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© The Rockefeller University Press,
0021-9525/2000//683 $5.00
The Journal of Cell Biology, Volume 149, Number 3,
, 2000 683-696
Original Article |
Analysis of Notch Lacking the Carboxyl Terminus Identified in Drosophila Embryos
wesleyc{at}rockvax.rockefeller.edu
The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate N
Cterm.
Key Words: Notch • Delta • neurogenesis • daughterless • differentiation
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
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During lateral inhibition, the ligand Delta (Dl) binds the extracellular domain of N, leading to transmission of signals to the nucleus by the intracellular protein, Suppressor of Hairless (Su(H)). Cells that respond to these signals by turning on the expression of Enhancer of split Complex genes (E(spl)C), and turning off the expression of the proneural Achaete scute Complex genes, become the epidermal precursor cells; cells that do not turn on the expression of E(spl)C but continue to express Achaete scute Complex genes, become the neuronal precursor cells (see Artavanis-Tsakonas et al. 1999). N function continues to be required during differentiation of neurons from the neuronal precursor cells (Giniger et al. 1993; Giniger 1998) and epidermis from the epidermal precursor cells (Hoppe and Greenspan 1990; Couso and Martinez-Arias 1994; Wesley 1999). Requirement of N function at successive stages is also observed during differentiation of tissues like the adult compound eyes and sensory bristles (Cagan and Ready 1989; Guo et al. 1996; Wang et al. 1997). This implies that N is required continuously during differentiation of a cell lineage to maintain the cell fates specified during lateral inhibition and/or generate additional differentiation signals at post-lateral inhibition stages.
Su(H) activity is affected by some proteins that also bind the N intracellular domain. Deltex contributes to the Su(H)-mediated N signaling pathway (Matsuno et al. 1995), while Numb, Dishevelled, and Hairless antagonize this pathway (Axelrod et al. 1996; Frise et al. 1996; Guo et al. 1996; Spana and Doe 1996; Wang et al. 1997). On the other hand, Disabled, which functions with N during differentiation of neurons from neuronal precursor cells (i.e., after lateral inhibition), is not known to affect Su(H) activity (Giniger et al. 1993; Giniger 1998). Su(H) interacts with the RAM 23 region and the CDC10/Ankyrin repeats region in the N intracellular domain (Fortini and Artavanis-Tsakonas 1994; Tamura et al. 1995; see Fig. 1). Deltex interacts with the CDC10/Ankyrin repeats region (Diederich et al. 1994; Matsuno et al. 1995), Numb with the RAM 23 and PEST regions (Guo et al. 1996), Dishevelled with the unique region carboxy-terminal of the CDC10/Ankyrin repeats (Axelrod et al. 1996), and Disabled with the RAM 23 region (Giniger 1998). The binding site of Hairless has not been mapped (Wang et al. 1997; see Fig. 1 a). These different activities and affinities suggest that regulation of activities of different proteins that bind the intracellular domain might be an important component of N functions at successive stages of differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NPCR antibody was generated against the intracellular segment of N, amino acids 2,115–2,536, between the CDC10/Ankyrin repeats and the OPA repeats (Lieber et al. 1993; Kidd et al. 1998; numbering of the 2,703–amino acid-long N protein is according to Kidd et al. 1986). The N203 antibody was generated in rats against a glutathione-S-transferase fusion peptide including N EGF-like repeats 1–3 (amino acids 59–177) following standard procedures (Harlow and Lane 1988). N203 immunoprecipitates and detects only N forms from embryos and S2-Notch cells. It gives N immunostaining patterns in embryos, imaginal discs, and larval brains that is indistinguishable from other published N staining patterns. All the N antibodies used in this study are N-specific antibodies: they do not give signals in N– embryos or N molecules recognized by each are recognized by at least two other independently generated N antibodies (Kidd et al. 1989, Kidd et al. 1998; Lieber et al. 1993). These polyclonal antibodies also recognize N only when their respective epitope regions are included and even a small terminal segment of the epitope region is sufficient for recognition by the respective antibody. Immunostaining procedure described in Lieber et al. 1993 was followed and signals detected with HRP. An anti–β-galactosidase antibody made in rabbit and alkaline phosphatase reactions were used to sort out FM7 lac-Z or TM6 lac-Z chromosome carrying embryos laid by N264-47/FM7 lac-Z or DlX/TM6 lac-Z flies. Embryos shown in Fig. 2, a–i and j–q were collected in separate batches and samples within each batch were processed identically.
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50–100-µl vol of dechorionated embryos, of appropriate ages (laid by circadian cycle entrained flies to minimize age variance in embryos), were crushed using a loose fitting pestle in a 1-ml Wheaton Dounce Grinder, in the presence of ice-cold pbBSS + protease inhibitors + 0.75% Triton X-100 (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; protease inhibitors: 20 ng/ml each of leupeptin, pepstatin, trypsin inhibitor, and E-64, 5 ng/ml of aprotinin, 2 mM phenylmethylsulfonyl fluoride). After 20 min of incubation on ice, deoxycholate was added to a final concentration of 0.5% and incubated on ice for 25 min. The extract was precleared for 2 h at 4°C with GammaBind Plus beads (Amersham Pharmacia Biotech), and incubated overnight at 4°C with the immunoprecipitation antibody. Immunocomplexes were captured with GammaBind Plus beads, the beads rinsed four times with 1 ml of cold pbBSS + protease inhibitors + 0.1% Triton X-100. Bound complexes were eluted with 40 µl of 1x Laemmli buffer + protease inhibitors, boiled for 6 min, separated by SDS-PAGE in 4% gels, Western blotted according to standard procedures (Harlow and Lane 1988; Sambrook et al. 1989), and signals were detected with the ECL kit (Amersham Pharmacia Biotech).
