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First published online 13 April 2005
doi: 10.1242/dev.01825
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1 Department of Molecular and Cell Biology, Howard Hughes Medical Institute, 545
Life Sciences Addition, University of California at Berkeley, Berkeley, CA
94720-3200, USA
2 Department of Physiology and Biochemistry, Howard Hughes Medical Institute,
1550 4th Street, Room GD481, University of California, San Francisco, Box
0725, San Francisco, CA 94143-0725, USA
3 Fox Chase Cancer Center, Cellular and Developmental Biology Program, 333
Cottman Avenue, Philadelphia, PA 19111, USA
* Author for correspondence (e-mail: lai{at}fruitfly.org)
Accepted 3 March 2005
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). We further demonstrate that D-mib can functionally
replace Neuralized in multiple cell fate decisions that absolutely require
endogenous Neuralized, a testament to the highly similar activities of these
two unrelated ubiquitin ligases in regulating Notch signaling. We conclude
that ubiquitination of Delta and Serrate by Neuralized and D-mib is an
obligate feature of DSL ligand activation throughout Drosophila
development.
Key words: Mind bomb, Neuralized, Serrate, Delta, Notch signaling, Endocytosis
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Indeed, there is scarcely a developmental process that does
not involve Notch signaling in some reasonably direct fashion. Its central
cell surface components are a Delta/Serrate/LAG-2 (DSL)-type ligand and the
receptor Notch, both of which are type I transmembrane proteins that interact
via their extracellular domains. Ligand-induced activation of Notch triggers
the cleavage of the intracellular domain of Notch, which subsequently
translocates to the nucleus and functions as a transcriptional co-activator
for a CSL-type DNA-binding protein (Lai,
2004).
There are two DSL ligands in Drosophila, Delta and Serrate, which
have both overlapping and distinct functions during development. For example,
lateral inhibition of neural precursors is mediated largely by Delta
(Heitzler and Simpson, 1991),
whereas embryonic segmental patterning is mediated by Serrate
(Wiellette and McGinnis,
1999). However, asymmetric cell divisions during peripheral sense
organ development (Zeng et al.,
1998) and leg joint specification
(Bishop et al., 1999;
de Celis et al., 1998;
Rauskolb and Irvine, 1999)
require both ligands. Distinct patterns of DSL ligand expression help to
explain why some settings require one or the other ligand. In fact, rescue
experiments involving ectopically expressed ligands demonstrate a certain
degree of functional interchangeability between Delta and Serrate
(Gu et al., 1995;
Klein and Arias, 1998). In
certain settings, however, Delta and Serrate are co-expressed but have
non-overlapping function. For example, lateral inhibition amongst proneural
clusters of the adult peripheral nervous system requires only Delta, even
though proneural clusters express both ligands.
The degree to which components of Notch signaling are regulated at the
post-translational level has only become fully apparent in the last few years.
Various components are subject to proteolysis, glycosylation, ubiquitination
and phosphorylation, which collectively have a tremendous range of
consequences on the efficacy of Notch signaling
(Schweisguth, 2004). This
variety is well illustrated by ubiquitination, a functionally versatile
protein modification that can promote protein degradation, influence protein
localization, or modulate protein activity
(Hershko and Ciechanover,
1998; Hicke and Dunn,
2003; Zhang,
2003).
Ubiquitination is mediated by the stepwise activity of several enzymes
(Hershko and Ciechanover,
1998). First, an ubiquitin-activating enzyme (E1) activates the
76-amino-acid ubiquitin molecule by an ATP-dependent mechanism and transfers
it to an ubiquitin-conjugating enzyme (E2). Then, an ubiquitin ligase (E3)
facilitates transfer of ubiquitin from E2 to the appropriate substrate. Three
protein motifs display intrinsic, biochemically demonstrable, ubiquitin ligase
activity: the HECT domain, the RING finger and a structural relative of the
RING finger termed the U box (Hatakeyama
and Nakayama, 2003; Jackson et
al., 2000; Joazeiro et al.,
1999; Scheffner et al.,
1993). As the E3 is responsible for target specificity, the number
of E3 enzymes is far greater than the number of either E1 or E2 enzymes. A
typical eukaryotic genome encodes a single E1 and perhaps 
20 E2s, but
>100 E3s. In some cases, including in the prototypical SCF complex,
ubiquitin ligase and substrate recognition domains reside in different
proteins that associate as a multisubunit E3. In most cases, though, the E3 is
a single protein that binds the substrate and catalyzes its
ubiquitination.
At least five different components of the Notch pathway are regulated by
ubiquitination [including the ligand Delta, the epsin Liquid facets (Lqf), the
receptor Notch, the Notch regulator Numb, and some bHLH repressor-encoding
products of the HES genes], and ubiquitination can either negatively- or
positively-regulate Notch signaling depending on the particular substrate and
situation (Chen et al., 2002;
Hirata et al., 2002;
Itoh et al., 2003;
Lai, 2002). In some cases, the
same component is directly targeted by multiple E3 ubiquitin ligases. For
example, membrane-localized Notch is regulated by Su(dx)/Itch
(Cornell et al., 1999;
Qiu et al., 2000) and possibly
by Sel-1 (Grant and Greenwald,
1997), whereas nuclear Notchintra is regulated by
Sel-10/Ago (Gupta-Rossi et al.,
2001; Hubbard et al.,
1997; Oberg et al.,
2001; Wu et al.,
2001).
