Dev Genes Evol (2003) 213:65–72 DOI 10.1007/s00427-003-0296-x

ORIGINAL ARTICLE

V
ronique Morel · Roland Le Borgne · Franois Schweisguth

Snail is required for Delta endocytosis and Notch-dependent activation of single-minded expression Received: 30 October 2002 / Accepted: 10 December 2002 / Published online: 5 February 2003
Springer-Verlag 2003

Abstract In the Drosophila embryo, the mesectoderm corresponds to a single row of cells abutting the mesoderm. It is specified by the expression of the single-minded (sim) gene. The information that precisely positions the sim-expressing cells along the dorso-ventral axis is incompletely understood. Previous studies have shown that Dorsal and Twist activate sim expression in a large ventral domain, while two negative regulators, Snail (Sna) and Suppressor of Hairless [Su(H)], repress sim expression in the mesoderm and neuroectoderm, respectively. Repression by Su(H) is relieved in the presumptive mesectoderm by Notch signaling. In this paper, we show that Sna also has a positive regulatory function on sim expression in the presumptive mesectoderm. This positive effect of Sna depends on the Su(H)-binding sites within the sim promoter, suggesting that Sna regulates Notch signaling. In addition, we find that Delta is endocytosed together with the extracellular domain of Notch. The endocytosis of Delta is only seen in the mesoderm and requires the activity of the sna and neuralized (neur) genes. Interestingly, the Neur-mediated endocytosis of Delta has recently been shown to be sufficient for the non-autonomous activation of Notch target genes in wing imaginal discs. We therefore propose a novel model in which Sna positions the mesectoderm via its dual regulatory activity. In this model, Sna cell-autonomously represses sim expression in the mesoderm and relieves Su(H)-dependent repression in a cell non-autonomous fashion by promoting the Neur-dependent endocytosis of Delta in the mesoderm. Edited by C. Desplan V. Morel · R. Le Borgne · F. Schweisguth ()) Ecole Normale Suprieure, CNRS UMR 8542, 46 rue d’Ulm, 75230 Paris Cedex 05, France e-mail: [email protected] Tel.: +33-1-44323923 Fax: +33-1-44323887 V. Morel Department of Genetics, University of Cambridge, Cambridge, CB2 3EH, UK

Keywords Notch · Delta · Mesoderm · Neuralized · Snail

Introduction Pattern formation along the dorso-ventral (DV) axis of the Drosophila embryo depends on a gradient of nuclear localization of the transcriptional regulator Dorsal in the preblastoderm embryo. Interpretation of this gradient at the blastoderm stage results in the subdivision of the embryo in large territories of gene expression along the DV axis (Rusch and Levine 1996). High levels of Dorsal in ventral nuclei activate the expression of the twist (twi; Roth et al. 1989) and snail (sna) genes (Ip et al. 1992b; Leptin 1991) which specify the mesoderm, while intermediate levels of nuclear Dorsal in lateral nuclei specify the neuroectoderm. In contrast to these large territories, the mesectoderm is specified as a one-cell row abutting the mesoderm that expresses the single-minded (sim) selector gene (Nambu et al. 1990, 1991). The mechanism by which positional information selects in a precise and reproducible way a single row of cells along the DV axis is only partly understood. Previous studies have shown that the expression of the sim gene is negatively regulated in the mesoderm by Sna, while it is positively regulated by low levels of Dorsal and Twi in more lateral cells (Kasai et al. 1992, 1998; Leptin 1991). The expression of the sim gene is also regulated both positively and negatively by the components of the Notch signaling pathway. Specifically, we have shown earlier that Su(H) represses the expression of the sim gene in the neuroectoderm and that Notch activation relieves this repression (Morel and Schweisguth 2000). The activation of Notch triggers a Su(H)-dependent transcriptional switch by which corepressors, such as the HairlessdCtBP complex (Morel et al. 2001), are displaced away by the activated nuclear form of Notch (Bray and Furriols 2001). This molecular switch is suggested to only occur in the presumptive mesectoderm. Thus, we had proposed earlier that Notch receptors are activated at the contact of the mesoderm (Morel and Schweisguth 2000). Consistent

