Drosophila receptor tyrosine kinase signaling

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1 Development 129, (2002) Printed in Great Britain The Company of Biologists Limited 2002 DEV MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling Rachel K. Smith, Pamela M. Carroll*, John D. Allard and Michael A. Simon Department of Biological Sciences, Stanford University, 385 Serra Mall, Stanford, CA , USA *Present address: Department of Applied Genomics, Bristol-Myers Squibb, P.O. Box 5400, Princeton, NJ , USA Present address: Inflammatory Diseases Unit, Roche Bioscience, 3401 Hillview Ave, Palo Alto, CA , USA Author for correspondence ( msimon@stanford.edu) Accepted 10 October 2001 SUMMARY The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway. Key words: Receptor tyrosine kinase, Corkscrew, Ankyrin repeat, KH domain, Photoreceptor development, Drosophila INTRODUCTION Receptor tyrosine kinases (RTKs) play important roles in mediating cell-cell communication during a variety of cellular processes, including cell proliferation, cell viability, cell fate specification and cell motility [reviewed by Schlessinger (Schlessinger, 2000)]. These receptors use a common set of signaling molecules to transduce messages from the cell surface to the nucleus. These common effectors include members of the Ras/MAPK pathway and the protein tyrosine phosphatase Shp2/Corkscrew (CSW), as well as the PI3 kinase and PLCγ pathways, all of which are highly conserved in metazoan organisms [reviewed by Pawson (Pawson, 1995)]. One system that has proved useful in the study of RTK signaling is the specification of photoreceptors in the developing Drosophila eye [reviewed by Raabe (Raabe, 2000)]. Proper Drosophila eye development requires signaling through the RTKs EGFR and Sevenless (SEV) (Hafen et al., 1987; Freeman, 1996). The adult eye is composed of approximately 800 clusters of cells called ommatidia arrayed in a uniform repeating pattern. Each ommatidium contains six outer photoreceptors (R1-R6), two inner photoreceptors (R7 and R8), and a fixed number of accessory cells. Cell fate specification begins with the selection of the R8 photoreceptor, followed by a series of cell-cell communication events resulting in the recruitment of R1-R7 [reviewed by Tomlinson and Ready (Tomlinson, 1988; Ready, 1989)]. The differentiation of R1-R7 requires signaling through EGFR, with the R7 also requiring additional signals through Sevenless (Freeman, 1996). In addition to its role in cell fate specification, EGFR activity is also necessary for promoting proliferation and preventing apoptosis among the undifferentiated cells after patterning has begun (Baker and Yu, 2001). Through the study of signal-dependent processes such as photoreceptor differentiation, the roles of many components of the RTK signaling pathway have been well characterized [reviewed by Schlessinger (Schlessinger, 2000)]. Upon ligand binding, the receptor dimerizes and auto-phosphorylates, initiating signaling through the canonical Ras/MAPK pathway. In addition to recruiting adapter molecules that stimulate Ras signaling, tyrosine phosphorylation also creates docking sites on scaffolding molecules such as Daughter of Sevenless (DOS/Gab1) [reviewed by Huyer and Alexander (Huyer and Alexander, 1999)]. These scaffolding proteins provide additional binding sites for signaling components, including the protein tyrosine phosphatase CSW/Shp2 [reviewed by Pawson and Scott, and Huyer and Alexander (Pawson and Scott, 1997; Huyer and Alexander, 1999)].

2 72 R. K. Smith and others The mechanism by which CSW/Shp2 contributes to the transduction of the RTK signal is not completely understood. While CSW/Shp2 is required for RTK signaling and MAPK activation, genetic and biochemical experiments have not placed CSW/Shp2 activity at any one specific point within the linear Ras/MAPK pathway [reviewed by Huyer and Alexander (Huyer and Alexander, 1999)]. One proposed mechanism for CSW/Shp2 signal transduction is that it serves as an adapter between the receptor and DRK/Grb2, leading to Ras activation (Li et al., 1994; Bennett et al., 1994; Cleghon et al., 1998, Ronnstrand et al., 1999). However, several lines of evidence demonstrate that CSW/Shp2 has additional signaling roles. First, the putative DRK/Grb2 binding site of CSW/Shp2 may be removed without affecting signaling (Allard et al., 1998; O Reilly and Neel, 1998). Second, the effects of dominantly activated Raf can be blocked with a dominant negative CSW, suggesting a role for CSW downstream of or in parallel to Ras/Raf signaling (Allard et al., 1996). Third, the phosphatase activity of CSW/Shp2 is essential for signaling (Tang et al., 1995; Allard et al., 1998; Deb et al., 1998; Frearson and Alexander, 1998). Therefore, CSW/Shp2 must have a role in addition to that of an adapter and must have substrates whose dephosphorylation is critical for signal transduction. In order to elucidate the role of CSW during RTK signaling and identify potential targets, we conducted a sensitized genetic screen using the Drosophila eye. This screen identified mutations that dominantly modify the phenotype caused by the expression of a dominant negative form of CSW. Many known