RESULTS

To identify genes that may encode architectural factors that support activation by the remote wing margin enhancer in cut, we screened for mutations that diminish cut expression. Most wing margin cells are lost when the wing margin enhancer is blocked by a gypsy insertion. Intermediate phenotypes with nicks in the wing margin occur when the wing margin enhancer is partially blocked by gypsy. The screens exploited the intermediate phenotype produced by the ct^L-32 gypsy insertion (Figure 1) when partially suppressed by the leaky su(Hw)^e2 mutation (Morcillo et al. 1996, 1997). These flies display ∼0.01 wing margin nicks per fly, indicating that cut gene activity in the wing margin is less than half wild type. We reasoned that partial loss of enhancer-binding proteins or architectural factors that facilitate communication with the promoter should decrease activation by the enhancer and increase the number of wing margin nicks. In the screens for dosage-sensitive modifiers, which are described elsewhere (Morcillo et al. 1996, 1997), mutagenized ct^L-32; su(Hw)^e2 males were mated to ct^L-32; su(Hw)^e2 females, and progeny with two or more wing margin nicks were tested for the presence of heritable mutations.

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Figure 1.

—Lesions affecting the remote wing margin enhancer in the cis regulatory region of the Drosophila cut locus. The 2.7-kb fragment containing the cut wing margin enhancer (hatched box labeled “wm”; Jacket al. 1991) is ∼85 kb upstream of the promoter (angled arrow) in the absence of a gypsy insertion (7.5 kb). The gypsy long terminal repeats (LTRs) are indicated by open boxes, and the Su(Hw) insulator protein-binding region by a filled circle. The extents of the ct^2s and ct^53d deletions are shown underneath by thick lines. ct^2s displays an extreme recessive cut wing phenotype (Mogilaet al. 1992). ct^53d deletes ∼0.5 kb and displays a weak recessive cut wing phenotype (Jack 1985). The ct^L-32 gypsy insertion blocks the wing margin enhancer and displays a strong recessive cut wing phenotype in the presence of wild-type su(Hw).

Most mutations recovered in these screens are recessive lethal. Screening of ∼30,000 progeny (∼8,000 by EMS and ∼22,000 by γ-ray mutagenesis) identified the sd, mam, and Chip genes (Morcilloet al. 1996). An additional screen of ∼220,000 progeny by γ-ray mutagenesis was used to isolate additional Chip alleles (Morcilloet al. 1997). The larger screen also identified several additional mam alleles, three vestigial (vg) mutations, and ∼30 alleles of a complex locus on chromosome 2 that we named Nipped. To understand the functions of Nipped, we characterized its multiple lethal complementation groups and their genetic interactions with cut.

Nipped mutations affect four lethal complementation groups near the chromosome 2 centromere: The abilities of the Nipped mutant chromosomes to enhance the ct^L-32; su(Hw)^e2 cut wing phenotype are tightly linked with recessive lethal mutations. Dominant enhancement of the cut wing phenotype could not be separated from recessive lethality after multiple backcrosses to the parental ct^L-32; su(Hw)^e2 stock. As determined by segregation, several Nipped alleles are also translocations between chromosomes 2 and 3 (Table 1). As described below, where tested, the dominant effects of Nipped mutations on cut expression are mimicked by a deficiency, implying that the Nipped mutations are loss-of-function alleles.

Multiple Nipped mutations were mapped by recombination based on the recessive lethality to a position near the centromere on chromosome 2. Complementation tests with known deficiencies confirmed this location (Figure 2). All Nipped alleles are lethal over Df(2R)M41A10 and Df(2R)M41A8 and viable over Df(2R)A″. This places Nipped in the distal portion of 41A, near the heterochromatin-euchromatin boundary.

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Figure 2.

—Genetic map of the 41A region and the Nipped locus. The chromosome 2 centromere is indicated by an open circle. All Nipped mutations are lethal over Df(2R)M41A10 and Df(2R)M41A8, and viable over the Df(2R)A² deficiency, placing them near the heterochromatin-euchromatin boundary. Nipped-A mutations complement all lethal mutations except l(2)41Ah (Hilliker 1976) and Nipped-C, D, and E mutations. Nipped-B mutations complement all except Nipped-C, D, and E mutations. Nipped-C alleles complement l(2)41Ae and l(2)41Af mutations (Hilliker 1976), and fail to complement Nipped-A [l(2)41Ah] and Nipped-B mutations. Nipped-D alleles complement l(2)41Ae mutations, and fail to complement l(2)41Af, Nipped-A, and Nipped-B mutations. Nipped-E mutations are lethal over l(2)41Ae, l(2)41Af, Nipped-A, and Nipped-B mutations.

Three lethal complementation groups, l(2)41Ae, l(2)41Af, and l(2)41Ah, were previously identified in the portion of 41A containing Nipped (Figure 2; Hilliker 1976). The Nipped mutations were tested for which lethal complementation groups they disrupt by crossing them to each other and to representative alleles of the 41A lethals. Most Nipped mutations are lethal over each other. However, two smaller groups, Nipped-A and Nipped-B, complement each other and produce viable progeny. All other Nipped alleles are mutant for both Nipped-A and Nipped-B. Nipped-B mutations complement all three previously known 41A lethal mutations, and Nipped-A mutations complement all except l(2)41Ah. We conclude that Nipped-B is a unique complementation group and that Nipped-A is identical to l(2)41Ah.

