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. 2007 Feb;175(2):659–669. doi: 10.1534/genetics.106.063966

Antagonizing Scalloped With a Novel Vestigial Construct Reveals an Important Role for Scalloped in Drosophila melanogaster Leg, Eye and Optic Lobe Development

Ankush Garg 1, Ajay Srivastava 1, Monica M Davis 1, Sandra L O'Keefe 1, Leola Chow 1, John B Bell 1,1
PMCID: PMC1800616  PMID: 17110491

Abstract

Scalloped (SD), a TEA/ATTS-domain-containing protein, is required for the proper development of Drosophila melanogaster. Despite being expressed in a variety of tissues, most of the work on SD has been restricted to understanding its role and function in patterning the adult wing. To gain a better understanding of its role in development, we generated sd47M flip-in mitotic clones. The mitotic clones had developmental defects in the leg and eye. Further, by removing the VG domains involved in activation, we created a reagent (VGΔACT) that disrupts the ability of SD to form a functional transcription factor complex and produced similar phenotypes to the flip-in mitotic clones. The VGΔACT construct also disrupted adult CNS development. Expression of the VGΔACT construct in the wing alters the cellular localization of VG and produces a mutant phenotype, indicating that the construct is able to antagonize the normal function of the SD/VG complex. Expression of the protein:protein interaction portion of SD is also able to elicit similar phenotypes, suggesting that SD interacts with other cofactors in the leg, eye, and adult CNS. Furthermore, antagonizing SD in larval tissues results in cell death, indicating that SD may also have a role in cell survival.

THROUGHOUT the course of development a single transcription factor may often be used to control the patterning of different tissues. For example, in Drosophila melanogaster, the paired domain protein Eyeless is required for the proper development of the adult compound eye (Halder et al. 1995; Sheng et al. 1997) as well as of the adult central nervous system (Callaerts et al. 2001), while the Drosophila GATA factor Pannier is required for sensory organ development (Ramain et al. 1993; Heitzler et al. 1996), cardioblast differentiation (Gajewski et al. 1999, 2001), and dorsal closure (Herranz and Morata 2001). The Drosophila TEA/ATTS domain (TEAD) protein, Scalloped (SD), is a transcription factor that is expressed in several different tissues throughout development. The existance of lethal alleles of sd (Campbell et al. 1991, 1992) implies that this gene is vital for proper development.

The TEAD is a highly conserved DNA-binding domain that recognizes the M-CAT motif (5′-TCATTCCT-3′) (Hwang et al. 1993; Stewart et al. 1994). TEAD-containing proteins generally bind directly to tissue-specific transcriptional intermediary factors (TIFs) to properly function as a specific transcription factor (TF) complex (Halder et al. 1998; Simmonds et al. 1998; Vaudin et al. 1999; Jiang et al. 2000; Maeda et al. 2002; Chen et al. 2004a,b; Mahoney et al. 2005). Since TEAD-containing proteins, such as SD, often lack an activation domain (Hwang et al. 1993; Vaudin et al. 1999), the associated TIFs may provide this function (Halder et al. 1998; Simmonds et al. 1998; Vaudin et al. 1999; Maeda et al. 2002; Chen et al. 2004a,b). Alternatively, the activation domains of TEAD-containing proteins may associate with TIFs that can act as coactivators or corepressors. To date, two TIF-interacting domains have been identified for TEAD proteins: the Vestigial interacting domain (VID) (Halder et al. 1998; Simmonds et al. 1998) and the C-terminal YAP/TAZ-transactivating domain (TD) (Vassilev et al. 2001; Fossdal et al. 2004). GST pull-down experiments with the human homolog transcription enhancer factor-1 (TEF-1) loosely position the VID and the TD between amino acids 221 and 329 (Vassilev et al. 2001) and 224 and 426 (Vassilev et al. 2001), respectively. The VID interacts with the Vestigial protein (VG) (Halder et al. 1998; Simmonds et al. 1998) and with Vestigial-like proteins (VGL) (Vaudin et al. 1999; Halder and Carroll 2001; Mielcarek et al. 2002; Chen et al. 2004a,b) in flies and mammals, respectively. To date, the TD has been shown to be functionally important only in the human TEAD proteins and to interact with the yes-associating protein 65 (YAP65) (Vassilev et al. 2001) and with the Yap65 homolog TAZ (Mahoney et al. 2005).

The gene scalloped (sd) is the only TEAD-encoding gene in the Drosophila genome. Enhancer trap studies reveal that sd is first expressed at stage 14 in the peripheral nervous system (PNS), the antennomaxillary complex, and the supraesophageal ganglion. By stage 16, sd expression is expanded to the anterior sense organs and the sense organs of the gnathal regions (Campbell et al. 1992). SD is also thought to be expressed in embryonic cardiac cells (Bidet et al. 2003). In third instar larvae, sd is present in the optic lobes and in a few discrete cells of the cerebral hemisphere and the ventral nerve cord. In the wing imaginal disc, sd is expressed in the wing blade, scutellum, and the mesopleura, while in the eye imaginal disc, staining is restricted to cells behind the morphogenetic furrow (Campbell et al. 1992).

