1 Department of Genetics, Washington University in St Louis, School of Medicine,
660 S. Euclid Avenue, St Louis, MO 63110, USA
2 Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N
1N4, Canada
* Author for correspondence (e-mail: jskeath{at}genetics.wustl.edu)
Accepted 17 September 2004
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SUMMARY |
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Key words: C. elegans Drosophila, Epsins, Notch
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Introduction |
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Epsins are evolutionarily conserved proteins that appear to promote
endocytosis. Binding of epsins to clathrin and AP2 protein complexes
stimulates assembly of clathrin-coated vesicles and promotes vesicle
internalization during endocytosis (Chen
et al., 1998; Rosenthal et
al., 1999
; Wendland et al.,
1999
). Epsin family members harbor multiple motifs with putative
endocytic functions and associate with the membrane via their epsin N-terminal
homology domain and ubiquitin-binding motifs (UIMs)
(Wendland, 2002
;
Itoh et al., 2001
;
Aguilar et al., 2003
). Although
epsins were first thought to play a general role in endocytosis, recent work
suggests epsins target specific proteins for regulated endocytosis
(Aguilar et al., 2003
;
De Camilli et al., 2002
),
consistent with the idea that epsins modulate the strength of specific
signaling pathways. In this context, epsins have been proposed to use UIMs to
target specific ubiquitinated-membrane proteins for regulated endocytosis
(Shih et al., 2002
;
Wendland, 2002
). However,
epsins have not been shown to modulate the activity of any signaling
pathway.
The Notch pathway is one of a handful of signaling pathways that
act reiteratively to control the development of higher metazoans.
Notch signaling is activated when a member of the DSL (Delta, Serrate
LAG-2) family of Notch ligands on a signaling cell binds to the Notch receptor
on the receiving cell (Mumm and Kopan,
2000). Ligand binding activates a series of proteolytic cleavage
events to the Notch receptor that result in the release of its intracellular
domain (Notch[intra]) from the membrane. Notch[intra] translocates into the
nucleus where it complexes with transcription factors of the CSL
(CBF, Su(H) and LAG-1) family and Mastermind
or LAG-3 to activate target gene transcription
(Mumm and Kopan, 2000
).
Genetic and molecular studies in Drosophila indicate that
Notch signaling requires Delta endocytosis in signaling cells. The
link between Delta endocytosis and Notch activation arose from work that
showed Delta internalization correlates with Notch signaling
(Parks et al., 2000),
presentation of secreted and intracellular truncated forms of ligand
antagonize Notch signaling
(Hukriede and Fleming, 1997
;
Hukriede et al., 1997
;
Sun and Artavanis-Tsakonas,
1996
; Sun and
Artavanis-Tsakonas, 1997
) and genetic abrogation of endocytosis
blocks Notch signaling (Seugnet
et al., 1997
).
Ubiquitination of Delta appears necessary for endocyosis of Delta. Two
members of the Notch pathway, neuralized and mind
bomb, encode E3-ubiquitin-ligases, ubiquitinate Delta and appear to be
required for Delta internalization as loss of neuralized or mind
bomb function causes excessively high levels of Delta at the cell
membrane and blocks Notch signaling
(Deblandre et al., 2001;
Itoh et al., 2003
;
Lai et al., 2001
;
Pavlopoulos et al., 2001
;
Yeh et al., 2001
). Recently,
the Drosophila homolog of epsin, liquid facets
(lqf), has also been shown to promote Delta internalization
(Overstreet et al., 2003
),
suggesting lqf may regulate Notch signaling even though the
functional ramifications of lqf on Notch pathway activity
have not been investigated.
Notch regulates sequential events during Drosophila heart
development. The heart and dorsal somatic musculature arise from the
dorsal-most region of the mesoderm. The cardiogenic mesoderm produces two
types of heart cells: cardioblasts and pericardial cells. Cardioblasts are the
contractile cells of the heart and coalesce to form the heart tube.
