INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The transmembrane protein Notch (N) regulates cell fates in Drosophila melanogaster during the development of tissues from all three germ layers (6, 11, 14, 24, 42, 83, 88). For example, embryos without zygotically contributed N produce excess neuroblasts at the expense of epidermoblasts (11, 66, 90). In this instance, N appears to function by suppressing a default fate in some members of a population of physically interacting cells. Delta (Dl), also a transmembrane protein, has been identified as the ligand for this function of N, known as lateral inhibition (1, 14, 24, 32, 46, 50, 86).

The extracellular domain of N, where extracellular ligands or factors regulating intracellular N activities are expected to bind, is made up of 36 epidermal growth factor-like (EGF-like) repeats (42, 88). In vitro analyses of deletions affecting different segments of the extracellular domain of N have shown that Dl binds N in the region of EGF-like repeats 11 and 12 (68). Serrate (Ser), the only other identified ligand of N but with functions similar to that of Dl, also binds the same two EGF-like repeats (25, 29, 68). A single-amino-acid substitution in this region can produce an embryonic lethal phenotype (18). However, these two repeats are not sufficient for wild-type N function: loss of the remaining extracellular sequence blocks formation of the embryonic cuticle (52), and single-amino-acid substitutions affecting the 2nd (nd³), 14th (spl), 24th (Ax⁹, Ax^59b, Ax^59d), 25th (Ax¹), 27th (Ax^71d), 29th (Ax¹⁶, Ax^E2), or 32nd (N^ts1) EGF-like repeats or the lin12/Notch repeats [l(1)N^B] produce lethality or aberrant Notch function (41, 54, 91). Since most of these mutations alter the structure of N EGF-like repeats, it was likely that these extracellular regions mediate interactions with alternative ligands. These alternative ligands could be associated with other N functions, such as induction of cell fates observed in the development of the compound eye (2, 13, 28) or differentiation of the epidermis (15, 36). Therefore, I explored the functional significance of EGF-like repeats of N other than those involved in binding of Dl or Ser.

Interspecific sequence comparisons identified two possible ligand binding sites in the region containing EGF-like repeats 19 to 36. A cell surface screen of embryonic cDNA-derived peptides identified Wingless (Wg) as a possible ligand of N. In vitro analysis showed that Wg associates with N in this region. In vitro and in vivo gene expression analyses showed that Wg is associated with regulation of expression of the Dfrizzled2, patched, shaggy, and hairy genes through two distinct forms of N: the full-length form and a form of N lacking 18 or more of the amino-terminal EGF-like repeats (thereby also the Dl binding repeats). These two forms of N produce different ligand-independent and ligand-dependent gene activities in cells expressing them.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Sequence analysis. Extracellular N sequences of D. virilis and of D. pseudoobscura were obtained by reverse transcription-PCR with Taq polymerase and degenerate primers. Plots were generated by using the PILEUP and PLOTSIMILARITY programs in the Genetics Computer Group sequence analysis program (27). There are only 10 single-residue, 4 two-residue, and 1 eight-residue changes in the EGF-like repeats region between N and human Notch1.

Biopanning. A 6- to 12-h Drosophila embryonic cDNA library was constructed in the Surfzap vector (Stratagene). Biopanning was performed as recommended by the manufacturer. Approximately 5 × 10⁸ lambda phage (~130 times the number of primary plaques) were used for mass excision of phagemids. About 6 × 10⁸ excised phagemids were used for filamentous phage preparation (in the presence of 1% glucose). Phage precipitate was resuspended in balanced salt solution (BSS) (89). A total of 4 × 10⁶ to 6 × 10⁶ heat shock-induced S2-N cells were washed twice with BSS and blocked first for 30 min at 4°C with 1 ml of BSS-5% protease-free bovine serum albumin (BSA) and then for 30 min at 4°C with 1 ml of BSS-5% BSA-100 µl of ~10¹⁴ M13mp8 phage per ml in BSS solution. Approximately 10¹³ to 10¹⁴ biopan-ready filamentous phage, mixed with 60 µl of M13mp8 phage solution, were added, and the tubes were shaken for 25 min at 4°C. The cells were washed twice with 10 ml of cold BSS-5% BSA followed by four times with 10 ml of cold BSS. The bound phages were eluted in 200 µl of 0.1 M triethylamine with protease inhibitors (1 µl each of benzamidine [0.1 mg/ml], trypsin inhibitor [10 mg/ml], pepstatin [10 mg/ml], leupeptin [10 mg/ml], and aprotinin [2.2 mg/ml] and 5 µl of phenylmethylsulfonyl fluoride [10 mg/ml]), the solution was neutralized with 200 µl of 1 M Tris-HCl (pH 7.5), and the phagemids were amplified in Escherichia coli. A total of ~10¹², ~10¹⁰, and ~10⁸ amplified phage were used in the second, third, and fourth rounds of biopanning, respectively. In the fifth and sixth rounds, ~10⁷ and ~10⁶ phagemids, respectively, were biopanned four times for 30 min consecutively on ~10⁷ heat shock-induced S2 cells (each time), and the supernatant was subsequently biopanned once on heat shock-induced S2-N cells. Then 1,000 to 2,000 eluted, biopanned phagemids were screened with probes of various genes by standard procedures (73). Phages from purified wg-carrying phagemids were prepared as specified by the manufacturer (Stratagene).