The complementation tests divided several of the Nipped alleles mutant for both Nipped-A and Nipped-B into three classes: Nipped-C, Nipped-D, and Nipped-E. Nipped-C alleles fail to complement Nipped-A and Nipped-B mutations, but complement the l(2)41Af and l(2)41Ae mutations. Nipped-D mutations fail to complement Nipped-A, Nipped-B, and l(2)41Af mutations, but complement the l(2)41Ae mutation. Nipped-E mutations fail to complement all four lethal groups (Figure 2). Table 1 lists all the Nipped-A and Nipped-B mutations isolated in the screens and the Nipped alleles tested for complementation of all four lethal groups.

Only one of the characterized Nipped alleles displays ambiguous complementation behavior. Nipped²⁵ fails to complement all Nipped-B mutations but is lethal over some, but not all, Nipped-A alleles. Nipped²⁵ is lethal over T(2;3)Nipped-A^394.2 and Nipped-A^222.3, semilethal over Nipped-A^34-12, and viable over l(2)41Af ^45-72 and l(2)41Ae^34-14. Because Nipped²⁵ is lethal over more than one Nipped-A allele, we classify it as a Nipped-C allele. Because it may be only weakly mutant for Nipped-A, we avoided use of Nipped²⁵.

Nipped mutant chromosomes do not contain mutations in known wing development genes: It was important to confirm that the Nipped mutant chromosomes do not contain other mutations that affect wing development. Therefore, we tested all Nipped alleles for complementation of mutations in the known wing development genes on chromosome 2, including apterous (ap), vg, wingless (wg), Suppressor of Hairless [Su(H)], mam, and Chip. Only one Nipped mutant chromosome has a second mutation in a known gene. Nipped-A^226.1 is lethal over the Su(H)⁸ and Su(H)² null alleles, and the Su(H)¹⁶ gain-of-function allele. Like gain-of-function Su(H) alleles and unlike Su(H) loss-of-function mutations (Fortini and Artavanis-Tsakonas 1994), Nipped-A^226.1 suppresses the lethality caused by negative complementation between two Abruptex (Ax^e2 and Ax^9B2) alleles of Notch (not shown). Other Nipped mutations do not suppress the Ax negative complementation (not shown), leading us to conclude that the Nipped-A^226.1 chromosome also contains a Su(H) gain-of-function mutation. We therefore avoided use of Nipped-A^226.1 in genetic experiments.

Nipped products are essential during embryonic and larval development: The complementation tests do not distinguish whether the Nipped locus consists of multiple genes or whether it is a single transcription unit that produces multiple products. However, they demonstrate that Nipped has multiple individual functions essential for viability. Nipped is required for viability prior to expression of cut in the wing margin, which begins late in third instar larval development.

Three of the five Nipped-A alleles, including the 2;3 translocation, are lethal at the second to third instar larval transition (Table 1). Nipped-A^222.3/Nipped-E³³⁸ heterozygotes also die at this stage. Two Nipped-A alleles, Nipped-A^357.2 and Nipped-A^226.1 (which also contains a Su(H) mutation), are primarily embryonic lethal, but produce a few larval escapers. The Nipped-A^34-12 allele is embryonic lethal, but this chromosome has been balanced for many years, and it may have acquired other lethals.

All four Nipped-B alleles, including the two translocations, are lethal at the second to third instar molt (Table 1). Two of the three Nipped-C alleles, including the translocation, are lethal at the same stage, as are Nipped-C^160.1/ Nipped-E⁴³ heterozygotes. Because Nipped-C alleles are mutant for both Nipped-A and Nipped-B, this confirms that the second to third instar molt is the primary lethal phase for both Nipped-A and Nipped-B. As shown below for Nipped-B, it is possible that maternally supplied product allows survival to this stage.

All Nipped-D and Nipped-E alleles are homozygous embryonic lethal as are the two Nipped-D/Nipped-E combinations tested (Table 1). Because both Nipped-D and Nipped-E alleles are mutant for l(2)41Af, this indicates that embryogenesis is the lethal phase for the l(2)41Af complementation group. We are uncertain of the l(2)41Ae lethal phase because the only Nipped alleles mutant for l(2)41Ae are the Nipped-E alleles, which are also mutant for l(2)41Af. Although the l(2)41Ae allele is embryonic lethal, this chromosome has been balanced for many years and may have acquired additional lethals.

Nipped mutations magnify the effects of a gypsy transposon insertion in cut: To ascertain the roles of the Nipped locus in regulating cut, heterozygous Nipped alleles were compared for their abilities to alter expression of different mutations in the cis regulatory region of cut (Figure 1; Table 2). The goal was to determine if any of the lethal groups in Nipped specifically magnify insulation by gypsy and Su(Hw). We quantitatively compared the abilities of several Nipped mutations to magnify the partially suppressed ct^L-32; su(Hw)^e2 cut wing phenotype. To ensure accuracy, we avoided the Nipped alleles that have a second mutation in the same chromosome (Nipped-A^226.1), an ambiguous complementation pattern (Nipped-C²⁵), or an atypical lethal phase (Nipped-A^357.2 and Nipped-A^34-12). Because the su(Hw)^e2 mutation could be lost from chromosome 3 during the balancing crosses, we also could not unambiguously test the translocation alleles [T(2;3)Nipped-A^394.2, T(2;3)Nipped-B⁴, T(2;3)Nipped-B^359.1, and T(2;3)Nipped-C^138.2] with the gypsy insertion. These constraints allowed us to compare two of the five Nipped-A alleles (Nipped-A³²³ and Nipped-A^222.3) and two of the four Nipped-B alleles (Nipped-B^292.1 and Nipped-B⁴⁰⁷). Of the Nipped mutations that affect multiple lethal groups, we were able to test one of the three Nipped-C alleles (Nipped-C^160.1), both of the Nipped-D alleles (Nipped-D^341.1 and Nipped-D^263.3), and all three Nipped-E alleles (Nipped-E^299.1, Nipped-E⁴³, and Nipped-E³³⁸).