For immunoprecipitation of N-Dl cross-linked complexes, 800-µl vol of dechorionated embryos of appropriate ages (laid by circadian cycle entrained flies) were 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 (Bis[sulfosuccinimidyl] suberate; Pierce Chemical Co.). 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 and 0.5% deoxycholate. The rest of the procedure was identical to that described for immunoprecipitation of Notch proteins from embryos except that the wash buffer included 10 mM Tris, pH 7.5. 100 µl of the monoclonal Dl was used per immunoprecipitation. The amounts of proteins in different extracts were standardized using absorbance values at 280 nM and the BioRad DC protein assay kit. See also Wesley 1999 for description of these immunoprecipitation procedures.
Western Blot Analyses
Embryos.
Populations of flies were transferred to the appropriate temperature, eggs collected for 2 or 3 h (or 6 h at 18°C), and reared for the indicated period of time at the indicated temperatures (with appropriate corrections for differences in developmental rate).
Cultured Cells.
Cells were heat-shocked for 30 min at 37°C, allowed to synthesize proteins for 1 or 2 h at room temperature, and washed 2x in Shields and Sang's M3 media plus antibiotics.
N and Dl Cell Aggregates.
1 x 106 S2-N, S2-N60g11, S2-N1–2155, or S2-N2262 cells were mixed with 1.5 x 106 S2-Dl or S2 cells, transferred to 14-ml round-bottom Falcon tubes or siliconized Falcon multiwell plates, and shaken gently for 1 or 2 h. Total proteins from embryos, cells, or cell aggregates were extracted in pbBSS + protease inhibitors + 0.75% Triton X-100 + 0.5% deoxycholate as described above for immunoprecipitation of N molecules. Proteins were separated in either 4 or 8% SDS-PAGE, Western blotting was performed as described (Harlow and Lane 1988; Sambrook et al. 1989), and signals were detected with the ECL kit (Amersham Pharmacia Biotech). The amount of proteins in different extracts was standardized using absorbance values at 280 nM and the BioRad DC protein assay kit. The Western blotting procedures followed are also described in Wesley 1999.
Cloning of N60g11.
Nco1-Nar1 (amino acids 1,996 and 2,323, respectively) fragment was Pfu-PCR amplified from N60g11/FM7 lac-Z DNA and cloned into pGEM7z vector (Promega). Clones carrying the 175-bp N60g11 fragment including the site of mutation (see Lyman and Young 1993) were distinguished from the 154-bp wild-type fragment carrying clones (derived from the FM7 lac-Z chromosome) by PCR analysis. The Nco1-Nar1 fragment of the N60g11 gene was checked by sequencing and used to replace the wild-type Nco1-Nar1 fragment in a hsCasper-N clone. Cloning of N1–2155 and N1–2262: N fragments were Pfu-PCR amplified with a primer 5' of the Nco1 site (amino acid 1,996) and with a primer containing a stop site either at the third BamH1 site (amino acid 2,155) or at the first EcoR1 site (amino acid 2,262). These fragments were sequenced to confirm that there were no PCR-related mutations and used to replace the Nco1-Nar1 fragment in a pGem7z-N clone. The whole N fragment was excised and cloned into the hsCasper vector. Cloning of the N2155–2703: the carboxy-terminal BamHI-XbaI fragment of the N gene was cloned into hsCasper vector. It was expressed transiently in S2 cells. UAS-Nintra1790 and UAS-N1893–2155 are described in Kidd et al. 1998 and Lieber et al. 1993, respectively. These were cotransfected into S2 cells with hsGal4 for transient expression. hsGal4 alone was transfected to obtain S2-hsGal4 only.
Northern Blot Analysis
Embryos.
0–24-h embryos laid by +/FM7 lac-Z x FM7lac-Z/Y crossed flies and N60g11/FM7 lac-Z flies, reared at 18°C (0–12-h staged embryos reared at 25°C) were used for Fig. 6 d. 0–6-h embryos laid by UAS-Nintra1790; hsGal4, UAS-N1893–2155; hsGal4, or yw Canton S embryos (collected at 25°C, heat-shocked at 37°C for 30 min, and incubated at room temperature for 45 min) were used for Fig. 6 e.
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Results |
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N203, which recognizes the amino terminus of N, and with NPCR, which recognizes the carboxyl terminus of N (see Fig. 1 and Materials and Methods for information about these antibodies). Stage 8-9 Canton S embryos immunostained with N203 showed relatively intense punctate staining in the region involved in lateral inhibition, whereas the embryos stained with NPCR showed a homogenous staining of the same region (Fig. 2, a–e). The intense punctate signals in embryos treated with N203 are derived from segregating neuroblasts: cell morphology identify them as neuroblasts and the pattern of N203 staining rapidly changed during this stage of embryogenesis (Fig. 2, compare b with c). Furthermore, N203 staining corresponded with the expression pattern of the proneural achaete gene accompanying neuroblast segregation (Campos-Ortega and Hartenstein 1985; Cabrera 1990; Skeath and Carroll 1992; data not shown). Differences in the staining patterns of NPCR and N203 were more striking at later stages of embryogenesis: NPCR antibody did not stain the commissures and connectives of the central nervous system (CNS; Fig. 2 f), while N203 showed strong staining of the same tissues as previous studies of N distribution have shown (Fig. 2 g; Kidd et al. 1989; Johansen et al. 1989; Fehon et al. 1991; Giniger et al. 1993; Kooh et al. 1993; Giniger 1998). The previous studies cited used antibodies made against the first two EGF-like repeats, the last six EGF-like repeats, extensive regions of the EGF-like repeats, or intracellular regions including both the CDC10/Ankyrin repeats and the unique region in the carboxyl terminus. None of these studies used an antibody that is specific to the unique region carboxy-terminal of the CDC10/Ankyrin repeats, like NPCR. At some other stages, the two antibodies gave similar patterns (Fig. 2h and Fig. i).