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with this model, cell transplantation experiments provided evidence for the non-autonomous activation of sim expression by mesodermal cells (Leptin and Roth 1994). Notch activation in the mesectoderm is thought to be triggered by Delta that is provided maternally (Kooh et al. 1993). In contrast, Serrate only becomes detectable at late embryogenesis (Thomas et al. 1991). However, because of a requirement for the activity of Delta in the germ-line during oogenesis (Lopez-Schier and St Johnston 2001), it has not been possible to determine the precise role of maternally-provided Delta for the expression of sim. Delta is initially localized uniformly at the plasma membrane in preblastoderm embryos and is specifically redistributed into dots in ventral cells at the end of cellularization (Kooh et al. 1993). Recent studies have shown that Delta is also redistributed into intracellular dots during wing and eye development. In these tissues, the intracellular dots of Delta correspond to large endocytic vesicles. Internalization of Delta requires the activity of neuralized (neur), a RING type E3-ubiquitin ligase that binds Delta and that is required for Notch signaling (Lai et al. 2001; Pavlopoulos et al. 2001; Yeh et al. 2001). Genetic analysis indicates that one function of Neur is to promote the endocytosis of Delta-Notch complexes in signal-producing cells. Interestingly, neur is required for the expression of the sim gene and is expressed at a high level in the mesoderm (Boulianne et al. 1991; Martin-Bermudo et al. 1995). sna encodes a transcriptional repressor expressed in a sharp ventral domain corresponding to the mesoderm. It contributes to mesoderm specification by directly repressing mesectodermal and neuroectodermal gene expression, such as snail and Delta, respectively (Ip et al. 1992a; Kosman et al. 1991; Leptin 1991). Ectopic expression of Sna also induces sim expression in a few cells containing low levels of the activators Twist and Dorsal (Cowden and Levine 2002). This suggests that Sna also plays a positive role in the regulation of sim expression within a lateral domain that is competent to respond to Notch activation (Cowden and Levine 2002; Morel and Schweisguth 2000). Here we confirm that Sna positively regulates the expression of sim in the mesectoderm. We further show that this activity of Sna depends on the Su(H)-binding sites, suggesting that Sna regulates Notch signaling. Consistently, we show that sna is required for the Neur-dependent endocytosis of Delta in the mesoderm, suggesting that Sna regulates the ability of mesodermal cells to signal and activate Notch receptors.

Materials and methods Drosophila stocks The sim-lacZ and sim mut-lacZ constructs are described in Morel and Schweisguth (2000). Transgenes on the third chromosome were used to obtain the following stocks: snaIIG/CyO ftz-lacZ; sim-lacZ (or sim mut-lacZ), twiIIH07/CyO ftz-lacZ; sim-lacZ (or sim mutlacZ), twiIIH07,snaIIG/CyO ftz-lacZ; sim-lacZ (or sim mut-lacZ). N55e11 germ-line clones were obtained as described in Morel and

Schweisguth (2000). The deficiency Df(2L)TE116GW11 (a gift of M. Leptin) deletes the sna locus. The neur1/TM3 Sb, Kr-Gal4, UAS-GFP (Pavlopoulos et al. 2001) and neurIF65/TM3 Sb,hb-lacZ (Lai et al. 2001) have been used as neur null alleles. Immunochemistry and in situ hybridization Embryos were fixed and stained as previously described (The et al. 1999). Anti-b-galactosidase antibodies were used to genotype mutant embryos. Antibodies were used at the following dilutions: mouse (1/500) and guinea-pig (1/3,000) anti-Delta (gifts from M. Muskavitch), mouse anti-extracellular Notch (1/3,000; DSHB), rabbit anti-Twi (1/2,000; gift from S. Roth), rabbit anti-b-galactosidase (1/2,000; Cappel), guinea-pig anti-Hrs (1/600; gift of H. Bellen). Anti-mouse biotinylated and anti-streptavidin-Cy3 were from Molecular Probes. Anti-streptavidin-Alexa-488 and antirabbit-Alexa-488 were purchased from Jackson’s Laboratories. The synthesis of DIG-labeled RNA probes was as previously described (Lecourtois and Schweisguth 1995). For the fluorescent staining, the sim RNA probe was revealed with the tyramiderhodamine amplification system (New England Nuclear).

