with the with LvNumb morpholino to determine the effectiveness of the LvNumb1 morpholino in blocking target mRNA translation. In addition, a control morpholino was injected as a control against any nonspecific effects due to toxicity in the morpholino solution. The LvNumb1 morpholino was injected at 1.5 mM and the LvNumb2 morpholino at 0.5 mM in a solution containing 25% glycerol. The two morpholinos produced identical phenotypes, and were rescued by expression of Numb protein from mRNA not containing the sequence recognized by the morpholinos.
In situ hybridization
In situ hybridization was performed on staged embryos using a protocol
adapted from Harland (1991). A lvnumb sequence lacking the PTB
domain, but containing the 3' polyA tail, was cloned into pGEMT-Easy and
linearized with SpeI. The probe was synthesized with T7 RNA
polymerase (New England Biolabs). Control lvnumb sense probe was used
to verify the specificity of the anti-sense probe hybridization (data not
shown). Anti-sense and sense probes were incubated for the same amount of time
in each experiment.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The LvNumb transcript is expressed during blastula and gastrula stages in the endomesoderm, and the protein is expressed within the presumptive non-skeletogenic mesoderm
Developmental northern analysis showed that a single lvnumb
transcript accumulated at hatched blastula stage and remained expressed
throughout development (Fig.
2A). The spatial location of lvnumb transcripts was
determined by whole-mount in situ hybridization and whole-mount
immunofluorescence with an antibody generated against LvNumb
(Fig. 2B; see Fig. S1A in the
supplementary material). In the egg and early cleavage stages, there was
little to no accumulation of lvnumb transcripts
(Fig. 2B, parts a,b). At the
hatched blastula and mesenchyme blastula stages, lvnumb expression
localized to the vegetal plate endomesoderm
(Fig. 2B, parts c,d). From
early- to mid-gastrula, lvnumb mRNA localized throughout the
invaginating endoderm, with reduced expression in delaminating secondary
mesenchyme cells (Fig. 2B, part
e). Later in gastrulation, transcripts localized to regions corresponding to
the foregut and the blastopore of the embryo, and lvnumb transcripts
were reduced in the midgut (Fig.
2B, part f). A similar profile of expression was observed when
embryos were stained with an antibody against the LvNumb protein. Following
reception of the Delta ligand, Notch protein is removed from the plasma
membrane of the NSM (Sherwood and McClay,
1997; Sherwood and McClay,
1999). At the vegetal pole in hatched blastula stage embryos, the
Notch receptor is missing after signaling there, and Numb protein is present
in the area vacated by the Notch receptor, with a small overlap on either side
with the remaining surface Notch (Fig.
2C, parts a-c) (Sherwood and
McClay, 1997; Sherwood and
McClay, 1999). The spatial and temporal expression of LvNumb
continues to be coincident with LvNotch expression throughout gastrulation
(Fig. 2), indicating that the
expression of LvNumb is appropriate for it to act as a modifier of Notch
signaling during SMC specification. Notch signaling does not appear to
activate lvnumb expression in the vegetal plate, as QPCR and
whole-mount in situ hybridization analysis of embryos injected with either
activated or dominant-negative forms of LvNotch show minimal changes in the
expression levels of lvnumb (see Fig. S1D in the supplementary
material).
LvNumb is necessary for non-skeletogenic mesoderm specification
Based on previous studies in Drosophila and vertebrates, we
hypothesized that LvNumb would function as a negative regulator of Notch
signaling in the sea urchin. Previous studies in the sea urchin showed that
injecting mRNA encoding the intracellular domain of the LvNotch receptor
(LvNACT) constitutively activated Notch signaling, causing
an increase in all four NSM subtypes. Conversely, expression of a
dominant-negative Notch (LvNNEG) construct caused a
decrease in all NSM subtypes (Sherwood and
McClay, 1999). Therefore, manipulating LvNumb activity should give
predictable phenotypes if sea urchin Numb functions as reported in other
systems. Accordingly, we designed two antisense morpholino oligonucleotides to
interfere with the translation of endogenous lvnumb and injected
these into fertilized embryos. Both morpholinos produced the same phenotype.