components of the RTK signaling pathway were identified, including DOS (Herbst et al., 1996). Since DOS plays a key role in activating CSW in response to RTK signaling, other genes isolated through this screen may also encode novel components of this pathway, perhaps including those involved in transducing the signal through CSW. Here we describe a second protein identified through this screen. MASK (Multiple Ankyrin repeats Single KH domain) is a novel, predominantly cytoplasmic protein that genetically interacts with components of RTK signaling pathways. MASK is crucial for photoreceptor differentiation, cell survival and cell proliferation in a manner similar to other signaling molecules that transduce the EGFR and SEV signals. However, unlike other pathway components, MASK is not absolutely required for the activation of MAPK, and potentially acts downstream of MAPK or defines a new branch of the RTK signaling pathway. MATERIALS AND METHODS Genetics Fly culture and crosses were performed using standard procedures. The csw CS screen was described previously (Herbst et al., 1996). mask alleles isolated from the screen were mask 10.22, mask 10.18, mask 8.7, mask 7.29, mask 7.13, mask 7.2, mask 6.3, and mask 5.8. The genotypes of flies used to test for genetic interactions were w 1118; P[sevhs-csw CS ], TM3/TM6b (Herbst et al., 1996), w 1118 ; P[sev-sev S11 ]/Cyo (Basler et al., 1991), w 1118, P[sevhs-csw src90 ] (Allard et al., 1996), P[sevras V12 ]/TM3 (Fortini et al., 1992), w 1118 ; P[sev-ras N17 ] (Allard et al., 1996), and w 1118 ; P[sev-rl SEM ]/Cyo (Brunner et al., 1994b). Clonal analysis Homozygous mutant clones in the eye were generated as previously described using FRT sites and the hs-flp recombinase (Xu and Rubin, 1993) or the ey-flp recombinase (Newsome et al., 2000). Homozygous wild-type tissue was eliminated using the l(3)cl-r3 1 allele (Newsome et al., 2000). Non-clonal tissue in the eye imaginal disc contained GFP. Non-clonal tissue in the adult eye contained pigment granules due to the presence of a functional w + transgene. The comparison between the homozygous mutant clone and homozygous wild-type twin spot was quantified using the Histogram function of Adobe Photoshop. Follicle cell clones were generated as previously described (Duffy et al., 1998). Eggs were collected on molasses/agar plates and prepared as previously described (Wasserman and Freeman, 1998). Histology Scanning electron microscopy was performed as previously described (Kimmel et al., 1990). Tangential sections (2 µm) of adult eyes were prepared as described previously (Tomlinson and Ready, 1987). Immunohistochemistry Third instar larval eye discs were dissected and stained essentially as described previously (Gaul et al., 1992) and mounted in Vectashield (Vector Laboratories, Inc.). Constructs and SL2 cells expressing SEV S11 and SEV S11 kinase dead were described previously (Basler et al., 1991; Simon et al., 1993). Cells were maintained and heat shock expression of SEV S11 was induced as described previously (Herbst et al., 1999). Cells were fixed in PLP on hydrophobic slides with a cell adhesion surface (Denville Scientific) and stained essentially as described for eye discs. Primary antibodies used were: rat monoclonal anti-elav (provided by G. M. Rubin), mab BP104 (Hortsch et al., 1990), mouse or rabbit anti-gfp (Molecular Probes), anti-phosphohistone H3 (Upstate Biotechnology), anti-diphosphorylated-erk1 and 2 (Sigma), and anti-mask. The MASK antibody was generated by injecting rabbits with a glutathione S-transferase/MASK fusion protein that contained amino acids 1047 to 1171 of the full-length MASK protein (Covance Research Products, Inc.). Secondary antibodies were obtained from Jackson Immunoresearch. All immunostained tissues were observed with a Biorad MRC 1024 and Nikon Eclipse E600 confocal microscope. TUNEL staining TUNEL staining kits used were the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics GmbH) and the In Situ Cell Death Detection Kit, TMR red (Boehringer Mannheim GmbH). Eye discs were stained as described above with modifications: the secondary antibody was diluted in the TUNEL reaction solution as prepared from the kits, and the discs were incubated for 3 hours at 37 degrees. Third instar larvae were cut open, and stained with TUNEL as described for eye discs. Cloning The mask alleles were mapped by recombination between P[w+] elements located at chromosomal positions 95E1 and 95F14. A cosmid genomic library was used to isolate clones from this region. Probes from the genomic cosmids were used to identify restriction fragment length polymorphisms (RFLPs). Recombination events between the RFLPs and mask were used to reduce the region known to contain mask to about 35 kb. Overlapping fragments of mask cdnas were isolated from the LD cdna library (Berkeley Drosophila Genome Project) using probes from this small genomic region. Nucleotide changes in mutant alleles were identified by sequencing PCR amplified sections of the coding region from heterozygous, genomic DNA (Schlag and Wassarman, 1999). mask approximately corresponds to the predicted transcripts CG18671, CG6268, and CG6313 in the annotated Drosophila genome.

