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Genetics, Vol. 170, 1775-1795, August 2005, Copyright © 2005
doi:10.1534/genetics.105.043125
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* The Burnham Institute, La Jolla, California 92037
Neuroscience Program, University of Michigan, Ann Arbor, Michigan 48109
Biological and Health Sciences Division, University of Pittsburgh, Bradford, Pennsylvania 16701
1 Corresponding author: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.
E-mail: rolf{at}burnham.org
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The Drosophila melanogaster gene cut is a neural selector gene, which establishes the developmental program directing external sensory (ES) organ identity. The peripheral nervous system is composed of diverse types of sensory organs, including ES organs (cuticular mechanosensory and chemosensory sensilla) and chordotonal organs (subcuticular proprioceptive organs). Although morphologically dissimilar, these sensory organs share molecular and developmental similarities suggestive of a common evolutionary origin (reviewed in LAI and ORGOGOZO 2004). During embryonic and pupal development Cut is specifically expressed in the neuroepithelial-derived sensory organ precursor cells (SOP cells) from which emerge the lineage-related cells of individual ES organs (BLOCHLINGER et al. 1990). Loss of cut function results in the morphological and molecular transformation of ES organs into chordotonal organs (BODMER et al. 1987; MERRITT 1997). Conversely, the ubiquitous misexpression of Cut transforms chordotonal organs into ES organs (BLOCHLINGER et al. 1991). The overexpression of Cut directs ES organ identity only in cells predetermined with proneural character (i.e., ES and chordotonal organ SOP cells) and acts in concert with factors common to these early precursor cells to direct ES organ identity. Thus cut represents a neural selector gene, the presence or absence of which is sufficient to direct alternative sensory organ fates. It is not known what downstream targets or realizator genes Cut regulates to instruct ES organ identity. The only putative Cut transcriptional target implicated in sensory organ specification is the gene bereft (bft), which is required for bristle morphogenesis (HARDIMAN et al. 2002).
cut is involved in the development of several embryonic and adult tissues, including the Malpighian tubules (LIU et al. 1991; LIU and JACK 1992), posterior spiracles (HU and CASTELLI-GAIR 1999), egg chamber (JACKSON and BLOCHLINGER 1997; JACKSON and BERG 1999), flight muscles (SUDARSAN et al. 2001), and wing margin (MICCHELLI et al. 1997). Additionally, the level of Cut expression regulates the degree of dendritic branching in a subset of multiple dendritic neurons (GRUEBER et al. 2003). It is not clear if cut acts as a selector gene in the development of these tissues (LIU and JACK 1992; HU and CASTELLI-GAIR 1999). In the developing wing, cut is required for proper patterning of the wing margin via complex interactions with multiple signaling pathways, including the Wingless (Wg) and Notch pathways (MICCHELLI et al. 1997). The absence of Cut activity leads to the nonautonomous degeneration of wing tissue, producing the classical "cut" wing phenotype (JACK et al. 1991). Degeneration of margin cells prefigures the development of several rows of ES organs arrayed along the anterior wing margin. It is conceivable that Cut is required early to convey a survival signal, most likely via the maintenance of Wg expression (JOHNSTON and SANDERS 2003), in addition to a later role in the specification of margin ES organ identity.
cut encodes a highly conserved homeodomain transcription factor with three novel DNA-binding domains, termed CUT repeats (BLOCHLINGER et al. 1988; ANDRES et al. 1994; MOON et al. 2000). Vertebrate cut homologs, including mouse Cux1 and Cux2 (VALARCHE et al. 1993; QUAGGIN et al. 1996; ZIMMER et al. 2004) and human CDP (NEUFELD et al. 1992), are postulated to regulate cell growth and terminal differentiation. In diverse systems, Cut homologs functionally interact with the regulatory regions of developmentally active genes, including human histone H4 (VAN WIJNEN et al. 1996; GUPTA et al. 2003), lactoferrin (KHANNA-GUPTA et al. 1997, 2003), myeloid cytochrome heavy chain (gp91-phox; SKALNIK et al. 1991; LIEVENS et al. 1995), and DNA polymerase 
(TRUSCOTT et al. 2003), as well as mouse N-CAM (VALARCHE et al. 1993) and immunoglobulin heavy chain (IgH) (WANG et al. 1999), among others. The targeted disruption of murine Cux1 disrupts normal growth control and dermal tissue development (ELLIS et al. 2001; SINCLAIR et al. 2001; LUONG et al. 2002). Constitutive overexpression of Cux1 results in multiple organ hyperplasia, a phenotype partially attributable to the downregulation of the cyclin kinase inhibitor p27kip1 (LEDFORD et al. 2002). Consistent with these results, the DNA-binding ability of CDP/Cux1 is post-translationally regulated in a cell-cycle-dependent manner (COQUERET et al. 1998; MOON et al. 2001; SANTAGUIDA et al. 2001; GOULET et al. 2004), suggesting that CDP/Cux1 may act as part of a transcriptional network controlling the G1/S phase transition. Less is known about the function of murine Cux2. However, the dynamic expression pattern of Cux2 mRNA in the central nervous system, particularly in the subventricular zone and upper cortical layers of the developing telencephalon, suggests that Cux2 regulates a pool of cycling precursor cells predetermined to generate upper-layer cortical neurons (ZIMMER et al. 2004). Interestingly, murine Cux1 and human CDP have been shown to functionally substitute for Drosophila Cut during embryonic development (LUDLOW et al. 1996; GRUEBER et al. 2003), signifying a high degree of structural and functional conservation. It is not clear, however, if mammalian CDP/Cux1 genes act as selector genes in their native developmental context.