Embryos injected with LvNumb morpholino showed a large decrease in the number
of pigment cells, blastocoelar cells and muscle cell fibers produced by the
embryo (Fig. 3B,E,J)
(Table 1), whereas embryos
injected with the control morpholino showed no defects in these cell types
(Fig. 3A,D,G)
(Table 1). Overexpression of
lvnumb mRNA did not increase the number of primary mesenchyme cells
(Fig. 3K), but did increase the
number of NSM (Fig. 3L),
including pigment cells and blastocoelar cells
(Fig. 3C,F)
(Table 1). Thus, embryos
injected with the LvNumb morpholino or lvnumb mRNA had similar
phenotypes to embryos injected with LvNNEG or
LvNACT mRNA, respectively. This result was surprising
because it indicated that LvNumb does not antagonize Notch signal mediated
specification of the NSM in sea urchin development. Instead, LvNumb acts
similarly to Notch, contradicting our hypothesis based on the previous
detailed studies on Numb and Notch interactions in Drosophila.
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). To test this hypothesis, we removed micromeres from normal
host embryos at the 16-cell stage and then transferred micromeres from embryos
injected with LvNumb2 morpholino. Whole embryos injected with LvNumb2
morpholino lacked pigment cells (Fig.
4B), whereas the control host embryos that received micromeres
bearing the LvNumb morpholino developed with pigment cells
(Fig. 4D). Thus, the Numb
morpholino in the micromeres does not affect the transfer of the Delta to the
overlying macromere progeny.
During the subdivision of the NSM subtypes, pigment cell specification
requires the first Delta signal from the micromeres. Soon after invagination
of the archenteron, these first NSM subtypes delaminate from the tip of the
archenteron, migrate within the blastocoel and then intercalate between
ectoderm cells (Gibson and Burke,
1985). It was possible that the ectoderm of embryos injected with
the LvNumb2 morpholino compromised the pigment cells and prevented them from
differentiating with pigment. To test this hypothesis, we halved normal
60-cell staged embryos and embryos injected with LvNumb2 morpholino and
combined the two halves (Fig.
4E). Although embryos injected with Numb morpholino developed
without pigment cells, the chimeric embryos had pigment cells from the control
half, and some of those migrated into the ectoderm of the half injected with
the LvNumb2 morpholino (Fig.
4F; green half). This result suggests that the ectoderm is not
affected by the Numb morpholino and that the observed lack of pigment cells in
embryos injected with the Numb morpholino is due to a reduction in NSM
specification.
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). Instead, expression of the PTB domain increased the number
of pigment cells and blastocoelar cells
(Fig. 5B,E)
(Table 1), whereas expression
of the PRR domain construct had no effect on SMC specification
(Fig. 5C,F). The PRR domain
protein was expressed and stable during NSM specification as seen by injecting
embryos with Myc-PRR domain mRNA. Western blot analysis of samples taken at
the early gastrula stage shows that the PRR domain was not degraded during the
NSM specification window (Fig.
5G). Thus, expression of the PTB domain produced a phenotype that
is opposite to the LvNumb morpholino phenotype. The phenotype was similar to
both the LvNumb overexpression and the constitutively active Notch phenotypes,
suggesting that the PTB region of LvNumb is the domain involved in NSM
specification. The observation that the PRR domain has no effect on NSM
specification suggests that the function of sea urchin LvNumb may not involve
the endocytic machinery. Alternatively, the PRR domain may require the PTB to
function in endocytosis.
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LvNumb requires the LvNotch signaling pathway to function during NSM specification
To test more directly the relationship between Numb and Notch, we designed
a set of experiments to test whether the Notch and Numb pathways interact. We
first asked whether overexpressing the PTB domain could rescue pigment cell
specification in embryos expressing dominant-negative Notch, and therefore
unable to respond to the Delta signal. LvNNEG blocks Notch
signaling by expressing an extracellular domain of Notch only, thereby binding
Delta without an intracellular domain to transduce the signal. In three
separate experiments, we injected one set of embryos with 25% glycerol
(Fig. 7A), one set with the
ptb domain mRNA (2 pg/pl) (Fig.