As expected, all Nipped alleles isolated in the screens dominantly increase the severity of the ct^L-32; su(Hw)^e2 cut wing phenotype (Table 2). However, the two Nipped-B mutations give ∼3- to 12-fold more wing margin nicks (1.2 and 4.8 nicks per fly) than the strongest Nipped-A mutation (0.4 nicks per fly). It is unlikely that the Nipped-A alleles are weaker mutations than the Nipped-B alleles because, as described below, these Nipped-A alleles have stronger effects than the Nipped-B alleles on other cut mutations. The l(2)41Ae^34-14 and l(2)41Af ^45-72 mutations have no detectable effects on the ct^L-32; su(Hw)^e2 phenotype, which explains why mutations disrupting only these lethal groups were not isolated in our screens. None of the Nipped-C, D, and E alleles, which disrupt multiple lethal groups, magnify the ct^L-32; su(Hw)^e2 cut wing phenotype more than the strongest Nipped-B allele. Indeed, only one, Nipped-E^299.1 (1.9 nicks per fly), has a slightly larger effect than the weaker Nipped-B allele (1.2 nicks per fly). We deduce, therefore, that disruption of the Nipped-B lethal group causes most of the magnification of the gypsy insertion phenotype by the Nipped-C, D, and E alleles. The weaker effects of the Nipped-C^160.1 and Nipped-E³³⁸ alleles relative to both Nipped-B mutations suggest that they may not be fully mutant for Nipped-B. Because the Nipped mutations were recently isolated in the same genetic background, do not contain mutations in the known wing development genes, and were crossed to the same ct^L-32; su(Hw)^e2 stock, the differences between the Nipped alleles are unlikely to be genetic background effects.

Nipped-B mutations amplify the effect of a gypsy insertion in Ultrabithorax: To examine the possibility that Nipped mutations may also magnify the effect that gypsy insertions have on other genes, we tested to see if heterozygous Nipped mutations increase the effect of a gypsy insertion in Ultrabithorax (Ubx). The bx^34e gypsy insertion is in a transcribed region (Peifer and Bender 1986), but blocks activation by remote enhancers located in the abx/bx region, ∼50 kb downstream of the Ubx promoter (Simonet al. 1990; Qianet al. 1993). Like gypsy insertions in cut, bx^34e is partially suppressed by the su(Hw)^e2 mutation (Dorsett 1993), providing a sensitive intermediate phenotype made more severe by partial loss of Chip activity (Morcilloet al. 1996).

The effects of Nipped mutations on su(Hw)^e2 bx^34e were compared with their Nipped⁺ siblings. Neither of the Nipped-A mutations tested significantly alters the bithorax phenotype (Table 3; Figure 3). In contrast, both Nipped-B alleles dramatically increase the severity of the mutant phenotype three- to fourfold. Furthermore, the Nipped-C allele, both of the Nipped-D alleles, and two of the three Nipped-E alleles significantly amplify the mutant phenotype. Although the Nipped-E³³⁸ allele has little effect, this Nipped-E allele also has the weakest effect on the gypsy insertion in cut (Table 2). None of the Nipped-C, D, or E alleles is more effective than the Nipped-B alleles, indicating that Nipped-B is responsible for the increased severity of the bithorax phenotype.

We conclude that relative to the other Nipped lethal complementation groups, mutations in Nipped-B more strongly intensify the effects of the gypsy insertions in both cut and Ubx. However, we do not think that Nipped-B regulates expression of su(Hw) or gypsy. As described below, Nipped-B mutants display weak cut wing phenotypes in the absence of gypsy insertions.

Nipped mutations amplify the effects of a deletion in the cut wing margin enhancer: Although Nipped-B has greater effects on the gypsy insertions in cut and Ubx than other Nipped lethal groups, it was feasible that Nipped-B products might simply be more limiting for cut expression than other Nipped products. If so, then Nipped-B mutations should also have stronger effects on other types of cut mutants. To test this we quantitatively compared heterozygous Nipped alleles for their ability to magnify the severity of the partial cut wing phenotype of ct^53d, a 0.5-kb deletion in the wing margin enhancer (Figure 1). Hemizygous ct^53d males display ∼7 nicks per fly in Nipped⁺ backgrounds (Table 2; Figure 4). This partial phenotype presumably results from changes in the quantity or composition of activation complexes that form on the enhancer.

In contrast to their strong effects on the cut gypsy insertion, the Nipped-B^292.1 and Nipped-B⁴⁰⁷ mutations do not magnify the ct^53d mutant phenotype (Table 2). The two Nipped-B translocations also do not magnify the ct^53d mutant phenotype. In contrast, the l(2)41Af ^45-72 mutation and three of four Nipped-A mutations increase the severity of the ct^53d cut wing phenotype (Table 2; Figure 4). The l(2)41Ae^34-14 mutation has little effect on ct^53d (Table 2; Figure 4). These results indicate that opposite to what is observed with the ct^L-32 gypsy insertion, Nipped-A or l(2)41Af products are more limiting than Nipped-B products for cut expression in ct^53d mutants.

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Figure 3.