In recent years, two types of E3 ubiquitin ligase were shown to target
Delta and regulate its localization and signaling activity
(Le Borgne and Schweisguth,
2003a). Both E3s were identified through the study of neurogenic
mutants, which display excess neural differentiation. This phenotypic class
includes mutations in all central components of Notch signaling.
neuralized (neur) is a fly neurogenic that was discovered
over twenty years ago (Lehmann et al.,
1983; Wieschaus et al.,
1984). Neur is absolutely required in some settings of Notch
signaling in flies, but is dispensable in others. For example, Neur restricts
neural precursors, R8 photoreceptors and muscle precursors, and controls
asymmetric cell divisions within neural lineages, but is dispensable for wing
margin specification, eye growth and restriction of wing vein thickness
(Corbin et al., 1991;
Lai and Rubin, 2001a;
Lai and Rubin, 2001b;
Lehmann et al., 1983;
Yeh et al., 2000). Mind
bomb (mib) is a fish neurogenic that also displays defective
Notch signaling in certain other developmental settings, including somite
formation and vascular development (Jiang
et al., 1996; Lawson et al.,
2001; Schier et al.,
1996; van Eeden et al.,
1996). Neur and Mib each contain a RING finger at their respective
C termini, and both have been biochemically demonstrated to directly
ubiquitinate Delta (Deblandre et al.,
2001; Itoh et al.,
2003; Lai et al.,
2001; Price et al.,
1993). Aside from this motif, however, these proteins are
completely unrelated.
As is the case for a number of other transmembrane proteins,
monoubiquitination of Delta induces its endocytosis and subsequent degradation
(Deblandre et al., 2001;
Itoh et al., 2003;
Lai et al., 2001;
Pavlopoulos et al., 2001).
Curiously then, Neur and Mib non-autonomously stimulate Notch activation by
Delta-expressing, signal-sending cells. The evidence for this is that
mib-mutant fish cells are deficient in their ability to send a
lateral inhibitory signal in neural tube cell transplantation experiments
(Itoh et al., 2003), that
neur mutant fly cells are preferentially inhibited from adopting the
neural fate at mosaic clone borders
(Pavlopoulos et al., 2001) and
display non-autonomous defects during asymmetric neural lineage divisions
(Le Borgne and Schweisguth,
2003b), and that co-expression of Neur with Delta potentiates the
ability of a cell to activate Notch signaling in neighboring cells
(Pavlopoulos et al.,
2001).
Although both Neur and Mib have been evolutionarily conserved in diverse
metazoans, a genetic requirement for both ubiquitin ligases in Notch signaling
in any single organism has not yet been demonstrated. In addition, it has not
yet been established whether signaling by the Drosophila DSL ligand
Serrate is regulated by endocytosis. In this study, we characterize the
Drosophila ortholog of Mind bomb (D-mib). We find
that D-mib is essential for a large number of
neur-independent, Notch pathway-mediated developmental processes,
allowing us to classify it as a vital component of Drosophila Notch
signaling. We find that D-mib directly associates with and targets both
Serrate and Delta for endocytosis and degradation, and is able to generally
influence Notch signaling through its ability to regulate DSL ligand activity
(see also Le Borgne et al.,
2005). Finally, we show that ectopic D-mib is able to rescue
multiple aspects of the neur mutant phenotype, demonstrating that
Neur and D-mib have highly overlapping functions in vivo.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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); UAS-neur,
UAS-neur
RF (Lai and
Rubin, 2001a).
D-mib structure-function analysis
We amplified the desired portions of D-mib, using the cDNA SD05267
as template (gift of the Berkeley Drosophila Genome Project), and cloned them
into TOPO-D-ENTR (Invitrogen). Primer sequences are available upon request.
Following sequence verification, these DNAs were cloned into UAS-HM-Gate, a
Gateway-compatible vector that creates N-terminal in-frame fusions to 6
xHis and 3 xMyc tags (gift of Cynthia Hsu and Brian McCabe). These
were injected into Drosophila embryos using standard protocols, and
multiple independent insertions were established and analyzed for each
construct.
Exogenous Delta assay
These experiments used the hs-Delta construct of Struhl
(Struhl and Adachi, 1998). We
selected Tb+ larvae of the following crosses:
hs-Delta xdpp-Gal4, UAS-D-mib/SM-TM6B, Cy, Tb
and hs-Delta xUAS-D-mibRF;
dpp-Gal4/SM-TM6B, Cy, Tb. Larvae were heat-shocked at
38°C in a circulating water bath and then allowed to recover at 25°C
for the desired length of time.
Neur rescue experiments
We made neur mutant clones using the FRT82B,
neurA101 and FRT82B, neurIF65 chromosomes
(Lai and Rubin, 2001a), using
ubx-FLP and the MARCM system (Lee
and Luo, 2001), and drove expression of transgenes within mutant
clones using sca-Gal4. ubx-FLP reliably creates mutant notum clones
in FRT-homozygous flies, so that 50% of the appropriate progeny will carry
mutant clones; the neur mutant phenotypes are themselves completely
penetrant. We inferred rescue of neurA101 by
UAS-neur from the observation that 100% of the appropriate progeny
were wild type. A similar observation applies to the rescue of
neurIF65 by UAS-D-mib, although in this case, the
presence of rescued, mildly tufted bristles is also a positive assessment of
amelioration of the neurIF65 balding phenotype. Crosses
were as follows.
neur rescue of neurA101: yw, ubx-FLP/+; sca-Gal4, UAS-pon-GFP, UAS-tau-GFP/CyO; FRT82B, tub-Gal80/TM6B, P(y+) x w/Y; UAS-neur/+; FRT82B, neurA101.