Pericardial cells associate with cardioblasts and appear to filter the
hemolymph (Bodmer and Frasch,
1999). The use of a Notch temperature-sensitive
allele, Nts-1, identified Notch as a critical
regulator of heart development. During stage 11-12, Notch regulates
the initial commitment of cells to the cardioblast and pericardial cell fate,
as loss of Notch function leads to significant overproduction of both
cell types (Hartenstein et al.,
1992
). Notch also appears to regulate the choice between
the cardioblast and pericardial cell fate, as removal of Notch
function later in development produces a heart with excess cardioblasts and
few pericardial cells (Hartenstein et al.,
1992
).
C. elegans contains two Notch homologs: glp-1
and lin-12 (Austin and Kimble,
1989; Yochem and Greenwald,
1989
). Here, we focus on glp-1. glp-1 promotes germ cell
proliferation in the distal region of the gonad
(Austin and Kimble, 1987
;
Seydoux and Schedl, 2001
).
GLP-1 protein is found on germ cells
(Crittenden et al., 1994
)
while its ligand, LAG-2, is expressed on the somatic distal tip cell (DTC)
(Fitzgerald and Greenwald,
1995
; Henderson et al.,
1994
). The close proximity between the DTC and the germ cells is
thought to bring ligand and receptor into contact and maintain distal germ
cells in the proliferative state. As germ cells move proximally, away from the
influence of the DTC, they leave the proliferative state and enter meiotic
prophase. In wild-type animals, germ cells enter meiosis at
19 germ cell
diameters from the DTC (Crittenden et al.,
1994
; Hansen et al.,
2004
) we refer to the region between the DTC and the
meiotic cells as the proliferative zone. Demonstration that glp-1
promotes the proliferative state of germ cells comes from loss- and
gain-of-function glp-1 alleles. Loss of glp-1 causes
premature entry of germ cells into meiosis in early larval development and
thus no proliferative zone is present in adult animals
(Austin and Kimble, 1987
;
Lambie and Kimble, 1991
). Weak
hypomorphic glp-1 alleles reduce but do not eliminate the
proliferative zone. Conversely, glp-1 gain-of-function alleles
increase proliferation and expand the proliferative zone
(Berry et al., 1997
;
Pepper et al., 2003
).
Our work on heart development led us to identify a functional link between lqf and Notch signaling. In a genetic screen we identified lqf as an inhibitor of cardioblast development. Our phenotypic studies support a model in which lqf acts on fusion-competent myoblasts to prevent their acquisition of the cardioblast fate. lqf and Notch exhibit similar heart phenotypes and our genetic studies reveal a broad role for lqf to promote specifically Notch signaling in Drosophila. Consistent with the model that epsins play an evolutionarily conserved role to potentiate Notch pathway activity, we found that the C. elegans lqf ortholog of epsin appears to mediate Notch/glp-1 activity in the DTC during C. elegans germline development.
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Materials and methods |
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The following double mutant flies were made by standard methods: lqfARI Dl3/Tm3-ftzlacZ, lqfARI neur1/Tm3-ftzlacZ, lqfARI htl42/Tm3-ftzlacZ, flbIF26/CyO-ftzlacZ;lqfARI/Tm3-ftzlacZ, wgcx4/CyO-ftzlacZ;lqfARI/Tm3-ftzlacZ, Nts-1/Y;lqfARI/+, lqfARI Dl3/lqfARI +, lqfARI neur1/lqfARI +, Nts-1/+;lqfARI/+ and + Dl3/lqfARI +.
C. elegans strains used were: N2 (wild type),
glp-1(bn18) (Kodoyianni
et al., 1992) and rrf-1(pk1417)
(Sijen et al., 2001
).
