Correspondence to: Cedric S. Wesley, Department of Genetics, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Tel:(212) 327-8233 Fax:(212) 327-7420 E-mail:wesleyc{at}rockvax.rockefeller.edu.
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Abstract |
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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 NCterm.
Key Words: Notch, Delta, neurogenesis, daughterless, differentiation
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Introduction |
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Notch (N)1 is required throughout development of Drosophila melanogaster for differentiation of tissues as diverse as the nervous systems, cuticle, internal organs, and muscles (for a review of Notch signaling, see
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
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 (
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In this study, we describe results showing that a truncated form of N lacking the sequence carboxy-terminal of the CDC10/Ankyrin repeats is produced during embryogenesis. This truncated receptor, which would lack the Dishevelled and one of the two Numb-binding sites, can function as a receptor for Dl. Its differential accumulation in interacting cells may play a role in choice of cell fates during lateral inhibition and regulation of activities of different proteins that bind the N intracellular domain.
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Materials and Methods |
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Immunostaining of N Protein in Embryos
NPCR antibody was generated against the intracellular segment of N, amino acids 2,1152,536, between the CDC10/Ankyrin repeats and the OPA repeats (
N203 antibody was generated in rats against a glutathione-S-transferase fusion peptide including N EGF-like repeats 13 (amino acids 59177) following standard procedures (
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 (
Immunostaining procedure described in
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Immunoprecipitations
For immunoprecipitation of N molecules from embryos, ~50100-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 (
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
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-N112155, 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 (
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
Northern Blot Analysis
Embryos.
024-h embryos laid by +/FM7 lac-Z x FM7lac-Z/Y crossed flies and N60g11/FM7 lac-Z flies, reared at 18°C (~012-h staged embryos reared at 25°C) were used for Fig 6 d. 06-h embryos laid by UAS-Nintra1790; hsGal4, UAS-N18932155; 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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Cultured Cells.
Cells were heat-shocked for 30 min at 37°C and allowed to synthesize proteins for 1 or 2 h. The cells were washed 2x in Shields and Sang's M3 media plus antibiotics (M3 medium), and resuspended in M3 medium at a concentration of 107 cells/ml. 0.7 ml of S2, S2-Dl, S2-N, S2-N60g11, S2-N112155, or S2-N2262 cells were mixed with 0.7 ml of S2 cells or S2-Dl cells. The mixtures were transferred to siliconized Falcon multiwell tissue culture plates and gently rotated for 2 h. UAS-Nintra1790, hsGal4, UAS-N18932155, hsGal4, and hsN21552703 cells were heat-shocked for 30 min and allowed to synthesize proteins for 45 min. Total RNAs from embryos and cells were extracted using RNAzol B (Tel-test, Inc.) according to manufacturer's protocol. 20 µg (UAS and yw embryos) or 40 µg of total RNA was loaded in each lane. Standard Northern blot procedures were followed (
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Results |
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An Antibody Made against the Carboxyl Terminus of N Does Not Stain Certain Embryonic Tissues Expressing N
Immunostaining experiments were done with 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, ae). 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 (
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;
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
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
NPCR as these tissues were stained with
N203 (Fig 2 g) and the nervous systemspecific 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,
Cterm 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 (
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 N
Cterm is not recognized by this antibody.