Immunocytochemistry on cultured cells. S2, S2-N, S2-N^I, S2-N^EGF19-36, and S2-N^EGF1-18 cells were heat shock induced before use. S2-Dfz2 cells (8) were grown in the presence of copper for 12 to 14 h before use. The cells were washed twice with cold Shields and Sangs M3 medium (M3 medium; Sigma), resuspended with 400 µl of cold medium conditioned with growth of S2-Wg or S2 cells (see below), and incubated for 15 min at 4°C. The cells were washed twice with cold M3 medium, fixed in 4% paraformaldehyde-1× phosphate-buffered saline, and processed for immunofluorescence as described previously (24, 51). pv9 1.1 S2-Wg cells were used to make Wg conditioned M3 media as described previously (69). Unconcentrated medium was used for all experiments.

Immunoprecipitations. (i) From cultured cells. N and Dl proteins were induced by heat shock. Dfz2 was induced by growing S2-Dfz2 cells for 12 to 14 h in the presence of copper. For N and Dl experiments, 10⁷ cells each of S2-N and S2-Dl cells, respectively, were incubated in M3 medium for 15 min for formation of aggregates (51) and used for each immunoprecipitation (i.e., each lane). The number of S2-N or S2-Dl cells was approximately the same wherever a mixture of cell lines was used, with the remainder being made up by the other cell line or S2 cells. For N and Wg experiments, 3 × 10⁷ S2 or S2-N cells were used per immunoprecipitation. For experiments with N^EGF1-18, N^EGF19-36, and S2-Dfz2 cells, 5 × 10⁶ cells were used per immunoprecipitation. The cells were washed twice in cold serum-free M3 medium and resuspended in 250 µl of cold M3 medium conditioned with growth of S2 cells (S2 media) or pv9 1.1 S2-Wg cells (S2-Wg media) plus protease inhibitors (20 ng each of leupeptin, pepstatin, trypsin inhibitor, and E-64 per ml, 5 ng of aprotinin per ml, and 2 mM phenylmethylsulfonyl fluoride). Where required, EGTA was added to a final concentration of 15 mM. Then 10 µl of ~1.25 mM bis(sulfosuccinimidyl suberate) (BS³) cross-linker resuspended in 1 ml of cold phosphate-buffered BSS (pbBSS: 55 mM NaCl, 40 mM KCl, 15 mM Mg₂SO₄, 10 mM CaCl₂, 20 mM glucose, 50 mM sucrose, 0.74 mM KH₂PO₄, 0.35 mM Na₂HPO₄) was added to appropriate samples, and the cells were incubated for 30 min at 4°C. The cells were pelleted, resuspended in 1/10 pbBSS-10 mM Tris-HCl (pH 7.5) (to quench cross-linking)-protease inhibitors, and incubated on ice for 10 min. The membranes were pelleted, resuspended in 400 µl of cold pbBSS-60 mM Tris (pH 7.5)-0.8% Triton X-100-protease inhibitors, and incubated for 20 min on ice. A 20-µl volume of 10% deoxycholate was added, and incubation was continued for 90 min to 2 h. The extract was precleared with GammaBind Plus beads (Pharmacia) for ~2 h at 4°C and incubated overnight at 4°C with the immunoprecipitation antibody where appropriate [100 µl of the monoclonal anti-Dl antibody, 1 µl of the polyclonal anti-Wg (rb) antibody, 1 µl of the polyclonal anti-Hedgehog antibody, 4 µl of the anti-Patched antibody, or 20 µl of the anti-Dfz2 monoclonal antibody]. Immunocomplexes were captured with GammaBind Plus beads, and the beads were rinsed four times with 1 ml of cold pbBSS-protease inhibitors-10 mM Tris (pH 7.5)-0.1% Triton X-100. Bound complexes were eluted with 40 µl of 1× Laemmli buffer-protease inhibitors, boiled for 6 min, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 4% polyacrylamide gels (for anti-Dl or anti-Wg immunoprecipitations) or in 6% polyacrylamide gels (for anti-Dfz2 immunoprecipitations). Western blot analyses were performed by to standard procedures (30, 73), and signals were detected with the ECL kit (Amersham).