To identify genes that interact with Drosophila cut, we conducted complementary gain-of-function and loss-of-function genetic suppressor screens. For this purpose, we created >2000 new Drosophila lines, each carrying a unique insertion of the modular UAS/GAL4-based Gene Search (GS) vector (TOBA et al. 1999). The GS vector contains bidirectional upstream activating sequences (UAS), which bind the transcriptional activator Gal4. Under the control of wing-margin-specific Gal4 expression, genes located near the GS vector insertion site were misexpressed and scored according to their ability to suppress the adult cut wing phenotype. Additionally, 158 deficiency chromosomes (second and third chromosomes) covering 
50% of the genome were tested for the ability to dominantly suppress the cut allele ctK. Of the genes that were identified through these screens, the BTB-domain zinc-finger gene longitudinals lacking (lola) and several genes encoding subunits of the Brahma chromatin-remodeling complexes were investigated further with regard to their interaction with cut during wing-margin development. The genetic interactions between these genes and cut suggest that they are involved in modulating the level of Cut expression and thus act together with Cut to pattern wing-margin tissues.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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500 bp and 1.6 kb, respectively, of the cut wing-margin enhancer, which is positioned –80 kb upstream of the first exon (LIU et al. 1991; MOGILA et al. 1992). The ctK and ct6 alleles result from insertions of the gypsy retrotransposon between the wing enhancer and the first exon. The ctK insertion is located –6 kb upstream of the 5'-most exon (LIU et al. 1991), whereas the ct6 insertion is located proximal to the wing enhancer (DORSETT 1993). Descriptions of the other gene mutations and deficiency chromosomes can be found on FlyBase (http://flybase.boi.indiana.edu). The C96-Gal4 driver line carries a P{GawB} insertion at the 70D locus near the C96 gene; a gene required for viability and imaginal disc development (GUSTAFSON and BOULIANNE 1996; KIM and BOULIANNE 1998). Flies homozygous for the P{GawB}C96 insertion are homozygous viable and wing development is normal. The P{GawB}C96 insertion directs Gal4 expression in a broad stripe straddling the dorsoventral boundary of the wing imaginal disc, which corresponds to the anlage of the adult wing margin and the cells expressing Cut. P{GawB}C96-driven Gal4 expression is unaffected by hypomorphic cut mutations.
Deficiency screen:
Prior to initiating the deficiency screen, the ctK stock was isogenized for the second and third chromosomes by first crossing females from a wild-type Oregon-R stock to males from the double balancer stock ctK/Y; Pin/CyO; D/TM6B. Individual F1 +/ctK; +/CyO; +/TM6B females were backcrossed to ctK; Pin/CyO; D/TM6B males. Stable isogenic ctK; +/+; +/+ stocks were maintained by crossing balanced F2 ctK; +/CyO; +/TM6B siblings derived from individual F1 females. The ctK stock was again isogenized after the initial screen and interacting deficiencies were rechecked. The use of two different isogenic stocks controlled for any phenotypes caused by differences in genetic background. To test for genetic interactions, female flies homozygous for ctK were crossed to male flies containing the deficiency chromosome (Df) over a marked balancer. The wings of male progeny that were ctK/Y; Df/+ were examined for a decrease in the penetrance of the ctK phenotype (see below).
Generation and screening of GS vector insertion lines:
The GS vector as described by TOBA et al. (1999) contains two copies of the sequence UAS (originating form Saccharomyces cerevisiae) adjacent to a core promoter. UAS/core promoter sites are proximal to the terminal inverted repeat sequences located at either end of the P-element vector and oriented as to mediate transcription outward in both directions. Independent GS vector insertion lines were generated by mobilizing the GS vector, located on a CyO chromosome, with 
2-3 transposase (ROBERTSON et al. 1988). The reinsertion of the mobilized GS vector was identified via the expression of the mini-white gene. Independent reinsertion events in the second and third chromosomes were balanced with SM5-TM6, a reciprocal translocation balancer. Stable stocks were maintained over the SM5-TM6 balancer or, if possible, in a homozygous state.
To identify genetic loci that suppress the ctK wing phenotype, three male flies from each of 2066 individual GS lines, with insertions on the second or third chromosomes, were crossed to six females of the genotype w,ctK;C96-Gal4. The penetrance of the cut wing phenotype of male progeny of the genotype ctK/Y;GS*/C96-Gal4 was compared to ctK/Y;C96-Gal4/UAS-lacZ and ctK/Y;C96-Gal4/+ controls. The controls showed a completely penetrant (>99%) ctK wing phenotype, as demonstrated by numerous discontinuities (i.e., gaps) in the regular array of wing-margin sensory bristles and frequent incisions of wing tissue. The expressivity of the ctK wing phenotype was identical for both controls. In the initial testing of all 2066 GS lines and the 158 deficiencies, 20–40 males of the genotypes ctK;GS*/C96-Gal4 or ctK;Df/+ were examined. In addition, females were examined for dominant effects. Due to the prevalence of dominant effects that enhanced the ctK phenotype resulting from the overexpression of the various GS insertions, we opted not to characterize these lines further. Those lines (GS or deficiency) that were found to suppress the ctK phenotype were retested. GS lines were retested by crossing to both ctK; C96-Gal4 and ctK alone to determine if suppression resulted from overexpression or from gene disruption. In all cases, >100 ctK males were screened in the retests. Identical but independent crosses produced similar results in the majority of cases. To confirm the interaction with cut, GS lines were secondarily tested for the ability to interact with ct53d; C96-Gal4. The presence of discontinuities in the anterior wing margin of ct53d/Y;C96-Gal4/UAS-lacZ and ct53d/Y;C96-Gal4/+ controls were less penetrant (50–60%) than with ctK, allowing increases (enhancement) or decreases (suppression) in penetrance to be scored. To maintain consistency, all crosses were performed with cut alleles in the presence of the P{GawB}C96 insertion. The presence of P{GawB}C96 did not influence the interaction between any of the loss-of-function mutations and cut.