3 MASK, a novel Drosophila RTK signaling protein 73 RESULTS mask was identified as a modifier of the csw CS phenotype In order to identify novel components of RTK signaling a sensitized genetic screen was conducted using a dominant negative form of the protein tyrosine phosphatase Corkscrew (CSW) (Herbst et al., 1996). This dominant negative isoform, CSW CS, contains a single amino acid change in the phosphatase catalytic domain, rendering the protein catalytically inactive. Expression of csw CS under the control of the sevenless promoter in the developing eye reduces the level of EGFR signaling in the presumptive R3 and R4 cells, and reduces the level of both EGFR and SEV signaling in the presumptive R7 cell. A complete block in CSW signaling in R3, R4 and R7 causes these cells to fail to differentiate, resulting in ommatidia that contain only four photoreceptors (Allard et al., 1996). Expression of CSW CS causes only a partial block in signaling and an intermediate average of 5.5 outer photoreceptors per ommatidium (Fig. 1D,E). The resulting phenotype is a roughened eye in which the normal regular array of ommatidial clusters has been disrupted (Fig. 1A,B). In a screen of 75,000 F 1 mutagenized progeny, eight EMS-induced mutations were identified that enhanced the CSW CS rough eye phenotype and represented alleles of the same gene based on a failure to complement for recessive lethality (Fig. 1C,F). These alleles defined a novel genetic locus we now call mask. Removal of one copy of mask in eyes expressing CSW CS reduced both the number of outer photoreceptors per ommatidium (from an average of 5.5 to 5.1) as well as the percentage of ommatidia that contain an inner R7 photoreceptor (from 93 percent to 68 percent) (Fig. 1G,H). Owing to differences in their ability to enhance the CSW CS rough eye phenotype, some alleles such as mask 5.8 and mask are considered strong loss-offunction alleles, and other alleles such as mask 6.3 and mask 7.29 are considered weaker hypomorphic alleles (data not shown). In addition to potentially mediating RTK and CSW signaling, mask is also an essential gene. Most mask homozygous and transheterozygous animals die during first instar larval development, however, the hypomorphic transheterozygous combination mask 6.3 /mask 7.29 produces some animals that survive to the third instar larval stage of development. Therefore, MASK is likely to be involved in RTK signaling or other essential signaling events during larval development. mask genetically interacts with members of the receptor tyrosine kinase signaling pathway Dominant negative and activated forms of known components of the receptor tyrosine kinase signaling pathway can affect photoreceptor differentiation when expressed in the developing eye (Basler et al., 1991; Fortini et al., 1992; Allard et al., 1996). If MASK is a component of the RTK signaling pathway, removal of one copy of the gene might be expected to modify these photoreceptor phenotypes. As described above, mask was identified owing to its ability to significantly enhance the photoreceptor phenotype of csw CS. In addition, the alleles mask and mask 5.8 suppressed the rough eye and ectopic R7 phenotypes caused by increased Fig. 1. mask was identified as a modifier of the csw CS phenotype. (A-C,I-L) Scanning electron micrographs of adult eyes. (D-F) Tangential sections of adult eyes. (A,D) w. (B,E) w; sevcsw CS /+. (C,F) w; sev-csw CS /+; mask /+. (G,H) The number of inner and outer photoreceptors per ommatidium was tabulated in w; sev-csw CS /+ (white bars) and w; sev-csw CS /+; mask /+ (black bars) eyes. (I-L) mask genetically interacts with ras. (I) ras V12 /+ (J) ras V12 / mask (K) w; ras N17 /+ (L) w; ras N17 /mask All constructs are expressed under the control of the sev promoter. In all figures, anterior is to the left, posterior is to the right. signaling through an activated SEV (sev S11 ) and an activated CSW (csw src90 ) (data not shown, Table 1). Furthermore, removal of one copy of mask weakly suppressed the rough eye phenotype caused by the expression of an activated Ras (ras V12 ) and weakly enhanced the rough eye phenotype caused by the expression of a dominant negative Ras (ras N17 ) (Fig. 1I,J,K,L). These results suggest that MASK plays a positive role in transducing the signal downstream of receptor tyrosine kinases.

4 74 R. K. Smith and others homozygous wild-type cells that are generated simultaneously, these genetically different tissues can be compared directly. The mask clones were significantly smaller than the accompanying homozygous wild-type clonal tissue, or twin spots (Fig. 2C,D). Since the mutant cells did not appear to be significantly smaller than wild-type cells (see Fig. 4C,F), this difference in tissue size may be due to reduced cell proliferation or increased cell death. Fig. 2. mask tissue is reduced in size. (A) Wild-type (w) third instar eye imaginal disc stained with α-bp104, a membrane marker for differentiating photoreceptors (Hortsch et al., 1990). (B) w; mask 6.3 /mask 7.29 third instar eye imaginal disc stained with α-bp104. (C) Loss-of-function mask clones lacking GFP were much smaller than the wild-type twin spots containing two copies of the GFP transgene. (D) For 21 clones examined, the size of the mutant clone was on average 20.8% the size of the twin spot. mask tissue is reduced in size In order to characterize the developmental phenotype of mask, third instar transheterozygous mask 6.3 /mask 7.29 larvae were examined. These animals were significantly smaller in size than typical wild-type third instar larvae (data not shown). Upon dissection, the eye-antennal imaginal discs of the mask 6.3 /mask 7.29 larvae were smaller (Fig. 2A,B). However, progression of the morphogenetic furrow proceeded, with some photoreceptor differentiation occurring posterior to the furrow (Fig. 2A,B). In order to compare further the development of mask tissue with the development of wild-type tissue, homozygous mask clones were generated using hs-flp in the