—Dominant enhancement of the su(Hw)^e2 bx^34e bithorax phenotype by some Nipped mutations. Dorsal views of representative flies with the indicated genotypes are shown. Nipped-A^222.3/+; su(Hw)^e2 bx^34e (left) displays a weak bithorax phenotype indistinguishable from Nipped⁺ controls (not shown). The arrow points to extra dorsal bristles. Nipped-D^341.1/+ (middle) and Nipped-B⁴⁰⁷/+ (right) are strongly enhanced, with increases in thoracic cuticle and bristles between the thorax and abdomen (arrows). See Table 3 for quantitated phenotypes with these and other Nipped alleles.

Comparison of the strongest Nipped-C, D, and E alleles suggests that the effects of the individual Nipped lethal complementation groups on ct^53d are additive and confirms that l(2)41Ae has little or no effect (Table 2; Figure 4). We postulate that the Nipped mutations are hypomorphic and that the alleles with the strongest effects are the most mutant. Confirming this idea, Df(2R)M41A8, which deletes all four lethal groups (Figure 2), has the strongest effect, increasing the number of nicks more than 4-fold (Table 2). T(2;3)Nipped-A^394.2 is the strongest Nipped-A allele, giving a 1.8-fold increase in the number of wing margin nicks over the controls. T(2;3)Nipped-C^138.2, which disrupts both Nipped-A and Nipped-B, increases the number of nicks ∼2.5-fold over the controls. Nipped-D^263.3, which is mutant for l(2)41Af, Nipped-A, and Nipped-B, has a stronger effect, increasing the number of nicks ∼3.5-fold. Nipped-E³³⁸, which is mutant for all four lethal groups, has a similar effect as Nipped-D^263.3, confirming that l(2)41Ae has little or no effect on ct^53d. We conclude, therefore, that the effects of Nipped-A and l(2)41Af mutations on the ct^53d enhancer deletion are additive.

It was possible that the effects of Nipped mutations on ct^53d may depend on the particular sequences deleted from the wing margin enhancer. ct^2s is a larger deletion that removes virtually all of the enhancer (Figure 1, Mogilaet al. 1992). Homozygous ct^2s females display an extreme cut wing phenotype, while heterozygous females have a wild-type phenotype. We previously observed that loss-of-function mutations in sd, which encodes a protein that binds the enhancer (Morcilloet al. 1996), and null alleles of mam, which encodes a protein that binds chromosomes in the vicinity of cut (Bettleret al. 1996), both dominantly enhance heterozygous ct^2s females to produce cut wing phenotypes (Morcilloet al. 1996). Presumably, these phenotypes occur because reducing the amount of enhancer-binding protein reduces activation of cut transcription by the solo wild-type enhancer present in ct^2s heterozygotes.

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Figure 4.

—Dominant enhancement of the ct^53d partial cut wing phenotype by some Nipped mutations. Representative wings from ct^53d males with the indicated genotypes are shown. A control ct^53d wing with a few margin nicks is shown in the upper left (+/+). Little or no effect is observed with Nipped-B⁴⁰⁷ (middle left), while the number of margin nicks is increased with Nipped-A^222.3 (top right), l(2)41Af ^45-72 (middle right), Nipped-D^341.1 (bottom left), and Nipped-E³³⁸ (bottom right). See Table 2 for quantitated phenotypes with these and other Nipped alleles.

All four Nipped-B alleles exhibit weak but significant cut wing phenotypes in the presence of heterozygous ct^2s (0.02-0.09 nicks per fly; Table 2). Importantly, this verifies that Nipped-B regulates cut expression in the absence of a gypsy insertion. However, two of the three heterozygous Nipped-A mutations tested display more severe cut wing phenotypes (0.25 and 0.94 nicks per fly, Table 2) in the presence of heterozygous ct^2s, indicating that Nipped-A is more limiting for cut expression than Nipped-B. Nipped-A³²³ does not show a phenotype with ct^2s, but this allele also has a weaker effect on ct^53d, suggesting that it is a hypomorph. The Nipped-C^160.1 mutation, which has no detectable effect on ct^53d, also displays a weak cut wing phenotype with heterozygous ct^2s (0.08 nicks per fly; Table 2), suggesting that it is weakly mutant for Nipped-A. Surprisingly, T(2;3)Nipped-C^138.2, which has a strong effect on ct^53d, displays a weak cut wing phenotype in combination with ct^2s (0.06 nicks per fly; Table 2). This is the only Nipped allele affecting multiple lethal groups, however, in which the effect on ct^53d does not correlate with the effect on ct^2s. Thus, both Nipped-D and all three Nipped-E alleles magnify the effect of the ct^53d lesion and display strong cut wing phenotypes with heterozygous ct^2s (0.43-0.76 nicks per fly; Table 2). None of the Nipped-D and E alleles has stronger effects on heterozygous ct^2s than the strongest Nipped-A alleles, suggesting that most of the effect is the result of disrupting Nipped-A. Confirming this idea, l(2)41Af ^45-72 has only a weak effect on ct^2s (Table 2). Combined, the results with ct^53d and ct^2s confirm that Nipped-B is more limiting for cut expression than the other Nipped products only when there is a gypsy insulator insertion between the enhancer and promoter.

Nipped mutations do not cause bithorax phenotypes with heterozygous deletions in Ubx: The observation that several Nipped mutations cause cut wing phenotypes in combination with heterozygous ct^2s led us to consider the possibility that some Nipped mutations might also cause bithorax phenotypes in combination with heterozygous deletions in Ubx. However, none of the Nipped mutations tested, including Nipped-A^222.3, Nipped-A³²³, Nipped-B^292.1, Nipped-B⁴⁰⁷, Nipped-D^341.1, and Nipped-E³³⁸, resulted in mutant phenotypes in combination with heterozygous bx^34ePartRev and pbx², both of which cause strong bithorax phenotypes (8 to 10) as homozygotes.