Most of our understanding of SD function is derived from work done with the wing imaginal disc, where SD interacts with the TIF, VG (Halder et al. 1998; Simmonds et al. 1998). VG does not contain a DNA-binding motif, but contains a SD-interacting domain (SID) and two regions important for activation of downstream genes, located at the N- and C-terminal ends of the protein (Vaudin et al. 1999; MacKay et al. 2003). These three regions are required for the proper development of the wing blade (MacKay et al. 2003). Binding of SD to VG is necessary for the formation of a functional transcription complex that is able to specifically activate the expression of downstream wing genes (Halder et al. 1998; Simmonds et al. 1998). Ectopic expression of VG in cells expressing SD but not VG causes activation of downstream wing genes and directs the developmental fate of that tissue into a wing (Kim et al. 1996; Halder et al. 1998). SD is also required to localize VG to the nucleus (Halder et al. 1998; Simmonds et al. 1998; Srivastava et al. 2002). In vitro experiments have shown that the binding of VG to SD can alter the DNA motif that SD recognizes. These experiments have also shown that the interaction of a truncated form of VG, containing only the SID, with SD is able to disrupt the complex from binding to wing-specific DNA-binding motifs. Proper recognition of the wing-specific DNA-binding motifs by SD requires the SID and at least one of the VG domains involved in activation (Halder and Carroll 2001). This is consistent with the findings that the protein encoded by a fusion of the sd TEAD domain with a full-length vg gene can rescue sd and vg wing mutations (Srivastava et al. 2002). In the absence of VG, SD is still able to bind to DNA, but recognizes a different motif (Halder and Carroll 2001).

The function of SD in nonwing tissues is poorly understood. Several lethal alleles of sd have been isolated (Campbell et al. 1991). The lethal alleles of sd fall into two classes: embryonic and pupal lethal (Campbell et al. 1991; Srivastava et al. 2004). Since wings are not essential to the survival of laboratory stocks, SD likely plays a vital role in nonwing tissues. To gain a better understanding of the role that SD plays in development, we generated mitotic clones using an embryonic lethal allele of SD. Loss of SD caused blistering in the wings (Liu et al. 2000), alterations in eye bristle shape and patterning, and truncation of the legs. We further assessed the role of SD by antagonizing its function with a novel allele of VG. By expressing a truncated form of VG, VGΔACT (Figure 1), that is able to bind to SD but not activate transcription, we were able to recapitulate all the phenotypes observed with the flip-in clones as well as cause defects in the optic lobe and promote fusions between ommatidia. Despite the VGΔACT protein's ability to antagonize SD in larval tissues, it did not affect the development of the embryonic PNS, central nervous system (CNS), or cardiac cells. To test whether the phenotypes seen in the affected tissues are due to the inability of SD to form a functional TF, we also expressed a truncated form of SD, SDΔ200 (Figure 1), that is unable to bind DNA but retains the TIF-interacting domains (Chow et al. 2004). The SDΔ200 protein should be able to bind to any TIFs that normally interact with the C-terminal 244 amino acids of the SD protein. Expression of the SDΔ200 protein induces phenotypes similar to those caused by the flip-in clones and the VGΔACT protein, indicating that SD likely binds to currently unknown TIFs in the eye, leg, and optic lobe. Through these studies we show that the level of SD is important in these tissues and that the SD/TF complex is involved in promoting cell survival in the leg imaginal disc.

Figure 1.—

Figure 1.—

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Schematic of the VG and SD constructs used herein. Within the 453 amino acid (aa) VG protein, two putative activation domains, ACT-1 (aa 1–65) and ACT-2 (aa 335–453), and a Scalloped-interacting domain, SID (aa 281–335), have been identified. The two activation domains are removed in the VGΔACT construct. The 440-aa SD protein contains a TEA/ATTS DNA-binding domain, TEAD (aa 88–159); a Vestigial-interacting domain, VID (aa 220–344); and an NLS (aa 144–162). The TEAD is no longer functional in the SDΔ88-123 construct while the TEAD and the NLS are nonfunctional in the SDΔ88-159 and SDΔ200 constructs.

MATERIALS AND METHODS

Drosophila stocks:

All crosses were done at 25°. The UASsd, UASsdΔ200, UASsdΔ88-123, and UASsdΔ88-159 constructs are described in Chow et al. (2004). The ptcGal4 and vgGal4 strains were a gift from S. Carroll, the sd47M FRT 18A was a gift from K. Irvine, and w+, ry+ 2[p-Myc] FRT18A; Sb FLP/Tb was a gift from S. Hughes. The 24BGal4, 109(2)80Gal4, 167YGal4, 179YGal4, C147Gal4, c698aGal4, ActinGal4, atoGal4, ChaGal4, CQ2Gal4, DllGal4, eyGal4, GMRGal4, Pan-R7Gal4, PdfGal4, RN2Gal4, sd72b, and sevEPGal4 stocks were obtained from the Bloomington Stock Center. The sd72b allele is a deficiency stock containing a deletion the spans the entire sd gene.