D-mib rescue of neurIF65: yw, ubx-FLP/+; sca-Gal4, UAS-pon-GFP, UAS-tau-GFP/CyO; FRT82B, tub-Gal80/TM6B, P(y+) x w, UAS-D-mib/Y; +/+; FRT82B, neurIF65/+. Female Cy+, Tb+ adults were selected.
Note that in second cross, only female progeny will show rescue, as an X-linked UAS-D-mib insertion was used. We exploited this in the analysis of D-mib rescue of neur mutant pupal clones, by separating male from female larvae in this cross, and selecting 36 hours after puparium formation (APF) pupae for patches of GFP expression on the notum, which indicated the presence of neurIF65 mutant MARCM clones. These were then fixed and stained.
D-mib:Delta and D-mib:Serrate co-immunoprecipitation
The construct for expressing Delta with two C-terminal polyoma tags was
described previously (Lai et al.,
2001). A similarly tagged Serrate expression construct was
generated by PCR using LP24305 (Berkeley Drosophila Genome Project) and cloned
into pcDNA3.1/TOPO (Invitrogen). Myc-tagged D-mib expression vectors contained
the coding regions of UAS-HM-D-mib constructs cloned into pcDNA3.1/TOPO using
PCR (Invitrogen). Plasmids were transiently transfected into 293T cells using
Fugene 6 (Roche). After incubation for 48 hours, cells were lysed in 50 mM
Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 0.5% Triton X-100 and EDTA-free
Complete Protease Inhibitors (Roche). 200 µg or 250 µg protein lysate
was used in Serrate and Delta co-immunoprecipitations, respectively; lysates
were incubated with mouse anti-Myc, mouse anti-Delta C594 (Developmental
Studies Hybridoma Bank, DSHB) or agarose-conjugated goat anti-Myc (NOVUS, San
Diego) in 1 ml lysis buffer. The captured proteins were then precipitated with
protein A/G-plus agarose (Santa Cruz biotechnology) and recovered by boiling
in Laemmli sample buffer. The immunoprecipitated proteins or 5 µg total
protein lysate were separated on SDS-PAGE gels (BioRad) and transferred to
Hybond-C extra membrane (Amersham Pharmacia Biotech). The membranes were
probed with anti-Myc, anti-Delta or anti-polyoma, and detected with ECL-plus
reagents (Amersham Pharmacia Biotech).
Immunofluorescence
We used the following primary antisera, all of which were previously
described: guinea pig anti-Senseless (1:5000, gift of Hugo Bellen), rat
anti-Su(H) (1:1500, gift of Francois Schweisguth), mouse anti-Cut (1:100,
DSHB), mouse anti-Delta (1:100, DSHB), guinea pig anti-Delta (1:2500, gift of
Marc Muskavitch), rat anti-Serrate (1:2000, gift of Ken Irvine), mouse
anti-Myc (1:5000, ascites), rat anti-ELAV (1:100). We detected proteins as
described previously (Lai and Rubin,
2001a).
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). All Mib proteins share a common domain structure that
we divide into three regions (Fig.
1). The N-terminal region consists of a zz zinc finger that is
flanked by a pair of domains shared between Mib and HERC2 (Mib/HERC2 domains),
followed by a repeated sequence specific to Mib (the Mib domain). The middle
region is characterized by eight ankyrin repeats. Finally, the C-terminal
region contains three RING finger domains, of which only the final RING finger
fully resembles the canonical sequence known to mediate ubiquitin ligation.
There are two predicted Mib homologs in Drosophila. Of these, CG5841
is more closely related to Mind bomb at the amino acid level and is thus the
true Drosophila ortholog of Mind bomb (D-mib). We refer to CG17492 as
Drosophila Mind bomb-like (D-mibl), but will not consider it further
in the present study.
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).
We could revert its lethality by precise excision of the transposon,
indicating that this insertion affects D-mib function. Accordingly,
we refer to it as D-mib1. Concurrent studies by the
Schweisguth group further indicate that D-mib1 behaves as
a genetic and protein null allele, and can be rescued by transgenic expression
of D-mib (Le Borgne et al.,
2005). We examined homozygous pharate D-mib1
flies and found that they were eyeless, had vestigial wings, and displayed
squat legs lacking joints (Fig.
2A-F). We examined the basis of the wingless phenotype by staining
for markers of wing development in the third instar imaginal disc.
D-mib1 mutant discs displayed a severely reduced wing
pouch, as marked by Nubbin (Fig.
2G,H). This is due to a loss of Cut expression in the wing pouch,
indicating a failure to specify the wing margin
(Fig. 2I,J). These phenotypes
closely resemble those caused by Notch pathway loss of function, thus strongly
implicating D-mib as a component of Drosophila Notch signaling.
However, lateral inhibition was largely unaffected in
D-mib1 discs, as most sensory organ precursors (as marked
by Senseless) were singularized normally
(Fig. 2K,L). Therefore, D-mib
is required for only a subset of Notch-dependent processes. Interestingly, the
ubiquitin ligase Neuralized (Neur) is essential for lateral inhibition of
sensory precursors, but is not required for wing margin, leg joint or eye
specification (Lai and Rubin,
2001a; Lai and Rubin,
2001b; Yeh et al.,
2000). Therefore, Neur and D-mib appear to have complementary
functions in regulating Drosophila Notch signaling.