Results Function of Su(H) in the mesoderm Sna has previously been shown to directly repress the expression of sim in the mesoderm (Kasai et al. 1992, 1998). Consistently, we found that sim is ectopically expressed in ventral cells in embryos homozygous for a small deficiency deleting the sna locus (Df(2L)TE116GW11, designated here as Df(sna); Fig. 1 a–c). Expression in ventral cells is seen all along the antero-posterior axis with the exception of a posterior gap (Fig. 1 c). The ectopic expression of sim appears to be less dramatic in embryos mutant for snaIIG, a sna allele classified as a strong loss of function (compare Fig. 1 c, d), suggesting that the mutant protein encoded by this EMS-induced allele might retain some activity (Hemavathy et al. 1997). We used this partial loss-of-sna-function mutation to examine whether Su(H) represses sim expression in ventral cells. We compared the expression pattern of a sim-lacZ construct which faithfully reproduces the expression of the endogenous gene with that of the sim mutlacZ construct which has its Su(H)-binding sites deleted (Morel and Schweisguth 2000; see Fig. 2a, b). In snaIIG mutant embryos, the pattern of sim-lacZ was similar to that of the endogenous sim gene, i.e. ventral cells close to the cephalic furrow express sim-lacZ while cells located more posteriorly do not (Fig. 1 d, e). By contrast, the sim mut-lacZ construct was expressed in a larger domain (Fig. 1 f). Therefore, the deletion of the Su(H)-binding sites led to the ectopic expression of a sim reporter construct in a larger territory. This suggests that Su(H) represses the expression of sim in ventral cells, at least in this sna mutant background.

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Fig. 1 Snail (Sna) and Suppressor of Hairless [Su(H)] repress single-minded (sim) expression in the mesoderm. Ventral (a, b, e) and ventro-lateral (c, d, f) views of stage 6–8 wild-type embryos (a, b), Df(sna) (c) and snaIIG mutant embryos (d–f) showing the expression of endogenous sim (a–d), sim-lacZ (e) and sim mut-lacZ (f). Expression of sim or lacZ is monitored by in situ hybridization. Stages are as described in Campos-Ortega and Hartenstein (1997). a–c The complete loss of sna function results in ectopic expression of sim in ventral cells, indicating that Sna represses sim expression in the mesoderm. d, e The derepression of sim expression in ventral cells is weaker in snaIIG mutant embryos compared to Df(sna) mutant embryos. The snaIIG mutation affects the sim-lacZ transgene (e) and the endogenous sim gene similarly (d). f Mutations of the Su(H)-binding sites lead to a lateral expansion of the ectopic expression of the sim-lacZ transgene in the snaIIG background (compare e and f), indicating that Su(H) represses sim expression in ventral cells. e', f' A schematic representation of sim-lacZ and sim mut-lacZ. The sim-lacZ (e') contains multiple binding sites for Dorsal, Twist (Twi), and Su(H), but only one site is represented for simplicity. Deletion of all ten Su(H)-binding sites in sim mut-lacZ (f') is indicated by an X

Sna promotes sim expression To test whether sna regulates the Su(H)-dependent expression of sim, we next analyzed the expression profile of both sim-lacZ and sim mut-lacZ constructs in either double twi, sna or single twi mutant embryos. In twi,sna double mutant embryos, neither the endogenous sim gene nor the sim-lacZ transgene were expressed (Fig. 2a, c; see also Leptin 1991). Likewise, no expression of the sim mut-lacZ construct was observed (Fig. 2b, d). This indicates that Dorsal alone, which directly binds to the sim regulatory region (Kasai et al. 1992, 1998), is not sufficient to activate the sim promoter, even in the absence of Su(H)-mediated repression. Consistent with this finding, Dorsal was recently shown to function

Fig. 2 sna positively regulates the expression of sim. Lateral (a–d, f) and ventro-lateral (e) views of stage 5–6 embryos showing the expression of the sim-lacZ (a, c, e) and the sim mut-lacZ (b, d, f) constructs in wild-type (a, b), twiIIH07 snaIIG (c, d) and twiIIH07 (e, f) mutant embryos. The sim-lacZ transgene is not expressed in sna twi double mutant embryos (c) while is expressed in twi mutant embryos (e), indicating that Sna has a positive function in the regulation of sim. The schematic representation of the sim reporter constructs shown on the right is as in Fig. 1. The presence of the proteins Dorsal (blue), Twi (orange) and Sna (green) illustrates the genotypes

cooperatively with Notch for the activation of sim (Cowden and Levine 2002). In twi mutant embryos, both the endogenous sim gene and the sim-lacZ transgene were similarly expressed in two irregular rows of cells (Fig. 2e; see also Rao et al. 1991). Consistent with the reduction in size of the domain of sna expression seen in twi mutant embryos (Kosman et al. 1991; Leptin 1991), we observed that these two rows were less spaced in twi mutant than in wild-type embryos (Fig. 2a, e). By contrast, the sim mutlacZ transgene was not expressed (Fig. 2f). Thus, the activity of the sna gene is necessary to promote the