Immunohistochemistry and immunofluorescence analysis
Immunolabeling analyses were performed as described previously
(Skeath, 1998). We used the
following antibodies in our analysis of heart development: guinea-pig
anti-Zfh1 (1:1000); anti-Mef2 (Lilly et
al., 1995
); anti-Eve (Frasch
et al., 1987
); anti-Lmd (Duan
et al., 2001
) and anti-FasIII and anti-MHC (Developmental Studies
Hybridoma Bank, University of Iowa).
Antibody staining of dissected C. elegans gonads is described
(Jones et al., 1996).
Anti-REC-8 (Pasierbek et al.,
2001
) and anti-HIM-3 (Zetka et
al., 1999
) antibodies were used to detect proliferative and
meiotic nuclei respectively (Hansen et al., 2004b).
Molecular cloning of Ce-epn-1 RNAi feeding vector
We identified the C. elegans ortholog of epsin, Ce-epn-1
(T04C10.2) through a BLAST search of the C. elegans genome using the
Lqf protein sequence. Ce-epn-1 was also identified computationally by
other groups (De Camilli et al.,
2002; Kay et al.,
1999
). Sequence corresponding to amino acids 1-238 was amplified
from the first strand cDNA pool of all stage worm tissues. We cloned the
amplified PCR fragment into pGEMT (Promega), and then excised and cloned it
into the standard RNAi feeding vector pPD129.36
(Timmons and Fire, 1998
).
Double strand RNA interference (RNAi) in C. elegans
Wild-type (N2), glp-1(bn18) and rrf-1(pk1417);
glp-1(bn18) eggs were placed on plates with bacteria expressing either
Ce-epn-1 or GFP double-stranded RNA at 20°C, the permissive
temperature for glp-1(bn18). Initially, adult animals,
rather than eggs, were placed on the RNAi plates with the intention of scoring
the progeny, however the glp-1(bn18) animals grown on the
Ce-epn-1 plates produced dead embryos, preventing analysis of adult
germline phenotypes. This probably results from reduction of Ce-epn-1
activity enhancing the temperature-sensitive embryonic lethal phenotype of
glp-1(bn18) (Austin and Kimble,
1987; Priess et al.,
1987
), however this phenotype was not analyzed in detail. By
placing eggs on RNAi feeding plates, the dsRNA is not administered until the
animal hatches and begins feeding (postembryonic RNAi), thereby bypassing the
embryonic requirement for Notch signaling. The hatched animals were
allowed to grow to one day past the fourth larval stage and then dissected,
fixed and stained for analysis of the germline phenotypes. We noticed that
both N2 and glp-1(bn18) animals grown on Ce-epn-1-expressing
bacteria grew more slowly than when grown on GFP-expressing bacteria,
requiring one additional day to reach the fourth larval stage.
![]() |
Results |
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|
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lqf promotes the development of fusion-competent myoblasts
To investigate whether the excess cardioblasts in lqf embryos
develop at the expense of a different mesodermal cell type we analyzed the
development of the other derivatives of the dorsal mesoderm
pericardial cells, visceral mesoderm and dorsal somatic muscles. Markers
specific for pericardial cells (Zfh-1) and visceral mesoderm (FasIII) revealed
grossly normal development of these tissues in lqf embryos, although
pericardial cell organization was slightly disrupted
(Fig. 1B,E;
Table 1; not shown for visceral
mesoderm). In contrast, we observed a decrease in the size but not pattern of
dorsal somatic muscles (Fig.
2A,C) while the pattern and size of ventral and lateral muscles
appear normal (data not shown). Thus, in addition to restricting cardioblast
development, lqf appears to promote dorsal somatic muscle
development.
|
To follow FCM development directly we assayed lame duck
(lmd) expression in wild-type and lqf embryos. lmd,
a Gli super family member, promotes FCM development and is expressed in all
FCMs (Duan et al., 2001).
Relative to wild-type embryos, we observe a moderate reduction of Lmd
expression in the dorsal region of the somatic mesoderm in stage 12/13
lqf mutant embryos (data not shown) consistent with the idea that
lqf promotes FCM development in the dorsal mesoderm.
