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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Dev Biol. 2014 Mar 29;390(2):170–180. doi: 10.1016/j.ydbio.2014.03.012

The Drosophila Wilms’ Tumor 1-Associating Protein (WTAP) Homolog is Required for Eye Development

Abigail M Anderson 1, Brandon P Weasner 1, Bonnie M Weasner 1, Justin P Kumar 1,*
PMCID: PMC4063124  NIHMSID: NIHMS588578  PMID: 24690230

Summary

Sine Oculis (So), the founding member of the SIX family of homeobox transcription factors, binds to sequence specific DNA elements and regulates transcription of downstream target genes. It does so, in part, through the formation of distinct biochemical complexes with Eyes Absent (Eya) and Groucho (Gro). While these complexes play significant roles during development, they do not account for all So-dependent activities in Drosophila. It is thought that additional So-containing complexes make important contributions as well. This contention is supported by the identification of nearly two-dozen additional proteins that complex with So. However, very little is known about the roles that these additional complexes play in development. In this report we have used yeast two-hybrid screens and co-immunoprecipitation assays from Kc167 cells to identify a biochemical complex consisting of So and Fl(2)d, the Drosophila homolog of human Wilms’ Tumor 1-Associating Protein (WTAP). We show that Fl(2)d protein is distributed throughout the entire eye-antennal imaginal disc and that loss-of-function mutations lead to perturbations in retinal development. The eye defects are manifested behind the morphogenetic furrow and result in part from increased levels of the pan-neuronal RNA binding protein Embryonic Lethal Abnormal Vision (Elav) and the RUNX class transcription factor Lozenge (Lz). We also provide evidence that So and Fl(2)d interact genetically in the developing eye. Wilms’ tumor-1 (WT1), a binding partner of WTAP, is required for normal eye formation in mammals and loss-of-function mutations are associated with some versions of retinoblastoma. In contrast, WTAP and its homologs have not been implicated in eye development. To our knowledge, the results presented in this report are the first description of a role for WTAP in the retina of any seeing animal.

Keywords: sine oculis, fl(2)d, elav, lozenge, eye development, Drosophila

Introduction

In Drosophila, the Sine Oculis (So) homeobox transcription factor is a critical member of the retinal determination (RD) network and it plays a central role in the development of the eye (Cheyette et al., 1994; Serikaku and O’Tousa, 1994; Pignoni et al., 1997; Weasner et al., 2007; Kumar, 2009; Anderson et al., 2012; Atkins et al., 2013; Weasner and Kumar, 2013). It appears to have dual roles in regulating gene expression within the retina. On the one hand, So promotes eye development via transcriptional activation of several RD genes including itself, eyeless (ey), eyes absent (eya) and dachshund (dac: Halder et al., 1998; Pauli et al., 2005; Pappu et al., 2005), the patterning gene hedgehog (hh: Pauli et al., 2005) and several cell fate genes such as atonal (ato) and lozenge (lz: Yan et al., 2003; Zhang et al., 2006). However, So is simultaneously required to repress the expression of head capsule and antennal selector genes such as cut (ct) and Lim1 during regional specification of the eye-antennal disc (Salzer and Kumar, 2009; Anderson et al., 2012; Wang and Sun, 2012; Weasner and Kumar, 2013). And behind the morphogenetic furrow, So stops promoting ey expression and instead is required to inhibits its transcription (Atkins et al., 2013). The ability of So to modulate transcription of downstream target genes is dependent upon interactions with Eyes Absent (Eya) and Groucho (Gro) (Pignoni et al., 1997; Kenyon et al., 2005; Anderson et al., 2012). These interactions are conserved in vertebrate systems as well (Ohto et al., 1999; Kobayashi et al., 2001; Zhu et al., 2002). However, the So-Eya and So-Gro complexes do not fully account for all So-dependent activities in either Drosophila or vertebrates. Over the last decade several yeast two-hybrid screens have identified approximately 25 additional factors that could also form biochemical complexes with So (Pignoni et al., 1997; Giot et al., 2003; Kenyon et al., 2005; Neilson et al., 2010). While these complexes are likely to make significant contributions to tissue specification and pattern formation, very little is know about their roles in regulating development in any experimental system.

Here, we report the identification of a biochemical complex containing So and Fl(2)d, the fly homolog of Wilms’ Tumor 1-Associating Protein (WTAP: Penalva et al., 2000). During sex determination, Fl(2)d plays an important role in the female-specific splicing of both Sex-lethal (Sxl) and transformer (tra) pre-mRNA transcripts (Granadino et al., 1990; 1992; 1996; Ortega et al., 2003). Outside of the sex determination pathway, Fl(2)d is also required for the proper alternate splicing of Ultrabithorax (Ubx) pre-mRNA transcripts in both sexes (Burnette et al., 1999). Mechanistically, Fl(2)d physically interacts with several early splicing factors to promote the alternate splicing of these mRNAs (Penn et al., 2008). This function appears to be evolutionarily conserved, as human WTAP has been isolated from spliceosome complexes (Zhou et al., 2002). Sequence analysis of the Fl(2)d protein has identified long stretches of histidine and glutamine residues with the N-terminal region of the protein. Similar stretches are found within the activation domains of many transcription factors (Ptashne and Gann, 1997; Penalva et al., 2000). Therefore it is possible that, in addition to its role in splicing, Fl(2)d may also function to co-regulate transcription of target genes.