As immunoprecipitations were done with a buffer approximating physiological conditions, it is possible that physiological NCterm 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. N
Cterm was detected by
NI (as expected) but not by
NPCR (Fig 3 b) indicating that non-recovery of N
Cterm 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 N
Cterm lacked the carboxyl terminus sequence. To determine whether N molecules truncated to remove the carboxyl terminus
NPCR epitope region migrate alongside N
Cterm in SDS-PAGE, and to get a rough estimate of how much of the carboxyl terminus region is lost in N
Cterm, the following cell lines were generated: S2-N112155 cells producing N molecules truncated after amino acid 2,155, immediately after the CDC10/Ankyrin repeats, and S2-N112262 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;
Cterm migrates alongside N112155 and N60g11, but faster than N112262 (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, N
Cterm 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 N
Cterm 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 N
Cterm because it lacks the carboxyl terminus (half of the intracellular domain); and the form migrating between NFull and N
Cterm 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-PAGEbased 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
Cterm do not distinguish between the colinear and the proposed heterodimeric forms of the receptors. Therefore, NFull and N
Cterm 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 (
The monoclonal anti-Dl antibody used here (mAb 202,
N immunoprecipitated by anti-Dl antibody from 0- to 3-h embryonic extracts was recognized by NPCR,
NI, and
NT (the last antibody was made against the first two EGF-like repeats,
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 14), 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 03-h embryos, it was expressed at very low levels in the 36-h-old embryos (Fig 4 b, lanes 1 and 2; N350.2 and NCterm are present at similar levels in 36-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 03-h embryos is NFull and the form of N associated with Dl in 36-h embryos is N
Cterm. The form of N associated with Dl in 03-h embryos is unlikely to be N350.2 (which is also recognized by
NPCR) because it is present at equivalent levels in both 03- and 36-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 36-h embryos and the association of Dl with N
Cterm in embryos of the same age are consistent with the observations that a form of N not recognized by
NPCR is enriched in 36-h embryos (Fig 2, ae) and that both N and Dl are required for neurogenesis after lateral inhibition (
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 (
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 36-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 13-h embryos (Fig 4 b, lanes 7 and 8). Furthermore, NFull is required for lateral inhibition (
Cterm-like protein (at 18°C) but have maternally contributed NFull are still deficient in lateral inhibition (
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 (
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 N
CtermTMintra 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 N
CtermTMintra (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-membranetethered Nintra1790 (Fig 5 b). Both Nintra and N
CtermTMintra 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. N
CtermTMintra was not observed in this experiment as its accumulation requires ~2 h. These experiments did not show accumulation of N
Cterm (data not shown).
The ~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 (CtermTMintra is not recognized by
NPCR, just like N
Cterm. Mobility in SDS-PAGE indicates that N
CtermTMintra lacks ~500 amino acids in the carboxyl terminus of the intracellular domain, also like N
Cterm. Expression of Nintra1790 fails to produce N
CtermTMintra (Fig 5 b). Longer expression periods, longer exposure to film, or expression of membrane-tethered Nintra failed to show even a trace of N
CtermTMintra or smaller molecules (not shown). These observations strongly suggest that N
CtermTMintra 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 1,7452,145) would be ~45 kD. The size of ~55 kD for N
CtermTMintra suggests that it contains the transmembrane domain. Therefore, we have tentatively designated it N
CtermTMintra (see Fig 1 b). As the cell surface N molecules are proposed to be a heterodimers of the extracellular domain and the intracellular domain (
CtermTMintra could very well be the intracellular domain of heterodimeric N
Cterm receptor. In all experiments with S2 cells, the N extracellular domain (Nextra) detected by our antibodies (i.e., the ~250300-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 N
Cterm might function as a receptor for Dl. We examined this possibility in cultured cells. S2-N cells express NFull, whereas S2-N60g11, S2-N112155, and S2-N112262 express N
Cterm-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
Cterm-like receptors. Expression of both NFull and N
Cterm-like receptors in S2 cells suppressed da expression (Fig 6 a, lanes 13, 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 N
Cterm-like receptors with Dl promoted accumulation of da RNA, while treatment of NFull did not (Fig 6 a, lanes 25 and 811). Another comparison of NFull and N
Cterm-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 (
Cterm-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-N112155 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 N112155 declines rapidly. The results show that a ~40-kD intracellular molecule, designated N
Ctermintra, accumulates in S2-N60g11 and S2-N112155 cells in response to Dl, and the expected Nintra accumulates in S2-N cells (Fig 6 b, lanes 18; see later for the basis for the name N
Ctermintra). S2-N112262 cells treated with S2-Dl cells do not accumulate the ~40-kD molecule but instead accumulate a 5255-kD molecule (Fig 6 b, lanes 911, see band marked with an asterisk). The 1215-kD size difference between this molecule and N
Ctermintra is approximately the difference between the carboxyl termini of N112155 and N112262. This indicates that N
Ctermintra is produced by a proteolytic cleavage amino-terminal of the CDC10/Ankyrin repeats. The ~40-kD size suggests that N
Ctermintra 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 N
Ctermintra (see Fig 1 b).
NCtermintra is closest in size to the ~35-kD intracellular fragment containing just the CDC10/Ankyrin repeats, N18932155 (data not shown) suggesting that the CDC10/Ankyrin repeats, with little flanking sequence, transduces the signals from N
Cterm. If just the CDC10/Ankyrin repeats fragment is the activated signaling molecule associated with N
Cterm receptor, then N18932155 was expected to promote expression of da in the absence of Dl. We tested this expectation. Results show that N18932155 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-N18932155 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, N21552703, does not suppress da expression as strongly as Nintra1790 (Fig 6 c).