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expression of the sim-lacZ construct in the absence of twi activity. This indicates that Sna positively regulates sim expression. Moreover, the lack of sim mut-lacZ transgene expression in twi mutant embryos leads us to conclude that the Su(H)-binding sites are required for this Snamediated positive regulation. Importantly, sim and sna are expressed in nonoverlapping patterns. Thus, positive regulation of sim expression by Sna must be non cell-autonomous. We therefore hypothesized that Notch, a known activator of sim expression that requires the Su(H)-binding sites, mediates this positive effect of Sna. Together, these data indicate that Sna plays a dual role. On one hand, Sna cellautonomously acts to directly repress the expression of the sim gene in the mesoderm and, on the other hand, acts in a cell non-autonomous manner to activate Notch signaling in the presumptive mesectoderm. The endocytosis of Delta into Hrs-positive vesicles is restricted to the mesoderm We next hypothesized that Sna may regulate the signaling activity of Delta in a cell-autonomous manner, which would in turn act in a cell non-autonomous manner to activate sim expression in the presumptive mesectoderm. This function of Sna is formally similar to the one recently proposed for Neur. Indeed, Neur has recently been shown to act in a cell-autonomous manner to promote the endocytosis of Delta, which in turns activates, in a non-autonomous manner, Notch target genes. Interestingly, Delta has previously been shown to redistribute from the plasma membrane to poorly characterized cytoplasmic dots in ventral cells prior to gastrulation (Kooh et al. 1993; Fig. 3 a–a3). Our above hypothesis suggests that these dots represent endocytic vesicles and further predicts that this internalization of Delta should be mesoderm-specific and Sna-dependent. We first studied the subcellular distribution of Delta in embryos stained for sim transcripts (Fig. 3 b') and Delta proteins (Fig. 3 b). This double staining experiment revealed that Delta remains uniformly localized at the plasma membrane in the neuroectoderm and in the simexpressing mesectodermal cells and is redistributed into dots only in the mesoderm (Fig. 3 b3). We therefore conclude that the subcellular redistribution of Delta is restricted to the mesoderm. The nature of those mesodermal dots remains largely elusive. They have previously been interpreted as intracellular endocytic vesicles (Kooh et al. 1993). Consistent with this interpretation, previous studies have indicated that Delta and the extracellular domain of Notch were internalized in cells sending the signal following ligandreceptor interaction (Parks et al. 2000). Using antibodies specific to the intracellular domain of Notch, we find that accumulation of the intracellular domain of Notch at the plasma membrane is reduced and that the intracellular domain of Notch does not accumulate in these vesicles (data not shown; see also Fehon et al. 1991). By contrast,

Fig. 3 Mesoderm-specific endocytosis of Delta. Lateral views of wild-type stage 6 embryos showing the localization of Delta (a, b; green in a3, b3), Twi (a'; red in a3) and sim RNA (b; red in b3). The mesoderm boundary is indicated by a yellow line in b and b'. The dots of Delta are seen in all mesodermal cells, but not in the sim-expressing mesectodermal cells. Dorsal is up