Flip-in clones:

The sd47M allele is a 157-bp deletion (Srivastava et al. 2004) and homozygous individuals exhibit an embryonic lethal phenotype (Campbell et al. 1991). The sd47M allele was recombined onto a w, P(ry, neoFRT)18A chromosome (Liu et al. 2000). Mitotic clones of this allele were generated by flipase-mediated mitotic recombination (Golic and Lindquist 1989; Xu and Rubin 1993) by repeated daily heat shocking at 39° for 45-min intervals.

Construction of VGΔACT:

Using a polymerase chain reaction (PCR) protocol, the nucleotides that encode amino acids 171–335 of VG were amplified with the following primers: 5′-TCG AGG CCT CAC ACA CAC ACG CAT ACG-3′ and 5′-GGG CTC GAG TTA GTG CAC GTA ATT GCT GTT-3′. The PCR products were digested with XhoI and StuI and cloned into a p131 vector (Abu-Shaar et al. 1999).

PCR conditions:

A Taq:Pfu (20:1) mix was used in standard PCR conditions. The template was allowed to initially denature at 94° for 5 min. DNA was amplified with 30 cycles at 94° for 30 sec (denaturing) followed by 55° for 30 sec (annealing) and 68° for 90 sec (extension).

Micro-injections:

Micro-injections were performed as described in Rubin and Spradling (1982) using the VGΔACT construct in pUAST and a helper Δ2-3 plasmid provided by S. Campbell. Four independent transgenic lines were isolated and tested. All the results shown make use of the VGΔACT-22 line.

Scanning electron microscopy:

Adult flies were fixed in a 2% glutaraldehyde, 1× PBS solution for 1 hr at room temperature. Samples were washed twice in 1× PBS before being dehydrated in increasing concentrations of ethanol. Ethanol was removed by bathing the samples in increasing concentrations of hexamethyldisilazane:ethanol solutions. Samples were dried and gold coated before visualization by scanning electron microscopy (SEM).

Silver staining:

Adult flies were fixed overnight in a 10% formalin solution, dehydrated, and embedded in paraffin. Samples were sectioned at 15 μm and mounted on charged glass slides, after which the paraffin was removed and the samples were rehydrated. Silver staining of samples was performed as described in Naoumenko and Feigin (1967).

Acridine–orange staining:

Imaginal discs were dissected in 1× PBS and incubated in a 1.6 × 10−6 m solution of acridine–orange solution for 10 min. Discs were rinsed and mounted in a 1× PBS solution.

RESULTS

SD47M mitotic clones affect the development of bristles and legs:

To gain a better understanding of the role that sd plays in development, we generated flip-in mitotic clones using an embryonic lethal allele of sd, sd47M. The sd47M allele contains a 157-bp deletion spanning intron 8 and exon 9 of the gene (Srivastava et al. 2004). Because the deletion spans a splice site, it presumably disrupts the VID and the C-terminal region of the protein. The induction of sd47M mitotic clones causes defects in the wing (Liu et al. 2000), eye bristles, and the leg. Similar to the wing, clones in the eyes were very small. No w, ry clones were visible in the eye. In wild-type eyes, bristles are found at alternating vertices of each ommatidium (Figure 2, A and B). The effect of clonal induction on bristles varied. SEM analysis revealed that a few of the bristles were either smaller (Figure 2C) or mispatterned (Figure 2D). Wild-type legs are made up of three basic parts: the femur, the tibia, and the tarsus (Figure 2E). The distal end of the fly leg contains a metatarsal claw (Figure 2E, arrow and inset). Inducing sd47M clones leads to truncations in the leg (Figure 2F; arrow and inset emphasize the absence of the metatarsal claw).

Figure 2.—

Figure 2.—

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Mitotic clones of sd47M cause defects in the eye and leg. (A) Wild-type ommatidia (×2000). (B) Illustration of normal bristle patterning in the eye. Hexagons represent individual ommatidia and circles represent bristles. In wild-type flies, bristles occur at alternating vertices of the ommatidium. (C) Bristles are sometimes shorter (asterisk) in sd47M mitotic clones. (D) Duplications and mispatterning (asterisk) of bristles also occurs in sd47M mitotic clones. (E) Wild-type T1 leg. Arrow indicates the metatarsal claw shown in the inset. The femur (fem), tibia (tib), and tarsus (tar) are marked. (F) Leg tissue and the metatarsal claw is lost (arrow and inset) in sd47M mitotic clones.

