Epsin potentiates Notch pathway activity in Drosophilaand C. elegans

Endocytosis modulates the output of different signaling pathways and also maintains morphogen gradients during development(Gonzalez-Gaitan and Stenmark,2003; Seto et al.,2002). For example, endocytosis positively regulates the Decapentaplegic (Dpp) and Notch pathways as trans-endocytosis of Dpp, a TGF-β family member, facilitates its spread(Entchev et al., 2000) and endocytosis of Delta appears essential for Notch signaling(Parks et al., 2000). Conversely, it is well established that endocytosis of cell surface receptors leads to signal downregulation, as for receptor tyrosine kinase and TGF-βsignaling (Seto et al., 2002),and receptor-mediated endoctyosis followed by lysosomal clearing restricts the spread of Wingless (Piddini and Vincent,2003).

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, N^ts-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 Notchfunction later in development produces a heart with excess cardioblasts and few pericardial cells (Hartenstein et al.,1992).

C. elegans contains two Notch homologs: glp-1and 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-1promotes 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. lqfand Notch exhibit similar heart phenotypes and our genetic studies reveal a broad role for lqf to promote specifically Notchsignaling 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 epsinappears to mediate Notch/glp-1 activity in the DTC during C. elegans germline development.

The following fly stocks were used: Oregon R or w¹¹¹⁸– as wild-type, lqf^ARI/Tm3-ftzlacZ,lqf^AG/Tm3-ftzlacZ, Df(1)N^81K, N^ts-1,N^55e11, Dl³/Tm6B, Dl⁷/Tm2,neur¹/Tm3, flb^IF26/CyO, wg^cx4/CyO,mam^NN86/CyO and htl⁴²/CyO.

The following double mutant flies were made by standard methods: lqf^ARI Dl³/Tm3-ftzlacZ, lqf^ARIneur¹/Tm3-ftzlacZ, lqf^ARI htl⁴²/Tm3-ftzlacZ,flb^IF26/CyO-ftzlacZ;lqf^ARI/Tm3-ftzlacZ,wg^cx4/CyO-ftzlacZ;lqf^ARI/Tm3-ftzlacZ,N^ts-1/Y;lqf^ARI/+, lqf^ARIDl³/lqf^ARI +, lqf^ARIneur¹/lqf^ARI +,N^ts-1/+;lqf^ARI/+ and +Dl³/lqf^ARI +.

C. elegans strains used were: N2 (wild type), glp-1(bn18) (Kodoyianni et al., 1992) and rrf-1(pk1417)(Sijen et al., 2001).

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).

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).

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-1activity 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.

We identified lqf as an inhibitor of cardioblast development in a screen of ∼2000 third chromosome lethal P-element lines from the Hungarian P-element collection. Homozygous embryos from l(3)0110/2 display an approximate doubling of cardioblasts (Fig. 1D). Molecular analysis localized the P element insertion site in l(3)0110/2 to the first intron of lqf. We confirmed that loss of lqf function produces an excess cardioblast phenotype by showing that embryos homozygous mutant for each of four additional lqf alleles, lqf^ARI, lqf^AG,lqf^be25and lqf^be428(Cadavid et al., 2000; Overstreet et al.,2003) yield an excess cardioblast phenotype. The phenotypic severity of these alleles varied consistent with the following allelic series: lqf^ARI × lqf^AG > lqf^be25 > lqf^be428 > lqf^l(3)0110/27 (data not shown). Based on these data, we used the strong lqf^ARI and lqf^AGalleles in our studies and found that 200±41 cardioblasts(n=9) develop in lqf^ARI mutant embryos compared to 104±7 cardioblasts (n=18) in wild-type embryos(Fig. 1, Table 1). The presence of excess cardioblasts in lqf mutants demonstrates that lqfnormally functions to oppose cardioblast development.

The Drosophila heart is composed of two distinct types of cardioblasts: Tinman-positive cardioblasts (Tin-CB) and Svp-lacZ-positive cardioblasts (Svp-CB) (Bodmer and Frasch,1999). To determine if the lqf excess cardioblast phenotype arises due to a preferential expansion of one type of cardioblast,we compared the relative frequency of Tin-CBs and Svp-CBs in wild-type versus lqf embryos. In the wild-type heart, 104 cardioblasts develop of which 30±0 (n=8) are Svp-CBs, the remainder being Tin-CBs. Although on average 200 cardioblasts develop in lqf embryos, the number of Svp-CBs remains essentially unchanged (32± 3; n=10)demonstrating that loss of lqf function appears preferentially to enlarge the pool of Tin-CBs.

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.

Muscle size correlates with the number of fusion-competent myoblasts (FCMs)that fuse with a given muscle founder cell, while the presence of individual muscles depends on the formation of appropriate muscle founder cells(Baylies et al., 1998). Thus,the reduced size of dorsal muscles suggests FCM development is impaired in lqf embryos. Even-skipped (Eve) labels all nuclei in the dorsal DA1 muscle and thus, can reveal the number of FCMs that contribute to this muscle. To assay whether fewer FCMs contribute to dorsal muscles in the lqfembryos, we used Eve to count the number of nuclei in DA1. In wild type, DA1 contains 11.1±1.8 (n=42) FCMs relative to 7.6±2.2 FCMs in lqf embryos (n=67; P<0.01; Fig. 2B,D; Table 1). Thus, lqfappears to promote either the development of FCMs or their ability to fuse with muscle founder cells.

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.

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 TGFβsignaling 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 N^ts-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 lqf^ARI mutant embryos exhibit an excess cardioblast phenotype. However, removal of one copy of Deltaor neur from lqf^ARI 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 N^ts-1 background dominantly enhances the weak N^ts-1 excess cardioblast phenotype (compare Fig. 3E to 3C). The near identity of the lqf and Notchheart phenotypes, together with the dominant genetic interactions between lqf and Notch pathway members, suggest that lqfpromotes Notch pathway function during heart development.