using antibodies specific to the extracellular domain of Notch, we find that Delta colocalizes in these mesodermal vesicles with the extracellular domain of Notch (Fig. 4a– c). Furthermore, most of the mesodermal vesicles containing the extracellular domain of Notch are positive for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate; Fig. 4d–f). Hrs is a FYVE-domain containing protein that localizes to endosomes and is involved in the sorting of endocytic cargoes into vesicles that bud into the lumen of multi-vesicular bodies (MVB), a late endosomal compartment (Lloyd et al. 2002; Raiborg et al. 2002). A few vesicles containing the extracellular domain of Notch appear to be negative for Hrs. These vesicles may be en route towards the Hrs-positive compartment. Importantly, Hrs-positive vesicles are uniformly seen both within and outside the mesoderm, consistent with the notion that sorting to MVBs is active in all cells at the blastoderm stage. In Drosophila, Hrs has recently been demonstrated to down-regulate signaling by the EGF and Torso tyrosine kinase receptors, presumably by promoting the degradation of activated receptors (Lloyd et al. 2002). Whether Hrs also participate in Delta-Notch signaling, particularly during mesectoderm specification, remains to be investigated. We note that the formation of these Deltacontaining vesicles does not strictly require Notch signaling, since Delta accumulates in dots in the mesoderm of Notch mutant embryos derived from germ-line clones (Fig. 5 a). Although endocytosis of Delta does not strictly require active Notch signaling, it most likely marks the cells that signal via Delta.

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Fig. 4 Endocytosis of Delta-extracellular-Notch complexes into Hrs-positive vesicles. Colocalization of the extracellular domain of Notch (green in a, d, c, f) with Delta (red in b, c) or with Hrs (red in e, f). These lateral views of wild-type stage 5–6 embryos show

that Delta-extracellular-Notch complexes are specifically endocytosed into Hrs-positive vesicles in the mesoderm. Insets in topright corners are high-magnification views of the boxed areas

Together, these data indicate that Delta-extracellularNotch complexes are internalized from the plasma membrane and targeted to the MVBs, and that these endocytotic events are mesoderm-specific.

neur is required for the endocytosis of Delta

sna is required for the endocytosis of Delta Our observation that the Notch-dependent regulation of sim expression involves a positive regulation by sna led us to study the distribution of Delta in sna mutant embryos. We found that Delta showed a uniform membrane distribution in ventral cells in stage 6 mutant embryos (Fig. 5 b–b3). This phenotype is not seen in twi mutant embryos (Fig. 5 c). Thus, the defective endocytosis of Delta seen in sna mutant embryos does not simply result from a defect in mesoderm specification or in ventral cell invagination (Leptin 1999; Leptin and Grunewald 1990). We therefore conclude that sna is specifically required for the endocytosis of Delta.

Regulation of Delta endocytosis by Sna must be indirect since Sna is a transcriptional repressor. Thus, one hypothesis is that Sna inhibits the expression of a regulator of Delta endocytosis. Recent studies have shown that the endocytosis of Delta requires the activity of the neur gene (Lai et al. 2001; Pavlopoulos et al. 2001). Neur binds to Delta and promotes its internalization, possibly by conjugating a ubiquitin acting as a signal for endocytosis to Delta (Hicke 2001; Lai et al. 2001; Pavlopoulos et al. 2001). We therefore investigated whether neur regulates the subcellular localization of Delta. We found that Delta is uniformly localized at the cell membrane in neur mutant embryos (Fig. 5 d–d3), indicating that neur is required for the endocytosis of Delta in the mesoderm. We also confirmed here that neur is required for the Notch-dependent expression of sim in the mesectoderm (Fig. 6b; Martin-Bermudo et al. 1995). In contrast, the Notch-independent expression of sim at the posterior pole was unaffected by a complete loss of neur activity (Fig. 6b). We conclude that the neur gene is required both for the endocytosis of Delta in the

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Fig. 5 sna and neur are required for the endocytosis of Delta. Lateral views of N55e11 (a) and twiIIH07 mutant embryos (c) showing the subcellular localization of Delta and ventral views of a Df(sna) and neur1 mutant embryos showing the distribution of Delta (respectively b and d; green b3 and d3) and Twi (respectively b' and d'; red in b3 and d3). The dots of Delta are observed in Notch

and twi mutant embryos, but not in embryos carrying the sna deletion neither in neur1 mutant embryos. The embryo in c is at stage 5, embryos in a, b and d are at stage 6. Genotype is followed by anti-b-galactosidase staining which marks the CyO balancer. Anterior is left and dorsal is up

up-regulation of the expression of the neur gene in the mesoderm.