7B), another set injected with LvNNEG mRNA (3
pg/pl) (Fig. 7C) and finally
one set of embryos was co-injected with ptb domain mRNA (2 pg/pl) and
LvNNEG mRNA (3 pg/pl)
(Fig. 7D). Embryos injected
with dominant-negative Notch lacked pigment cells, whereas embryos injected
with ptb domain mRNA showed an increase in pigment cells. The PTB
domain had no effect on pigment cell formation when co-injected with
dominant-negative Notch, as none of the double-injected embryos displayed
pigment cells (n=40) (Fig.
7D). This result suggests that LvNumb requires activation of the
Notch signal to function during pigment cell specification. We next asked
whether expression of activated Notch could rescue pigment cell specification
in embryos injected with LvNumb morpholino
(Fig. 7E-H). Similar to the
above experiments, we performed three separate experiments in which one set of
embryos was injected with control morpholino (1.8 mM)
(Fig. 7E), one set with
LvNACT mRNA (2 pg/pl)
(Fig. 7F), a third set with
LvNumb morpholino (1.8 mM) (Fig.
7G) and finally a fourth set was injected with LvNumb morpholino
and LvNACT mRNA (Fig.
7H). Embryos injected with activated Notch mRNA showed an increase
in pigment cells, but fewer than 10% of embryos injected with LvNumb
morpholino showed pigment cell specification (n=49). However, pigment
cell formation occurred in more than 86% of the embryos co-injected with
activated Notch mRNA and LvNumb morpholino (n=46)
(Fig. 7H). Thus, expression of
the Notch intracellular domain overcomes the lack of Numb expression,
suggesting that a constitutively active intracellular Notch signal can
overcome, or bypass, the absence of Numb during NSM specification.
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). Consistent with the observations described above, our
molecular analysis showed that gcm expression is attenuated at early
mesenchyme blastula stage in embryos injected with LvNumb2 morpholino
(Fig. 7J-K). This result
indicates that LvNumb is necessary in the macromeres for the activation of a
direct target of Notch signaling at the time Delta-Notch is known to initiate
specification of pigment cells, further strengthening support for the
hypothesis that LvNumb and Notch work synergistically to specify
non-skeletogenic mesenchyme. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Ruiz Gomez and Bate, 1997;
Skeath and Doe, 1998).
However, whether Numb has a positive or negative impact on Notch signaling in
vertebrate embryos is less clear. In fact, the function of Numb during
vertebrate neurogenesis suggests that it may depend on the cellular context
(Cayouette and Raff, 2002;
Wakamatsu et al., 1999;
Zhong et al., 2000;
Zhong et al., 1997;
Zilian et al., 2001). For
example, in the developing chicken central nervous system, neural progenitor
cells divide asymmetrically to produce one progenitor cell and one neural
cell. Notch signaling is necessary in the apical cell to specify progenitor
cell fate and chicken Numb (cNumb) asymmetrically localizes to the basal cell.
In that context it appears that cNumb inhibits the Notch signal in the basal
daughter cell, promoting neural cell fate
(Wakamatsu et al., 1999). By
contrast, mouse Numb (mNumb) is localized to the apical progenitor cell (the
Notch signal-requiring cell) in the developing mouse central nervous system
(Zhong et al., 1997).
Moreover, mNotch knockouts and mNumb knockouts have remarkably similar
phenotypes, suggesting that they are both necessary for progenitor cell fate
in the apical cell (de la Pompa et al.,
1997; Lutolf et al.,
2002; Zhong et al.,
2000). These results suggest that both mNumb and mNotch signaling
may function as positive regulators of progenitor cell fate in this context, a
result that suggests the sea urchin is not alone in using Numb as a positive
regulator of Notch signaling.
NSM specification depends on LvNumb; however, it is unclear whether LvNumb
acts by associating with the intracellular domain of the Notch receptor to
facilitate its release from the membrane after 
-sequestrase activity,
the transport of the Notch intracellular domain to the nucleus or by another
mechanism. Several recent studies present evidence suggesting that Numb has
functions independently of the Notch pathway. In cultured mouse neurons, Numb
was shown to localize at the tip of growing axons where it is necessary for
growth by way of its endocytosis of L1, a neuronal cell-adhesion molecule
(Nishimura et al., 2003). In
mouse neuroepithelial progenitor cells, Numb was shown to interact with
several proteins at the adherens junctions, including E-cadherin, N-cadherin
and catenins. These interactions appear to be necessary for the integrity of
the neural epithelium, which is disrupted in Numb mutants
(Rasin et al., 2007). In the
sea urchin, LvNumb is expressed progressively in the presumptive NSM field
where the Notch receptor is degraded from the plasma membrane, and LvNumb
protein expression occurs at sites where Notch is actively transducing a Delta
signal. Numb is then retained for a period of time in the sites where Notch
has previously signaled. This expression pattern is consistent with a role in
Notch signaling and perturbation studies reinforce this conclusion as the Numb
morpholino knocks down expression of the earliest known Notch target, GCM.