VGΔACT is able to antagonize SD function in the wing imaginal disc:

In the wing, induction of mitotic clones causes only blistering, whereas homozygous viable mutant alleles of sd cause a loss of wing tissue. Because the clones are surrounded by a population of wild-type cells, it is possible that the loss of sd in the eye and leg is partially masked. To create a more robust phenotype, we created a reagent, VGΔACT, which is able to bind to SD but is not able to activate transcription. The VGΔACT construct was generated by removing the two activation domains, ACT1 (amino acids 1–65) and ACT2 (amino acids 356–453) (Halder and Carroll 2001; MacKay et al. 2003), from vg (Figure 1), leaving the entire scalloped interacting domain intact. Thus, the expectation is that the VGΔACT protein will cause a dominant-negative phenotype. To test if the VGΔACT construct can cause a dominant-negative effect, we first expressed it in the wing. Using the vgGAL4 driver, we expressed the VGΔACT transgene along the dorsal–ventral axis of the wing disc. This ectopic expression causes a loss of wing bristles and wing tissue (compare Figure 3B to Figure 3A). The extent of the phenotype varies considerably from almost a complete loss of the wing (not shown) to small patches of missing bristles in the anterior margin and loss of wing tissue along the posterior margin (Figure 3B). To test whether the observed effects are due to the VGΔACT protein antagonizing the endogenous SD protein, we attempted to rescue the dominant-negative phenotype by coexpressing full-length SD. Coexpression of full-length SD and VGΔACT rescued the bristle and the wing margin phenotypes (Figure 3C).

Figure 3.—

Figure 3.—

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VGΔACT interacts with SD and disrupts SD function. (A) A wild-type wing. (B) Wing bristles and wing tissue are lost in a vgGAL4/+; UAS VGΔACT/+ fly. (C) Rescue of the vgGAL4/+; UAS VGΔACT/+ phenotype by coexpression of full-length SD.

VGΔACT is not able to antagonize SD function in the embryo:

Since the VGΔACT protein is able to cause a dominant-negative phenotype in the wing, it may be able to have a similar effect in other tissues where SD is expressed. To test the ability of VGΔACT to antagonize endogenous SD in other cells, we expressed the transgene in the embryo using an actin GAL4 driver and a muscle-specific 24BGAL4 driver (Kidd et al. 1999). Overexpression of SD using the 24BGAL4 driver has been shown to cause embryonic lethality and to have an effect on the development of cardiac cells (Bidet et al. 2003). Furthermore, we found that tinman-expressing cells are mispatterned in a sd null background (data not shown). Neither embryonic lethality nor tinman mispatterning is seen when the VGΔACT or SD deletion transgenes are expressed under the control of either driver. However, larval lethality is seen when the transgenes are expressed using the actin GAL4 driver.

VGΔACT is able to antagonize SD function in tissues outside the wing disc:

Driving the VGΔACT transgene with the widely expressed ptcGAL4 driver causes more robust defects in the fly (Figure 4) than those seen with the mitotic clones. Antagonizing SD in the eye causes the eyes to protrude (compare Figure 4B to Figure 4A). We also observe more severe defects that affect bristle patterning, number, and morphology. In VGΔACT/+; ptcGAL4/+ flies, bristles can occur at adjacent vertices (Figure 4C; the ommatidium marked with an asterisk has bristles at four vertices) or they can be absent (Figure 4D, arrow). Duplications of bristles are also visible (Figure 4C, arrowhead) and in some of the more severe cases as many as three extra bristles at a single vertex are seen (not shown). Expression of the VGΔACT protein also affects bristle size (Figure 4C; note the size differences between the bristles identified by the arrows). Interestingly, bristle defects are more frequent along the tip of the eye protrusion. In some eyes, defects in the ommatidia are also present. Changes in ommatidia vary from increases in size (data not shown) to fusions between adjacent ommatidia (Figure 4D; compare fused ommatidia marked by a single asterisk to wild-type ommatidia marked by a double asterisk).

Figure 4.—

Figure 4.—

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Antagonizing SD in tissues outside the wing. (A) Wild-type eye (×200). (B) Eyes protrude (arrowhead) in a ptcGAL4/+; UAS VGΔACT/+ fly (×200). (C and D) Bristle pattern is affected in a ptcGAL4/+; UAS VGΔACT/+ fly (×2000). (C) Bristles are duplicated (arrowhead), and the size of the bristles is affected (arrows). Patterning defects are seen in the ommatidium marked by an asterisk. Four vertices, rather than three, of this ommatidium have bristles. (D) Bristles can also be missing (arrow) and ommatidia may be fused [compare the fused ommatidia (single asterisk) to the normal ommatidium (double asterisk)]. (E) A silver-stained horizontal section of a wild-type eye. The structures of the eye are labeled: lo, lobula; lo p, lobula plate; me, medulla; lam, lamina; and om, ommatidia. (F) An ectopic cluster of cells is present between the lamina and the ommatidia (arrowhead) in a ptcGAL4/+; UAS VGΔACT/+ fly eye. The ectopic cluster of cells coincides with the protrusion in the eye (arrow). (G) Leg tissue and the metatarsal claw is lost (arrow and inset) in a DllGAL4/+; UAS VGΔACT/+ fly. Legs may also contain a kink within the tibia (arrowhead). (H) Single leg showing that it has been duplicated at the base of the joint in a ptcGAL4/+; UAS VGΔACT/+ fly (arrowhead). The duplicated structures are also truncated.

Four structures make up the optic lobe: lobulla, lobulla plate, medulla, and lamina (Figure 4E) (Armstrong et al. 1995). In wild-type flies, the lamina makes direct contact with the basal membrane of ommatidia (Figure 4E). Silver staining of horizontal sections from VGΔACT/+; ptcGAL4 flies reveals an ectopic cluster of cells between the lamina and the basal membrane (Figure 4F, arrowhead) that correlates with the location of the protrusion (Figure 4F, arrow). All of the components of the optic lobe are present and appear normal in these flies. To try to identify the cells affected in the eye and brain, we expressed our construct with a variety of drivers (see Table 1). Only the eyeless GAL4 driver was able to cause optic lobe and eye phenotypes. None of the other eye- or brain-specific drivers had any effect.