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). This contrasts with the highly restricted expression of
neur in sensory organ precursors of the wing imaginal disc
(Boulianne et al., 1991). To
gain further insight into the activity of D-mib, we ectopically expressed
full-length and truncated D-mib proteins in transgenic Drosophila
(Figs 1,
3). The activities of
full-length D-mib and D-mibRF (lacking only its most C-terminal RING
finger) exactly parallel those of corresponding Neur proteins
(Lai and Rubin, 2001a;
Lai and Rubin, 2001b) in that
the full-length proteins hyperactivate Notch signaling, while RING-finger
deleted versions inhibit Notch signaling. Thus, misexpression of D-mib induces
mild loss of macrochaete bristles and campaniform sensilla
(Fig. 3B,H), loss of sensory
organ precursors (as marked by Sens expression,
Fig. 3K), as well as loss of
wing veins (Fig. 3E). By
contrast, D-mibRF induces mild bristle tufting and multiplication of
sensory precursors (Fig.
3C,I,L), potently inhibits wing margin development
(Fig. 3F), eliminates
expression of wing margin markers (including Cut,
Fig. 3O), inhibits restriction
of wing vein thickness (Fig.
3F,I), and inhibits eye disc growth (data not shown). Some effects
of D-mibRF phenocopy D-mib loss of function (such as loss of
wing margin and absent retinal development); however, D-mib and D-mibRF
affect other settings of Notch signaling that only weakly require, or are
independent of, D-mib. Therefore, as is the case for Neur
(Lai and Rubin, 2001a;
Lai and Rubin, 2001b), D-mib
can generally affect Notch signaling when expressed ectopically. This is
particularly so for the RING-finger deleted, dominant-negative isoforms,
NeurRF and D-mibRF.
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RF is consistent with
the observation that the only two missense alleles of zebrafish Mib alter
amino acids in the most C-terminal RING finger of Mib
(Itoh et al., 2003), which we
may infer to be the RING finger most critical for Mib function. A construct
that lacks all three C-terminal RING fingers (D-mib3RF) also strongly
antagonizes Notch signaling, but its activity is less potent relative to
D-mibRF in all settings examined (summarized in
Fig. 1, and data not shown).
The developmental effects of ectopic D-mib, D-mibRF and D-mib3RF
are confined entirely to Notch-regulated patterning events. However, further
truncation of the ankyrin repeats resulted in an isoform (D-mib-N) with
weakened ability to induce vein thickening, and also potentially nonspecific
activity, manifested by wing blistering
(Fig. 1 and data not shown).
Finally, misexpression of the ankyrin repeats and RING finger domains had no
detectable effects on development (Fig.
1 and data not shown).
D-mib physically associates with both Delta and Serrate
Our loss- and gain-of-function analyses indicate that the major function of
D-mib is to regulate Notch signal transduction. As Delta is a bona fide
substrate of zebrafish Mib (Chen and
Corliss, 2004; Itoh et al.,
2003), we tested for a physical association of D-mib and Delta by
co-immunoprecipitation. We transfected 293T cells with Delta and various D-mib
expression vectors, and performed co-immunoprecipitation in both directions.
Although Delta did not successfully co-immunoprecipitate full-length D-mib, it
did associate with all isoforms that contain the D-mib N terminus and lack the
C-terminal RING finger (namely D-mib-N, D-mib3RF and D-mibRF,
Fig. 4A, lanes 2-4).
Conversely, these same D-mib isoforms efficiently co-immunoprecipitated Delta
(Fig. 4B, lanes 12-14);
full-length D-mib also showed modest association with Delta in this direction
(Fig. 4B, lane 11). We
consistently observed that the presence of full-length D-mib reduced Delta
levels (Fig. 4B, lane 16),
which might account for why this interaction is poorly detected. Notably,
D-mib-N showed the strongest interaction with Delta. In fact,
immunoprecipitated D-mib-N brought down both full-length Delta and cleaved
DeltaIC (Fig. 4B,
lane 12), consistent with a direct interaction between the N terminus of D-mib
and the intracellular domain of Delta. A truncated D-mib protein lacking the
N-terminal domain (D-mib-C) showed no binding to Delta
(Fig. 4A, lane 5, and
Fig. 4B, lane 15),
demonstrating that this region is crucial for association with Delta.
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Our in vitro data correlate well with our in vivo studies, in that all
RING-finger-deleted D-mib isoforms that retain the ability to associate with
DSL ligands (D-mib-N, D-mibRF and D-mib3RF) have at least some
ability to inhibit Notch signaling. However, full specificity and activity of
D-mib requires inclusion of the ankyrin repeats and the two non-canonical RING
fingers. Curiously, there is no significant similarity at the primary amino
acid level between the intracellular domains of Delta and Serrate. In this
regard, it is relevant to note that Xenopus Neur (X-Neur) robustly regulates
Drosophila Delta in vivo
(Deblandre et al., 2001), even
though there is no significant similarity between the intracellular domains of
Delta and X-Delta. D-mib and Neur may therefore recognize a more hidden,
possibly structural, feature that is shared by DSL ligands.