Lack of staining of commissures and connectives of the CNS by NPCR was not because this antibody fails to recognize N in the embryos: (a) omission of NPCR from the immunostaining procedure resulted in complete loss of signals in the embryos (Fig. 2, compare k with j); (b) NPCR failed to generate any signals in the neurogenic N264-47/Y embryos which have lost expression of N (see Kidd et al. 1989), but generated strong signals in the neurogenic Dlx/Dlx embryos which continue to express N (Fig. 2l and Fig. m); (c) NPCR generates a patchy staining pattern in Nts1 embryos raised at 30°C (Fig. 2n and Fig. o; patchy loss of N in Nts1 embryos is expected since only 70% of these embryos fail to complete embryogenesis at the restrictive temperature of 30°C, see Shellenbarger and Mohler 1978); and (d) non-recognition of N in connectives and commissures of the CNS was a property of NPCR as these tissues were stained with N203 (Fig. 2 g) and the nervous system–specific anti-HRP antibody (Fig. 2, compare q with p stained with NPCR). If both N203 and NPCR antibodies recognized the same N molecules at all stages of development, similar staining patterns would be expected at all stages. Instead, only N203 showed higher levels of N in the connectives and commissures of the developing CNS (Fig. 2b, Fig. c, Fig. e, and Fig. g, compare with NPCR staining in a, d, and f). The pattern of N expression in E(spl)C– embryos deficient in lateral inhibition signaling was the same as in Dl– embryos (detected by N203 and NPCR antibodies): expression of N is higher than in Canton S embryos and limited to the neurogenic region (data not shown). These results indicated that a subset of differentiating tissues that express N, produced after lateral inhibition signaling, are enriched for a form of N that either does not contain the region known to be present carboxy-terminal of the CDC10/Ankyrin repeats, or has masked the antibody epitopes in that region.
Embryos Produce Notch Molecules Lacking Sequence Carboxy-terminal of the CDC10/Ankyrin Repeats
SDS-PAGE analysis of N immunoprecipitated from Canton S embryonic extracts showed that N203 and NI recover a triplet of N proteins in the 350-kD range (Fig. 3 a, lanes 1 and 2; NI is made against the intracellular region between the transmembrane domain and the end of CDC10/Ankyrin repeats, Lieber et al. 1993; see Fig. 1). The three forms are referred to as NFull, N350.2, and NCterm in increasing order of electrophoretic mobilities (see later for the basis for these names). Similar forms of N have been reported previously, detected using an antibody made against the last six EGF-like repeats (Johansen et al. 1989). However, NPCR, made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats, immunoprecipitated only NFull and N350.2 (Fig. 3 a, lane 3) indicating that NCterm is not recognized by this antibody.
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Cterm masked NPCR epitopes and this prevented immunoprecipitation by NPCR. To evaluate this possibility, N was immunoprecipitated from Canton S embryos with NI (which recovers all three forms), two equal aliquots of the immunoprecipitates were separated by SDS-PAGE, and the resultant Western blots probed with NI and NPCR. NCterm was detected by NI (as expected) but not by NPCR (Fig. 3 b) indicating that non-recovery of NCterm with NPCR is due to absence, rather than masking, of NPCR epitopes.
The absence of NPCR epitopes and the faster SDS-PAGE migration (compared with NFull containing the NPCR epitopes) suggested that NCterm lacked the carboxyl terminus sequence. To determine whether N molecules truncated to remove the carboxyl terminus NPCR epitope region migrate alongside NCterm in SDS-PAGE, and to get a rough estimate of how much of the carboxyl terminus region is lost in NCterm, the following cell lines were generated: S2-N1–2155 cells producing N molecules truncated after amino acid 2,155, immediately after the CDC10/Ankyrin repeats, and S2-N1–2262 cells producing N molecules truncated after amino acid 2262. Extracts from these cells were separated in SDS-PAGE alongside extracts from embryos, from S2 cells expressing N, and from S2 cells expressing N60g11. N60g11 is N protein produced from the mutant N60g11 allele. N60g11 contains a frame shift mutation that results in deletion of the intracellular region carboxy-terminal of amino acid 2,123 (580 amino acids are deleted and 19 random amino acids added before termination; Lyman and Young 1993). Western blotting analysis showed that NCterm migrates alongside N1–2155 and N60g11, but faster than N1–2262 (Fig. 3 c). The migration of all N molecules in SDS-PAGE reflected the size of truncation in the carboxyl terminus (see diagram in Fig. 3 d). A difference in mobility due to a difference of 107 amino acids (in 2,300 amino acids) is clearly apparent in SDS-PAGE (Fig. 3 c, lanes 1, 2, 4, and 5). Thus, NCterm is not recognized by NPCR because it is truncated for 500 amino acids in the carboxyl terminus and therefore lacks the NPCR epitope region. The nature of differences between N350.2 and NFull and between N350.2 and NCterm are presently unknown. The slowest migrating 350-kD form is named NFull because it appears to contain the complete sequence; the fastest migrating 350-kD form is called NCterm because it lacks the carboxyl terminus (half of the intracellular domain); and the form migrating between NFull and NCterm is named N350.2 because it is the second of three forms in the 350-kD range.