(ii) From embryos. Approximately 800 µl of dechorionated embryos of appropriate ages and strains, laid by circadian cycle-entrained flies (to minimize age variance in embryos), was partially crushed, with a loose-fitting pestle in a 1-ml Wheaton Dounce grinder, in the presence of 400 µl of ice-cold pbBSS-protease inhibitors, with or without ~2 mM BS³. After 45 min of incubation on ice, 12 µl of cold 2 M Tris-HCl (pH 7.5) was added to quench the cross-linking reaction. Membrane proteins were extracted in 0.75% Triton X-100-0.5% deoxycholate. The rest of the procedure was identical to that described for immunoprecipitation from cultured cells. Anti-Dl, anti-Wg, and anti-Ser immunoprecipitates (see Fig. 4A and B) were separated by SDS-PAGE in 4% polyacrylamide gels; anti-Wg, anti-Hh, and anti-Ptc immunoprecipitates (see Fig. 4C) were separated by SDS-PAGE in 6% polyacrylamide gels.

Northern analyses. Total RNAs were extracted from cultured cells or embryos by using RNAzol B (Tel-test, Inc.). I used 0- to 20-h dechorionated embryos, collected at the indicated temperatures (with appropriate corrections for developmental times). A total of 2 × 10⁷ cells of different cell lines (grown to confluence) were heat shocked and incubated for 2 h at room temperature before use. S2-Dfz2 cells were grown in either the presence or absence of copper for 12 to 14 h before use. The cells were washed twice with serum-free M3 medium with antibiotics, and equal volumes were aliquoted to two 1.5-ml Eppendorf tubes, pelleted, and resuspended in 600 µl of S2 medium or S2-Wg medium. After 2 h of gentle shaking at room temperature, RNAs were extracted. Then 40 µg of total RNA was loaded in each lane. Standard Northern blot procedures were used (73). For generation of N^{EGF1-18/Ax59d} molecules, the fragment including NheI (nucleotide 4324) (42) and BglII (nucleotide 5160) was PCR amplified (with Pfu enzyme) from Ax^59d/FM7 flies, cut with NheI and BglII, and used to replace the same fragment in N^EGF1-18. Samples were sequenced fully in the replaced region, and clones carrying the Ax^59d mutation was transfected into S2 cells. Expression of protein was confirmed by Western blotting and immunocytochemistry.

Western blot analyses. For assessment of Armadillo protein in the cytoplasm, about 3 × 10⁶ heat shock- or metal-induced cells were processed as described by Bhanot et al. (8). The same blot was stripped and stained for ~16 h with India ink. For analyses of proteins from embryos, proteins were extracted with 0.75% Triton X-100-0.5% deoxycholate or with SDS lysis buffer (43). The amounts of proteins in different embryonic extracts were standardized by using absorbance values at 280 nm and the Bio-Rad D_C protein assay kit. The proteins were separated by SDS-PAGE in 4% polyacrylamide gels. For analysis of S2-N, S2-N^EGF1-18, S2-N^{EGF19-36 and LN rpts}, and S2-N^EGF1-36 proteins, heat shock-induced cells were dissolved in 1× Laemmli buffer and separated by SDS-PAGE in 4% polyacrylamide gels. Western blotting in all cases was performed by standard procedures (30, 73) and signals were detected with the ECL kit.

In situ RNA hybridization. Batches of wild-type and mutant embryos were grown under identical conditions at 25 or 18°C and processed simultaneously under the same conditions at all steps of the procedure. For each comparative experiment, the batches of wild-type and mutant embryos were divided just before addition of probe, and different probes were added to the divided batches of embryos. A double RNA-protein hybridization procedure, described by Corbin et al. (14), was used. An anti--galactosidase antibody made in mouse was used to sort out FM7 or TM6 chromosome carrying embryos laid by N^264-47/FM7, spl Ax^59d/FM7, Ax^9B/FM7, Ax^59d/FM7, or Dl^X/TM6 flies.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

EGF-like repeats 23 to 27 and 31 to 34 are potential ligand binding sites. A chimeric Drosophila N protein containing EGF-like repeats 10 to 13 from Xenopus physically associates with the Drosophila Dl protein (68). Therefore, the Dl binding region in N is expected to be conserved between homologous sequences. If there are additional ligands associated with different functions of Notch and if they interact with regions other than the Dl binding region, these regions are expected to be conserved between homologous sequences also. To determine whether such conserved regions exist in the extracellular domain of N, the DNA sequences coding for the extracellular portions of N proteins in D. virilis and D. pseudoobscura were compared by plotting the running averages of conservation between sequences of these invertebrate homologs and between sequences of D. melanogaster N and the human homolog, hNotch 1 (Fig. 1A). Figure 1 shows that, as expected, a peak with high conservation is centered on EGF-like repeats 11, 12, and 13, which include the Dl binding region. Besides that region, there are two additional regions that are as conserved as EFG-like repeats 11 to 13 in both comparisons. In the D. melanogaster-D. virilis comparison, the comparison relevant to N function in Drosophila, one region extends from EGF-like repeats 23 to 27 and encompasses most of the domain affected by the Ax mutations of N (17, 41). A second conserved region extends from EGF-like repeats 31 to 34. The functional importance of all these conserved regions is underscored by inclusion of at least one lethal mutation in each (boldface type in Fig. 1A). As EGF-like repeats 11 and 12 bind Dl (68) and are evolutionarily conserved (Fig. 1A), the regions containing EGF-like repeats 23 to 27 and 31 to 34 were hypothesized to be conserved because they are additional ligand binding regions in the extracellular domain of N. 