Nipped displays dosage-sensitive interactions with other cut regulators: To further define the roles of the different Nipped lethal groups in regulating cut, we compared dosage-sensitive interactions between Nipped mutations and other wing development mutations. These include mutations in ap, which defines the dorsal-ventral boundary at which the margin will form (Diaz-Benjumea and Cohen 1993; Blairet al. 1994), and Su(H), vg, and wg, which act at the margin prior to and during the time of cut expression (reviewed in Cohen 1996).

None of the Nipped mutations displays a mutant phenotype when combined with heterozygous loss-of-function alleles of Su(H) (Su(H)² and Su(H)⁸ (not shown), or ap or wg (Table 4). In contrast, l(2)41Af^45-72 and certain Nipped-D and Nipped-E alleles exhibit significant mutant phenotypes when transheterozygous with vg¹, with up to ∼1 wing margin nick per fly (Table 4). Nipped-B alleles display little or no mutant phenotype with vg¹, while some Nipped-A and Nipped-C alleles display a weak cut wing phenotype (∼0.1-0.2 nicks per fly). Therefore, l(2)41Af and Nipped-A display dosage-sensitive interactions with vg and Nipped-B does not.

Several Nipped mutations also display dosage-sensitive interactions with mam and Chip mutations. The strongest Nipped-A mutations result in 2.5-3 nicks per fly when transheterozygous with mam^g2.1, and 1-2 nicks per fly when transheterozygous with Chip^e5.5 (Table 4). In contrast, Nipped-B mutations display fewer nicks, from 0.2 to 0.5 nicks per fly with mam^g2.1, and <0.2 nicks per fly with Chip^e5.5. Nipped-C mutations, which are mutant for both Nipped-A and Nipped-B, display interactions with mam and Chip mutations similar to those displayed by Nipped-A mutations. Nipped-D and Nipped-E mutations have stronger interactions than the Nipped-A mutations, showing 3.5 to nearly 6 nicks per fly with heterozygous mam^g2.1, and 2 to nearly 7 nicks per fly with heterozygous Chip^e5.5 (Table 4). The stronger effects of Nipped-D and Nipped-E mutations are likely the result of disrupting both Nipped-A and l(2)41Af. The l(2)41Af ^45-72 mutation displays ∼1 nick per fly with mam^g2.1 and ∼0.4 nicks per fly with Chip^e5.5.

The dosage-sensitive interactions between Nipped mutations and the vg, mam, and Chip mutations have noteworthy parallels in the interactions between Nipped mutations and the ct^53d enhancer deletion. In both cases, l(2)41Af and Nipped-A show stronger interactions than Nipped-B mutations, and the effects of Nipped-A and l(2)41Af are additive. The cut wing phenotypes exhibited by flies heterozygous for both Nipped and mam mutations, or by Nipped and Chip transheterozygotes, are similar in strength to those displayed by flies transheterozygous for Chip and mam, Chip, and sd, or sd and mam mutations (Morcilloet al. 1996). These results indicate that l(2)41Af and Nipped-A cooperate closely with Chip, mam, and vg to regulate cut expression in the wing margin, and further confirm that Nipped-B plays a unique role.

Nipped mutations modify Notch mutant phenotypes: The genes that display dosage-sensitive interactions with Nipped function downstream of Notch in the wing margin, leading us to consider the possibility that Nipped may function widely in Notch receptor signaling. We therefore tested the ability of Nipped mutations to modify the phenotypes displayed by various Notch receptor mutants.

The hypomorphic nd¹ mutation alters the intracellular domain of the Notch receptor (Xuet al. 1990) and displays a wing margin phenotype similar to that displayed by ct^53d, with a few nicks per wing (Figure 5). Heterozygous Nipped-A^222.3 (Figure 5) and Nipped-A³²³ (not shown) enhance this phenotype to give several more nicks. Nipped-B⁴⁰⁷ has no effect (Figure 5), while Nipped-B^292.1 may slightly reduce the number of nicks (not shown). Strikingly, heterozygous Nipped-D^341.1 and Nipped-E³³⁸ strongly enhance the nd¹ phenotype to give strap-like wings (Figure 5). The strong effect of Nipped-D and Nipped-E mutations is likely the result of disrupting l(2)41Af because l(2)41Af ^45-72 exhibits a similar phenotype, while l(2)41Ae^34-14 has no effect (Figure 5). Because loss of cut expression does not cause loss of wing blade, we conclude that l(2)41Af has functions in wing development beyond regulation of cut.

The ability of heterozygous l(2)41Af mutations to give strap-like wings with nd¹ is similar to the effects of heterozygous vg (Rabinow and Birchler 1990; Figure 5), Chip^e5.5 (Figure 5), and Su(H) gain-of-function mutations (Fortini and Artavanis-Tsakonas 1994; Figure 5). Chip protein interacts with Apterous protein and is required for Apterous activity in the wing (Morcilloet al. 1997; Milanet al. 1998; Shoreshet al. 1998; P. Morcillo and D. Dorsett, unpublished results). Apterous activity is required for expression of Serrate protein (Diaz-Benjumea and Cohen 1995), which serves as the dorsal ligand for Notch at the wing margin (Cousoet al. 1995; Diaz-Benjumea and Cohen 1995; Kimet al. 1995; De Celiset al. 1996; Neumann and Cohen 1996). However, heterozygous ap^56f, in contrast to Chip^e5.5, has no detectable effect on the nd¹ phenotype (Figure 5), indicating that Chip has roles in wing development beyond mediating Apterous activity and regulation of cut.

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Figure 5.