NFull, N350.2, and NCterm are colinear N molecules as they are recognized by an amino terminus antibody (N203) and at least one of the intracellular antibodies (NI and NPCR) in SDS-PAGE–based Western blot analysis. Therefore, these colinear forms may be substrates of Kuzbanian or Furin-like Convertase enzymes for production of heterodimeric cell surface molecules as proposed by Pan and Rubin 1997, Blaumueller et al. 1997, and Logeat et al. 1998. Our data related to activities of NFull and NCterm do not distinguish between the colinear and the proposed heterodimeric forms of the receptors. Therefore, NFull and NCterm would refer to the colinear receptors on Western blots but to both the colinear and the proposed heterodimeric receptors with regard to activities. N, without any numbers, acronyms, or abbreviated names, will be used to refer to the N protein in general (inclusive of all forms). The proposed or inferred structures of the various forms of N referred to in this study and the caveats, if any, associated with inference of their structures or usage of names are shown in Fig. 1 b.
NCterm Is Associated with Delta during Embryogenesis
Anti-Dl immunoprecipitations were performed from different stages of embryos to determine whether NCterm is associated with Dl during embryogenesis. Embryos laid by circadian cycle entrained adult flies were used to minimize age variance and maximize chances for detection of any developmental stage-specific recovery of different forms of N. Proteins interacting at the cell surfaces were cross-linked, and the complexes immunoprecipitated by anti-Dl antibody were analyzed with antibodies made against different regions of N. The cross-linking/immunoprecipitation procedure employed recovers only complexes of proteins known to interact at cell surfaces during Drosophila embryogenesis (Wesley 1999).
The monoclonal anti-Dl antibody used here (mAb 202, Fehon et al. 1990) does not recover detectable levels of NFull in the absence of cross-linkers (Fig. 4 a, lanes 1 and 2; see also Wesley 1999). It does not recover even the intracellular and extracellular fragments of the proposed heterodimeric NFull (data not shown). This may be due to disruption of N-Dl complexes when cells are lysed for immunoprecipitation (Fehon et al. 1990; Wesley 1999) or due to inefficient recovery of NFull by this anti-Dl antibody. Also, none of the anti-N antibodies produced in our laboratory (six have been tested), nor anti-Dl antibodies tested, detect or recover significant levels of the intracellular domain of the proposed heterodimeric NFull receptor, either from cultured cells expressing N or wild-type embryos (Kidd et al. 1998; Wesley 1999). Significant levels of a 250–300-kD N extracellular fragment is detected in Western blots or immunoprecipitations with antibodies made against the extracellular domain (Wesley, C.S., personal observation). The 180-kD extracellular domain fragment described in Blaumueller et al. 1997 is not detected by these extracellular N antibodies. We do not know the reason for this. Failure to recover the proposed intracellular domain of the heterodimeric NFull receptor may be due to the fact that most NFull molecules in these cells or embryos are nonfunctional (see Struhl and Adachi 1998; Schroeter et al. 1998). In the cross-linking/immunoprecipitation procedure employed here, the intracellular and extracellular fragments composing the heterodimeric cell surface receptor are expected to be cross-linked along with the ligand.
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NPCR, NI, and NT (the last antibody was made against the first two EGF-like repeats, Kidd et al. 1989; see Fig. 1 for their epitope regions), indicating that N in these complexes contains all domains of N (Fig. 4 a, lanes 5, 7, 9). N immunoprecipitated from 3–6-h embryonic extracts was recognized by NI and NT, but not by NPCR (Fig. 4 a, lanes 6, 10, 8) suggesting that this form of N is not recognized by NPCR. Immunoprecipitation in the absence of cross-linkers, or without the anti-Dl antibody, failed to recover any N containing complexes (Fig. 4 a, lanes 1–4), indicating that the complexes recovered in these experiments contained both N and Dl. Recognition of N by NPCR in one extract and not in the other (when both were extracted at the same time, with the same procedure) ruled out any experimental variation influencing antibody recognition and indicated that N molecules in the two complexes are indeed different.
Western blot analysis of a 3-h interval sampling of proteins showed that while NFull was the predominant form in 0–3-h embryos, it was expressed at very low levels in the 3–6-h-old embryos (Fig. 4 b, lanes 1 and 2; N350.2 and NCterm are present at similar levels in 3–6-h extracts and migrate close to each other in 4.25% SDS-PAGE gels). This suggested that the form of N associated with Dl in 0–3-h embryos is NFull and the form of N associated with Dl in 3–6-h embryos is NCterm. The form of N associated with Dl in 0–3-h embryos is unlikely to be N350.2 (which is also recognized by NPCR) because it is present at equivalent levels in both 0–3- and 3–6-h embryos (see Fig. 4 b) and would have been recovered from both embryos if it associated with Dl. The low level of NFull in 3–6-h embryos and the association of Dl with NCterm in embryos of the same age are consistent with the observations that a form of N not recognized by NPCR is enriched in 3–6-h embryos (Fig. 2, a–e) and that both N and Dl are required for neurogenesis after lateral inhibition (Giniger et al. 1993; Giniger 1998). The majority of embryos in the 3–6-h sample will be between 4 to 5 h of development at 25°C and past the lateral inhibition stage (see Campos-Ortega and Hartenstein 1985). The low levels of NFull in 3–6-h embryos also indicate that most of NPCR signals in embryos at these stages (Fig. 2, a and d) derive from N350.2 and N200. N200 is a form of N lacking >18 amino-terminal EGF-like repeats (thereby the Dl-binding region) and associates with Wingless during embryogenesis (Wesley 1999).