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Search for new N ligands. (A) Interspecific sequence comparisons reveal possible ligand binding sites in the extracellular domain of N. Each plot represents a running average of sequence conservation between two homologs, over sliding blocks of 40 amino acids (aa). At the top, the corresponding EGF-like repeats of N are graphically represented. lin12 = lin12/N repeats (42, 88). The line in the middle of each plot represents average similarity between the two sequences compared (the maximum value is 1.5). This average would include sequence conservation due to sequence elements common to all EGF-like repeats. The plot of D. melanogaster N and its D. pseudoobscura homolog is similar to that of N and the D. virilis homolog (differing only in the level of overall conservation). The between-lineage comparison identifies evolutionarily stable conserved regions. dmN, D. melanogaster N; hN1, human homolog of N (23); dvN, D. virilis N. Sites of mutations in nd3 and l(1)NB are from reference 54, NM1 are from reference 18, spl and the Ax alleles are from reference 41, and Nts1 are from 91. Lethal alleles are in bold letters. (B) Schematic representation of the biopanning screen used for identification of potential N ligands (see Materials and Methods).

Cell surface biopanning screen suggests a physical affinity between N and Wg. The two identified ligands, Dl and Ser, bind N only at the region including EGF-like repeats 11 and 12 (68). This specificity suggested that if the regions including EGF-like repeats 23 to 27 and 31 to 34 bound ligands, these are likely to be novel ligands. To identify such novel N ligands, if any, phagemid biopanning (55, 74) was performed on the surfaces of live S2 cells expressing full-length N proteins (S2-N). The procedure used is schematically shown in Fig. 1B. Phagemids encoding the known N ligands, Dl and Ser, were specifically enriched by this biopanning. Enrichment was also observed for Wg, N, Big Brain, Pecanex, and Fringe phagemids but not for Scabrous and Star phagemids (Table 1). Genes encoding these proteins are known to genetically interact with N (5, 15, 22, 31, 40, 47, 56, 64, 67). Enrichments were not detected for hedgehog (hh), patched (ptc), and slit genes, whose products function on cell surfaces (35, 45, 49, 57, 70, 81), or for several genes whose products are cytoplasmic proteins (Table 1 footnote). The high enrichment of Pecanex phagemids (from 0.3/10⁵ before biopanning to 25,600/10⁵ after biopanning), enrichment of phagemids of known ligands of N (Dl and Ser), enrichment of phagemids representing only a subset of genes showing interaction with the N gene, and lack of enrichment of EGF-like repeat sequence containing Slit phagemids indicated specificity in the enrichment process (the after-biopanning phagemid population was estimated to be composed of phagemids representing only about 15 genes).

Soluble Wg proteins form two molecular complexes with N proteins. To confirm that enrichment of wg phagemids on S2-N cells was due to binding of Wg protein (expressed on the surfaces of phagemids) to the surfaces of S2-N cells, immunocytochemical analyses was performed with soluble Wg and S2 cells expressing the following N molecules (see reference 52 for a complete description of these molecules): full-length N (S2-N), N lacking EGF-like repeats 19 to 36 (S2-N^EGF19-36), N lacking EGF-like repeats 1 to 18 (S2-N^EGF1-18), and N lacking the intracellular domain (S2-N^I). Wg was detected on the surfaces of S2-N cells (Fig. 2B), S2-N^EGF1-18 cells (Fig. 2C), and S2-N^I cells (Fig. 2E) but not on surfaces of S2 cells (Fig. 2A) and S2-N^EGF19-36 cells (Fig. 2D). More than 2 × 10⁶ cells were processed on each slide, and several such slides were examined for each cell type. Immunofluorescence signals were not detected on S2 and S2-N^EGF19-36 cell slides. Double staining with antibodies against N and Wg showed that only N-expressing cells bound Wg (Fig. 2F). Comparable frequencies of Wg-positive cells were obtained with S2-Dfz2 cells (8) treated in the same way as N-expressing cells (Fig. 2G). Dfz2 is known to bind Wg (8).

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Soluble Wg binds N in the region containing EGF-like repeats 19 to 36. Wg binds surfaces of S2-N (B), S2-NEGF1-18 (C), and S2-NI cells (E) but not surfaces of S2 (A) and S2-NEGF19-36 cells (D). Only N-expressing cells bind Wg (F), and the frequency of N-expressing cells binding Wg is comparable to the frequency of S2-Dfz2 cells binding Wg (G). (A to E and G) The photograph on the left shows anti-Wg immunofluorescence in a microscopic field of cells, and the photograph on the right shows Nomarski illumination of the same microscopic field of cells. (F) The photograph on the left shows immunofluorescence generated by the anti-Wg antibody, and the photograph on the right shows immunofluorescence generated in the same microscopic field of cells by an anti-N antibody. Cells were treated with unconcentrated culture medium conditioned by growth of S2-Wg cells. Wg on cell surfaces was detected immunocytochemically with anti-Wg (rb), an antibody made in rabbit (69), and a rhodamine-conjugated secondary antibody. N on cell surfaces was detected with NI (52) and a fluorescein-conjugated secondary antibody. None of the cells incubated with culture medium conditioned by growth of S2 cells showed any detectable signals. Only cells expressing high levels of N are apparent in the photographs. An actual count of all immunofluorescent cells indicates a Wg-positive frequency of ~40% (N is expressed by only 50% of the cells stably cotransfected with the hygromycin gene). A short binding period was used because N was found to be lost from the cell surfaces within minutes of treatment with Wg.