Discussion Fig. 6a, b neur is required for sim expression. Expression of sim in wild-type (a) and neur1 mutant (b) embryos. Expression of sim in the mesectoderm is lost in the absence of neur activity. By contrast, expression of sim at the posterior pole of expression, which is not dependent on Notch (Morel and Schweisguth 2000), remains unchanged. The activity of neur is thus required for the Notchdependent expression of sim

mesoderm and for the Notch-dependent expression of sim in the mesectoderm. At the late blastoderm stage, both the sna and neur genes are co-expressed in the mesoderm (Boulianne et al. 1991; Price et al. 1993; data not shown). Since Sna is a transcriptional repressor, it is conceivable that Sna regulates neur expression in a double-negative linear pathway. We found, however, that loss of sna activity did not change the expression of the neur gene (data not shown). This indicates that Sna regulates the endocytosis of Delta by a mechanism that does not simply rely on the

Our results indicate that Sna has a dual function in the regulation of sim expression. First, Sna acts as a direct repressor in the mesoderm where it prevents activation by Twist and Dorsal. Second, Sna promotes the expression of sim in the presumptive mesectoderm. This second function of Sna requires the Su(H)-binding sites within the cisregulatory sequences of the sim gene, suggesting that Sna stimulates Notch signaling. Sna therefore appears to play a major role in positioning the mesectoderm by autonomously repressing sim expression and non-autonomously promoting Notch signaling. A similar conclusion has recently been obtained in an independent and complementary study by Cowden and Levine (2002). In their study, an even-skipped (eve) stripe2-sna transgene was used to direct the ectopic expression of sna in a single stripe along the anteroposterior axis of the embryo. This ectopic expression of sna was shown to induce the ectopic expression of sim in a few cells containing low levels of the activators Twist and Dorsal and located next to the eve stripe 2 (Cowden and Levine 2002). This observation therefore suggests

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that Sna may act non-autonomously, presumably via Notch, to up-regulate sim expression within a lateral domain that is competent to respond to Notch activation (Cowden and Levine 2002; Morel and Schweisguth 2000). An important issue is to understand how Sna promotes Notch signaling in the mesectoderm. Interestingly, the ability of Sna to regulate sim expression in a nonautonomous fashion depends on its dCtBP interaction motifs (Cowden and Levine 2002). This therefore indicates that stimulation of Notch signaling must depend on the ability of Sna to function as a transcriptional repressor. Based on these findings, Cowden and Levine (2002) have proposed that Sna positions Notch signaling by repressing Notch target genes as well as potential regulators of Notch signaling. This model assumes that Sna functions as a gradient repressor in two consecutive steps. Initially, the domain of sna expression extends into the future mesectoderm. At this early stage, Sna would down-regulate negative regulators of Notch signaling, thereby promoting expression of Notch target genes, such as sim, in a broad ventral domain. During cellularization, the expression of sna then becomes restricted to the mesoderm, and sna is no longer expressed in the presumptive mesectoderm. At this later stage, Sna would repress Notch target genes, thereby restricting the expression of these genes to the mesectoderm. The authors also suggest that a potential negative regulator of Notch that gets down-regulated by Sna is the Notch ligand Delta. Indeed, Delta has the ability to act cellautonomously to inhibit Notch signaling (Sakamoto et al. 2002). Furthermore, Sna inhibits the zygotic expression of the Delta gene in the mesoderm and the stripe2-sna transgene reduces the accumulation of Delta in the neuroectoderm (Cowden and Levine 2002). However, this model involving a sna-dependent repression of the Delta gene is not consistent with the observation that a complete loss of zygotic Delta function has no effect on the expression of sim in the mesectoderm (MartinBermudo et al. 1995). Also, this model does not easily explain the non-autonomous activation of sim expression induced by mesodermal cells that is revealed by cell transplantation experiments (Leptin and Roth 1994). We propose here another model in which sna regulates signaling by Delta and acts in a non cell-autonomous manner to activate sim expression in the mesectoderm. We have shown here that sna and neur are both required for the endocytosis of Delta in the mesoderm. These two genes are expressed in the presumptive mesoderm and participate in the transcriptional activation of sim in the mesectoderm. Moreover, the neur-dependent endocytosis of Delta has recently been shown to promote Notch signaling in a non-autonomous manner (Pavlopoulos et al. 2001). Finally, we have found that the internalized Deltaextracellular-Notch complexes co-localize with Hrs. Interestingly, it has recently been shown that Hrs contains one ubiquitin interacting motif (UIM) that could possibly interact with Delta following its Neur-dependent ubiquitination (Lloyd et al. 2002). Together, these observations