Next, we examined whether NCterm-like receptor, N60g11, and N18932155 increase da expression in vivo. Northern blot analysis of RNA extracted from N60g11 embryos showed that overexpression of the N
Cterm-like receptor results in overproduction of da RNA (Fig 6 d). As observed in S2 cells, expression of N18932155 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.
The differential response of da and the E(spl)C might be due to expression of N60g11 and N18932155 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 (Cterm promoting da expression and not the full-length N (as in S2 cells). Activation of E(spl)C by N
Cterm 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 N
Cterm, 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 (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 N
Cterm 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 N
Cterm (data not shown). The overexpressed form in nd3 embryos (25°C) is N
Cterm 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 N
Cterm 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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A higher percentage SDS-PAGE analysis of extracts prepared from 25°C reared embryos revealed that ~55- and ~40-kD N intracellular fragments, having the same SDS-PAGE migration properties as NCtermTMintra and N
Ctermintra from cultured cells, are also overexpressed in nd3 (25°C) embryos (Fig 7 b). Overexpression of these and N
Cterm molecules in nd3 (25°C) embryos suggest that the processes producing N molecules lacking the carboxyl terminus are interrelated and N
Cterm is the source of N
CtermTMintra and N
Ctermintra. Nintra is not clearly detected in embryonic extracts (see Fig 7 b, lanes 46). This may be because very low amounts of Nintra molecules are sufficient to transduce the Dl-mediated lateral inhibition signal in vivo (
CtermTMintra and N
Ctermintra molecules from among the many minor N molecules generally detected in a N Western blot.
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Discussion |
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Our analysis of N molecules in embryos and S2 cells show the following: (a) whereas the cells undergoing lateral inhibition in the developing embryo are enriched for N molecules recognized by both the amino and carboxyl terminus antibodies, the cells and tissues produced subsequent to lateral inhibition are enriched for N molecules not recognized by the carboxyl terminus antibody (Fig 2). (b) Correspondingly, Dl forms complexes with the full-length N during lateral inhibition period, and with the N molecule lacking the carboxyl terminus in the period after lateral inhibition (Fig 4). (c) N molecules lacking the carboxyl terminus (NCterm, N
CtermTMintra, and N
Ctermintra) are produced during embryogenesis (Fig 3 and Fig 5 Fig 6 Fig 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, N
CtermTMintra (Fig 5). (e) N
Cterm is the most likely substrate for production of N
CtermTMintra (Fig 5 and Fig 7). (f) N
Cterm functions as a receptor for Dl, with the N
Ctermintra (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 N
Cterm become the neuronal precursor cells and cells expressing higher levels of NFull become the epidermal precursor cells. NFull disappears in neuronal precursor cells and N
Cterm, 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 N
Cterm, continue the same process during differentiation of the epidermis. Advance from signaling by NFull to signaling by N
Cterm would mean that those cells have attained a degree of irreversibility in their differentiation process. For example, once N
Cterm 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 (
Ctermintra lacking the Su(H)-binding sites from N
Cterm receptor might promote neuronal fates by promoting activities of Hairless or Numb or Achaete (through Daughterless;
Cterm and not NFull. Thus, production and functions of NFull and N
Cterm might provide directionality to N functions at successive stages of differentiation. All these properties of NFull, N
Cterm, 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 (
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 N
Cterm 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. N
Cterm regulates da, not NFull. Daughterless protein, is an activator of proneural proteins (
Cterm 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 (
Ctermintra 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 (
Cterm 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 N
Cterm 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 Cterm 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 (
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 N
CtermTMintra. 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 (
Cterm and Daughterless protein (
Cterm is involved in this upregulation of da expression. Accordingly, nd3 embryos which overproduce N
Cterm also overproduce da RNA in the neuroblasts (data not shown).
In the embryo, da is expressed at low levels in almost all cells (Cterm receptors had lower levels of da RNA than S2 cells without N. In response to Dl, only S2-N
Cterm 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 N
Cterm 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 N
Cterm may also be important for development.
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Footnotes |
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1 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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Acknowledgements |
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We thank Michael Young for his support of the study; Toby Lieber and Simon Kidd for generous sharing of research materials; and Justin Blau, Umadevi Wesley, and Monica Roth for comments on the manuscript. We are also thankful for the excellent comments and suggestions of two anonymous reviewers.
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
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References |
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