EGF1-18

View larger version (56K): [in this window] [in a new window]   FIG. 3.   Two different Wg- and N-containing complexes are recovered from N-expressing S2 cell surfaces. (A) N- and Dl-containing complexes are recovered from S2-N and S2-Dl cell aggregates in the presence of cross-linkers. Dl-containing cross-linked complexes were immunoprecipitated by the monoclonal anti-Dl antibody, MAb 202 (24) and analyzed by Western blotting with anti-NI antibody. (B) Wg- and Dfz2-containing cross-linked complexes are recovered from S2-Dfz2 cells treated with Wg medium containing cross-linkers, in the presence or absence of EGTA (lanes 1 and 2). A mouse monoclonal anti-Dfz2 antibody (kindly provided by R. Nusse) was used for immunoprecipitation (lane 4) and for detection of Wg-Dfz2 complexes by Western blotting. Wg-Dfz2 complexes were not recovered from S2-Dfz2 cells treated with medium conditioned by growth of S2 cells (not shown). (C) Two Wg- and N-containing cross-linked complexes (arrows) are immunoprecipitated from S2-N cells (lanes 7 and 9), and only one is immunoprecipitated from S2-NEGF1-18 cells (lanes 14 and 16), treated with Wg-containing medium. N- and Wg-containing complexes were immunoprecipitated with anti-Wg(rb) and detected by Western blotting with the indicated antibodies (W-Ab). Lanes 7 and 9 and lanes 14 and 16 show reaction of the same blots with anti-Wg(rb) and anti-NI antibodies. (D) Wg and N containing cross-linked complexes are recovered from S2-NEGF1-18 cells (lane 3) in the absence of EGTA (lane 8) but not from S2-NEGF19-36 cells (lane 4). S2-NEGF1-18 or S2-NEGF19-36 cells were treated with Wg medium containing cross-linkers, immunoprecipitation was performed with anti-Wg(rb) antibody, and the Western blots were probed with the indicated antibodies. ppt, immunoprecipitated complexes eluted from GammaBind beads; Super, an aliquot of the protein extract after the last pelleting of the GammaBind beads (see Materials and Methods); IP-Ab, immunoprecipitation antibody; W-Ab, Western blotting antibody; cross-linker, BS3. Wg, medium conditioned by growth of S2-Wg cells; S2, medium conditioned by growth of S2 cells. For panels A, C, and D, 4% polyacrylamide gels were used; for panel B, 6% polyacrylamide gels were used. Only proteins or protein complexes migrating slower than a 120-kDa marker protein are resolved in panels A, C, and D. The tops of all the blots shown coincide with the top of the resolving gel of the discontinuous SDS-PAGE gels.

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Wg-N complexes similar to those recovered from cultured cells are recovered from Canton S embryonic extracts. To determine whether the two Wg-N complexes produced in cultured cells treated with Wg are also produced in vivo, immunoprecipitations were performed with proteins extracted from Canton S embryos. The most frequent and predominant Wg-N complex recovered from young embryos (0 to 3 h or 0 to 6 h) was Wg complexed with a truncated N (N lacking the anti-NT epitope) (Fig. 4A, lanes 10 to 13, see Fig. 4D for epitopes of N antibodies). The electrophoretic mobility of this complex was similar to that of the faster-migrating Wg-N complex (i.e., near the ~220-kDa marker protein) recovered from cultured cells (Fig. 3C). From aliquots of the same embryonic extracts, Dl was found complexed with N recognized by all three N antibodies (Fig. 4A, lanes 7 to 9). This N in the Dl-N complex is apparently the full-length N. 