suggest that Neur and Sna promote Delta signaling by regulating its endocytosis in the mesoderm. Activation of Delta signaling in the mesoderm would lead to the activation of Notch both in the mesoderm and in the adjacent presumptive mesectoderm. Together with the cell-autonomous Sna-mediated repression of sim in the mesoderm, this would lead to the expression of sim in a single row of cells abutting the mesoderm. This model led us to speculate that at least one of the direct transcriptional targets repressed by Sna should inhibit the neur-dependent endocytosis of Delta. In sna mutant embryos, this target gene is predicted to be ectopically expressed and to dominantly block the function of Neur on the endocytosis of Delta. Finally, it is of interest to note that sna, as well as the sna-related genes escargot and wormiu, are also expressed in neural precursor cells during early neurogenesis (Ashraf et al. 1999; Ashraf and Ip 2001; Cai et al. 2001). Moreover, Neur also appears to accumulate in neural precursor cells (Boulianne et al. 1991; Yeh et al. 2000). These observations strongly suggest that Neur and Sna might participate together in the regulation of Delta endocytosis during lateral inhibition, thereby promoting activation of Notch in neighboring cells. Thus, our model based on a Sna-dependent repression of a Neur inhibitor may be of more general significance. Acknowledgements We thank H. Bellen, C. Delidakis, E. Lai, M. Leptin, M. Muskavitch, S. Roth, G. Rubin, D. St. Johnston, the Bloomington Stock Center, the Tbingen stock center and the Developmental Studies Hybridoma Bank (University of Iowa) for providing fly stocks and antibodies. We thank A. Martinez-Arias and M. Leptin for helpful discussions. We also thank Y. Bellache, S. Lee, V. Orgogozo and I. Stttem for critical reading. This work was supported by specific grants from the Centre National de la Recherche Scientifique, the Ministre de la Recherche (ACI Program) and the Association pour le Recherche contre le Cancer (ARC 5575).

References Ashraf SI, Ip YT (2001) The Snail protein family regulates neuroblast expression of inscuteable and string, genes involved in asymmetry and cell division in Drosophila. Development 128:4757–4767 Ashraf SI, Hu X, Roote J, Ip YT (1999) The mesoderm determinant snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis. Embo J 18:6426–6438 Boulianne GL, de la Concha A, Campos-Ortega JA, Jan LY, Jan YN (1991) The Drosophila neurogenic gene neuralized encodes a novel protein and is expressed in precursors of larval and adult neurons. Embo J 10:2975–2983 Bray S, Furriols M (2001) Notch pathway: making sense of Suppressor of Hairless. Curr Biol 11:R217–R221 Cai Y, Chia W, Yang X (2001) A family of snail-related zinc finger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions. Embo J 20:1704–1714 Campos-Ortega JA, Hartenstein V (1997) The embryonic development of Drosophila melanogaster. Springer, Berlin Heidelberg New York Cowden J, Levine M (2002) The Snail repressor positions Notch signaling in the Drosophila embryo. Development 129:1785– 1793