However, these data do not rule out a function independent of Notch.
Interestingly, the Notch receptor is still somewhat cleared from the
presumptive NSM in Numb morphants (see Fig. S1F-I in the supplementary
material). Notch receptor clearance from the NSM has been shown to be a
consequence of Notch signaling (Sherwood
and McClay, 1999; McClay et al., 2001), suggesting that some
aspects of Notch signaling may still be intact in Numb morphants. Furthermore,
expression of the Notch intracellular domain (NICD) rescues the Numb
morpholino phenotype, suggesting that a high level of Notch signaling
overcomes a LvNumb requirement. Nevertheless, overexpression of LvNumb, under
conditions that normally augment Notch signaling, fails to rescue the
dominant-negative Notch phenotype, suggesting that a Notch signal must be
triggered for augmentation to occur. Thus, LvNumb works synergistically with
the Notch signal in initiating NSM specification.
Endocytic-independent function of LvNumb
Many studies in Drosophila and vertebrates have linked the
function of Numb to an association with the endocytic machinery: Numb has an
EH domain that interacts with the endocytic machinery, Numb can interact with
-adaptin (a member of the A2 endocytic complex), Numb localizes to
clathrin-coated pits, and it is associated with endocytic organelles
(Berdnik et al., 2002;
Jafar-Nejad et al., 2002).
Furthermore, in a study focusing on Numb and L1 interactions in axon growth,
the PTB domain and the PRR domain both acted as dominant-negative versions of
Numb because they prevented endogenous Numb from connecting L1 to the
endocytic pathway (Nishimura et al.,
2003). Interestingly, however, in Drosophila, the
elimination of the binding motifs for endocytic proteins does not affect the
ability of Numb proteins to specify cell fates
(Tang et al., 2005). Similarly
in the sea urchin, the PTB domain of LvNumb rescues pigment cell specification
in LvNumb morpholino-injected embryos, which contain little to no endogenous
LvNumb, whereas expression of the PRR domain had no effect on NSM
specification. As it is likely that the ability of Numb to interact with the
endocytic machinery resides in the PRR domain, specifically in the EH domain
(Salcini et al., 1997;
Santolini et al., 2000;
Smith et al., 2004), this
result suggests that the function of LvNumb need not include interactions with
the endocytic machinery. Rather, it appears that the function of LvNumb
resides in its interactions with other proteins via the PTB domain. Indeed, as
suggested by data shown in Fig. S1F-I in the supplementary material, in the
presence of the Numb morpholino the Notch signal begins with Delta binding to
Notch, and as a consequence the Notch extracellular domain is lost as normal
(see Fig. S1H in the supplementary material). However, the signal transduction
is not completed because the Numb morpholino causes a failure in the
specification of pigment cells (see Fig. S1J in the supplementary
material).
The present results raise important questions about the function of Numb, in particular its role as a Notch pathway agonist. It is possible that the function of Numb has changed during the course of evolution. Although LvNumb is still closely associated with Notch signaling in the sea urchin, the PTB domain may have changed to the point that it activates or protects the Notch intracellular domain on its way to the nucleus. Alternatively, LvNumb may bind other factors in cells via its PTB domain and as a consequence Notch signaling is positively affected. This is the second study to suggest that Numb has a function exclusive of endocytosis and that the PTB domain alone is able to specify cell fates, suggesting that this may be a conserved mechanism in both protostomes and deuterostomes. Thus, further studies are merited to identify the proteins that interact with the LvNumb domains. Identification of such factors will not only help clarify NSM specification in the sea urchin, but will have implications for Notch and Numb signaling in general.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/14/2445/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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