View larger version (56K): [in this window] [in a new window]   FIG. 4.   Wg and N form complexes during embryogenesis. (A) Two Wg- and N-containing cross-linked complexes, similar to those recovered from cultured cells, are immunoprecipitated from Canton S embryonic extracts. Anti-Dl, is monoclonal antibody MAb 202; anti-NT is described in reference 43; anti-NPCR is described in reference 52; and anti-Wg(rt) was kindly provided by A. Martinez-Arias (See panel D for epitope regions for N antibodies). I used 0- to 3-h embryos for lanes 1 to 16, 6- to 12-h embryos for lane 17, and 10- to 16-h embryos for lanes 18 and 19. Arrow 1 shows Wg complexed with full-length N (lanes 17, 18, and 19); arrow 2 shows Wg complexed with a truncated N (lanes 10 to 17). The asterisk marks the Wg complex not containing N (lanes 10 and 19). A single blot was probed sequentially with the indicated antibodies to form lanes 10 and 11; 12, 13, and 14; 15 and 16; and 19 and 18 (numbers also indicate the sequence of probing). The same embryonic extract was used for lanes 1 to 4 and 7 to 14; lanes 5 to 6, 15, 17, and 18 are derived from different embryonic extracts. (B) Ser-N cross-linked complexes are also recovered from cross-linked embryonic extracts. Complexes were immunoprecipitated with anti-Ser antibody (kindly provided by Elizabeth Knust). Complexes migrating faster than a ~120-kDa marker protein were not analyzed in panels A and B. (C) The procedure recovering Wg-N, Dl-N, and Ser-N complexes also recovers Wg-Dfz2 (lane 1) and Hh-Ptc complexes from cross-linked embryonic extracts (lanes 3 to 6). For lanes 5 and 6, equal volumes of anti-ptc immunoprecipitate was separated in two different lanes and probed with the indicated antibodies. IP-Ab, immunoprecipitation antibody; cross-linker, BS3; W-Ab, Western blotting antibody; AEL, after egg laying. For panels A and B, 4% polyacrylamide gels were used; for panel C, 6% polyacrylamide gels were used. The tops of all the blots shown in panels A, B, and C coincide with the top of the resolving gel of the discontinuous SDS-PAGE gels. (D) Diagram showing the N epitopes used to produce the N antibodies used in the study.

Wg regulates expression of Dfrizzled2, hairy, patched, and shaggy genes in S2 cells expressing N and N^EGF1-18. The cell surface binding and immunoprecipitation experiments described above showed that N and Wg form physical complexes in vitro and in vivo. But does Wg alter the physiological state of cells through N? This question is not easily answered for the in vivo case because N is required for production of epidermal precursor cells through lateral inhibition functions associated with Dl (11, 66, 79, 90) and is also subsequently required for production of epidermis from these epidermal precursor cells (15, 36). Of these two successive developmental events, Wg is required only for production of epidermis from the epidermal precursor cells (4, 7). Any perturbation of the lateral inhibition functions of N (associated with Dl) is expected to mask epidermal functions of N associated with Wg. Therefore, I explored the response of N-expressing S2 cells to Wg in the medium and tested wild-type and mutant N embryos for comparable responses. Since regulation of endogenous gene activities in response to exogenous factors in the medium is a good indicator of the signaling effects of cell surface ligand-receptor interactions, the expression of a selected sample of genes that are linked to the N and Wg signaling pathways was assessed in N-expressing S2 cells treated with Wg.

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View larger version (60K): [in this window] [in a new window]   FIG. 5.   N and NEGF1-18 have different ligand-independent activities and respond differently to Wg. (A to D) Expression of Dfz2, h, ptc, and sgg in S2-N and S2-NEGF1-18 cells are regulated by Wg. en, wg, ac, hh, and m5 and m8 of E(spl)C were not detected in any of the experiments. The sizes of transcripts of all genes were similar to published reports. Dfz2 (8), ptc (35), h (37), rp49 (61), and sgg (77). The largest sgg RNA corresponds in size to the embryonic transcript, while the smallest sgg RNA has a size expected for the ovarian transcript (77). sgg, wg, ac, and m5 and m8 of E(spl)C are known to genetically interact with N (15, 33, 71, 72, 85); Dfz2, ptc, wg, and sgg, are involved in epidermal patterning (4, 7, 8, 20, 35, 57, 65); h is a negative regulator of ac (38, 79, 84). (E) The extracellular domain of NEGF1-18 is required for regulation of Dfz2, sgg, ptc, and, to a lesser extent, h expression. (F) Ax59d mutation in NEGF1-18 abolishes Wg-mediated down regulation of Dfz2 expression in S2-NEGF1-18 cells. The two autoradiographs were derived from the same blot with different exposure times. (G) S2-Dfz2 cells (S2-pMK 33 cells [8]) do not down regulate expressions of ptc, sgg, and h in response to Wg, with or without copper induction. (A to G) Total RNAs were extracted from the indicated cells treated with M3 medium conditioned by growth of S2 cells (S2 medium) or S2-Wg cells (Wg medium) and analyzed by Northern blotting. The same batch of S2 or Wg medium was used for all of the studies. Gene sequences used as probes are indicated on the right of each panel. rp49 was used to indicate the relative levels of RNA in different lanes. The individual blots are exposed to film for different periods. Exposure times: rp49 < Dfz2 < ptc < sgg < h. (H) S2-NEGF1-18 cells do not accumulate Arm in the cytoplasm in response to Wg. The bottom panel (*) shows a India ink-stained protein band visible in all lanes of the blot to indicate the amount of samples loaded. An anti-Arm antibody made in rabbits (kindly provided by Laurent Ruel) was used to detect Arm.