72 Fehon RG, Johansen K, Rebay I, Artavanis-Tsakonas S (1991) Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J Cell Biol 113:657–669 Hemavathy K, Meng X, Ip YT (1997) Differential regulation of gastrulation and neuroectodermal gene expression by Snail in the Drosophila embryo. Development 124:3683–3691 Hicke L (2001) Ubiquitin and proteasomes: protein regulation by monoubiquitin. Nat Rev Mol Cell Biol 2:195–201 Ip YT, Park RE, Kosman D, Bier E,. Levine M (1992a) The dorsal gradient morphogen regulates stripes of rhomboid expression in the presumptive neuroectoderm of the Drosophila embryo. Genes Dev 6:1728–1739 Ip YT, Park RE, Kosman D, Yazdanbakhsh K, Levine M (1992b) dorsal-twist interactions establish snail expression in the presumptive mesoderm of the Drosophila embryo. Genes Dev 6:1518–1530 Kasai Y, Nambu JR, Lieberman PM, Crews ST (1992) Dorsalventral patterning in Drosophila: DNA binding of snail protein to the single-minded gene. Proc Natl Acad Sci USA 89:3414– 3418 Kasai Y, Stahl S, Crews S (1998) Specification of the Drosophila CNS midline cell lineage: direct control of single-minded transcription by dorsal/ventral patterning genes. Gene Express 7:171–189 Kooh PJ, Fehon RG, Muskavitch MA (1993) Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 117:493–507 Kosman D, Ip YT, Levine M, Arora K (1991) Establishment of the mesoderm-neuroectoderm boundary in the Drosophila embryo. Science 254:118–122 Lai EC, Deblandre GA, Kintner C, Rubin GM (2001) Drosophila neuralized is a ubiquitin ligase that promotes the internalization and degradation of delta. Dev Cell 1:783–794 Lecourtois M, Schweisguth F (1995) The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev 9:2598–2608 Leptin M (1991) twist and snail as positive and negative regulators during Drosophila mesoderm development. Genes Dev 5:1568– 1576 Leptin M (1999) Gastrulation in Drosophila: the logic and the cellular mechanisms. Embo J 18:3187-3192 Leptin M, Grunewald B (1990) Cell shape changes during gastrulation in Drosophila. Development 110:73–84 Leptin M, Roth S (1994) Autonomy and non-autonomy in Drosophila mesoderm determination and morphogenesis. Development 120:53–859 Lloyd TE, Atkinson R, Wu MN, Zhou Y, Pennetta G, Bellen HJ (2002) Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila. Cell 108:261– 269 Lopez-Schier H, St Johnston D (2001) Delta signaling from the germ line controls the proliferation and differentiation of the somatic follicle cells during Drosophila oogenesis. Genes Dev 15:1393–1405 Martin-Bermudo MD, Carmena A, Jimenez F (1995) Neurogenic genes control gene expression at the transcriptional level in

early neurogenesis and in mesectoderm specification. Development 121:219–224 Morel V, Schweisguth F (2000) Repression by Suppressor of Hairless and activation by Notch are required to define a single row of \textit{single-minded} expressing cells in the Drosophila embryo. Genes Dev 14:377–388 Morel V, Lecourtois M, Massiani O, Maier D, Preiss A, Schweisguth F (2001) Transcriptional repression by suppressor of hairless involves the binding of a hairless-dCtBP complex in Drosophila. Curr Biol 11:789–792 Nambu JR, Franks RG, Hu S, Crews ST (1990) The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63:63–75 Nambu JR, Lewis JO, Wharton KA Jr, Crews ST (1991) The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development. Cell 67:1157–1167 Parks AL, Klueg KM, Stout JR, Muskavitch MA (2000) Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127:1373–1385 Pavlopoulos E, Pitsouli C, Klueg KM, Muskavitch MA, Moschonas NK, Delidakis C (2001) neuralized encodes a peripheral membrane protein involved in delta signaling and endocytosis. Dev Cell 1:807–816 Price BD, Chang Z, Smith R, Bockheim S, Laughon A (1993) The Drosophila neuralized gene encodes a C3HC4 zinc finger. Embo J 12:2411–2418 Raiborg C, Bache KG, Gillooly DJ, Madshus IH, Stang E, Stenmark H (2002) Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes. Nat Cell Biol 4:394–398 Rao Y, Vaessin H, Jan L, Jan Y (1991) Neuroectoderm in Drosophila embryos is dependent on the mesoderm for positioning but not for formation. Genes Dev 5:1577–1588 Roth S, Stein D, Nusslein-Volhard C (1989) A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell 59:1189–1202 Rusch J, Levine M (1996) Threshold responses to the dorsal regulatory gradient and the subdivision of primary tissue territories in the Drosophila embryo. Curr Opin Genet Dev 6:416–423 Sakamoto K, Ohara O, Takagi M, Takeda S, Katsube K (2002) Intracellular cell-autonomous association of Notch and its ligands: a novel mechanism of Notch signal modification. Dev Biol 241:313–326 The I, Bellaiche Y, Perrimon N (1999) Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. Mol Cell 4:633–639 Thomas U, Speicher SA, Knust E (1991) The Drosophila gene Serrate encodes an EGF-like transmembrane protein with a complex expression pattern in embryos and wing discs. Development 111:749–761 Yeh E, Zhou L, Rudzik N, Boulianne GL (2000) Neuralized functions cell autonomously to regulate Drosophila sense organ development. Embo J 19:4827–4837 Yeh E, Dermer M, Commisso C, Zhou L, McGlade CJ, Boulianne GL (2001) Neuralized functions as an E3 ubiquitin ligase during Drosophila development. Curr Biol 11:1675–1679