Fig. 4 b reveals an interesting feature of N: the level of NFull appears to fluctuate significantly in relation to levels of N350.2 and NCterm. The depletion of NFull in 3–6-h embryos is not due to depletion of maternal contribution. Zygotic contribution appears to start at 1.5 h of embryogenesis as the level of NFull increases in 1–3-h embryos (Fig. 4 b, lanes 7 and 8). Furthermore, NFull is required for lateral inhibition (Lieber et al. 1993; Struhl et al. 1993; Axelrod et al. 1996; Struhl and Adachi 1998; Kidd et al. 1998), embryos lacking the zygotically contributed N (but having maternally contributed N) are deficient in lateral inhibition (Cabrera 1990; Skeath and Carroll 1992), and N60g11 embryos which produce NCterm-like protein (at 18°C) but have maternally contributed NFull are still deficient in lateral inhibition (Lyman and Young 1993; Brennan et al. 1997; Wesley, C.S., unpublished observation). The depletion in levels of NFull is observed again in 8–11-h embryos (Fig. 4 b, lanes 5). Equal amounts of total proteins were loaded in all the lanes of Fig. 4 b. Since 8–11-h embryos express the highest level of N (see Johansen et al. 1989; Kidd et al. 1989), the blot containing lanes 4–6 was exposed to film for a shorter period than the blot containing lanes 1–3. Thus, it appears that NFull is produced intermittently during embryogenesis. If NFull was replenished continuously in embryos, or if the embryos were not synchronized in their development by circadian entrainment, the variation in levels of NFull would not have been apparent.
An Intracellular Fragment of Notch Lacking the Carboxyl Terminus Accumulates when S2 Cells Expressing NFull Are Treated with S2-Dl Cells
To determine whether NCterm or the intracellular domain of this cell surface receptor is produced when Dl binds NFull, in vitro experiments were performed with S2-Dl and S2-N cells. N and Dl produced in S2 cells bind each other (Fehon et al. 1990; Rebay et al. 1991). Most of N produced in S2-N cells is NFull (see later and Wesley 1999). S2 cells (untransfected) and S2-Dl cells do not express N and the Notch gene in S2 cells is rearranged (Wesley, C.S., unpublished data; Fehon et al. 1990; Ye et al. 1999).
S2-N cells were treated with S2-Dl cells or S2 cells, and protein extracts analyzed by Western blotting with NI and NPCR antibodies. S2-N cells treated with S2-Dl cells for 2 h accumulated higher levels of a 120-kD fragment (designated Nintra) and a 55-kD fragment (designated NCtermTMintra that are recognized by NI (Fig. 5 a, lanes 1 and 2; see later for the basis for these names). The same blot probed with NPCR recognized Nintra but not NCtermTMintra (Fig. 5 a, lanes 3 and 4). Nintra, recognized by both NI and NPCR (see Fig. 5 a, lanes 2 and 4), is the full-length N intracellular domain. It migrates alongside the non-membrane–tethered Nintra1790 (Fig. 5 b). Both Nintra and NCtermTMintra were not recognized by any of the extracellular domain antibodies (data not shown). Since Nintra1790 is rapidly depleted in cells, S2-N cells in this experiment were treated with Dl for only 45 min so that comparable levels of Nintra and Nintra1790 were obtained. NCtermTMintra was not observed in this experiment as its accumulation requires 2 h. These experiments did not show accumulation of NCterm (data not shown).
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120-kD Nintra produced in response to Dl is most likely the 120-kD N intracellular domain that accumulates in embryos in a Dl-dependent manner (Kidd et al. 1998; Struhl and Greenwald 1999; Ye et al. 1999). We have therefore tentatively designated it Nintra (see Fig. 1 b). NCtermTMintra is not recognized by NPCR, just like NCterm. Mobility in SDS-PAGE indicates that NCtermTMintra lacks 500 amino acids in the carboxyl terminus of the intracellular domain, also like NCterm. Expression of Nintra1790 fails to produce NCtermTMintra (Fig. 5 b). Longer expression periods, longer exposure to film, or expression of membrane-tethered Nintra failed to show even a trace of NCtermTMintra or smaller molecules (not shown). These observations strongly suggest that NCtermTMintra is not derived from Nintra but derived from the full-length N molecules also present in the cells. The N segment from the amino terminus of the transmembrane domain to the carboxyl terminus of the CDC10/Ankyrin repeats (amino acids 1,745–2,145) would be 45 kD. The size of 55 kD for NCtermTMintra suggests that it contains the transmembrane domain. Therefore, we have tentatively designated it NCtermTMintra (see Fig. 1 b). As the cell surface N molecules are proposed to be a heterodimers of the extracellular domain and the intracellular domain (Blaumueller et al. 1997; Pan and Rubin 1997; Logeat et al. 1998), NCtermTMintra could very well be the intracellular domain of heterodimeric NCterm receptor. In all experiments with S2 cells, the N extracellular domain (Nextra) detected by our antibodies (i.e., the 250–300-kD fragment) did not enrich in response to Dl although its level relative to NFull increased (data not shown).