The formation of excess cardioblasts, combined with the reduction of FCMs
in lqf embryos, supports the model that ectopic cardioblasts arise at
the expense of FCMs. However, it remains possible that over-proliferation of
cardioblast progenitors produces the lqf cardioblast phenotype. To
investigate this, we blocked cell division in lqf embryos by using a
mutation in Rca1. Rca1 is a cell cycle regulator and loss of
Rca1 function blocks the division of cardioblast progenitors
(Han and Bodmer, 2003).
Embryos mutant for Rca1 contain 65±6 cardioblasts
(n=14) while Rca1;lqf double mutant embryos contain
111±16 cardioblasts (n=13). The near doubling of cardioblasts
in Rca1; lqf mutants relative to Rca1 mutants indicates that
over-proliferation of cardioblast progenitors does not play a primary role in
the lqf heart phenotype. Taken together, our phenotypic data are
consistent with a model in which lqf acts in or on presumptive dorsal
FCMs to inhibit the cardioblast fate and promote the FCM fate.
lqf acts through the Notch pathway to repress cardioblast fate
Recent studies link endocytosis to the regulation of different signaling
pathways. We therefore examined whether mutations in known signaling pathways
yield heart phenotypes similar to lqf and whether any of these
pathways genetically interact with lqf during heart development.
Mutations in the EGF, FGF, Wingless, Hedgehog and TGFß
signaling pathways exhibit heart phenotypes distinct from that of lqf
(reviewed by Bodmer and Frasch,
1999). However, Notch mutants display an excess
cardioblast phenotype similar to that of lqf with the exception that
Notch embryos also lack pericardial cells
(Hartenstein et al., 1992
). We
re-examined the Notch heart phenotype using
Nts-1/Df(1)81K embryos and found such embryos exhibit a
twofold increase in cardioblasts, confirming the role of Notch in
regulating cardioblast number (Fig.
1G); however, these embryos also exhibit grossly normal
pericardial development (Fig.
1H). Thus, the lqf and Notch heart phenotypes
appear essentially identical, supporting the idea that lqf and
Notch act together to regulate heart development.
While we failed to detect genetic interactions between lqf and the EGF or FGF (heartless) pathways during heart development (not shown), we observed dominant genetic interactions between lqf and three components of the Notch pathway. When raised at 18°C, 10% of lqfARI mutant embryos exhibit an excess cardioblast phenotype. However, removal of one copy of Delta or neur from lqfARI embryos raised at 18°C causes the majority of such embryos to develop excess cardioblasts (62/65 for Delta and 32/36 for neur) (Fig. 3B,D,F). Similarly, a 50% reduction of lqf function in a Nts-1 background dominantly enhances the weak Nts-1 excess cardioblast phenotype (compare Fig. 3E to 3C). The near identity of the lqf and Notch heart phenotypes, together with the dominant genetic interactions between lqf and Notch pathway members, suggest that lqf promotes Notch pathway function during heart development.
|
|
|
We focused our analysis on the role of Ce-epn-1 in germline
development and used RNAi-mediated gene interference
(Fire et al., 1998) to assay
the effect of postembryonic loss of Ce-epn-1 function on germline
development and to determine whether Ce-epn-1 genetically interacts
with glp-1. We tested the effect of reducing Ce-epn-1
function on germline development by placing wild-type C. elegans
embryos, as well as embryos carrying the weak temperature-sensitive
loss-of-function mutant glp-1(bn18) on plates with bacteria
containing the Ce-epn-1-encoding RNAi feeding vector at 20°C. As
controls, we performed parallel experiments using bacteria containing the RNAi
feeding vector with a GFP insert. At 20°C, glp-1(bn18) yields a
weak premature entry into meiosis phenotype with a slightly smaller
proliferative zone, and provides a sensitized background within which to assay
Ce-epn-1 function. We find that postembryonic RNAi-mediated reduction
of Ce-epn-1 function in wild-type individuals reduces the size of the
adult proliferative zone relative to control worms
(Fig. 6A,C). The size of the
proliferative zone is similar to that observed in weak glp-1 alleles
indicating that Ce-epn-1 acts in the same direction as glp-1
to promote germline proliferation. In addition, we find that postembryonic
RNAi-mediated reduction of Ce-epn-1 function in glp-1(bn18)
worms significantly enhances the glp-1(bn18) phenotype such that 95%
of such animals (n=43) lack a proliferative zone, phenocopying a
strong loss of glp-1 phenoytpe
(Fig. 6A,B). These results
suggest that Ce-epn-1 promotes GLP-1 signaling during C.