Individual GS insertion lines were scored according to their ability to suppress or enhance the penetrance of the cut wing phenotype. For our purposes, only a discrete region of the anterior wing margin, consisting of the region stretching from the proximal-most point of the anterior wing margin to the distally located intersection of the L1 and L2 wing veins (see Figure 1C), was scored. This region was chosen because it encompasses most of the triple row of innervated sensory bristles and can be easily examined in anesthetized intact animals with their wings tucked back in the resting position. In cases in which discontinuities were not observed within this region of individual experimental wings, the cut wing phenotype was considered suppressed. Conversely, in cases in which discontinuities were observed, regardless of the number of bristles affected, the cut wing phenotype was considered not suppressed. The scoring system is based on "all or none" suppression and therefore does not take into account individual variation in either the frequency or the severity of margin bristle loss of individual wings. The degree of penetrance correlated well with the degree of severity. In Table 2, "suppression of ctK" was calculated for each genotype by dividing the number of wings suppressed by the total number of wings scored. The ability of deficiency chromosomes to affect ctK and ct53d was scored in a similar manner. To quantify phenotypes, dissected wings of each genotype were dehydrated in absolute ethanol, mounted in Canada Balsam:methyl-salicylate (1:3), and photographed at 10x magnification using a digital camera (Canon Power Shot S45) mounted on a compound microscope (Zeiss, Axioplan). For each genotype, a representative image of a median wing phenotype was selected from a photographic series.
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In situ hybridization, immunohistochemistry, and X-Gal staining:
In situ hybridization to third instar wing discs was performed as described by STURTEVANT et al. (1993) using digoxigenin (DIG)-labeled RNA probes and visualized using alkaline phosphatase conjugated -DIG antibody (Roche; 1:200). To generate lola and pipsqueak (psq) RNA probes, coding DNA sequences from both loci were amplified by PCR using gene-specific primer sets lola[766bp] (lola[A]—5'-GTC CTC GTC ATC GCC TTG-3' and lola[B]—5'-GAA CAG TAC GAC AAA CAT CC-3') and psq[644bp] (psq[A]—5'-GTA GCG ATA GCG TGC CAG-3'; and psq[B]—5'-GCT GCT GAA ACA CGG ACG-3')]. PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI). Immunohistochemistry on third instar wing discs was performed according to standard techniques. Dissected third instar wing discs were fixed with 4% formaldehyde/NaPO4, washed with PBS/0.5% Triton X-100 (PBST), and blocked with PBST/4% BSA. Antibodies were used at the following dilutions: -Wg (1:50; 4D4, Developmental Studies Hybridoma Bank), -Ct (1:20; 2B10, DSHB), -ßGal (1:40; 40-1a, DSHB), and horseradish peroxidase conjugated -mouse (1:200; Bio-Rad, Richmond, CA). In situ detection of ß-galactosidase activity in dissected third instar wing imaginal discs was carried out as described by GLASER et al. (1986).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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The ctK, ct6, and ct2s alleles display a cut wing phenotype characterized by incised wing-blade tissue and decreased numbers of margin bristles (Figure 2, A–C). Whereas the ctK allele primarily disrupts margin bristle development, ct6 and ct2s disrupt both blade tissue and margin bristle development. The margin-specific overexpression of UAS-cut directed by the C96-Gal4 driver significantly rescues the cut wing phenotype (Figure 2, D–F). In hemizygous ctK/Y mutant males, the large discontinuities in the regular array of anterior margin bristles are rescued by Cut overexpression, but the total number of margin bristles remains less than that of wild type (Figure 2D). This may be interpreted as an incomplete rescue. However, since a similar reduction in margin bristle number is observed when Cut is overexpressed in heterozygous ctK/+ females (Figure 2J), which under normal conditions have wings of wild-type morphology, we prefer the alternative interpretation that a complete rescue is confounded by a dominant negative effect resulting from Cut overexpression (see also LUDLOW et al. 1996). Cut overexpression also partially restores blade tissue and margin bristles of the ct6 and ct2s alleles (Figure 2, E and F). In particular, the number of stout mechanosensory bristles is significantly rescued (Table 1).
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Gain- and loss-of-function suppressor screens of the cut wing phenotype:
Having determined the dual requirement of cut to maintain margin cell survival and to specify margin bristle identity, we carried out complementary loss-of-function and gain-of-function suppression screens of the ctK phenotype to identify genes that interact with cut during wing-margin patterning. ctK is classified as a Lethal I cut allele, as defined by its failure to complement all cut mutant alleles except for the kinked femur class (JACK 1985). Placed in trans to a cut null allele, ctK is characterized by both semilethality and the transformation of embryonic ES organs into chordotonal organs. Unlike other Lethal I alleles, however, adult males that are hemizygous for the ctK allele are viable and display a completely penetrant cut wing-margin phenotype (Figure 2A). Because of these characteristics, we reasoned that ctK would provide a uniquely sensitized background in which genetic suppressor screens could be designed to identify genes involved in both wing-margin patterning and sensory bristle specification.