NCterm Promotes Expression of daughterless in Response to Dl
The staining pattern shown in Fig. 2 indicates that NCterm is involved in development of commissures and connectives of the CNS. This raised the possibility that NCterm might function as a receptor for Dl. We examined this possibility in cultured cells. S2-N cells express NFull, whereas S2-N60g11, S2-N1–2155, and S2-N1–2262 express NCterm-like receptors (see Fig. 3 c). All N molecules have the complete extracellular domain and form aggregates with S2-Dl cells indicating that they bind Dl (data not shown; see Rebay et al. 1991). We treated S2-N, S2-N60g11, S2-N1–2155, and S2-N1–2262 with S2-Dl cells and assayed RNA extracted from these cells for expression of numerous genes known to interact genetically with Notch and Delta. The RNAs of Achaete Scute Complex, Enhancer of Split Complex, and wingless were not detected in our experiments. RNA of many other genes were not responsive to Dl treatment. However, the expression of the daughterless (da) gene was responsive to NCterm-like receptors. Expression of both NFull and NCterm-like receptors in S2 cells suppressed da expression (Fig. 6 a, lanes 1–3, 8, and 10). This indicated that the presence or absence of sequence carboxy-terminal of the CDC10/Ankyrin repeats per se does not affect da expression. Treatment of NCterm-like receptors with Dl promoted accumulation of da RNA, while treatment of NFull did not (Fig. 6 a, lanes 2–5 and 8–11). Another comparison of NFull and NCterm-like receptor, N60g11, treated with Dl is shown in Fig. 6 a, lanes 6 and 7. Non-response of da to NFull receptor is consistent with the observation that mammalian full-length Notch suppresses the activity of a da related gene in mammalian cell lines (Ordentlich et al. 1998). The dependence on Dl for promotion of da expression indicated involvement of a ligand-induced, activated N intracellular molecule for signal transduction from the NCterm-like receptors.
To identify the ligand activated signaling molecule of NCterm-like receptors, Western blot analysis was performed after treatment of S2-N60g11 and S2-N1–2155 cells with S2-Dl cells. These two N molecules are indistinguishable in Western blots (differing in length by only 12 amino acids). The cells were treated for only 1 h as the expression of N60g11 and N1–2155 declines rapidly. The results show that a 40-kD intracellular molecule, designated NCtermintra, accumulates in S2-N60g11 and S2-N1–2155 cells in response to Dl, and the expected Nintra accumulates in S2-N cells (Fig. 6 b, lanes 1–8; see later for the basis for the name NCtermintra). S2-N1–2262 cells treated with S2-Dl cells do not accumulate the 40-kD molecule but instead accumulate a 52–55-kD molecule (Fig. 6 b, lanes 9–11, see band marked with an asterisk). The 12–15-kD size difference between this molecule and NCtermintra is approximately the difference between the carboxyl termini of N1–2155 and N1–2262. This indicates that NCtermintra is produced by a proteolytic cleavage amino-terminal of the CDC10/Ankyrin repeats. The 40-kD size suggests that NCtermintra does not contain the transmembrane domain. Since this molecule is produced in response to Dl, just like Nintra from NFull, we have tentatively designated it NCtermintra (see Fig. 1 b).
NCtermintra is closest in size to the 35-kD intracellular fragment containing just the CDC10/Ankyrin repeats, N1893–2155 (data not shown) suggesting that the CDC10/Ankyrin repeats, with little flanking sequence, transduces the signals from NCterm. If just the CDC10/Ankyrin repeats fragment is the activated signaling molecule associated with NCterm receptor, then N1893–2155 was expected to promote expression of da in the absence of Dl. We tested this expectation. Results show that N1893–2155 indeed promotes da expression in S2 cells in the absence of Dl, while Nintra1790, just like N, does not (Fig. 6 c). In several repetitions of the experiment, expression of da in S2-N1893–2155 cells was consistently higher than in the control cells (S2 cells transfected with hsGal4 only) and always lower in S2-Nintra1790 cells. Expression of the N intracellular sequence carboxy-terminal of the CDC10/Ankyrin repeats, N2155–2703, does not suppress da expression as strongly as Nintra1790 (Fig. 6 c).
Next, we examined whether NCterm-like receptor, N60g11, and N1893–2155 increase da expression in vivo. Northern blot analysis of RNA extracted from N60g11embryos showed that overexpression of the NCterm-like receptor results in overproduction of da RNA (Fig. 6 d). As observed in S2 cells, expression of N1893–2155 in embryos promotes expression of da, while expression of Nintra1790 does not (Fig. 6 e). As embryos in an early stage of embryogenesis were used here, only the expression of the maternal transcript is prominent.
Struhl and Adachi 1998 have shown that molecules like N1893–2155 (NCDC10MYR-NLS-) localize in the nucleus, rescue epidermal development in Notch– embryos, rescue epidermal development in the absence of Dl, and suppress neuroblast segregations in early Notch+ embryos (i.e., antineurogenic effect). These activities are similar to those of Nintra and related to expression of E(spl)C (see also Roehl and Kimble 1993; Kidd et al. 1998). This suggested that despite the different effects on da expression, both Nintra and N1893–2155 should overproduce E(spl)C RNA. Probing of the same blots with m5 and m8 genes of E(spl)C shows that these genes are overexpressed in both (Fig. 6 e). As expected, m5 and m8 genes of E(spl)C are also overexpressed in N60g11 embryos (Fig. 6 d).