D-mib promotes the signaling activity of DSL ligands
We have shown that D-mib is positive component of Drosophila Notch
signaling that physically interacts with both DSL ligands. To further explore
its function in the Notch pathway, we assayed the ability of D-mib to
influence DSL ligand activity in vivo. Misexpression of Delta along the
anteroposterior compartment boundary of the wing disc using dpp-Gal4
strongly induces disc overgrowth and ectopic wing margin in the dorsal wing
pouch (Doherty et al., 1996;
Panin et al., 1997)
(Fig. 5A,B,G,H). By contrast,
Delta does not induce ectopic margin ventrally
(Fig. 5B). Co-misexpression of
D-mib with Delta strongly potentiated Delta signaling, resulting in increased
ventral disc overgrowth and ectopic wing margins that span the ventral
compartment, both anterior and posterior to the dpp-Gal4 domain
(Fig. 5C,I). Conversely,
co-misexpression of D-mibRF with Delta completely suppressed the
activity of exogenous Delta, so that no ectopic margin or disc overgrowth was
seen (Fig. 5D,J) In fact,
misexpression of Delta was unable to rescue the loss of endogenous wing margin
induced by D-mibRF (compare Fig.
3O with Fig. 5D).
We conclude that D-mibRF simultaneously inhibits endogenous and
exogenous Delta activity.
|
RF
(Fig. 5F,L). This is consistent
with the observation that ectopic Serrate fails to rescue wing margin in
D-mib discs (Le Borgne et al.,
2005). Thus, both DSL ligands appear to be nonfunctional in the
presence of D-mibRF, a finding that reinforces the essential nature of
ubiquitination to DSL ligand activity in Drosophila.
D-mib induces DSL ligand internalization and degradation
Le Borgne and colleagues have recently shown that D-mib mutant
cells display a selective defect in DSL ligand internalization. Specifically,
their antibody uptake assays demonstrated that D-mib is required for
Serrate, but not Delta, endocytosis in living epithelial cells of wing
imaginal discs (Le Borgne et al.,
2005). In addition, they observed that D-mib cells show
elevated accumulation of Serrate at the apical plasma membrane and a decrease
in Serrate+ vesicles, whereas no corresponding alteration in Delta
accumulation or localization was seen (Le
Borgne et al., 2005). We verified that D-mib1
imaginal discs show a primary defect in Serrate, but not Delta accumulation
(Fig. 6A-D).
|
). We find that D-mib similarly decreases the overall level of
endogenous Delta, particularly at the apical plasma membrane
(Fig. 6J). D-mib decreases the
level of Serrate to a lesser extent; nevertheless, it was clear that the level
of Serrate at the apical plasma membrane is reduced in the presence of ectopic
D-mib (Fig. 6I). Conversely,
D-mibRF strongly increased the steady state levels of both Delta and
Serrate (Fig. 6K,L),
demonstrating that D-mib requires ubiquitin ligase function to induce the
internalization of DSL ligands.
Although an abnormally large amount of Serrate and Delta accumulates at the
apical plasma membrane in the presence of D-mibRF, their
internalization was not completely blocked. Consistent with this, it was
recently shown that vesicular Serrate can still be detected in D-mib
mutant cells (Le Borgne et al.,
2005). We also note that DSL ligands showed distinct behavior in
the presence of D-mibRF, as Serrate accumulates in extremely large
apical intracellular aggregates that co-localize only partially with Delta
(Fig. 6K,L, insets). As Serrate
and Delta are nonfunctional in the presence of D-mibRF
(Fig. 5), the internalization
of DSL ligands does not strictly correlate with their activation.
The observation of changes in the steady-state levels and localization of
DSL ligands does not by itself distinguish between transcriptional,
post-transcriptional and post-translational mechanisms. For example, the
increase in Delta induced by D-mibRF is likely to be partly a
consequence of the associated neurogenic defect
(Fig. 3), which should be
associated with increased transcription of Delta
(Schweisguth and Posakony,
1994). Even if the observed effects are the result of
post-translational modification of DSL ligands by D-mib, one cannot
confidently distinguish between mechanisms whereby internalization of
membrane-localized Delta is specifically affected, as opposed to aberrant
trafficking of Delta from the endoplasmic reticulum directly to endosomes.
Previous studies of zebrafish Mib employed static assays in transfected tissue
culture cells (Chen and Corliss,
2004; Itoh et al.,
2003), and also do not distinguish these possibilities.
We therefore employed a dynamic assay using exogenously expressed Delta
under the control of a heat-shock promoter. When induced with a 40-minute heat
shock, high levels of Delta accumulate at the plasma membrane of all cells
(Fig. 6M), and remain
detectable there for many hours. In the presence of Neur, exogenously
expressed Delta is correctly trafficked to the plasma membrane, but is rapidly
internalized into vesicles and is subsequently degraded
(Lai et al., 2001). We find
that D-mib has identical activity to Neur in this assay. Thirty-five minutes
into the heat-shock regime, ectopic Delta could be detected at apical cell
membranes in the presence of exogenous D-mib (see Fig. S1 in the supplementary
material), indicating that trafficking of Delta is normal in the presence of
elevated levels of D-mib. Strikingly, Delta is quickly internalized in the
D-mib-expressing domain, so that all of the Delta protein is vesicular by 40
minutes after heat shock-mediated induction of Delta expression
(Fig. 6N). By 90 minutes
post-induction, almost all of the ectopic Delta has been degraded
(Fig. 6O). The effect of D-mib
on DSL ligands is relatively specific, as double staining experiments showed
bulk localization of the Notch receptor to be largely unaffected at time
points when large amounts of Delta were actively being internalized and
degraded (see Fig. S2). As with the endogenous Delta assay, D-mibRF is
unable to mediate Delta endocytosis in this assay, and high levels of Delta
persist even at 120 minutes post-induction
(Fig. 6P). Therefore, D-mib and
Neur have identical abilities to induce the internalization and degradation of
Delta in a RING finger-dependent fashion.