In an initial approach, we carried out a dominant loss-of-function suppression screen using available cytological deficiencies covering 50% of the genome. Male flies from each deficiency line were crossed to females homozygous for ctK, and the cut wing phenotype of the resulting male progeny—hemizygyous for ctK and heterozygous for the deficiency chromosome—was scored. The capacity of each deficiency chromosome to dominantly suppress ctK was quantitatively assessed according to their ability to reduce the overall penetrance of ctK-associated discontinuities in the anterior wing-margin sensory bristles (MATERIALS AND METHODS). The results of this screen are summarized in Table 2. Df(2L)Prl, Df(3L)Cat, and Df(3R)p25-Df(3R)P2 completely suppressed ctK. Unlike the Df(3R)p25-Df(3R)P2 dual deficiency chromosome, neither Df(3R)p25 nor Df(3R)P2 alone was able to suppress ctK. It is possible that the two deficient genomic regions cooperate to suppress ctK, or an unrelated second-site mutation present only in the dual deficiency may be responsible for the suppression. A fourth deficiency, Df(3R)sbd105, partially suppressed ctK as assessed by a decrease in the severity of margin bristle loss, but did not reduce the overall penetrance of the phenotype. Df(3R)sbd105 covers moira (mor), a gene encoding a core subunit of the Brahma (BRM) chromatin-remodeling complex. Genetic interactions between cut and components of the BRM complex are examined below.
The ctK allele results from the insertion of a gypsy retrotransposon into the cut locus 6 kb upstream of the first exon, where it partially disrupts the regulation of embryonic and adult Cut expression (JACK 1985; JACK and DELOTTO 1995). In wing tissue, the gypsy element functions by insulating the activity of the distal wing-margin enhancer from the proximal promoter, resulting in the loss of Cut expression specifically in the wing margin. At least two genes are known to be directly required for gypsy-mediated gene insulation: Suppressor of Hairy wing [Su(Hw)] and modifier of mdg 4 [mod(mdg4)] (HOOVER et al. 1992; GAUSE et al. 2001; GHOSH et al. 2001). The ctK allele is unusual in that it contains a mutated gypsy insulator with a partial deletion of the Su(Hw)-binding region, which presumably makes it more sensitive to moderate decreases in the activity of Su(Hw) and mod(mdg4) (HOOVER et al. 1992). As part of the deficiency screen, two deficiencies, Df(3R)red1 and Df(3R)e-N19, respectively covering Su(Hw) and mod(mdg4), were tested for an interaction with ctK. Although loss-of-function mutations in both genes have been shown to dominantly suppress ctK (HOOVER et al. 1992; GAUSE et al. 2001), our screen failed to identify either deficiency. In the case of mod(mdg4), however, mutations that suppress ctK behave as antimorphic alleles in that they suppress the wing phenotype more strongly than null alleles do. This could account for why we did not identify Df(3R)e-N19 as a dominant suppressor of ctK. Similarly, the chromosome deficiencies Df(2R)vg-B, Df(2R)Px4, and Df(2R)nap1, covering loci previously shown to encode positive regulators of Cut expression and including the genes vestigial, Chip, and Nipped-B, respectively, were tested for an interaction with ctK. Only Df(2R)nap1 showed an interaction in the wing. Df(2R)nap1 enhanced the wing phenotype of hemizygous ctK males and produced a mild dominant cut wing phenotype in heterozygous ctK females. This is consistent with previous evidence suggesting that Nipped-B facilitates the activation of cut expression (ROLLINS et al. 1999, 2004). Deficiencies covering other regulators of cut expression, including scalloped and mastermind, were not tested (MORCILLO et al. 1996).
We also conducted a complementary gain-of-function screen using the modular GS system of misexpression (TOBA et al. 1999). The margin-specific Gal4 driver, C96-Gal4, was used to drive expression of genes located proximal to 2066 unique insertions of the GS vector (MATERIALS AND METHODS). The ability of individual GS lines to suppress ctK was scored as described above. In total, 3.8% of the GS vector insertions (79/2066), representing at least 42 distinct loci, were found to suppress the ctK phenotype (Table 2). Insertions at 35 loci suppressed the ctK phenotype in response to Gal4-dependent misexpression. The seven remaining loci suppressed without the C96-Gal4 driver and presumably act as dominant loss-of-function suppressors of ctK. In addition, a large number of gain-of-function GS lines (319/2066) enhanced the ctK phenotype, as determined by an increase in the severity of margin tissue loss. Due to the large number of ctK-enhancing loci, we opted not to characterize them further and instead focused on the suppressing loci.
As previously stated, it is possible that genes identified by the ability to suppress ctK may result from an interaction with the cut regulatory region or, alternatively, with the gypsy insulator or a gene required for gypsy insulator activity. To distinguish between these possibilities and to further characterize the interaction with cut, we examined the ability of the candidate GS suppressor lines to modify the wing phenotype of the weak ct53d allele. In contrast to ctK, the ct53d allele results from a partial deletion (500 bp) of the minimal cut wing-margin enhancer (defined as a region of 2.7 kb) and does not contain gypsy-derived elements (JACK et al. 1991; MOGILA et al. 1992). The ct53d allele disrupts Cut expression primarily in the presumptive wing tip, which corresponds to a severe loss of wing tissue in the distal-most region of the adult wing. The genetic interaction data with ct53d are summarized in Table 2.