—Dominant effects of Nipped mutations on the nd¹ Notch mutant phenotype. Representative wings from nd¹ males with the indicated genotypes are shown. A control nd¹ wing with margin nicks is shown on the left in the top row (+/+). Little or no effect is observed with l(2)41Ae^34-14 (third row, left), ap^56f (bottom left), and the Su(H)⁸ loss-of-function allele (bottom row, middle). Slight suppression of the mutant phenotype is observed with Nipped-B⁴⁰⁷ (top right). Enhancement of the mutant phenotype is observed with Nipped-A^222.3 (top center), Nipped-D ^341.1 (second row, left), Nipped-E³³⁸ (second row, center), l(2)41Af ^45-72, Chip^e5.5 (third row, center), vg¹ (third row, right; Rabinow and Birchler 1990), and the Su(H)¹⁶ gain-of-function allele (bottom right; Fortini and Artavanis-Tsakonas 1994).

Nipped mutations also modify the vein-shortening phenotype of the Ax^E2 gain-of-function allele of Notch, which encodes a Notch receptor with a lesion in epidermal growth factor (EGF)-like repeat 29 in the extracellular portion (Hartleyet al. 1987; Kelleyet al. 1987). Heterozygous Nipped-A³²³ (not shown) and Nipped-A^222.3 (Figure 6) weakly and moderately suppress the vein phenotype, while Nipped-B⁴⁰⁷ (Figure 6) and Nipped-B^292.1 (not shown) have no detectable effect. Nipped-D^341.1 (Figure 6), Nipped-E⁴³ (not shown), and Nipped-E³³⁸ (Figure 6) strongly suppress the vein-shortening phenotype. The strong suppression is presumably the result of disrupting l(2)41Af because l(2)41Af ^45-72 strongly suppresses the Ax^E2 mutant phenotype, while l(2)41Ae^34-14 has no effect (Figure 6). The effects of Nipped-A and l(2)41Af mutations on Ax^E2 are similar to those of Su(H) gain-of-function mutations (Figure 6). vg (Rabinow and Birchler 1990) and Chip mutations have no visible effects on the Ax^E2 vein phenotype (Figure 6).

The phenotypes displayed by the split (spl) allele of Notch, which encodes an amino acid substitution in EGF-like repeat number 14 (Hartleyet al. 1987; Kelleyet al. 1987), are also affected by Nipped mutations. spl displays small rough eyes, occasional twinned bristles, and missing bristles (Figures 7 and 8). l(2)41Ae^34-14 has no detectable effects on the spl eye phenotype (Figure 7). T(2;3)Nipped-A^394.2 (not shown) and Nipped-A^222.3 (Figure 7) also have no detectable effect on the rough eye phenotype, while Nipped-A³²³ slightly suppresses (not shown), making the eyes slightly larger and less rough. Nipped-B ⁴⁰⁷ (not shown), Nipped-B^292.1, and l(2)41Af ^45-72 also suppress the rough eye phenotype (Figure 7). Nipped-E³³⁸, Nipped-D^263.3 (not shown), and Nipped-D^341.1, which disrupt both Nipped-B and l(2)41Af, have similar effects to the Nipped-B alleles (Figure 7). The Nipped-A and Nipped-B alleles have little or no effect on the thoracic bristle phenotypes of spl (not shown and Figure 8), but Nipped-D ^263.3 (not shown), Nipped-D^341.1 (Figure 8), Nipped-E⁴³ (not shown), and Nipped-E³³⁸ (Figure 8), all of which disrupt l(2)41Af, make the thoracic bristles sparser, thinner, and shorter. Because the l(2)41Af ^45-72 chromosome contains a Pin mutation that affects bristle morphology, we could not evaluate the effect of this allele on bristles. We conclude that Nipped-B and l(2)41Af mutations suppress the spl eye phenotype, and infer that l(2)41Af mutations enhance the thoracic bristle phenotype of the spl allele of Notch.

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Figure 6.

—Dominant suppression of the Ax^E2 Notch mutant phenotype by some Nipped mutations. Representative wings from Ax^E2 males with the indicated mutations are shown. An Ax^E2 control wing with shortened veins is shown on the left in the top row (+/+). Little or no effect is observed with Nipped-B⁴⁰⁷ (top row, right), l(2)41Ae^34-14 (third row, left), Chip^e5.5 (bottom row, left), and vg¹ (bottom row, center; Rabinow and Birchler 1990). Lengthening of the veins (suppression) is observed with Nipped-A ^222.3 (top row, center), Nipped-D ^341.1 (second row, left), Nipped-E³³⁸ (second row, center), l(2)41Af ^45-72 (second row, right), and the Su(H)¹⁶ gain-of-function mutation (third row, right). Shortening of the veins (enhancement) is observed with the Su(H)² loss-of-function allele (third row, center).

The dominant effects of Nipped mutations on the nd¹, Ax^E2, and spl Notch mutant phenotypes indicate that l(2)41Af influences Notch receptor signaling, or Notch expression during development of the wing margin, wing veins, eye, and thoracic bristles. Nipped-A influences Notch phenotypes in both the wing veins and margin, while Nipped-B has influences primarily in the eye. The effects of l(2)41Af mutations in the wing margin, wing vein, and eye are similar to those gain-of-function mutations in Su(H). Similarly, the effects of Nipped-A mutations in the wing veins and wing margin, and the effect of Nipped-B mutations in the eye also mimic the effects of Su(H) gain-of-function mutations. Su(H) protein is a direct mediator of Notch signaling (Fortini and Artavanis-Tsakonas 1994) and a direct activator of vg (Kimet al. 1996a). We postulate that l(2)41Af mutations, Nipped-A mutations, and Su(H) gain-of-function mutant proteins downregulate Notch signaling in the wing margin. Su(H) gain-of-function mutant proteins may recruit transcription repressors, similar to the wild-type mammalian homologues of Su(H) (Kaoet al. 1998; Taniguchiet al. 1998).