eyeantennal imaginal disc (Xu and Rubin, 1993). Because mitotic recombination creates patches of homozygous mutant cells and Table 1. mask genetically interacts with components of the Sevenless signaling pathway Avg. no. of ectopic R7 Genotype cells per ommatidium No. of ommatidia scored w; csw src90 / w; csw src90 /+; mask / w; csw src90 /+; mask 5.8 / w; sev S11 / w; sev S11 /+; mask / w; sev S11 /+; mask 5.8 / Eyes were tangentially sectioned, and the number of R7 cells per ommatidium in the posterior section of each eye was counted. The reduction in the number of R7 cells per ommatidium when mask is heterozygous is similar to the effects seen with other members of the signaling pathway, and is consistent with a role for MASK in transducing this signal. Loss of MASK increases programmed cell death During normal development, scattered apoptotic cells are found in the third instar eye imaginal disc posterior to the morphogenetic furrow [reviewed by Bonini and Fortini (Bonini and Fortini, 1999)]. Loss of EGFR or Ras in clones in the developing eye results in elevated levels of apoptosis (Baker and Yu, 2001; Halfar et al., 2001). To assess whether loss of MASK also induces cell death, homozygous mask clones were generated in third instar eye imaginal discs and stained by terminal dutp nick end labeling (TUNEL), which marks apoptotic nuclei (Sgonc et al., 1994). Many cells in the mutant clones exhibited TUNEL staining when compared with the surrounding wild-type tissue, suggesting a requirement for MASK in maintaining cell viability (Fig. 3A-C). However, this increase in apoptosis may be explained by a deficiency in the growth and proliferation of the mask cells, resulting in their elimination in favor of the surrounding wild-type cells. To address this question, eye imaginal discs of third instar mask 6.3 /mask 7.29 larvae were examined with TUNEL staining. An extremely high level of TUNEL staining occurred among the cells in these mutant discs, which were not in competition with any wild-type cells (Fig. 3D,E). The alimentary canal and many other tissues throughout these hypomorphic mutant larvae also exhibited extremely elevated levels of TUNEL staining (Fig. 3F,G). mask enhances a novel apoptosis phenotype of csw CS Several members of the EGFR and Ras/MAPK signaling pathway are required for the maintenance of cell viability, however, CSW has not previously been implicated as a cell survival signaling molecule in the developing eye (Bergmann et al., 1998; Kurada and White, 1998; Baker and Yu, 2001; Halfar et al., 2001). mask was isolated as an enhancer of the dominant negative csw CS rough-eye phenotype. While this eye roughness is mainly due to a loss of photoreceptors, the concomitant reduction in eye size may indicate that removal of one copy of mask also enhances a previously unidentified cell death phenotype caused by the expression of a dominant negative CSW. In order to establish a requirement for CSW and MASK in the prevention of apoptosis, third instar eye imaginal discs were stained by TUNEL to mark apoptotic nuclei. Discs from males hemizygous for the loss-of-function allele csw C114 (Perkins et al., 1992) showed a significant increase in TUNEL staining (Fig. 3D,H). Discs expressing csw CS under the control of the sevenless promoter also showed a significant increase in TUNEL staining posterior to the morphogenetic furrow (Fig. 3I). In addition, a zone of TUNEL staining was seen anterior to the morphogenetic furrow, where apoptosis is not observed in wild-type discs but is seen in EGFR ts loss-of-function discs (Baker and Yu, 2001) (Fig. 3D,H,I). Removal of one copy of

5 MASK, a novel Drosophila RTK signaling protein 75 Fig. 3. Loss of MASK increases programmed cell death and reduces proliferation. (A-C) mask clones in third instar larval eye imaginal discs. Non-clonal cells are marked with GFP (green) and apoptotic nuclei are marked with TUNEL-TMR (red). (D,E) Third instar eye imaginal discs are stained with antibodies against Elav, which is expressed in photoreceptor nuclei (red), and TUNEL-fluorescein, which marks the nuclei of apoptotic cells (green). White arrows mark the morphogenetic furrow. (D) Wild type (w); (E) w; mask6.3/mask7.29. (F,G) Alimentary canal tissue from third instar larvae stained for apoptotic nuclei with TUNEL-fluorescein (green). (F) Wild type (w); (G) w; mask6.3/mask7.29. (H-J) Third instar eye imaginal discs. As above, α-elav marks photoreceptor nuclei (red) and TUNEL-fluorescein marks apoptotic nuclei (green). White arrows mark the morphogenetic furrow. H) w, csw114 In these mutants, the morphogenetic furrow does not progress farther than is shown. (I) w; sev-cswcs/+; (J) w; sev-cswcs/mask (K,L) Mutant clones (marked as cells not expressing GFP; green) in eye imaginal discs were stained with α-phosphohistone H3 (red) to visualize nuclei in M phase. (K) Clones generated using hs-flp were small and rarely contained any mitotic cells. (L) Clones generated using ey-flp were larger and contained more cells undergoing mitosis. White arrowheads mark zones of mitosis before and after the morphogenetic furrow. the mask gene in the cswcs background enhanced the number of cells exhibiting TUNEL staining, both posterior and anterior to the morphogenetic furrow (Fig. 3J). The ability of mask alleles to enhance the loss of photoreceptor differentiation in eyes expressing cswcs may be due to a reduction in viability of those photoreceptors. However, an extensive search for TUNEL staining of differentiating photoreceptors in cswcs and cswcs/mask discs revealed no neurons undergoing apoptosis (data not shown). These results indicate that MASK and CSW act in a crucial signaling pathway responsible for maintaining cell viability in undifferentiated cells. Loss of MASK reduces cell proliferation The reduction in mask tissue size could result from reduced cell proliferation as well as increased cell death. Ras and EGFR mutant clones exhibit reduced proliferation due to a phenomenon called cell competition, in which