Neur and D-mib are functionally interchangeable
Our studies, together with those of others, collectively demonstrate that
Neur and Mib possess very similar activities with respect to the regulation of
DSL ligands and to Notch signaling. However, it is a fact that the two have no
sequence or domain similarity apart from their C-terminal RING fingers.
Moreover, their in vivo requirements for DSL ligand internalization appear to
be distinct, with Neur being more important for Delta endocytosis and D-mib
being more important for Serrate endocytosis. Therefore, we were interested to
directly test the functional interchangeability of these two proteins and we
approached this in two ways.
The first assay tested the ability of ectopic Neur and D-mib to genetically
suppress the effects of their respective RING finger-deleted,
dominant-negative counterparts. When activated using dpp-Gal4, the
full-length proteins cause mild loss of veins and campaniform sensilla
(Fig. 7A-C), whereas the
dominant-negative proteins cause vein thickening, ectopic campaniform
sensilla, and loss of wing margin (Fig.
7D,G); the effects of D-mib and D-mibRF are stronger than
those of Neur and NeurRF, respectively. Ectopic Neur rescues the
phenotype of dpp-Gal4>NeurRF flies, as does
ectopic D-mib (Fig. 7E,F).
Conversely, both ectopic D-mib and Neur can suppress the phenotype of
dpp-Gal4>D-mibRF flies, although higher doses of
Neur are required for rescue of dpp-Gal4>D-mibRF
back to wild type (Fig. 7G-I
and data not shown). The latter result is especially notable because, as shown
in Fig. 5, neither ectopic
Delta nor Serrate are able to rescue the wing margin defect induced by
D-mibRF. In summary, both of these ubiquitin ligases can rescue the
mutant phenotypes induced by their respective dominant-negative
derivatives.
|
), We
reasoned that sca-Gal4 might direct spatially relevant expression of
transgenes, as its activity is elevated or exclusive to microchaete sensory
precursor cells and persists in the sensory lineage
(Abdelilah-Seyfried et al.,
2000).
Neur is required at multiple steps during the development of peripheral
sensilla. It is first required to restrict the sensory precursor fate amongst
proneural cluster cells, and is subsequently required to direct multiple
asymmetric cell fates in the sensory lineage. In the adult notum, clones of
the hypomorphic allele neurA101 display tufted bristles
(Fig. 8A) as a result of a mild
defect in sensory precursor restriction, and subsequent development of
supernumerary sensilla with normal cell complements. By contrast, similar
clones of the null allele neurIF65 are bald
(Fig. 8C) because of the
combined effects of strongly defective lateral inhibition, followed by
mis-specification of all sensory lineage cell fates as neurons
(Lai and Rubin, 2001a;
Yeh et al., 2000). We made
notum clones of both alleles using the MARCM system and ubx-FLP, and
tested the ability of full-length UAS-neur and UAS-D-mib to
rescue neur mutant clones that have activated sca-Gal4. In
Fig. 8A,B, we show that Neur
completely rescues the bristle tufting phenotype of
neurA101 clones, demonstrating the efficacy of this rescue
strategy.
|
We stained mutant sensory clusters that were positively marked by MARCM expression of sca-Gal4>UAS-pon-GFP. We first stained 36 hours after puparium formation (APF) pupae for Su(H), a marker of external socket sockets. neurIF65 mutant cells never express Su(H), whereas in the presence of ectopic D-mib, mutant sensory clusters displayed small groups of Su(H)+ cells (Fig. 8E-J). We next stained for the neuronal marker Elav. Individual rescued sensory organs show a strongly reduced neurogenic phenotype in which two to three Elav+ cells are present, instead of the large clusters present in neur mutant clusters (Fig. 8K-P, red). Finally, there is a strong reduction in the overall number of cells in each cluster (Fig. 8K-M, green). Together, these data directly demonstrate the rescue of pIIa specification (which gives rise to the outer sensory cells) and substantial rescue of the neur lateral inhibition defect (because clusters usually contained two to three cells of each lineage fate). We take the ability of D-mib to replace neur during multiple cell fate decisions in vivo as strong evidence for their functional similarity.
|
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The
presence of a clear Drosophila ortholog of Mind bomb was somewhat of
a surprise then, given that: (1) almost without exception, different species
display similar functional requirements for evolutionarily conserved
components of the Notch pathway (Lai,
2004); (2) this locus never emerged from any of the extensive
Drosophila genetic screens for neurogenic genes and components of the
Notch pathway (Abdelilah-Seyfried et al.,
2000; Fortini and
Artavanis-Tsakonas, 1994; Go
and Artavanis-Tsakonas, 1998;
Verheyen et al., 1996;
Xu and Artavanis-Tsakonas,
1990); and (3) it is a large locus that might be expected to have
been relatively easily hit, as has proven to be the case in zebrafish
(Golling et al., 2002;
Jiang et al., 1996). It was
therefore an open question as to whether D-mib was actually a component of
Notch signaling in Drosophila, a question that our present studies
allow us to answer in the affirmative.