Candidate suppressor loci were classified into three groups according to the genetic interaction with ctK and ct53d. Group A loci suppress both ctK and ct53d and are expected to represent candidate regulators or effectors of cut activity. The interaction with ct53d indicates that group A loci do not suppress the ctK wing phenotype simply by interfering with gypsy activity. In contrast, group B loci suppress ctK and enhance ct53d, suggesting a more complex interaction with the cut locus during wing-margin patterning. This may include direct interference with gypsy insulator activity, in addition to being required for cut wing enhancer activity. It should be noted that all group B loci, except a subgroup that suppressed the ctK equivalent to the UAS-Cut rescue, do not adversely affect wing development when misexpressed in heterozygous cut mutant females. Thus, enhancement of the wing phenotype in hemizygous ct53d mutant males is unlikely due to misexpression alone. Finally, group C includes candidate loci that suppress only ctK and are therefore presumed to interfere with gypsy activity. For instance, one gain-of-function suppressor contains a GS insertion near trithorax (trx), a gene previously shown to enhance gypsy insulator activity when mutated (GERASIMOVA and CORCES 1998).
Group A consists of four candidate loci that suppress both ctK and ct53d and includes brain tumor (brat), CyclinE (CycE), and lola. brat encodes a tumor-suppressor protein (FRANK et al. 2002) and CycE a cell cycle regulator controlling the G1/S phase transition (RICHARDSON et al. 1993, 1995). Thus, both may act to suppress the wing phenotype by influencing cell growth and proliferation. The genetic interaction with lola is explored further below.
Group B consists of 12 candidate loci, including two genes encoding components of the eukaryotic translation initiation factor 4F complex (eIF-4F), eIF-4A and eIF-4E (reviewed in GEBAUER and HENTZE 2004). eIF-4A and eIF-4E regulate translation downstream of the insulin/target of rapamycin signaling pathway and as such act globally to regulate cell growth and proliferation (MIRON et al. 2003). Overexpression of eIF-4E and eIF-4A may relieve putative cell growth or survival deficits associated with the loss of cut activity by enhancing the translation of Cut target genes. Similarly, group B candidate genes lesswright (lwr) and fussilli (fus) are also involved in regulating cell growth or proliferation of wing imaginal tissue. Some heterozygous mutants of lwr, a gene encoding a ubiquitin-like conjugating enzyme, exhibit wings severely reduced in size (EPPS and TANDA 1998). Lwr has been shown to be required for the nuclear import of Bicoid during early embryogenesis (EPPS and TANDA 1998). It is possible that Lwr plays a role in the nuclear import of Cut or its downstream targets. The RNA-binding protein Fus is involved in regulating cell growth in the wing disc and, similarly to eIF-4F, may affect the translation of Cut target genes (WAKABAYASHI-ITO et al. 2001; RAISIN et al. 2003). The differential interaction of group B candidates with various cut alleles likely reflects either direct or indirect effects on both gypsy insulator activity and cut wing-enhancer-mediated transcription. It will be of interest to determine if group B loci regulate gene insulation and transcription via common mechanisms.
A subgroup of gain-of-function candidate genes, including Hephaestus (heph), suppress the ctK wing-margin patterning defects to a degree comparable to the UAS-Cut rescue; the large discontinuities in the triple-row bristles are mitigated, but the total number of sensory bristles is less than normal. Heph is expressed in the presumptive wing and encodes a polypyrimidine tract binding protein that binds to and regulates RNA stability (DANSEREAU et al. 2002). Heph appears to attenuate Notch signaling downstream of the binding of the Notch ligand, Delta, and heph– clones cause the nonautonomous formation of wing-margin structures (DANSEREAU et al. 2002). How the overexpression of Heph and presumably the attenuation of Notch signaling suppresses ctK is not clear. It is possible that Heph may affect the activity of the gypsy insulator, since overexpression of Heph did not produce an appreciable alteration of the ct53d wing phenotype.
longitudinals lacking is required for cut-dependent wing-margin patterning:
Twenty-one GS vector lines with insertions at 12 unique locations proximal to the coding region of lola were identified by their ability to suppress the ctK allele (lolaGS[A916]; Figure 3, A and B). All lolaGS insertions require the C96-Gal4 driver, indicating that suppression results from GS-vector-mediated overexpression. lolaGS[A916]-mediated suppression of the ctK phenotype is robust with 94% of ctK mutant wings displaying a normal triple row of sensory bristles (Table 2). The ct53d, ct6, and ct2s alleles are also strongly suppressed by lolaGS[A916] (Figure 3, C–H, and Table 1), demonstrating that the interaction with cut in the wing margin is not allele specific. In addition to reversing the loss of sensory bristles, lolaGS[A916] suppresses the loss of blade tissue, a phenotype thought to result from a failure of Cut to maintain Wg expression (MICCHELLI et al. 1997). lolaGS[A916] may interact with cut both during Wg-dependent patterning of the wing margin and during the specification of margin bristles.
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Overexpression of lolaGS can rescue Cut expression and ectopically induce Wg:
The overexpression data are consistent with lola acting genetically downstream of cut in wing-margin patterning, but do not rule out the possibility that lola suppresses the wing phenotype by restoring Cut expression in the wing discs of cut regulatory mutants. To determine if the lolaGS line rescues Cut expression, we examined the pattern of Cut protein in ct53d wing imaginal discs in either the presence or the absence of driving lolaGS[A916] in the wing margin. In ct53d mutant discs, Cut expression is reduced throughout the wing margin and completely absent at the presumptive wing tip, corresponding to the region of the adult wing most visibly disrupted (Figure 4, D and E). Overexpression of lolaGS[A916] in the wing margin rescues Cut expression in ct53d mutants (Figure 4F), indicating that lola may be involved in regulating Cut expression.