Nipped-B protein is homologous to chromosomal adherins, which have cohesin-like activities: The in vivo observations indicate that the role of Nipped-B in regulation of cut and in Notch signaling differs significantly from those of the other Nipped lethal groups. Most importantly, Nipped-B is particularly antagonistic to the insulator activity of Su(Hw). We hypothesized, therefore, that Nipped-B products may participate in enhancer-promoter communication in cut and Ubx. To explore this idea we cloned and sequenced Nipped-B cDNAs.

Complementation tests revealed that the l(2)02047 P-element insertion (Berkeley Drosophila Genome Project) is allelic to Nipped-B. l(2)02047 flies are lethal over all Nipped-B alleles, and viable over l(2)41Ae^34-14, l(2)41Af ^45-72, and Nipped-A mutations. Nine of 13 induced excisions of the P element reverted the Nipped-B mutation in l(2)02047, confirming that the P insertion is the Nipped-B mutation.

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Figure 7.

—Dominant effects of some Nipped mutations on the spl Notch mutant eye phenotype. Representative eyes of spl mutant males with the indicated mutations are shown. All photographs are at the same magnification. In a wild-type background (+/+), spl mutant eyes are small and rough (top row, left). Little or no effect is observed with Nipped-A^222.3 (top row, middle), l(2)41Ae^34-14 (third row, left), Chip^e5.5 (third row, center), vg¹ (third row, right), and the Su(H)⁸ loss-of-function allele (bottom left). Larger, less rough eyes are observed with Nipped-B^292.1 (top right), Nipped-D^341.1 (second row, left), Nipped-E ³³⁸ (second row, center), l(2)41Af ^45-72 (second row, right), and the Su(H)¹⁶ gain-of-function allele (bottom row, center).

We rescued the P insertion from genomic DNA and cloned a 13-kb genomic region surrounding the insertion site (Figure 9A). In this region, only a 2.5-kb EcoRI-XbaI fragment located ∼4 kb from the insertion site (Figure 9A) detects transcripts in Northern blots. This probe hybridizes to 7- and 4-kb transcripts oriented toward the P-insertion site (Figure 9, B and C). The 7-kb transcript is undetectable in homozygous l(2)02047 second instar larvae, while the relative levels of the 4-kb transcript appear to increase (Figure 9B). The 7-kb transcript is reduced in size in homozygous T(2;3)Nipped-B^359.1 second instar larvae, while the 4-kb transcript is unaffected (Figure 9B). The 7-kb transcript is not affected by two Nipped-A mutants, but the levels are reduced to ∼50 and 30% wild-type levels in homozygous Nipped-C^160.1 and Nipped-B⁴⁰⁷ mutants (Figure 9B). Alterations in the size or reductions in the levels of the 7-kb transcript in multiple Nipped-B mutants demonstrate that the 7-kb transcript is a Nipped-B mRNA. These results also confirm that Nipped-B⁴⁰⁷ is a hypomorph. Nipped-B mRNA is expressed at all stages of development, but the highest levels are present in newly laid embryos, indicating that it is maternally loaded (Figure 9C).

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Figure 8.

—Dominant effects of some Nipped mutations on the spl Notch mutant bristle phenotype. Dorsal views of representative thoraces from spl mutant males with the indicated mutations are shown. In a wild-type background (+/+, top left), spl displays missing and occasional twinned bristles. Little or no effect is observed with Nipped-A^222.3 (top middle), Nipped-B ^292.1 (top right), Chip^e5.5 (middle row, right), vg¹ (bottom left), the Su(H)⁸ loss-of-function allele (bottom center), and the Su(H)¹⁶ gain-of-function (bottom right). Loss of scutellar bristles and shorter, thinner bristles are observed with Nipped-D^341.1 (middle row, left) and Nipped-E³³⁸ (middle row, center).

The probe detecting the Nipped-B mRNA was used to isolate overlapping cDNA clones from an imaginal disc library. The probe sequence hybridizes near the middle of the cDNAs (Figure 9A). One clone (6-1) contains a long ORF starting at the 5′ end, while an overlapping clone (3-1) contains the putative initiation codon. Combined, the clones give a complete ORF of 6159 nucleotides, encoding a protein of 2053 amino acids. Because the 2.5-kb EcoRI-XbaI genomic probe fragment is the only fragment in the cloned genomic region that hybridizes to the cDNA, and because it only hybridizes to a fragment near the middle of the cDNA, we can deduce that the 5′ end of the cDNA comes from an uncloned genomic region on one side of the P-insertion site in l(2)02047 and that the 3′ end of the cDNA comes from an uncloned region on the other side of the P-insertion site. Thus, we conclude that the P-insertion site is in an intron.

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Figure 9.