TABLE 1.

Eye and neural drivers used to express VGΔACT and SDΔ200

Driver Expression pattern Phenotype
Actin Gal4 Ubiquitous Larval lethal
Ato Gal4 In ato+ cells in the brain and SOP No effect
Cha Gal4 In all cholinergic neurons No effect
CQ2Gal4 In U/CQ neurons No effect
Dll GAL4 Distal region of the leg disc Distal truncation of legs
ey GAL 4 ey+ cells in the eye disc and the larval brain Protrusion of the eyes. Bristle defects and fusion of ommatidia.
GMR Gal4 GMR+ cells in the eye disc No effect
Pan-R7Gal4 In all R7 cells No effect
PdfGal4 In ventrolateral neurons of the brain No effect
RN2Gal4 In RP2, aCC and pCC neurons No effect
sevEPGal4 In sev+ cells No effect
24BGal4 Embryonic mesoderm No effect
109(2)80Gal4 Dendritic neurons No effect
167YGal4 Neuroblast in the central brain and ventral ganglion No effect
179YGal4 Outer proliferative center near central brain No effect
C147Gal4 Larval brain No effect
c698aGal4 In the third instar larval brain No effect
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Truncation can occur in all three leg segments when the VGΔACT transgene is under the control of the DllGAL4 driver (Figure 4G; arrow and inset emphasize the absence of the metatarsal claw). The degree of leg truncation varies from loss of only a few of the metatarsals (Figure 4G, arrow) to the loss of all the metatarsals, tarsus, and tibia (not shown). Sharp bends in the femur or tibia are also sometimes seen (Figure 4G, arrowhead). In addition to causing truncations as seen in the mitotic clones, the VGΔACT protein is also able to cause duplications of the leg. However, only the VGΔACT/+; ptcGAL4 flies show duplications of the leg (Figure 4H, arrowhead) and these occur only in the T2 and T3 legs.

SD likely binds to other cofactors:

To help determine if the VGΔACT protein hinders the binding of important cofactors to SD, we expressed a truncated form of sd (SDΔ200) that lacks the coding capacity for the first 200 amino acids of SD (Figure 1). Although the SDΔ200 protein is missing the TEA/ATTS DNA-binding domain, it should still be able to bind to TIFs and compete with endogenous SD for binding to them. Thus, the expectation is that if SD binds to and interacts with other cofactors via a domain located in the terminal 244 amino acids of the protein, the SDΔ200 transgene should induce a phenotype similar to those seen in the VGΔACT/+; ptcGAL4 flies. If, however, SD does not interact with cofactors, or interacts with cofactors via a domain located within the first 200 amino acids of the protein, the SDΔ200 transgene should have no effect. Expression of the SDΔ200 protein with the ptcGAL4 driver causes phenotypes similar to those observed with the VGΔACT construct (Figure 5). In the head, the SDΔ200 protein causes an eye protrusion (data not shown), alters bristle patterning (Figure 5A, arrow) and morphology (Figure 5B, arrow), causes duplication of bristles (Figure 5A, arrowhead), induces the fusion of ommatidia (Figure 5C, asterisk), and produces an ectopic cluster of cells between the lamina and ommatidia (Figure 5D). SDΔ200 is also able to cause duplications in the legs (Figure 5E) and a loss of wing bristles and tissue (Figure 5F).

Figure 5.—

Figure 5.—

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The SDΔ200 protein can also antagonize SD function. ptcGAL4/+; UAS SDΔ200/+ flies (×2000) have mutant phenotypes similar to ptcGAL4/+; UAS VGΔACT/+ flies. (A) Bristles are duplicated (arrowhead) or missing (arrow). Also note that several of the ommatidia have bristles at four of their vertices. (B) Alterations in bristle size are common (arrow). (C) Fusion of ommatidia (fused ommatidia is marked by asterisk). (D) A silver-stained horizontal section shows an ectopic cluster of cells (arrowhead) that coincides with the eye protrusion (arrow). (E) The legs are occasionally duplicated. (F) Wing tissue and bristles are also lost in a vgGAL4/+; UAS SDΔ200/+ fly.

SD likely functions as a TF in tissues outside the wing disc:

To determine if the sole function of SD in larval tissues is to transport proteins into the nucleus, we overexpressed SDΔ88-123 and SDΔ88-159 (Figure 1) using the ptcGAL4 driver. The protein encoded by the SDΔ88-123 transgene contains a deletion in the first half of the TEA/ATTS DNA-binding domain that does not compromise the nuclear localization signal (NLS), while the deletion in the SDΔ88-159 removes the entire TEA/ATTS DNA-binding domain and the majority of the NLS. If the sole function of SD is to transport proteins into the nucleus, expression of the SDΔ88-159 but not the SDΔ88-123 transgene should be able to induce phenotypes similar to those observed by expressing VGΔACT and SDΔ200. Expression of either the SDΔ88-123 or the SDΔ88-159 proteins is able to induce similar eye, brain, wing, and leg phenotypes (data not shown), indicating that the role of SD in these tissues is not limited to transporting proteins into the nucleus.