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N lacking amino-terminal EGF-like repeats is associated with Dfz2 expression in vivo. Full-length N and a truncated N were associated with Wg in S2 cells and embryos (Fig. 3 and 4). In S2 cells, the full-length N and the truncated N^EGF1-18 were active and responsive to Wg in different ways: N did not induce gene expression in the absence of ligands, whereas N^EGF1-18 did; N up regulated the expression of genes in response to Wg, whereas N^EGF1-18 down regulated the expression of genes in response to Wg (Fig. 5A to D). Thus, the two N-Wg complexes formed in embryos (Fig. 4) appear to have the potential to trigger different intracellular activities in vivo. To determine whether N^EGF1-18 shows the same activity in vivo that it showed in vitro, expression of Dfz2 was assessed in embryos expressing the transgenic N^EGF1-18. Dfz2 expression was assessed because in S2 cells it was regulated only by N^EGF1-18; S2-N cells did not induce or regulate Dfz2 expression (Fig. 5A). Figure 6A shows that N^EGF1-18 embryos overexpress Dfz2, indicating that N^EGF1-18 behaves similarly in vitro and in vivo.

View larger version (51K): [in this window] [in a new window]   FIG. 6.   NEGF1-18 and nd3 embryos show increased expression of genes regulated by NEGF1-18 in vitro, and an endogenous form of N resembling NEGF1-18 is overproduced in nd3 embryos. (A) Embryos carrying the heat shock-inducible NEGF1-18 transgene also overexpress Dfz2. We heat shocked 0- to 20-h Canton S (CS) (yw strain) and NEGF1-18 (in Canton S, yw strain) embryos for 30 min, allowed them to recover at room temperature for 45 min, and extracted total RNAs for Northern blot analysis. rp49 indicates relative levels of RNA in different lanes. (B) Dfz2, h, ptc, and sgg are overexpressed in nd3 embryos at 18°C, the temperature at which the overt mutant phenotype is observed. At 25°C, expression of these genes in nd3 does not differ from that in Canton S. Levels of expression in Canton S at 25 and 18°C are indistinguishable (expression at 18°C is shown). Total RNAs were extracted from 0- to 20-h Canton S and nd3 embryos, reared at 25 or 18°C (with appropriate correction for developmental times), and analyzed by Northern blotting. Gene sequences used as probes are shown at the right. Exposure times: rp49 < Dfz2 < ptc < sgg < h. (C) nd3 embryos at 18°C overproduce a ~200-kDa form of N, N200. Because signals from high-molecular-weight forms of N interfere with assessment of levels of the less abundant N200, total embryonic proteins extracted from 0- to 12-h Canton S and nd3 embryos (at 18 or 25°C) were incubated with anti-NT and cleared prior to SDS-PAGE. Anti-NT does not react with N200 (see below). Extracts containing equivalent levels of ~350-kDa N were used for lanes 1 and 2. N is detected with anti-NI. (D) N200 is truncated in the amino terminus. N was immunoprecipitated from 0- to 12-h Canton S or nd3 embryos (reared at 18°C) by using anti-NI and separated by SDS-PAGE (4% polyacrylamide), and the Western blots were probed with the indicated antibodies (W-Ab). See Fig. 4D for epitopes for N antibodies. nd3 embryos were used to determine missing epitopes because they produce higher levels of N200 than do Canton S embryos (compare lanes 1 and 3). (E) Different developmental stages of Canton S flies express N200, and N200 lacks more than 18 of the amino-terminal EGF-like repeats. Aliquots of total proteins extracted from Canton S embryos (0 to 3 h), one Canton S larva, one Canton S pupa, S2-N cells, S2-NEGF1-18 cells, S2-NEGF19-36 and LN rpts cells, and S2-NEGF1-36 cells were separated by SDS-PAGE (4% polyacrylamide) and analyzed by Western blotting with anti-NI. *1 is recognized by all of the N antibodies studied and is therefore considered to be the partially denatured form of N (43); *2 is not recognized by anti-NT and anti-NPCR (not shown) but is recognized by anti-NI.

EGF1-18

EGF1-18

EGF1-18

EGF1-18

EGF1-36

EGF1-18

EGF1-18

N and Ax mutant embryos show altered expression patterns of epidermal patterning genes that are consistent with expression patterns observed in vitro. A further test of involvement of N in expression of cuticle-patterning genes in vivo would be that N and Ax embryos (which do not overproduce N²⁰⁰) show low and high expression, respectively, of the same genes. Since N and Ax alleles are homozygous lethal, in situ hybridization rather than Northern blotting was used. In situ hybridization of nd³ embryos with Dfz2 and en probes (the latter serving as a control for levels of RNA in embryo) show that the results are qualitatively comparable to results obtained by Northern blotting (Fig. 7A to D).