Although C96-Gal4-driven expression at the presumptive margin is broader than the normal Cut expression domain (Figure 4A), ectopic Cut is not observed outside of the margin cells in response to lolaGS[916]. Similarly, when lolaGS[A916] was overexpressed along the anterior/posterior boundary using the patched-Gal4 driver (ptc-Gal4; Figure 4G), ectopic Cut expression was not observed (data not shown). In contrast, ptc-Gal4-directed lolaGS[A916] overexpression resulted in ectopic Wg protein in cells immediately adjacent to the dorsoventral boundary (Figure 4, G–I). Although lolaGS[A916] can be active in wing-blade cells, as shown by ectopic Wg expression, rescued Cut expression remains confined to the margin cells, suggesting that some unknown factor, other than Lola, is involved in restricting Cut expression to margin cells.
These results suggested that lola may be required for wild-type wing-margin morphogenesis. To test this, we generated somatic clones of lola mutant cells using the FLP/FRT method (XU and RUBIN 1993). In homozygous lola mutant clones located adjacent to or bisecting the wing margin, neither Cut expression nor the morphology of wing-margin bristles is disrupted (data not shown). Thus, it appears that lola, although sufficient to rescue decreased levels of Cut expression, is not absolutely required for Cut expression and margin development of otherwise wild-type wing discs, but strongly influences the development of wings with compromised cut activity.
Disruption of Brahma complex activity suppresses the ctK phenotype:
Among the GS lines able to completely rescue the ctK phenotype, we identified a GS vector insertion in the first exon of mor (designated morGS[A897]; Figure 5, A and B, and Table 3). On the basis of its failure to complement the lethality of hypomorphic mor1 mutants, morGS [A897] behaves genetically like a loss-of-function allele (data not shown). In addition, morGS[A897] suppressed ctK independently of the presence of the C96-Gal4 driver. To determine if a reduction of mor function is indeed responsible for suppression, we tested the ability of mor1 to interact with ctK. Adult males of the genotype ctK/Y; mor1/+ display a near-complete restoration of anterior wing-margin structures normally disrupted or missing in ctK mutants, including L1 wing-vein tissue and triple-row sensory bristles (Figure 5C and Table 3). Surprisingly, the deficiencies Df(3R)sbd105 (deficiency suppressor screen) and Df(3R)Exel7327 (Table 3), both covering the mor locus, only weakly suppress the severity of the ctK phenotype. It is not clear why the hypomorphic mor alleles suppress ctK more strongly than a mor deficiency does. Perhaps the cut wing phenotype is particularly sensitive to the level of Mor activity, or the deficiencies have accumulated modifier mutations that are not present in mor hypomorphs, which act to conceal the suppressive effect of reduced Mor function.
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To reduce Brm activity further, a dominant-negative brm transgene, UAS-brmK804R (ELFRING et al. 1998), was overexpressed specifically in the presumptive wing margin, using the C96-Gal4 driver. The BrmK804R protein is defective in its ability to hydrolyze ATP, but maintains an association with other BRM complex components. BrmK804R strongly suppresses the ctK-dependent loss of margin sensory bristles (Figure 5D and Table 3), suggesting that reducing energy-dependent BRM complex activity, without disrupting the interactions among components of the complex per se, suppresses the ctK wing-margin phenotype. Thus, manipulating the activity of the BRM complex components Mor and Brm strongly modifies ctK-dependent wing-margin loss.
Disruption of Brahma complex activity enhances the ct53d phenotype:
The gypsy retrotransposon inserted 5' to the cut coding region in ctK disrupts communication between the distal cut wing enhancer and the proximal core promoter (JACK et al. 1991), possibly by affecting higher-order chromatin structure (CHEN and CORCES 2001; BYRD and CORCES 2003). To determine if the disruption of BRM complex activity suppresses the ctK wing-margin phenotype via a gypsy-dependent or -independent mechanism, we examined the genetic interactions of mor and brm mutants with other gypsy and non-gypsy cut alleles. We find that both mor1 and brm2 heterozygote mutations interact with the non-gypsy ct53d allele. In contrast to the interaction with ctK, however, we observed aggravation rather than suppression of the ct53d phenotype (Table 3). Similarly, wing-margin-specific overexpression of BrmK804R severely enhanced the loss of anterior margin tissue in ct53d (Figure 5, E and F). The loss of wing-margin bristles observed in ctK and ct53d mutants likely reflects a decrease in Cut expression in the wing margin (Figure 6A). Cut expression is substantially restored in ctK/Y;UAS-brmK804R/C96-Gal4 wing imaginal discs (data not shown). Conversely, the aggravated loss of wing-margin bristles of ct53d/Y;UAS-brmK804R/C96-Gal4 correlates with a further decrease in the level of Cut protein throughout the presumptive wing margin in ct53d mutant discs (Figure 6B).
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Both BAP and PBAP interact with cut:
In Drosophila, there are two distinct Brm-containing complexes, BAP (Brahma-associated proteins) and PBAP (Polybromo-associated BAP). Both complexes contain the DNA-dependent ATPase Brm and seven core subunits, Mor/BAP155, BAP111, BAP74 (hsp70 cognate hsc4), BAP60, BAP55, actin/BAP47, and Snr1/BAP45 (MOHRMANN et al. 2004). Heterozygous loss-of-function mutations in BAP111, BAP60, BAP55, or Snr1/BAP45 did not modify the ctK or ct53d margin bristle phenotype (Table 3), suggesting that decreasing the expression level of these subunits is not limiting for BRM complex activity in vivo.