The differential response of da and the E(spl)C might be due to expression of N60g11 and N1893–2155 in both neuronal and epidermal precursor cells, and expression of Nintra1790 only in epidermal precursor cells (see Fig. 2). Only the neuronal precursor cells increase da expression during embryogenesis (Vaessin et al. 1994). However, the differential expression could be also due to NCterm promoting da expression and not the full-length N (as in S2 cells). Activation of E(spl)C by NCterm may have come about through the proneural genes rather than through lateral inhibition signaling (see Discussion). Thus, it is possible that genes like da are responsive to signals from NCterm, not from NFull, and genes like m5 and m8 of E(spl)C are responsive to signals from both receptors.
nd3 Embryos Overproduce NCterm and Related Molecules
nd3 is a temperature-sensitive, homozygous viable allele of N (Shellenbarger and Mohler 1975) with an amino acid replacing point mutation in the EGF-like repeat 2 (Lyman and Young 1993). In a screen of Notch mutants, we discovered that nd3 embryos reared at 25°C accumulate higher levels of a form of N that is recognized by NT and NI but not by NPCR, which migrates close to the full-length form (Fig. 7 a). 4% SDS-PAGE gels were used here as N that are recognized by NPCR (NFull and N350.2), migrate together in these gels and the levels of NCterm can be unambiguously determined. Embryos heterozygous or hemizygous for the null allele, N264-47, the homozygous viable allele, split, and several Abruptex alleles of Notch showed no alteration in levels of NCterm (data not shown). The overexpressed form in nd3 embryos (25°C) is NCterm because: (a) there is no other N molecule in D. melanogaster that migrates close to the full-length form and is recognized by NT and NI, but not by NPCR (Wesley, C.S., unpublished data); (b) it is recognized by NT made against the first two EGF-like repeats indicating that the amino terminus is intact in this form (Fig. 7 a); and (c) NPCR failed to immunoprecipitate a form of N migrating alongside NCterm from nd3 embryos (25°C) (expected if the faster mobility is due to a truncation in the amino terminus rather than in the carboxyl terminus, data not shown).
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55- and 40-kD N intracellular fragments, having the same SDS-PAGE migration properties as NCtermTMintra and NCtermintra from cultured cells, are also overexpressed in nd3 (25°C) embryos (Fig. 7 b). Overexpression of these and NCterm molecules in nd3 (25°C) embryos suggest that the processes producing N molecules lacking the carboxyl terminus are interrelated and NCterm is the source of NCtermTMintra and NCtermintra. Nintra is not clearly detected in embryonic extracts (see Fig. 7 b, lanes 4–6). This may be because very low amounts of Nintra molecules are sufficient to transduce the Dl-mediated lateral inhibition signal in vivo (Schroeter et al. 1998; Struhl and Adachi 1998; Kidd et al. 1998). The nd3 experiments indicate that (1) production of N molecules lacking the carboxyl terminus is a normal feature of the N gene that can be altered by a point mutation, just like any other functional aspect of a protein, and (2) EGF-like repeat 2 (the site of mutation in the nd3 allele; Lyman and Young 1993) regulates production of these truncated molecules. Of no less significance, nd3 allele provided us with a means to identify the putative in vivo NCtermTMintra and NCtermintra molecules from among the many minor N molecules generally detected in a N Western blot. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cterm, NCtermTMintra, and NCtermintra) are produced during embryogenesis (Fig. 3 and Fig. 5Fig. 6Fig. 7). (d) S2 cells expressing N receptors containing the carboxyl terminus (NFull) treated with S2-Dl cells accumulate an intracellular N molecule lacking the carboxyl terminus, NCtermTMintra (Fig. 5). (e) NCterm is the most likely substrate for production of NCtermTMintra (Fig. 5 and Fig. 7). (f) NCterm functions as a receptor for Dl, with the NCtermintra (comprised mostly of the CDC10/Ankyrin repeats) as its activated signaling molecule, and the da gene is responsive to its signals (Fig. 6).
Based on the results summarized above, we propose the following hypothetical model for N functions during embryogenesis. Lateral inhibition starts with NFull receptor containing the full signaling potential. The back and forth lateral inhibition signaling between interacting cells leads to carboxyl terminus processing of the full-length N molecules present inside the cells (i.e., those not involved in Dl binding) and production of the NCterm receptors. Cells expressing higher levels of NCterm become the neuronal precursor cells and cells expressing higher levels of NFull become the epidermal precursor cells. NFull disappears in neuronal precursor cells and NCterm, a secondary receptor with restricted signaling potential, functions during differentiation of the nervous system. Epidermal precursor cells expressing only NFull, or appreciable levels of both NFull and NCterm, continue the same process during differentiation of the epidermis. Advance from signaling by NFull to signaling by NCterm would mean that those cells have attained a degree of irreversibility in their differentiation process. For example, once NCterm becomes the sole N receptor in the neuronal precursor cells, these cells can only proceed along the neuronal differentiation path. N would continuously function in this manner to both specify and restrict cell fates during differentiation of a cell lineage.
NCterm would lack the Dishevelled-binding region, one of the Numb-binding regions, the OPA sequence, and the PEST sequence (see Fig. 1 a). Therefore, it is likely that loss of one or more of these features is involved in restricting the differentiation possibilities for a cell. Dishevelled and Numb are known to antagonize Su(H) activities (Axelrod et al. 1996; Frise et al. 1996; Guo et al. 1996; Spana and Doe 1996; Wang et al. 1997). Proteolytic removal of their binding sites is likely to eliminate antagonisms to Su(H) activities and promote activities of facilitators like Deltex. This might contribute to the lateral inhibition process and selection of precursor cells for neuronal fates. On the other hand, production of NCtermintra lacking the Su(H)-binding sites from NCterm receptor might promote neuronal fates by promoting activities of Hairless or Numb or Achaete (through Daughterless; Schweisguth and Posakony 1994; Schweisguth 1995; Frise et al. 1996; Guo et al. 1996; Spana and Doe 1996). It is also possible that Disabled, which functions with N during differentiation of the CNS after lateral inhibition, can signal from NCterm and not NFull. Thus, production and functions of NFull and NCterm might provide directionality to N functions at successive stages of differentiation. All these properties of NFull, NCterm, and the proteins interacting or not interacting with these two receptors, may be involved during differentiation of the adult sensory organ (bristle) wherein Su(H) activity is required for determination of some fates and not others (Schweisguth and Posakony 1994; Schweisguth 1995; Wang et al. 1997).