elegans germline development, and support the idea that epsins
play an evolutionarily conserved role to potentiate Notch
signaling.
|
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Discussion |
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The roles of liquid facets and Notch in cell-type specification in the heart
Our phenotypic studies of lqf embryos are consistent with the
model that lqf acts in a subpopulation of FCMs to inhibit their
acquisition of the cardioblast fate. How might lqf and Notch
inhibit cardioblast development? In lateral and ventral regions of the
mesoderm, Notch-mediated lateral inhibition helps select individual somatic
muscle progenitor cells from clusters of equipotential cells. Cells in these
clusters express lethal of scute (l'sc) and can adopt either
the muscle progenitor or FCM fate. Cells that retain l'sc become
progenitor cells while cells that lose l'sc expression become FCMs
(Carmena et al., 1995). In
these clusters, Notch inhibits l'sc expression and the
progenitor fate, thereby promoting the FCM fate
(Corbin et al., 1991
;
Hartenstein et al., 1992
;
Bate, 1993
;
Giebel, 1999
). We speculate
that lqf and Notch may act similarly to regulate the
cardioblast progenitor/FCM decision in the dorsal mesoderm with Notch
functioning to inhibit tin expression and the cardioblast progenitor
fate and, in so doing, promoting the FCM fate. In this model, loss of
lqf/Notch activity would lead to excess cardioblast
progenitors at the expense of FCMs. Consistent with this, clusters of
Tin-expressing cells in the dorsal mesoderm resolve to individual heart cells
during the stages when lqf and Notch inhibit cardioblast
development.
If lqf plays a general role to promote Notch activity, why do we observe defects only during heart development in lqf embryos? One explanation is that maternal lqf product masks earlier requirements for lqf during Notch-dependent events. Consistent with this, temperature shift experiments indicate lqf acts during late stage 12 to restrict cardioblast development (data not shown). Nearly all well-characterized Notch-dependent events in the embryo occur before stage 12. Thus, the apparent specificity of the lqf phenotype for the heart may simply arise due to the late stage at which this Notch-dependent event occurs combined with the masking effect of lqf maternal product. Unfortunately, we could not assay embryos devoid of maternal and zygotic lqf function as lqf germline clones failed to produce eggs.
In the case of postembryonic Ce-epn-1 RNAi treatment, we observed a weak glp-1 loss-of-function germline phenotype, which could be significantly enhanced in a glp-1 temperature-sensitive background at the permissive temperature. Strong lin-12 loss-of-function phenotypes were not observed in these experiments (unpublished observation). The weak glp-1 loss-of-function and absence of lin-12 phenotypes in a wild-type background is very likely because the postembryonic feeding RNAi treatment only partially depletes Ce-epn-1 mRNA. The isolation and characterization of a null mutation will greatly facilitate uncovering all roles of Ce-epn-1 in C. elegans.
Is liquid facets/epsin function specific to Notch pathway regulation?
As epsins appear to regulate endocytosis, and endocytosis regulates the
activity of most signaling pathways (reviewed by
Wendland, 2002), lqf
might act broadly to regulate the output of many signaling pathways rather
than acting specifically on the Notch pathway. However, existing data
support a specific interaction between lqf and Notch
activity. For example, we failed to detect genetic interactions between
lqf and the EGF or FGF pathways during heart
development, and lqf does not appear to interact with the dominant
Egfr[ellipse] allele during eye development (data not shown).