The BAP and PBAP complexes are distinguished by association with either Osa or Polybromo and BAP170, respectively (MOHRMANN et al. 2004). Thus, we examined the ability of osa, polybromo, and BAP170 loss-of-function mutations to enhance the ct53d wing phenotype. Flies heterozygous for the osa2 allele do not display wing defects alone, nor does osa2 cause a strong enhancement of the ct53d phenotype. However, the overexpression of the full-length UAS-osa transgene (COLLINS et al. 1999) specifically in the wing margin suppressed the loss of anterior margin bristles of the ct53d phenotype (Table 3), implying that an Osa-associated BRM complex interacts with cut during margin development by increasing its activity.
Specific mutations are not available for either polybromo or BAP170. Therefore, we used the deficiencies Df(3R)slo8 and Df(2R)ED1552, covering polybromo and BAP170, respectively, to explore the genetic interaction with ct53d. As with brm or mor mutations, both deficiencies enhanced margin bristle and tissue loss of ct53d (Table 3). Although it cannot be ruled out that one of the other genes disrupted by the deficiencies is responsible for the enhancement of the ct53d phenotype, these data support the idea that cut activity is sensitive to disruptions of PBAP complex components. It should be noted that neither osa mutations nor the polybromo or BAP170 deficiencies were able to modify the phenotype of ctK. Overall, the genetic data suggest that both Brm-containing chromatin-remodeling complexes, BAP and PBAP, may contribute to cut-dependent wing-margin development in a complex manner.
Osa may act as a transcriptional repressor in its interaction with ct53d:
An Osa-containing BRM complex has previously been implicated in the repression of Wg target genes during development of the wing imaginal disc (COLLINS and TREISMAN 2000). To study whether Osa acts as a transcriptional activator or repressor with regard to its interaction with cut, we examined the ability of obligatory activator and repressor forms of Osa (COLLINS et al. 1999) to modify the ct53d phenotype (Table 3). Wing-margin-specific overexpression of the Osa AT-rich interaction domain (ARID)-DNA-binding domain fused to the Engrailed repression domain (UAS-OsaRD[11c]) strongly suppressed the ct53d phenotype, but had no effect in a wild-type background. As previously stated, overexpression of full-length Osa also ameliorates the ct53d phenotype, consistent with the idea that Osa acts as a repressor. Conversely, overexpression of the Osa-ARID domain fused to the VP16 transcriptional activation domain (UAS-OsaAD[20e]) enhanced the wing-margin phenotype of ct53d. Together, these results suggest that the Osa-containing BAP complex must act as a transcriptional repressor to ameliorate cut-dependent wing-margin patterning defects. This is in accordance with the repressive activity of Osa on Wg target genes in the wing disc (COLLINS and TREISMAN 2000).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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As a means to further elucidate the role of cut in wing-margin patterning, we performed complementary loss- and gain-of-function genetic screens to identify other genes that modify the cut wing phenotype. Several classes of cut modifiers include loci near known genes that regulate processes influencing cell growth and proliferation, including brat, CycE, eIF4A, and eIF4E. The identification of these genes suggests that during wing-margin development Cut activity may be regulated in a manner dependent upon cell cycle phasing and/or may coordinate cell cycle progression with terminal specification of cell identity. This is consistent with the proposed activity of the vertebrate Cut homolog CDP/Cux1, the DNA-binding activity of which is modulated in coordination with cell cycle progression and is postulated to synchronize cell cycle exit with terminal cell differentiation (reviewed in NEPVEU 2001)
lola is required in the context of decreased Cut expression for wing-margin development:
lola is known for its role as a regulator of axon growth in Drosophila and is proposed to coordinately control the expression of multiple genes that execute axon guidance decisions (GINIGER et al. 1994; MADDEN et al. 1999; CROWNER et al. 2002). We identified a novel role for lola in wing-margin development, revealed by its gain- and loss-of-function genetic interactions with hypomorphic cut alleles. Overexpression directed by lolaGS insertions is sufficient to rescue the reduction in Cut expression of regulatory cut mutants and to suppress the hypoplastic cut wing phenotype. Conversely, loss of lola function aggravates the cut wing-margin defects. It is feasible that Lola modulates Cut expression by interacting directly or indirectly with the cut wing-margin enhancer or with other regulatory regions adjacent to or distant from this enhancer, which may also be involved in promoting Cut expression at the margin. The suppression of the cut wing phenotype by Lola misexpression is consistent with other alternative possibilities, such as that Lola may be involved in the regulation of an unknown Cut target gene, may be a Cut target itself, or both. Consistent with the ability to interact with cut during wing development, lola mRNA is expressed ubiquitously in the imaginal wing disc. However, the requirement for lola in wing development is evident only in cut mutants, since in lola null mutant cell clones Cut expression and wing-margin development is not disrupted. It may be that lola plays a nonessential role in the regulation of processes directing wing-margin development, which only becomes apparent when Cut activity is decreased, akin to the cryptic variations necessary for evolutionary adaptations (GIBSON and DWORKIN 2004).