Levels of SD are important in tissues outside the wing disc:

To assess whether the relative levels of SD are as important for the proper development of the eye, brain, and leg as they are in the wing (Halder et al. 1998; Simmonds et al. 1998), we overexpressed the full-length SD transgene (Figure 1) using the ptcGAL4 driver. Unfortunately, this causes pupal lethality. Removal of pharate adults from the pupal cases reveals phenotypes similar to those seen in the presence of the VGΔACT and the SDΔ200 proteins. Protrusion of the eyes (Figure 6A, arrowhead), truncations of the legs (Figure 6B, arrowheads), and an ectopic cluster of cells below the basal membrane of the ommatidia (Figure 6C) are all seen. In most of the pupae, the head of the fly is found in the abdomen (Figure 6D). Silver staining of the horizontal sections in these flies shows that the lamina and regions of the optic lobe are missing (Figure 6, C and E). The brain is also physically separated from the optic lobe (Figure 6E). However, the development of the ommatidia remains intact (Figure 6F).

Figure 6.—

Figure 6.—

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SD levels are important for proper development. Overexpression of wild-type SD in ptcGAL4/+; UAS SD/+ flies causes (A) eye protrusions (arrowhead) and (B) truncated legs (arrowheads). Overexpression of SD does not affect (C) the lobula (lo), medulla (la), or ommatidia (om), but does affect the lobula plate and lamina as these structures are missing. An ectopic cluster of cells is also present (arrowhead). In an extreme situation (D), the head remains inside the abdominal cavity (arrowhead). Silver staining of a horizontal cross section shows (E) that the brain (br) and the medulla (me) are located in the abdominal cavity and are no longer connected. The lamina and lobula are absent. The structure of the ommatidia (F) is preserved.

SD is required for cell survival in the leg disc:

One function of the SD/VG complex in the wing is to promote cell survival (Liu et al. 2000; Delanoue et al. 2004). To ascertain if SD also plays a role in promoting cell survival in other larval tissues, we looked for the presence of increased cell death in our transgenic flies using acridine–orange staining. In the wild-type wing disc, SD expression is restricted to the wing pouch and the periphery of the notum. Very little to no cell death is visible in the wing (Figure 7A) and leg (Figure 7B) imaginal discs of the wild-type fly. Staining of ptcGAL4; UAS SDΔ200 (Figure 7C, arrow) and ptcGAL4; UAS VGΔACT (data not shown) imaginal discs shows only increased acridine–orange staining along the anterior/posterior (A/P) boundary of the wing pouch. No increase in staining is seen along the A/P boundary of the notum (Figure 7C, arrowhead). Increased staining is also seen in the leg imaginal disc (Figure 7D). In addition to seeing an increase in staining in the leg discs, the leg discs may also be duplicated (Figure 7D). No notable change in acridine staining is visible in the larval optic lobes or in the eye-antennal imaginal disc (data not shown).

Figure 7.—

Figure 7.—

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SD promotes cell survival in the leg imaginal disc. Acridine–orange staining in a (A) wild-type wing imaginal disc, (B) wild-type leg imaginal disc, (C) ptcGAL4; UAS SDΔ200 wing imaginal disc, and (D) ptcGAL4; UAS SDΔ200 leg imaginal disc. Very little cell death is visible in the wild-type wing and leg imaginal discs. Ectopic expression of SDΔ200 induces cell death only in the (C) wing pouch (arrow) and not in the notum (arrowhead). Increased cell death is also visible in the (D) leg imaginal disc.

DISCUSSION

The generation of sd47M mitotic clones resulted in a mild phenotype in the eye and leg and had no effect in the optic lobe. Considering that induction of sd47M mitotic clones also caused a weak blistering phenotype in the wing (Liu et al. 2000), it is not surprising that they had a minor effect in tissues outside the wing. The weak phenotype is due to the inability of the clones to survive. No w, ry clones were seen in the eye, despite the fact that numerous blisters are present in the wing. However, the induction of clones is still able to cause a bristle phenotype, indicating that SD may be required for the survival of some of the eye cells.

To create a stronger phenotype, we attempted to antagonize the SD protein in the developing embryo and larva. This was accomplished by ectopically expressing a truncated form of VG, VGΔACT, which can still bind to SD but cannot activate transcription of downstream genes. Thus, we hoped that the interaction between the VGΔACT protein and SD would create an inert complex and consequently affect overall SD function. To test if the VGΔACT protein can antagonize SD function, we expressed it in the wing. Similar to sd mutant flies, the VGΔACT protein in the wing is able to induce wing-pouch-specific cell death (Figure 7C) (Liu et al. 2000) and to promote the loss of bristles and wing tissue (Figure 3B) (Campbell et al. 1992; Simmonds et al. 1998). These observations indicate that the VGΔACT protein likely binds to SD and disrupts its function in the wing.