View larger version (68K): [in this window] [in a new window]   FIG. 7.   Expression of Dfz2 is reduced in zygotic N embryos, while expression of en and wg are unaffected. (A to D) In situ hybridization of Canton S and nd3 embryos corroborates results from Northern blotting that Dfz2 is overexpressed in nd3 embryos but en is not (E to H, G', H') Dfz2 expression is lost in N264-47/Y embryos but not in comparable stages of Canton S or DlX/DlX embryos. (I to L) wg expression is not lost in N264-47/Y. (M to P) en expression is similar in Canton S and N264-47/Y embryos. CS, Canton S; nd3, nd3; N, N264-47/Y; Dl, DlX/DlX embryos; genes used as probes are indicated below the appropriate sets of embryos. Anterior is to the left of each embryo. N264-47/Y and DlX/DlX embryos were identified by lack of -galactosidase staining associated with the FM7 or TM6 balancer chromosomes (see Materials and Methods). Embryos A to D were processed simultaneously, and so were embryos E to P.

View larger version (71K): [in this window] [in a new window]   FIG. 8.   Ax embryos overproduce Dfz2 and sgg RNA. (A to L) Ax embryos overproduce Dfz2 RNA (A, C, E, G, I, and K) but not en RNA (B, D, F, H, J, and L). (M to R) Ax embryos overproduce sgg RNA (O to Q) but not Canton S (M and R) and N264-47/Y embryos (N). Due to low-level of expression in a general pattern, reduced sgg expression (as in panel N) and weak sgg overexpression (as in panel O) are more obvious in pools of embryos than in individual embryos. CS, Canton S; Ax9, Ax9B/Y; spl Ax59, spl Ax59d/Y; Ax59, Ax59d/Y; N, N264-47/Y. Anterior is to the left of each embryo. Homozygous Ax or N embryos were identified by the lack of -galactosidase staining associated with the FM7 balancer chromosomes (see Materials and Methods). Embryos A to H and M to P were processed simultaneously, and so were embryos I to L and Q to R.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

N Regions within EGF-like repeats 19 to 36 mediate interactions with Wg. Wg was identified as a putative ligand of N in a cell surface screen with phagemids carrying cDNA sequences from Drosophila embryos. Further analyses showed that Wg and N form molecular complexes, both in vitro and in vivo. EGF-like repeats 19 to 36 of N are required for the association of these two proteins (Fig. 2 to 4). These repeats of N include two strongly conserved regions, those containing EGF-like repeats 23 to 27 and EGF-like repeats 31 to 34 (Fig. 1A). Experiments with the Ax^59d allele (Fig. 5F and 8) showed that EGF-like repeat 24 is important for Wg-mediated down regulation of gene expression through N^EGF1-18. N²⁰⁰ is identified as the in vivo equivalent of N^EGF1-18. Since the nd³ embryos (overproducing N²⁰⁰), Ax^59d embryos, and S2-N^{EGF1-18/Ax59d} cells overexpress the same genes down regulated by Wg and N^EGF1-18 (Fig. 5F and 8), the region containing EGF-like repeats 23 to 27 might be involved in down regulation of gene expression by Wg and N molecules in vivo. The evolutionary conservation of this region in homologous N molecules might be due to the Wg-associated functions of N. Whether Wg associates with the same region in full-length N, for induction of sgg and ptc expression, is not known. Preliminary results suggest that Wg associates at a second site within the region containing EGF-like repeats 19 to 36 of N.

Two forms of Notch regulate expression of epidermal patterning genes. Immunoprecipitations from cultured cells and embryos recovered Wg complexed with the full-length N and a form of N lacking EGF-like repeats in the amino terminus (Fig. 3 and 4). In vitro experiments showed that two forms of N regulate expression of cuticle-patterning genes, i.e., sgg and ptc by the full-length N in response to Wg, and Dfz2, sgg, h, and ptc by a form of N lacking 18 amino-terminal EGF-like repeats, N^EGF1-18, both independent of ligands and in response to Wg. Thus, Wg behaves as a ligand for two different forms of N in vitro, eliciting different responses from cells expressing these two forms of N. The significant difference between N and N^EGF1-18 is the lack of the Dl binding region in N^EGF1-18. This appears to be true of N²⁰⁰ as well. N and N²⁰⁰ might therefore regulate genes in vivo in a manner comparable to gene regulations by N and N^EGF1-18 in vitro. The in vivo relative levels of the full-length N (capable of associating with Dl and Wg) and N²⁰⁰ (capable of associating with Wg) may therefore represent a differential commitment of N to Dl or Wg signaling during embryogenesis and a differential commitment of N to different kinds of Wg signaling. Since N is required not only for different developmental functions but also for sequential developmental functions (13, 76), it is quite possible that N and N²⁰⁰ constitute important components of the mechanism of N function at successive steps of differentiation. There is some indication that the activity of N^EGF1-18 requires the activity of N in a preceding step: expression of N^EGF1-18 in N^264-47/Y embryos prior to onset of the neurogenic phenotype results in overexpression of h and sgg (as expected, in the expected stage-specific pattern for h), but this does not occur in the neurogenic embryos (data not shown).

EGF1-18

EGF1-18

EGF1-18

EGF1-18