Next, we asked if lqf genetically interacts with the Notch pathway during other Notch-mediated developmental events. Notch signaling regulates wing vein formation and bristle development. For example, flies heterozygous for Delta exhibit mild vein thickening along the length and at the distal tip of L2, bifurcation of the posterior cross vein and the distal tip of L5(Fig. 4C,D) as well as thickening of L4 at its junction with the anterior cross vein (data not shown). Flies heterozygous for lqf exhibit wild-type veination. However, a 50% reduction of lqf dominantly enhances the expressivity and penetrance of all haploinsufficient Delta vein phenotypes(Fig. 4E,F). For example, 54.4%of Dl³/+ wings (n=33) exhibit vein thickening along L2 and 51.6% (n=33) exhibit bifurcation of the posterior cross vein and the distal tip of L5. In contrast, 100% of Dl³+/+lqf^ARI wings (n=36) exhibit more severe vein thickening along L2 and 91.7% (n=36) exhibit a more severe bifurcation of the posterior cross vein and the distal tip of L5. In addition,while only 12% of Dl³/+ wings (n=33) exhibit thickening of L4 at its junction with the anterior cross vein, 83.3% of Dl³ +/+lqf^ARI wings (n=36)exhibit more severe thickening of L4 in this region (data not shown). The dominant enhancement of Delta by lqf is not allele specific as we observe similar genetic interactions between lqf^AG,lqf^be428 and Dl⁷ (data not shown).

lqf and Notch also exhibit dominant genetic interactions during bristle development. Flies heterozygous for N^55e11,a hypomorphic allele of Notch, exhibit a wild-type bristle pattern(n=27) while 2.5% of flies heterozygous for lqf exhibit one extra notal bristle (n=40). However, 40% of flies doubly heterozygous for Notch and lqf contain extra notal bristles(n=45; Fig. 5B). The dominant genetic interactions of lqf with Notch and Delta indicate that lqf acts with the Notch pathway to promote bristle and wing development. Together with our results in the heart, these data suggest that lqf promotes Notch signaling during multiple developmental events in Drosophila, consistent with lqf playing a general role to promote Notch pathway activity.

Our experiments in Drosophila suggest lqf promotes Notch pathway function in most Notch-dependent developmental events. To test whether lqf has an evolutionarily conserved role in contributing to Notch signaling we investigated the functional relationship between the C. elegans lqf ortholog and Notchsignaling during C. elegans germline development. We identified the C. elegans lqf ortholog – T04C10.2 (referred to as Ce-epn-1) – and cloned a region of Ce-epn-1 (amino acids 1-238) into the standard C. elegans RNAi feeding vector, pPD129.36 (Timmons and Fire,1998). This region of Ce-epn1 bears no significant similarity to any other C. elegans gene and should specifically target Ce-epn-1 for RNAi-mediated gene interference.

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-1function on germline development by placing wild-type C. elegansembryos, 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-1to 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 epsinsplay an evolutionarily conserved role to potentiate Notchsignaling.

To distinguish whether Ce-epn-1 acts non-autonomously in the signaling cell, the somatic DTC, or autonomously in the germ line receiving cell we fed C. elegans embryos of the genotype rrf-1;glp-1(bn18) on either Ce-epn-1 RNAi bacteria or control GFP RNAi bacteria. The rrf-1 mutation renders somatic cells deficient for RNAi while the germline remains sensitive to RNAi(Sijen et al., 2001). Thus, a failure to observe enhancement of the glp-1(bn18) phenotype in an rrf-1 background would suggest that Ce-epn-1 acts in the somatic DTC, where RNAi does not work, as part of the signaling mechanism to promote germ cell proliferation. In agreement with this, postembryonic Ce-epn-1 RNAi treatment does not enhance the glp-1(bn18)proliferative zone phenotype in an rrf-1 background(Fig. 6B,C). Thus, Ce-EPN-1 appears to act in the soma to help transmit the LAG-2 signal to GLP-1-expressing germ cells. These data agree with the observation that lqf acts cell non-autonomously in the Drosophila eye(Cadavid et al., 2000).

Our work links epsins with Notch pathway activity. However, these results raise many questions with respect to epsin/Notch function during heart development and the molecular understanding of Notch pathway activation. For example, what is the precise developmental role of lqf/Notch during heart development? How specific is the role of epsins to Notch signaling? And, how do epsins regulate Notchsignaling at the molecular level?

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 Notchinhibit 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 Notchfunctioning 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 lqfembryos? 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 lqfphenotype 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.

As epsins appear to regulate endocytosis, and endocytosis regulates the activity of most signaling pathways (reviewed by Wendland, 2002), lqfmight 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 Notchactivity. 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 lqfalters the activity of other signaling pathways regulated by endocytosis, such as the TGFβ, 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.

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 Notchpathway, 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.

We are especially grateful to Alan Michelson and Janice Fischer for providing many helpful reagents and advice. We also thank Hanh Nyugen, Manfred Frasch and Zhi-Chun Lai for antibodies and fly stocks. We obtained the FasIII and MHC antibodies, generated in Corey Goodman's lab, from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We greatly appreciate the help of Min-Ho Lee and Swathi Arur in the isolation of Ce-epn1 and that of Heather Broihier for critical comments on the manuscript. As always we are indebted to Kathy Matthews and the Bloomington Stock Center for their help. This work was supported by a grant to J.B.S. from NSF (IBN-0077727) and NIH RO1 GM63310 to T.S.