While ectopic expression of full-length SD using either the actin or the 24BGal4 drivers causes embryonic lethality, the VGΔACT and the SD deletion proteins are unable to affect embryonic development. Several possibilities may explain why the VGΔACT protein is unable to disrupt SD function in the embryo: (1) proteins with which SD interacts in the embryo may utilize a domain that the VGΔACT protein is unable to affect or may bind to a region outside of the SDΔ200 protein, (2) the TIFs with which SD interacts in the embryo have a higher binding affinity than the VGΔACT protein, or (3) SD may have a TIF-independent role in the embryo. The inability of our construct to inhibit SD function in the embryo is consistent with the observation that lethal alleles of sd carrying a point mutation in either the VID or the putative TD do not cause embryonic lethality (Srivastava et al. 2004), but do cause pupal lethality. Insight into the regions of SD required for embryonic development may come from the fact that the SD human homolog TEF-1 is able rescue the embryonic lethal allele sdETX81 (Deshpande et al. 1997). These observations, taken together, indicate that the mechanism by which SD functions in the embryo is likely different from that in larval tissues.

The VGΔACT protein, however, is able to antagonize SD function in larval tissues. Ectopic expression of the protein is able to disrupt leg, eye, and brain development. In the leg, expression of the VGΔACT transgene is able to induce duplications and truncations (Figure 4, G and H). Several lines of evidence indicate that the phenotypes seen in the leg may be related to an induction of cell death, rather than to a defect in pattern formation. Previous reports have shown that high levels of apoptosis induced by ultraviolet (UV) light in the leg are able to induce splitting and duplication of the T2 and T3 but not T1 appendage (Arking 1974). Similar to the UV experiments, duplications caused by the VGΔACT protein in the leg disc are seen only in the second and third appendages. The lack of duplications seen in the T1 segment supports the idea that the phenomenon is related to cell death. Furthermore, dissections of VGΔACT/+; ptcGAL4/+ larvae show splitting of the leg imaginal disc and an increased level of cell death (Figure 7D). Finally, expression of the construct at the distal end of the leg with the DllGAL4 driver induces truncations at the tip of the appendage (Figure 4G).

Antagonizing the SD protein in the eye affects proper development of the bristles and ommatidia. Defects in bristles include changes in morphology and patterning. Previous reports have shown that SD is able to activate genes involved in sensory organ development (Halder et al. 1998; Srivastava and Bell 2003) and expression of the sdTEA∷VG fusion construct in the eye is able to alter bristle morphology (Srivastava et al. 2002). Thus, it is likely that SD has a role in patterning eye bristles. Another possible explanation for the bristle defects may be related to the cone cell phenotype. Pigment cell identity in the developing eye is based on cues provided by precursor cone cells (Cagan and Ready 1989a). The secondary and tertiary pigments can be sacrificed to form extra bristle cells (Cagan and Ready 1989b). Thus, it possible that the bristle phenotype may be due to defects in cone cell patterning. The ommatidial defects are seen on the surface of the fly eye and not in the horizontal sections. The inability to see these effects in the horizontal sections may be because ommatidial fusions occur at a low frequency in the eye (typically four to five fused ommatidia are seen in each eye) or because this inability is caused by cone-cell-patterning defects. Expression of the VGΔACT protein is also able to cause a protrusion in the adult eye (Figure 4B). Horizontal sections show that the protrusion is due to an unidentified ectopic layer of cells (Figure 4F). These cells are located between the lamina and the ommatidia (Figure 4F, arrowhead). Overexpression of SD induces similar, but more severe, phenotypes than when the VGΔACT or SDΔ200 proteins are expressed (Figure 6). In flies where SD is overexpressed, components of the optic lobe are lost (Figure 6, C and E). Thus, it is possible that the ectopic layer of cells originates from the optic lobe.

To determine if SD forms a TF complex in larval tissues, we expressed a truncated form of SD, SDΔ200 (Figure 1). The DNA-binding domain of SD and the NLS are deleted in the SDΔ200 transgene. However, the SDΔ200 protein should still bind to and potentially sequester SD-specific TIFs. Expression of the SDΔ200 protein (Figure 5) is able to induce phenotypes similar to those seen with the VGΔACT protein, suggesting that SD probably binds to cofactors in the eye, leg, and brain. In larval tissues, SD could interact with a putative TIF via the VID or perhaps even the TD. The existence of a TD domain in SD is supported by the observation that a mutation in the C-terminal end of the gene causes pupal lethality, but does not inhibit wing development (Srivastava et al. 2004). However, whether or not this association forms a TF complex is not clear. In the wing, SD is required to transport VG into the nucleus (Halder et al. 1998; Simmonds et al. 1998; Srivastava et al. 2002). Thus, it seemed possible that the sole function of SD in the leg, eye, and optic lobe would be to transport proteins into the nucleus. Expression of a truncated form of SD that contains an intact NLS, but is unable to bind to DNA, is still able to induce all the mutant phenotypes, indicating that SD function in larval tissues is not restricted to transporting proteins into the nucleus.