Although D-mib mutants have in fact been previously isolated, they
are only very weakly neurogenic (Le Borgne
et al., 2005; Melendez et al.,
1995). This might partially explain how it was missed in earlier
screens for components of the Notch pathway. By contrast, D-mib is
absolutely required for the execution of several other Notch-regulated
development events, including wing margin specification, eye growth and leg
joint specification. Misexpression of full-length and dominant-negative
truncations of D-mib affects Notch-mediated pattern formation even more
broadly, including many settings that do not normally require D-mib.
Biochemical and genetic experiments demonstrate that D-mib associates with
both Drosophila DSL ligands, and promotes their internalization and
signaling activity. However, dominant-negative D-mibRF binds Delta and
Serrate but interferes with their normal trafficking and inhibits their
signaling capacity. We infer that D-mibRF binding to both Delta and
Serrate occludes endogenous Neur and D-mib from ubiquitinating and activating
DSL ligands, which likely underlies the broad capacity of D-mibRF to
inhibit Notch activation in virtually all settings of Notch signaling.
Curiously, NeurRF potentiates Delta signaling during wing margin
induction just as full-length Neur does
(Pavlopoulos et al., 2001),
even though ectopic NeurRF otherwise strongly inhibits Notch signaling
(Lai and Rubin, 2001a;
Lai and Rubin, 2001b). We lack
an explanation for this difference between NeurRF and D-mibRF,
but it might hint at a functional difference between these DSL-regulating
ubiquitin ligases. In almost every other regard, however, the activities and
functions of D-mib/D-mibRF are highly reminiscent of
Neur/NeurRF. In fact, we showed that D-mib can functionally replace
Neur in a series of developmental decisions in vivo. Conversely,
contemporaneous studies show that Neur can functionally replace D-mib during
wing margin specification (Le Borgne et
al., 2005). Nevertheless, the essential endogenous requirements
for neur and D-mib are quite distinct, in that they are
genetically required for different developmental processes and the respective
mutants have differential effects on DSL ligands. Despite potent effects of
ectopic D-mib on Delta localization and activity, Delta is mislocalized
primarily only in neur mutant tissue
(Lai et al., 2001;
Pavlopoulos et al., 2001),
whereas Serrate is mislocalized primarily only in D-mib mutant tissue
(Le Borgne et al., 2005).
This apparent specificity is unexpected, as D-mib is expressed
ubiquitously, and is therefore present in all Delta-expressing cells. Does
endogenous D-mib normally regulate Delta as implied by its ability to
associate with Delta, induce Delta endocytosis, and potentiate Delta signaling
activity? A close examination of D-mib mutants reveals certain
phenotypes that are either stronger than those of Serrate mutants
(i.e. leg truncation) or are more suggestive of Delta loss of
function (i.e. wing vein deltas and a mildly neurogenic phenotype in the adult
thorax) (Le Borgne et al.,
2005). These observations collectively imply that another
ubiquitin ligase may co-regulate Delta and thereby partially compensate for
loss of D-mib. Neur is a possible, but relatively poor, candidate to
supply this function. Although it has a demonstrated role in regulating Delta,
neur expression in imaginal tissue is restricted mostly to neural
precursors and photoreceptors (Boulianne et
al., 1991). A more tantalizing candidate is D-mibl (CG17492),
which we suspect may also prove to regulate DSL ligands. In support of this,
systematic yeast two-hybrid screening has identified a specific interaction
between D-mibl and Delta
(http://pim.hybrigenics.com/pimriderext/droso/prflybase.html).
Therefore, the in vivo function of D-mibl with regard to the regulation of DSL
ligands deserves future investigation.
We have shown that both Drosophila DSL ligands are regulated by
ubiquitin ligases that promote ligand endocytosis. Still, the mechanism by
which endocytosis promotes DSL ligand activity is still unclear. An earlier
proposal was that Delta endocytosis might facilitate Notch proteolytic
processing by helping to unmask the S2 Notch cleavage site
(Parks et al., 2000). Other
models suggested that ligand endocytosis might promote ligand clustering or
clearance of extracellular NECD
(Le Borgne and Schweisguth,
2003a). Most recently, genetic studies of the epsin Liquid Facets
(Lqf), an apparently DSL ligand-specific endocytic component
(Overstreet et al., 2004;
Wang and Struhl, 2004), have
led to further insight into this mechanism. In particular, a provocative model
was put forth suggesting that Lqf directs Delta into an endocytic recycling
compartment, and that Delta recycling back to the plasma membrane is a
prerequisite for ligand activation (Wang
and Struhl, 2004). The finding that Serrate is similarly regulated
by endocytosis via D-mib (this study) (Le
Borgne et al., 2005) suggests further avenues for testing this
model. For example, it will be informative to ask whether lqf shows
defects in Serrate trafficking, or if the requirement of Serrate for
D-mib can be bypassed by shunting it through an endocytic recycling
pathway.