neighboring wild-type cells grow faster and therefore divide preferentially over mutant cells (Prober and Edgar, 2000; Baker and Yu, 2001). To examine proliferation directly in homozygous mask clones, eye imaginal discs were stained with α-phosphohistone H3 antibodies, which stain the nuclei of cells undergoing mitosis (Hendzel et al., 1997). Two waves of proliferation occur in wild-type third instar eye imaginal discs, one preceding the morphogenetic furrow, and a second wave following the onset of photoreceptor differentiation [reviewed by Wolff and Ready (Wolff and Ready, 1993)]. Small mask clones induced with the hs-flp very rarely contained cells in M phase (Fig. 3K). However, in larger mutant patches induced with the more efficient ey-flp the pattern of cell division within the mitotic zones continued through the tissue unaltered (Fig. 3L). These results suggest that the ability of a cell lacking MASK to undergo mitosis may depend on the genotype of its neighboring cells. In larger clones mask cells are next to other mask cells, and are able to proliferate. In smaller clones mask cells must compete with neighboring wild-type cells, and are only infrequently able to divide. Loss of MASK inhibits photoreceptor differentiation In addition to affecting tissue size, removal of MASK function within the developing eye also resulted in a loss of photoreceptor specification. Eye imaginal discs from transheterozygous mask6.3/mask7.29 larvae contained differentiating photoreceptors (Fig. 3B). However, when these discs were stained with α-elav, a marker for photoreceptor

6 76 R. K. Smith and others Fig. 4. Loss of MASK inhibits photoreceptor differentiation. (A,B) Third instar larval eye imaginal discs stained with α-elav to mark photoreceptor nuclei. (A) Optical section of a wild-type disc; each ommatidial cluster contains eight photoreceptor nuclei. (B) Optical section of a mask 6.3 /mask 7.29 disc. Yellow arrows indicate ommatidial clusters containing fewer than eight photoreceptor nuclei. (C-E) A third instar larval eye imaginal disc containing a mask clone. Arrows mark incomplete ommatidia. (C) Photoreceptor nuclei are stained with α-elav. (D) The clone is identified as those cells not expressing GFP. (E) Color merge: Elav is in red and GFP is in green. (F,G) Tangential sections of adult eyes. (F) Small mask clone induced by the expression of hs-flp. White arrows indicate ommatidia missing photoreceptors. (G) A portion of a large mask clone induced by the expression of ey-flp. Almost all ommatidia are missing at least one photoreceptor. nuclei, many ommatidia did not contain the normal complement of eight photoreceptors (Fig. 4A,B). In order to examine photoreceptor differentiation using a stronger mask allele, homozygous mutant clones of mask were analyzed. While some cells lacking MASK function began photoreceptor differentiation, the ommatidia within the clones did not contain all eight photoreceptors (Fig. 4C,D,E). This partial block in photoreceptor differentiation is consistent with a role for MASK in EGFR and SEV signaling. In order to assay the requirement for MASK function in individual photoreceptors, mask 6.3, mask 7.29 and mask clones were induced in flies with the genotype w,hs-flp; FRT82B, mask/frt82b, P[w+]. The adult eyes were tangentially sectioned, revealing a loss of outer and inner photoreceptors in many clonal ommatidia (Fig. 4F). Ommatidia along the borders of the mutant clones contained both wild-type and mutant photoreceptors, which could be distinguished by the presence or absence of pigment granules, respectively. The photoreceptors in these normally constructed, mosaic ommatidia were assayed for their ability to differentiate without MASK function. If a particular photoreceptor does not require MASK function for differentiation, one would expect that photoreceptor to be wildtype in 50% of the ommatidia scored and mutant in 50% of the ommatidia scored. However, in mask clones, fewer than 50% of photoreceptors scored in each class (R1-R8) were mutant, indicating a partial requirement for MASK function for their differentiation (Table 2). Strikingly, no R7 cells in mosaic ommatidia in clones made with mask were mutant. Only a small number of R7 photoreceptors in mosaic ommatidia in clones generated using the hypomorphic alleles mask 6.3 and mask 7.29 were mutant (Table 2). In addition, most R7 cells were missing from ommatidia within the clones. The numbers of mutant R1 and R6 photoreceptors were also significantly reduced in mosaic ommatidia from mask clones. From these data, the most stringent requirement for MASK is in the differentiation of the R7 photoreceptor, followed closely by R1 and R6. R1, R6 and R7 are all derived from cells that have undergone a round of cell division after the progression of the morphogenetic furrow and differentiation of R8, R2, R5, R3 and R4 [reviewed by Wolff and Ready (Wolff and Ready, 1993)]. Therefore, the increased sensitivity of R1, R6 and R7 to loss of MASK may be an indirect result of a reduced ability of mask mutant cells to proliferate. In order to determine whether R1, R6 and R7 truly require MASK for differentiation or are preferentially recruited from among the more abundant wild-type cells, large clones were created in which ommatidia were not developing near an abundance of wild-type cells that could be recruited as photoreceptors. Homozygous mutant clones were made in eyes of the genotype w,ey-flp; FRT82B, P[w + ]90E, l(3)cl-r3 1 / FRT82B, mask The l(3)cl-r3 1 is a cell-lethal mutation, causing the elimination of the homozygous wild-type twin-spot cells (Newsome et al., 2000). Mutant cells were able to proliferate and form much of the adult eye. However, many photoreceptors, including the majority of R7 cells, failed to differentiate (Fig. 4G). Taken Fig. 5. Loss of MASK affects RTK-dependent processes other than eye development. (A) Dorsal appendages produced by wild-type follicle cells. (B,C) Dorsal appendages produced by mask clones in the follicle cells.