—The Nipped-B⁰²⁰⁴⁷ P-element insertion [l(2)02047] and Nipped-B transcript. (A) Restriction maps of the l(2)02047 P-insertion site and Nipped-B cDNA. As described in the text, excisions of the P{RZ} element revert the Nipped-B mutation. At the top is a restriction map of the 13-kb genomic region surrounding the P-insertion site (E, EcoRI; X, XbaI; Sp, SpeI; S, SalI; B, BamHI; Sc, SacI; P, PstI). The 2.5-kb EcoRI-XbaI genomic fragment, marked with an arrow indicating the direction of transcription, is the only fragment in the 13-kb region that detects transcripts (7 and 4 kb) in Northern blots. None of the other fragments in the 13-kb genomic region hybridizes to transcripts. The EcoRI-XbaI genomic fragment was therefore used as a probe to isolate cDNAs. As indicated by the dashed line, the SalI-EcoRI fragment in the cDNA, marked with an arrow showing the direction of transcription, is the only fragment in the cDNA that hybridizes to the EcoRI-XbaI genomic fragment. Because the genomic DNA has not been sequenced, the extent of sequence overlap between the EcoRI-XbaI genomic fragment and the SalI-EcoRI cDNA fragment is unknown, but the EcoRI site at the upstream end of the EcoRI-XbaI genomic fragment is not the same EcoRI site present at the downstream end of the SalI-EcoRI cDNA fragment. Indeed, none of the restriction sites shown for the cDNA are present in the 13-kb cloned genomic DNA. Because the EcoRI-XbaI genomic fragment is the only genomic fragment that hybridizes to the cDNA, and because it only hybridizes to the SalI-EcoRI fragment in the middle of the cDNA, it is clear that the 5′ end of the cDNA comes from an uncloned genomic region to the left of the P-insertion site, and that the 3′ end of the cDNA comes from an uncloned genomic position to the right of the P-insertion site. Therefore, we conclude that the P-insertion site is in an intron. The filled region of the cDNA indicates the ORF. (B) Northern blots of total RNA isolated from second (L2) and third (L3) instar larvae. The blots were hybridized with antisense [³²P]RNA probe prepared from the 2.5-kb genomic EcoRI-XbaI fragment (top) and then stripped and probed with antisense rp49 probe as a loading control (bottom). On the left, the lanes contained 10 μg of RNA from wild-type second and third instar larvae (WT), homozygous l(2)024047 second instar larvae (Nip-B ⁰²⁰⁴⁷), and heterozygous l(2)02047 third instar larvae (Nip-B⁰²⁰⁴⁷/+). On the right, the lanes contained 10 μg of RNA from wild-type second instar larvae and second instar larvae homozygous for the indicated Nipped alleles: Nip-C ^160.1, Nip-A^222.3, Nip-A³²³, T(2;3)Nip-B^359.1, and Nip-B⁴⁰⁷. The 7-kb transcript is absent or altered in size in Nip-B⁰²⁰⁴⁷ mutants and reduced in size in T(2;3)Nip-B Nipped-B mRNA. Probes were as described above. The lanes contained 10 μg of total cellular RNA isolated from wild-type flies at different developmental stages: EE, 0 to 30 min after egg laying; LE, 30 min to 16 hr after egg laying; L1-L3, first to third instar larvae; P1-P4, first to fourth day of pupation; M, 0- to 1-day-old adult males; F, 0- to 1-day-old females.

Database searches reveal homologues of the Nipped-B protein in fungi, worms, and mammals. Only short expressed-sequence tags (ESTs) of Caenorhabditis elegans, mouse, and humans were identified. The human ESTs are from a variety of tissue-specific libraries, suggesting that the human homologues are widely expressed. The combined human ESTs, which do not represent a complete sequence, encode 411 amino acids. Residues 2-232 of the partial human protein overlap Nipped-B residues 1744-1994 with 34% identity and 52% similarity. In order of decreasing homology, the 2157-amino acid Rad9 protein of Coprinus cinereus, the 1583-amino acid Mis4 protein of Schizosaccharomyces pombe, and the 1493-amino acid Scc2 protein of Saccharomyces cerevisiae, are more distantly related. Rad9 residues 669-2071 display 21% identity and 41% similarity to Nipped-B residues 576-1887, Mis4 residues 780-1492 have 19% identity and 41% similarity to Nipped-B residues 1110-1818, and Scc2 residues 697-1291 display 19% identity with 39% similarity to Nipped-B residues 1103-1704. The fungal homologues show similar levels of homology between themselves, but it is evident that there is a large conserved domain among all the proteins (Figure 10).

Consistent with the idea that Nipped-B plays an architectural role in enhancer-promoter communication, the fungal homologues of Nipped-B all participate in regulating chromosome structure, with roles in DNA repair (Valentineet al. 1995; Furuyaet al. 1998), meiotic chromosome condensation (Seitzet al. 1996), or sister chromatid cohesion (Michaeliset al. 1997; Furuyaet al. 1998). It has been proposed that these three fungal proteins define a new class of chromosomal proteins and have been named adherins to distinguish them from the cohesins that have similar functions (Furuyaet al. 1998).

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Figure 10.

—The Nipped-B protein shares a central core of homology with fungal chromosomal adherins. The proteins are diagrammed with the regions of homology in gray and the lengths of the protein on the right. Comparisons were made using the Blastp program (www.ncbi.nlm.nih.gov/gorf/wblast2.cgi/; Altschulet al. 1997) with the BLOSUM62 matrix, a gap open penalty of 11, a gap extension penalty of 1, gap x_dropoff of 50, expect value of 10.0, a wordsize of 3, and no filtering. Nipped-B protein is compared with the Coprinus cinereus (C.c.) Rad9 protein, which is compared with the Schizosaccharomyces pombe (S.p.) Mis4 protein, which is compared with the Saccharomyces cerevisiae (S.c.) Scc2 protein. The numbers near the ends of the lines that connect the homologous regions indicate the amino acid endpoints of the regions of homology detected by the alignment program, and the numbers between the proteins indicate the percentage of identical residues in the homologous regions and the percentage of residues that are either identical or similar (positives).