Even though Neur and D-mib promote DSL ligand activity by stimulating
ligand endocytosis, they also efficiently induce ligand degradation. This
might conceptually be at odds with the proposition that ligand recycling back
to the plasma membrane underlies DSL ligand activation
(Wang and Struhl, 2004). These
activities might be reconciled if ubiquitination permits a portion of the DSL
ligand pool to enter the select Lqf-mediated recycling pathway, but directs
the bulk of DSL ligands for degradation. Consistent with this, Lqf is strictly
required for DSL ligand activation, but is not required for bulk endocytosis
of DSL ligands (Wang and Struhl,
2004). If ubiquitination is prerequisite for DSL ligand activation
but also makes DSL ligands prone to degradation, this would prevent endless
recycling of activated ligands and thereby limit the temporal extent of Notch
pathway activation. The strategy of coupling activation with downregulation is
seen with Notch itself. Ligand-induced Notch cleavage liberates activated
Notchintra, which is a potent regulator of gene expression as a
nuclear co-activator for Su(H). However, nuclear Notchintra also
becomes a substrate for ubiquitination by the ubiquitin ligase Sel-10, and is
rapidly degraded (Gupta-Rossi et al.,
2001; Hubbard et al.,
1997; Oberg et al.,
2001; Wu et al.,
2001). Coupled activation and downregulation allows for precise
temporal control of signaling by limiting the lifetime of activated signaling
components (Schweisguth,
2004).
Evolutionary flux in regulation of DSL ligands by ubiquitin ligases
The presence of Neur and Mib homologs in both fly and vertebrate genomes
suggests that both proteins were present and regulated DSL ligands in the
ancient common ancestor of these species. We have demonstrated surprising
functional overlap between these structurally unrelated ubiquitin ligases in
regulating DSL ligand activity. What, then, was the rationale of evolving such
different proteins to perform the same function?
As discussed earlier, the genetic implication that Neur and D-mib preferentially regulate Delta and Serrate, respectively, belies the ability of these enzymes to interact with and regulate the localization and signaling activity both DSL ligands. While it remains to be seen whether Neur regulates Serrate in addition to its documented substrate Delta, we showed that D-mib directly and efficiently regulates both Delta and Serrate. Therefore, these ubiquitin ligases did not obviously co-evolve with different classes of DSL ligands.
Another possible explanation lies in the curious observation that Neur is genetically required mostly in settings that involve `lateral inhibitory' Notch signaling, wherein Notch restricts a cell fate amongst equipotent cells. By contrast, D-mib is required largely in settings that involve `inductive' Notch signaling, which occurs between non-equivalent cell populations. This apparent division of labor raises the possibility that different ubiquitin ligases could help to specify the appropriate response to Notch activation in each developmental setting.
This hypothesis, however, is not particularly supported by the observations that D-mib and Neur can functionally replace each other in a variety of processes. Neither does this correlation hold up in other species, because Neur mediates lateral inhibition of neural precursors in flies, whereas Mib mediates lateral inhibition of neural precursors in fish. This latter finding highlights the plasticity in how these ubiquitin ligases have been deployed during evolution, and is consistent with a model in which fish Mib has subsumed the function of fly Neur during neurogenesis (or vice versa). This may have occurred by appropriate changes in the transcriptional regulation of these genes. Given this likely scenario, one wonders whether it might not have been more evolutionarily expedient to have diversified the function of duplicated, paralogous genes. There are indeed multiple neur and mib genes in vertebrates, and two mib genes in flies. Of course, it might be argued that a similar conundrum concerns the co-existence of the HECT domain and the RING finger/U box as unrelated protein domains that both catalyze E3 ubiquitin ligation.
How general is the requirement for DSL ligand endocytosis across evolution?
The neurogenic mutant phenotypes of Drosophila neur and zebrafish
mib, along with the involvement of Xenopus neur in lateral
inhibition, show that DSL ligand ubiquitination is required in both
invertebrates and vertebrates. However, thorough loss-of-function genetic
studies are incomplete in any organism and are complicated by the duplication
of neur and/or mib genes. For example, knockout of murine
Neur1 did not affect Notch signaling
(Ruan et al., 2001;
Vollrath et al., 2001),
possibly due to functional overlap with Neur2.
Our present work clearly demonstrates that the vast majority of
Notch-regulated settings during Drosophila development are strictly
dependent on either Neur or D-mib. Thus, DSL ligand ubiquitination and
endocytosis appears to be obligate in Drosophila. In light of this,
the situation in nematodes provides an interesting possible counter-example.
C. elegans lacks a recognizable Mind bomb ortholog, but does
possess a single Neur gene. However, in contrast to what has been
found in Drosophila, where intracellular deletions of the DSL ligands
have a dominant-negative activity (Sun and
Artavanis-Tsakonas, 1996; Sun
and Artavanis-Tsakonas, 1997), the extracellular domains of the
DSL ligands LAG-2 and APX-2 can fully rescue the lag-2 mutant and can
activate Notch signaling ectopically
(Fitzgerald and Greenwald,
1995). A more recent analysis actually revealed a large family of
putative secreted DSL ligands in the worm, at least one of which (DSL-1) is a
bona fide DSL ligand (Chen and Greenwald,
2004). This suggests that nematodes may have dispensed with
ubiquitination and endocytosis of DSL ligands in at least some settings of
Notch signaling. Nevertheless, a nematode ortholog of epsin/Lqf
(Ce-epn-1) participates in Notch signaling during germline
development (Tian et al.,
2004). The functional relationships amongst Ce-epn-1,
nematode DSL ligands and any potential DSL-regulating E3s in nematodes remain
to be determined.
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ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/10/2319/DC1
|
REFERENCES |
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|
TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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