7 MASK, a novel Drosophila RTK signaling protein 77 Fig. 6. MASK is not required for MAPK activation or translocation. (A) A stacked confocal image of a wild-type third instar eye imaginal disc stained with α-diphospho- MAPK. In all panels an arrow indicates the location of the morphogenetic furrow; arrowheads mark nuclei containing activated MAPK. (B) A single confocal slice showing cytoplasmic activated MAPK in a mask clone. (C) A single confocal slice showing nuclear activated MAPK in a mask clone. (B,C ) High magnification of α-diphospho-mapk staining. (B,C ) GFP (B,C ) Color merge. (D) mask suppresses the ectopic R7 phenotype of rl SEM. together, these data indicate that MASK activity is required to properly specify the fate of photoreceptors, particularly R7, in the developing eye. Loss of MASK affects RTK-dependent processes other than eye development Signaling through EGFR in the ovarian follicle cells is required for proper construction of the egg [reviewed by Ray and Schupbach (Ray and Schupbach, 1996)]. Follicle cells that are mutant for many components of the RTK pathway results in dorsal appendages that are spaced too closely, are completely fused, or are absent altogether (Wasserman and Freeman, 1998). In order to establish a requirement for mask in egg shell patterning, homozygous mask clones were generated in the ovarian follicle cells. Eggs from females containing mask clones had dorsal appendages that were close together or fused (Fig. 5). No fused dorsal appendages were seen in the absence of a mask allele (data not shown). A role for MASK in this EGFR-signaling-dependent process further supports MASK as a common component of this signaling pathway. Loss of MASK does not affect MAPK activation A major portion of the signal initiated by receptor tyrosine kinases is transduced through the Ras/MAPK pathway [reviewed by Guan (Guan, 1994)]. In the developing eye, EGFR and Sevenless signaling lead to the activation of Ras, which initiates a downstream chain of events resulting in the phosphorylation and nuclear translocation of MAPK [reviewed by Raabe (Raabe, 2000)]. CSW/Shp2, whose relationship to the Ras pathway is unclear, is also required for MAPK activation and translocation (Tang et al., 1995; Bennett et al., 1996; Deb et al., 1998; Frearson and Alexander, 1998; Shi et al., 1998; Ghiglione et al., 1999; Oh et al., 1999; Bjorbak et al., 2000; Lorenzen et al., 2001). In order to determine whether MASK is required for MAPK activation and translocation, homozygous mask clones were generated in third instar eye discs and stained with an antibody that specifically recognizes diphosphorylated, activated MAPK (Yung et al., 1997). In differentiating photoreceptors, activated MAPK is in the cytoplasm of cells in the morphogenetic furrow, and moves into the nucleus after the furrow has advanced several rows (Kumar et al., 1998) (Fig. 6A). Diphosphorylated MAPK was seen both in the cytoplasm and in the nuclei of cells within mask clones (Fig. 6B,C). While MAPK activation and localization appeared unaltered, the possibility remains that a few individual cells failed to activate MAPK. This result indicated that MASK function is not absolutely required for the activation or translocation of MAPK. Activated MAPK is produced by the rolled Sevenmaker (rl SEM ) allele, and induces the differentiation of ectopic R7 cells in many ommatidia in the developing eye (Brunner et al., 1994b). In order to further examine whether a reduction in MASK activity affects MAPK signaling, one copy of mask was removed in the rl SEM background. This reduction in MASK activity partially suppressed the activated MAPK phenotype, significantly reducing the average number of ectopic R7 cells per ommatidium from 1.6 to 1.2 (Fig. 6D). Because MASK genetically interacts with MAPK yet does not appear to be required for its activation, MASK may act downstream of or in parallel to MAPK. Photoreceptor class Table 2. mask photoreceptors within normally constructed mosaic ommatidia mask 6.3 mask 7.29 mask n=48 n=49 n=32 # mutant % mutant # mutant % mutant # mutant % mutant R R R R R R R R * Homozygous mutant clones were induced in flies of the genotype hs-flp; FRT82B, mask / FRT82B, P[w+]90E. Photoreceptors within normally constructed, mosaic ommatidia were examined for the presence or absence of pigment granules as a marker for the presence or absence of MASK function. The number of mutant photoreceptors in each class is an indication of the ability of that photoreceptor to differentiate without MASK function. mask 6.3 and mask 7.29 are hypomorphic alleles, mask is a strong allele. *n=35, n=37.

8 78 R. K. Smith and others Fig. 7. MASK is a large protein containing ankyrin repeats and a KH domain. (A) Genomic structure of the mask locus (GenBank accession number AF425651). The light gray boxes represent the stretches of ankyrin repeats, and the dark gray box represents the KH domain. Arrowheads indicate the positions of amino acids that have been changed to stop codons in mutant alleles. (B) Stretches of homology between MASK and predicted proteins from cdna sequences from other species. Dark gray shading indicates identical residues, and light gray shading indicates similar residues. MASK is a large protein containing ankyrin repeats and a KH domain The EMS-induced alleles of mask were mapped by recombination between P-elements and restriction fragment length polymorphisms (RFLPs). The mutations were localized to a small region within chromosomal bands 95E-F. Probes from this region were then used to isolate cdnas from a library, which were cloned and sequenced. Several overlapping cdnas were isolated and a long open reading frame of approximately 13 kb was identified. By sequencing mutant alleles, single nucleotide changes resulting in premature stop codons were found in mask 10.22, mask 5.8 and mask 8.7 (Fig. 7A). These three mask alleles with stop codons in the same gene confirmed the identity of this transcript as mask.