In other species, whenever a TEAD protein interacts with a TIF it forms a TF complex. In mammals, the TEAD protein (TEF-1) is known to interact with a variety of different TIFs, such as VGL-1 and VGL-3 in the placenta (Halder et al. 1998; Maeda et al. 2002), VGL-2 in skeletal muscles (Maeda et al. 2002), VGL-4 in the heart (Chen et al. 2004b), YAP65 (Vassilev et al. 2001), and p160 (Belandia and Parker 2000). In each case, the interaction between TEF-1 and its relevant TIF results in a TF complex. Thus, it is likely that in larval tissues the association between SD and putative TIFs also results in the formation of a TF complex (SD/TIF). The identity of putative TIFs with which SD may interact is currently unknown. Homology searches reveal three potential novel interacting partners for SD. The VGL-4 homolog in Drosophila is an unidentified gene, CG10741. To date there have been no studies done with CG10741. The p130 homolog, taiman, is widely expressed in the embryo and follicle cells in the larva (Bai et al. 2000). The YAP65 homolog, yorkie (yki), is another putative SD-interacting partner. Yki is known to control cell survival and cell proliferation (Huang et al. 2005). Yeast two-hybrid analysis shows with high confidence that SD and Yki interact with each other (Giot et al. 2003). Similar to VG, Yki contains an activation domain (Yagi et al. 1999); thus it is possible that Yki and SD interact with each other to form a functional TF complex. Presumably, this interaction occurs through the yet to be identified sd TD, as in humans YAP65 is known to interact with the TD of TEF-1. If Yki and SD form a complex, then it is possible that the Yki/SD complex may be the entity that is antagonized in the leg, eye, and/or brain.

One interesting property of SD is that its amount is important for proper wing development (Simmonds et al. 1998). A change in the ratio of SD to VG affects the development of the wing. Overexpressing SD causes phenotypes in leg, eye, and brain similar to those observed when the SD/TIF complex is antagonized. Why the levels of SD are important for its proper function remains unclear. In the wing, the SDVG complex requires two SD molecules and two VG molecules (Halder and Carroll 2001). SD can form homo-dimers (Halder et al. 1998) and thus the overexpression of SD may promote the formation of SD dimers as opposed to forming a tetrameric TF complex with its TIF. Another possibility may be that free SD may act as a repressor (personal communication with S. Veeraraghavan) and that overexpressing SD may lead to suppression of genes that are required to promote cell survival in the brain, eye, and leg.

When SD function is disrupted in the leg and the eye, a common phenotype observed is the loss of tissue. Bristles are lost in the eye (Figure 4D) and truncation can be seen in the legs (Figure 4G). Furthermore, the induction of clones in eye did not give rise to any w, ry clones. In the wing, SD has a crucial role in promoting cell survival (Liu et al. 2000; Delanoue et al. 2004) and this role may also be important in the eye and the leg. Acridine–orange staining of leg discs where SD is antagonized shows an increased amount of cell death (Figure 7D), suggesting that the complex is required to promote cell survival. Whether this is a direct relationship is currently unclear. The increased cell death may be because the cells in these tissues fail to properly differentiate, and consequently are unable to interpret the proper cues to survive. No increase in acridine–orange staining was observed in the eye disc (not shown). There is a high level of background acridine–orange staining (data not shown) and missing bristles occur only in a small percentage of the ommatidia, but this does not exclude the possibility that the SD/TIF complex is required to promote cell survival in these tissues.

Determining the role that SD has in promoting the fate of eye cells, leg cells, or optic lobe cells is difficult without knowing the identity of the possible TIFs with which it associates. Thus, the exact role for such a complex in these tissues must still be determined. However, we were able to gain some insight into the function of SD in these tissues by generating mitotic clones and expressing a truncated form of VG, VGΔACT. The VGΔACT protein is able to bind to SD and likely prevents TIFs from interacting with SD protein:protein interaction domains that include any of the amino acids between positions 220 and 344 (Vaudin et al. 1999). In the wing, the SD/VG complex is required to determine wing fate (Halder et al. 1998) and to promote cell survival (Liu et al. 2000; Delanoue et al. 2004). We have shown that the mechanism by which SD functions in the eye, leg, and brain is similar to that of SD in the wing imaginal disc. SD likely binds to tissue-specific TIFs to form a TF and this interaction occurs by a domain located in the C-terminal portion of the protein. The TF complex is also sensitive to the levels of SD present in the system and antagonizing SD function induces cell death. Thus, the SD/TIF complexes in larval tissues may be involved in promoting optic lobe, bristle, ommatidia, and leg fate or be involved in regulating cell proliferation and/or promoting cell survival. Studies aimed at identifying the TIFs or at determining the exact protein:protein interaction domains that these TIFs recognize may be able to identify the role of SD in these tissues.

Acknowledgments

We thank Ross Hodgetts and Andrew Simmonds for their help with the manuscript; Rakesh Bhatnagar and Jack Scott for their assistance with the confocal and scanning electron microscopes; and Randy Mandryk for his help with preparation and staining the fly horizontal sections. This work was funded by the National Science and Engineering Research Council (Canada) and the Alberta Ingenuity Fund.

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