We have no evidence, one way or the other, about involvement of Su(H) in transducing signals from NCterm. Regulation of expression of E(spl)C genes by NCterm seems to indicate that the canonical Su(H)-mediated lateral inhibition pathway is involved. However, E(spl)C genes expression could be regulated by an alternate pathway. NCterm regulates da, not NFull. Daughterless protein, is an activator of proneural proteins (Dambly-Chaudiere et al. 1988; Murre et al. 1989; Cabrera and Alonso 1991; van Doren et al. 1991) and proneural genes also activate expression of E(spl)C (Kramatschek and Campos-Ortega 1994; Singson et al. 1994). Some differences in the activities of the intracellular domains of NFull and NCterm seem very likely. One, the RAM23 region in the intracellular domain of N (see Fig. 1 a) is important for Su(H) activities related to NFull, Nintra and lateral inhibition (Fortini and Artavanis-Tsakonas 1994; Tamura et al. 1995; Matsuno et al. 1997). It appears that NCtermintra lacks most of this region, if not all. Two, the sequence carboxy-terminal of the CDC10/Ankyrin repeats is required for transcriptional activation upon binding DNA (Kidd et al. 1998). Since NCterm lacks this sequence, it might activate genes indirectly through inactivation of a constitutive repressor or stabilization of RNA. NFull containing the carboxyl terminus would activate genes directly from DNA. Thus, it is possible that NFull and NCterm might signal through different pathways with some shared outcomes at certain stages of development, like expression of E(spl)C genes. Su(H) might be functioning with both pathways, albeit in different ways.
Production of N receptors with restricted signaling potential may be important for another reason. NFull binds different ligands and regulates different genes in response to them (see Artavanis-Tsakonas et al. 1999; Wesley 1999). Removal of the carboxyl terminus after initiation of NFull signaling by one ligand might set the cell on a differentiation path specific to that ligand. For example, removal of the carboxyl terminus in neuronal precursor cells after Delta-specific lateral inhibition signaling might make NCterm in these cells either unresponsive to Wingless functioning in the epidermis differentiation pathway, or responsive to Wingless in the manner specific to neuronal differentiation pathway. Treatment of full-length N with Wingless results in accumulation of a N molecule lacking the Dl-binding region (Wesley 1999). This secondary N receptor may be produced during epidermogenesis to eliminate the antagonism to Wingless functions presented by the Dl-binding site. Non-response or pathway-specific response to a second ligand may be necessary for development given the broad overlap in distributions of different N ligands. Thus, expression of a particular secondary N receptor might indicate both the differentiation path taken by a cell and the degree to which this cell has differentiated from cells in the parent population.
The molecular phenotypes of nd3 allele suggest that EGF-like repeat 2 might be an important component in the regulation of NCterm production during embryogenesis. It seems possible that the EGF-like repeat array of N might include two classes of repeats, one containing repeats that bind ligands outside the cells and the other containing repeats that target Notch for different kinds of processing inside the cell. Such a function for EGF-like repeats might explain why Nintra do not produce NCtermTMintra. These molecules might lack the appropriate EGF-like repeats to target them to the right place for carboxyl terminus processing. An interesting extension of this possibility is that there are different targeting EGF-like repeats responsive to different ligands.
The regulation of da expression by NCterm may be significant for embryogenesis. da genetically interacts with Notch (Brand and Campos-Ortega 1988, Brand and Campos-Ortega 1990), it is required for development of the nervous system from neuroblasts but not for lateral inhibition (Caudy et al. 1988a,Caudy et al. 1988b; Vaessin et al. 1994), and the Daughterless protein promotes DNA-binding activities of the proneural Achaete-Scute Complex proteins (Dambly-Chaudiere et al. 1988; Murre et al. 1989; Cabrera and Alonso 1991; van Doren et al. 1991). Both NCterm and Daughterless protein (Vaessin et al. 1994) accumulate in segregating neuroblasts raising the possibility that NCterm is involved in this upregulation of da expression. Accordingly, nd3 embryos which overproduce NCterm also overproduce da RNA in the neuroblasts (data not shown).
In the embryo, da is expressed at low levels in almost all cells (Murre et al. 1989; CSW, personal observation) but is upregulated in certain cells including the segregating neuroblasts (Vaessin et al. 1994). In our experiments, S2 cells expressing NFull and NCterm receptors had lower levels of da RNA than S2 cells without N. In response to Dl, only S2-NCterm cells increased expression of da RNA, but only to the level observed in cells without N (Fig. 6 a). Therefore, it appears possible that with the expression of different forms of N, developing cells acquire an ability to differentially regulate the otherwise constitutive da expression. Such differential regulation might be important for suppressing the activities of Achaete-Scute Complex proteins in the developing epidermis where NFull is expected to function, but not in the developing nervous system where NCterm is expected to function. Since both N receptors have the ability to activate E(spl)C, the timing and sequence of expression of NFull and NCterm may also be important for development.
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Acknowledgments |
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This work was supported by National Institutes of Health GM 25103 to Michael Young.
Submitted: 15 September 1999
Revised: 25 February 2000
Accepted: 17 March 2000
Abbreviations used in this paper: CNS, central nervous system; Dl, Delta; da, daughterless gene; E(spl)C, Enhancer of split Complex genes; N, Notch; Su(H), Suppressor of Hairless protein.
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