9 MASK, a novel Drosophila RTK signaling protein 79 The mask gene spans 18 kb of genomic DNA and encodes a protein 4001 amino acids long, with a predicted mass of 423 kda (GenBank accession number AF425651). The MASK protein contains structural motifs found in other proteins; two blocks of ankyrin repeats and a KH domain (Fig. 7A). Ankyrin repeats are found in proteins with a wide variety of functions and are known to mediate protein-protein interactions [reviewed by Sedgwick and Smerdon (Sedgwick and Smerdon, 1999)]. KH domains bind to RNA or DNA, and are found in proteins that process RNA, mediate transcription, and associate with heterochromatin (Gibson et al., 1993; Siomi et al., 1994; Tomonaga and Levens, 1995; Davis-Smyth et al., 1996; Dejgaard and Leffers, 1996; Cortes and Azorin, 2000). In addition, MASK contains several long stretches of glutamine residues and a highly basic region. MASK does not show significant homology in sequence or overall structure to any protein of known function. However, a search of cdna databases revealed many transcripts in other species that may define a MASK-like family of proteins (Fig. 7B). In particular, a C. elegans cdna, R11A8.7, encodes a predicted protein that contains both ankyrin repeats and a KH domain, as well as other regions that are quite similar to MASK. MASK predominantly localizes to the cellular cytoplasm An antibody was raised against a portion of MASK that lies between the two blocks of ankyrin repeats. The specificity of the antibody was tested by staining third instar eye imaginal discs containing mask clones. This allele contains a stop codon before the sequence used as the antigen, and therefore clones should contain no protein recognized by the antibody. This antibody clearly stained wild-type tissue, but did not stain within the mask clones (Fig. 8A,B). Upon closer examination of eye imaginal discs stained with the α-mask antibody, MASK appeared to be ubiquitously expressed, and was localized primarily in the cytoplasm (Fig. 8C). Expression appeared to be somewhat higher in presumptive photoreceptors (Fig. 8D). In order to further characterize the localization of MASK, S2 cells containing an activated Sevenless (SEV S11 ) and S2 cells containing SEV S11 with a mutation rendering the kinase inactive were stained with the MASK antibody (Fig. 8E,F). In both cases, MASK was mainly localized in the cytoplasm. In order to confirm the ubiquitous expression of mask, animals throughout embryonic and early larval development were stained with the MASK antibody. Expression was ubiquitous throughout these stages (data not shown). Based on these data, MASK is expressed ubiquitously during development and is localized primarily in the cytoplasm. While localization does not change when the RTK signal is activated, expression appears to be higher in differentiating photoreceptors. DISCUSSION MASK is a novel protein involved in RTK signaling We have described the identification and characterization of the large ankyrin repeat and KH domain containing protein MASK. Several lines of evidence indicate that MASK plays a role in receptor tyrosine kinase signaling. First, mask was identified through a genetic screen that has successfully Fig. 8. MASK is localized in the cellular cytoplasm. (A,B) mask clones. The MASK antibody (A) only stains the GFP-marked cells containing wild-type MASK (B). (C) Third instar wild-type eye disc stained with α-mask. The morphogenetic furrow is marked with white arrows. (D) A section at high magnification showing increased α-mask staining around the nuclei of R8 photoreceptors. (E,F) S2 cells expressing signaling proteins, stained with α-mask. (E) hs-sev S11 (F) hs-sev S11 kinase dead. identified other components of the RTK/CSW signaling pathway. Second, mask genetically interacts with components of the SEV and EGFR signaling pathways in the Drosophila eye. Third, the homozygous mask phenotype affects photoreceptor differentiation, cell survival and proliferation in the developing eye in a manner similar to CSW, SEV, EGFR and other molecules that transduce the RTK signal. Finally, mask clones in follicle cells produced a dorsal appendage phenotype similar to that caused by loss of EGFR signaling in follicle cells. Loss of MASK compromises cell differentiation, cell survival and cell proliferation MASK activity is required for the differentiation of all developing photoreceptors, although the penetrance of this phenotype varies depending on the photoreceptor type examined. The strongest requirement for MASK appears to be in R1, R6 and R7. The R7 photoreceptor is often the most sensitive to decreases in RTK signaling, since its specification requires the activation of both EGFR and SEV (Freeman, 1996). R1, R6 and R7 are all recruited from cells that have undergone mitosis during the second mitotic wave, and these cells are often related (Hofbauer and Campos-Ortega, 1976). This relatedness may account for the apparent increased sensitivity of R1 and R6 to a reduction in signaling. EGFR and Ras are important in actively promoting cell survival in Drosophila and in mammalian systems [reviewed by McNeill and Downward (McNeill and Downward, 1999)]. EGFR is required in developing Drosophila eyes to maintain the viability of undifferentiated cells surrounding the ommatidial preclusters (Baker and Yu, 2001). Removal of Ras