Furthermore, lqf mutant clones in the Drosophila eye exhibit
phenotypes consistent with the specific loss of Notch activity
(Cadavid et al., 2000
;
Overstreet et al., 2003
;
Overstreet et al., 2004
).
Thus, lqf appears to display specific interaction with the
Notch pathway. Although it is important to assay whether lqf
alters the activity of other signaling pathways regulated by endocytosis, such
as the TGFß, Wingless, and Hedgehog pathways,
these data support a model in which Lqf plays a relatively specific role to
target a component of the Notch pathway for endocytosis and in so
doing promotes Notch signaling.
Are epsins core components of the Notch pathway?
Our work shows that lqf/epsins promote Notch pathway
activity in Drosophila and C. elegans. Notably, epsins
participate in Notch-mediated lateral inhibition signaling during bristle and
perhaps heart development, as well as Notch-mediated inductive signaling in
the C. elegans germline. These data argue that epsins are essential
evolutionarily conserved components of the Notch pathway, potentially
required for most if not all Notch-mediated processes.
Genetic studies have the potential to identify all components of a
signaling process, however, they do not necessarily differentiate between the
roles different genes play in a signaling process. Here, we distinguish
between core components of a signaling pathway those factors that
actively take part in the signal transduction event and factors that
set the stage for signal transduction but do not actively transmit the signal.
For example, Notch, DSL ligands, presenillins and CSL effectors are core
components of the Notch pathway as they actively transmit the signal
DSL ligands bind Notch, induce the metalloprotease-mediated S2
cleavage followed by the presenilin dependent intramembrane (S3) cleavage of
Notch that releases Notch[intra], which translocates to the nucleus and
complexes with CSL-class proteins to activate Notch target genes
(Mumm and Kopan, 2000).
However, many other proteins set the stage for signaling by ensuring each core
member of a pathway is present in the right location and correct form such
that signal transduction will occur given the proper stimulus. For example,
presentation of a functional Notch receptor on the cell membrane appears to
require S1-mediated cleavage of Notch by furin-type proteins (see
Mumm and Kopan, 2000
).
Although furins do not actively take part in the signaling event, furin
activity and its requirement for presentation of Notch is a prerequisite for
Notch signaling. Similarly, ras signaling requires Ras
localization to the cell membrane and prenylation of Ras constitutively
targets it to the cell membrane (Zhang and
Casey, 1996
).
In support of lqf/epsins as core components of the Notch
pathway, Delta endocytosis appears essential for Notch signaling
(Parks et al., 2000) and
lqf appears essential for Delta endocytosis
(Overstreet et al., 2003
).
Furthermore, epsins are thought to target ubiquitinated membrane proteins for
regulated endocytosis via their ubiquitin-interacting motif (UIM)
(Aguilar et al., 2003
;
Shih et al., 2002
) and
ubiquitination of Delta appears necessary for Delta endocytosis and active
Notch signaling (Deblandre et
al., 2001
; Lai et al.,
2001
; Pavlopoulos et al.,
2001
; Yeh et al.,
2001
). Thus, Lqf/epsins may act as part of a complex that
specifically targets Delta for internalization after ubiquitination and as
such be core members of the Notch pathway.
It remains possible, however, that lqf/epsin function is a prerequisite for Notch signaling. For example, some endocytic proteins function in protein transport in the secretory pathway and epsin1 family members could in principle enable transport of Delta to the membrane. In such a capacity, epsins would not be considered core components of the Notch pathway. Clearly, future experiments that test the requirement of specific domains of epsins, such as the UIM, for Notch signalling, as well as those that identify the protein complexes within which epsins act, should help elucidate the molecular basis through which lqf/epsins potentiate Notch signaling.
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ACKNOWLEDGMENTS |
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