Overexpression of Lola in cut mutants suppresses the margin loss phenotype presumed to result from a failure to maintain expression of the secreted factor Wg at the dorsal/ventral boundary of the wing disc. We demonstrate that Lola induces ectopic Wg expression at locations proximal to the dorsal/ventral boundary. Thus, it may be that Lola overexpression rescues wing-blade tissue in cut mutants via the induction of Wg expression. The suppression of sensory bristle loss, however, is likely independent of this effect on Wg expression. It will be interesting to determine if lola contributes to other tissue-specific aspects of cut activity.
The induction and refinement of both Wg and Cut expression at the wing dorsal/ventral boundary requires activation of the Notch signaling pathway (DIAZ-BENJUMEA and COHEN 1995; MICCHELLI et al. 1997). The induction of Cut and Wg expression in response to Lola overexpression implies that Lola may positively regulate Notch signaling in wing boundary cells. In the eye, however, Lola appears to act in the converse manner, where the loss of Lola function enhances the rough-eye phenotype resulting from the overexpression of a constitutively active form of Notch (VERHEYEN et al. 1996). Clonal analysis of amorphic lola mutations does not produce the incised wing-margin phenotype indicative of a loss of Notch function, suggesting that Lola activity is not required to regulate Notch signaling. Furthermore, although Cut expression is rescued in the wing-margin in response to broad Lola overexpression, it is not expanded outside of the boundary cells. This is in contrast to the observed expansion of Wg into cells adjacent to the boundary, indicating that the induction of Wg is independent of Cut. Since ectopic expression of both Wg and Cut is induced in the wing disc in response to activated Notch (DE CELIS et al. 1996), expanded Wg expression due to Lola overexpression may not involve Notch signaling.
The lola locus encodes a family of at least 20 BTB-zinc-finger transcription factors, expressed in partially distinct tissue-specific patterns. The functional significance of the diversity in Lola isoforms and their expression patterns is not entirely clear. In several instances, mutations inactivating a single Lola isoform affect only a subset of axon guidance defects associated with amorphic lola alleles (GOEKE et al. 2003). This led to the hypothesis that specific isoforms and interactions with cofactors contribute to the diversity in lola-dependent axon guidance decisions. Lola isoform F has been shown to physically interact in vitro with the chromosomal JIL-1 kinase (ZHANG et al. 2003). JIL-1 regulates chromatin structure by influencing the phosphorylation state of histone 3 (WANG et al. 2001). Amorphic lola alleles act as dominant modifiers of a hypomorphic JIL-1 allele, leading to an increase in embryonic viability (ZHANG et al. 2003). It is not clear, however, if Lola isoform F is responsible for the in vivo genetic interaction with JIL-1. Similarly, we were unable to determine which Lola isoform(s) is responsible for the interaction with cut in the wing margin. All amorphic lola alleles interact with cut (i.e., enhancement of bristle loss) in a similar manner. Interestingly, the axon guidance decision-selective lolaORE119 allele, thought to disrupt only isoform L, enhanced the cut wing phenotype, whereas the isoform K-specific lolaORC4 allele did not.
Brm-associated chromatin-remodeling complexes regulate multiple aspects of wing development:
In Drosophila, Brm and Brm-associated proteins regulate multiple aspects of wing development. Early in wing development, Brm and Osa modulate the activity of the dorsal wing compartment specific selector gene, Apterous, and the subsequent localization of the Wg-dependent organizer at the dorsal/ventral boundary (MILAN et al. 2004). Similarly, Mor is required for the expression of the posterior compartment specific selector gene engrailed (BRIZUELA and KENNISON 1997). The triune of Brm, Osa, and Mor is required to repress the Wg target gene nubbin, a gene required for the growth and patterning of the wing (COLLINS and TREISMAN 2000). Finally, Brm activity is required for the cell-type-specific activation and repression of genes involved in wing-vein elaboration (MARENDA et al. 2004). Our genetic analysis indicates that cut-dependent wing-margin patterning also relies upon the activity of Brm, as well as upon the activity of several Brm-associated subunits of both the BAP and the PBAP complex. Heterozygous loss-of-function mutations in the core subunits Brm and Mor, although individually having no effect on normal wing-margin development, enhance the loss of wing-margin tissue of the ct53d allele. Deficiencies covering the PBAP subunits, BAP170 and Polybromo, or the overexpression of BAP subunit Osa, exhibit similar interactions with ct53d. The enhancement of the ct53d wing phenotype correlates with a decrease in Cut expression in the presumptive wing margin, thus indicating that the BRM complex activity is required genetically upstream of cut. Together with other studies, our data support the idea that the BRM complex may globally regulate the expression of genes required for wing development.
How might the BRM complex regulate cut expression in the wing? The regulation of the distal cut wing enhancer requires the activity of both enhancer-binding and enhancer-facilitator proteins. Enhancer-facilitator proteins are proposed to structurally facilitate communication between distal enhancer elements and the proximal promoter and are different from enhancer-binding (co)activators in that they do not directly activate the initiation of transcription. A number of genes, including scalloped (sd), mastermind (mam), Chip (Chi), and Nipped-B, are involved in the regulation of cut expression (MORCILLO et al. 1996; ROLLINS et al. 1999). Genetic and biochemical data suggest that sd and mam encode cut wing-margin enhancer-binding transcriptional (co)activators. Consistent with their role as enhancer-binding activator proteins, loss-of-function sd and mam mutations enhance the severity of the cut wing phenotype resulting from deletions in the wing enhancer. In contrast, Chip and Nipped-B encode putative general enhancer-facilitator proteins (MORCILLO et al. 1996, 1997; ROLLINS et al. 1999, 2004) and primarily enhance the cut wing phenotype </