10 80 R. K. Smith and others in clones in the wing imaginal disc and the eye imaginal disc leads to an increase in apoptosis (Prober and Edgar, 2000; Halfar et al., 2001). Developmental apoptosis is promoted through the activity of three Drosophila genes: hid, grim and reaper [reviewed by Bangs and White (Bangs and White, 2000)]. Ras activity leads to the downregulation of Hid, both through transcriptional regulation and phosphorylation by MAPK (Bergmann et al., 1998; Kurada and White, 1998). The role of CSW in preventing apoptosis is not as clearly defined. However, recent studies in mammalian systems have implicated the CSW homolog Shp2 in signal-mediated cell survival (Chauhan et al., 2000; Wu et al., 2000). As demonstrated here, CSW and MASK also play an important role in maintaining cell viability. Whether this anti-apoptotic signal is mediated by Ras and Hid or defines a new regulatory mechanism remains to be seen. Recent studies have demonstrated that signaling through EGFR and Ras is important for proliferation in developing Drosophila imaginal discs (Prober and Edgar, 2000; Baker and Yu, 2001). Cells in clones lacking Ras in the wing imaginal disc do not grow or undergo division at the same rate as their wild-type neighbors (Prober and Edgar, 2000). An inability to transduce the Ras signal leads to a disadvantage among these competing cells. Similarly, studies of EGFR signaling in the developing eye imaginal disc have demonstrated that undifferentiated cells posterior to the morphogenetic furrow undergo cell division only if they receive a signal from neighboring developing photoreceptors (Baker and Yu, 2001). Such developmental regulation of cell division is essential for the proper patterning of the adult eye. Likewise, cells that lack MASK function seem to undergo mitosis at a lower rate than their neighbors, suggesting that the mask cells may be at a similar competitive disadvantage. This requirement for MASK activity in specifying cell fate, maintaining cell viability, and promoting cell proliferation is consistent with the known roles of RTK signaling during Drosophila eye development. How does MASK transduce the RTK signal? The phenotypic characterization of MASK presented here has not determined the mechanism by which MASK mediates signaling downstream of RTKs. MASK was isolated through a genetic screen designed to identify proteins that interact with CSW. While DOS, the first novel signaling component isolated through this screen, does interact directly with CSW, no evidence presented here suggests that MASK can as well (Herbst et al., 1996). mask alleles genetically interact with both dominant negative and activated alleles of ras and csw. However, unlike both Ras and CSW, MASK function does not seem to be required for normal levels of MAPK activation. Several lines of evidence suggest that Shp2, the vertebrate homolog of CSW, can transduce signals through mechanisms that do not affect MAPK activation. First, dissociation of Shp2 from the scaffolding protein Gab2 (a vertebrate homolog of DOS) prevents signal-induced transcription without affecting MAPK activation (Gu et al., 1998). Second, Shp2 is required for Insulin-like Growth Factor stimulated FAK dephosphorylation and cellular chemotaxis, processes that are unperturbed when MAPK activation is eliminated (Manes et al., 1999). Third, expression of activated Shp2 in Xenopus animal caps induces elongation without significantly increasing MAPK activity (O Reilly et al., 2000). Also, Shp2 negatively regulates gene expression induced by Leukemia Inhibitory Factor signaling in a MAPK-independent manner (Bartoe and Nathanson, 2000). In addition, Shp2 has been shown to act upstream of the small GTPase RhoA during filamentous actin remodeling in response to the activity of growth factor receptors (Schoenwaelder et al., 2000). Finally, mutations in Shp2 eliminate NF-κB signaling and IL-6 induction in response to IL-1α and TNF-α signaling, without affecting MAPK activation (You et al., 2001). MASK may mediate a part of the CSW/Shp2 signal that is downstream of or parallel to MAPK. Alternatively, MASK may respond to RTK signaling through an entirely new mechanism. MASK does not show overall homology to any protein of known function, although it does contain several wellcharacterized domains. The presence of the ankyrin repeats and KH domain, the predominantly cytoplasmic localization of the protein, and the largely unaffected MAPK activation allow for some speculation as to the function of this protein. Signaling through RTKs such as EGFR and SEV affect the Ets-domain transcription factors Yan (also known as AOP), a transcriptional inhibitor, and PNT, a transcriptional activator (Brunner et al., 1994a; O Neill et al., 1994; Rebay and Rubin, 1995). Since MASK is not absolutely required for MAPK activity it may act downstream, perhaps as a transcriptional cofactor of PNT or a repressor of Yan. Such a role in transcription is also suggested by the fact that MASK contains a KH domain, many long stretches of glutamines, and a highly basic region that could be a bi-partite nuclear localization sequence. One example of such a transcriptional cofactor is found in mammalian cells. The GA Binding Protein α (GABPα) is an Ets-domain transcription factor that is only active when bound to its partner GABPβ, an ankyrin repeat containing transcriptional cofactor (LaMarco et al., 1991; Thompson et al., 1991; Batchelor et al., 1998). The antibody staining presented here shows that MASK is localized predominantly in the cytoplasm, and therefore a nuclear function for this protein is unlikely. However, MASK levels in the nucleus may be extremely low or transient and therefore difficult to detect. Alternatively, the protein may be cleaved, with a portion not recognized by the antibody entering the nucleus. MASK could also act downstream of MAPK by binding to Yan while localized in the cytoplasm, sequestering the transcriptional repressor. Signaling may be transduced not only through transcriptional activation, but also through regulating the translation and mrna stability of downstream effectors. The nuclear signaling molecule Split ends (SPEN) contains RNA binding motifs and may act to downregulate Yan translation (Chen and Rebay, 2000; Kuang et al., 2000; Rebay et al., 2000). Since MASK contains a potential RNA-binding KH domain, it may also act post-transcriptionally, mediating the effects of RTK signaling on mrna in the cytoplasm. Alternatively, the presence of two blocks of ankyrin repeats suggests a role for MASK as a scaffolding protein, bringing together many signaling molecules, perhaps at particular DNA sequences or on particular mrnas. The exact role of MASK in transducing the RTK signal will be determined by further study. The presence of close homologs in other systems indicates that MASK is a conserved, essential signaling molecule. Understanding the role of this novel protein in

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