Mammalian WTAP was first identified in a yeast two-hybrid screen for proteins that interact with Wilms’ tumor-1 (WT1: Little et al., 2000). Mice lacking WTAP die between embryonic day 6.5 –10.5 and show dramatic defects in cell proliferation, which in turn leads to defects in endoderm and mesoderm formation (Horiuchi et al., 2006; Naruse et al., 2007; Fukusumi et al., 2008). At least one of its roles in proliferation appears to be to prevent the degradation of cyclin A2 mRNA transcripts. In cultured cells depletion of WTAP leads to a dramatic reduction in Cyclin A2 protein levels and as a consequence the cells are arrested in G2 (Horiuchi et al., 2006). Consistent with a role in blocking degradation of cyclin A2 transcripts, murine WTAP is found within a complex that contains proteins involved in mRNA stabilization, polyadenylation and mRNA transcript export (Horiuchi et al., 2013). Murine WTAP is likely to also play its traditional role in splicing as it was found to interact with serine/arginine (SR) proteins and members of the general splicing machinery (Horiuchi et al., 2013).

WT1 is expressed within the mammalian retina and is required for the expression of Pou4f2/Brn3-b, which is essential for the specification of retinal ganglion cells (Armstrong et al., 1993 Wagner et al., 2002a; 2003). The retinas of mice that lack WT1 display increased levels of cell death and are thus thinner and contain fewer retinal ganglion cells (Wagner et al., 2002a). Certain WT1 mutant alleles are also associated with some versions of retinoblastoma (Wagner et al., 2002b; Punnett et al., 2003). klumpfuss (klu), the Drosophila homolog of WT1, contributes to the development of the Drosophila retina by regulating cell death levels (Rusconi et al., 2004; Wildonger et al., 2005). In contrast, prior to this report neither WTAP nor any of its homologs have been previously implicated in retinal development within any seeing animal. Here, for the first time, we demonstrate a role for a WTAP homolog in the eye. We used yeast two-hybrid assays and immunoprecipitations from Kc167 cells to detect the formation of a So-Fl(2)d complex and to identify the domains within both proteins that mediate the physical interaction. We further show that Fl(2)d is distributed throughout the developing eye disc and that reductions in protein levels results in defects in photoreceptor number, cell fate and rhabdomere structure. Our data indicates that Fl(2)d regulates the levels of the pan-neuronal RNA binding protein Embryonic Lethal Abnormal Vision (Elav) and the RUNX class transcription factor, Lozenge (Lz). The structural defects that are seen in the adult eyes of fl(2)d mutants are caused in part by increased levels of both Elav and Lz proteins.

Materials and Methods

Fly Strains and Genetic Crosses

The following 20 stocks were used in this study: (1) y w ey-flp; (2) FRT42D fl(2)df01270/CyO; (3) FRT42D so3/CyO; (4) FRT42D Ubi-GFP/CyO; (5) UAS-fl(2)d RNAi; (6) UAS-dicer2; (7) UAS-fl(2)d; (8) UAS-so; (9) UAS-eya; (10) UAS-elav; (11) UAS-lz; (12) ey-GAL4; (13) UAS-lacZ; (14) GMR-GAL4; (15) DE-GAL4; (16) elav-GAL4; (17) lz-lacZ; (18) w1118 ; (19) y w ey-flp; FRT42D cl P[w+]; (20) UAS-GFP. All flies and genetic crosses were maintained at 25°C. GAL4 crosses that involved UAS-dicer2 and UAS-fl(2)d RNAi were compared to control crosses that contained UAS-GFP and UAS-fl(2)d RNAi constructs in order to ensure that any observed effect was not due to a dilution of the GAL4 protein. In all cases the control crosses looked nearly identical to the experimental crosses.

Antibodies and Microscopy

The following 17 antibodies were used in this study: (1) guinea pig anti-So (1:50, gift of Ilaria Rebay); (2) rat anti-Elav (1:100, DSHB); (3) mouse anti-Fl(2)d (1:100, DHSB); (4) mouse anti-Ct (1:100, DSHB); (5) mouse anti-Dac (1:5, DSHB); (6) mouse anti-Eya (1:5, DSHB); (7) mouse anti-Ey (1:250, DSHB); (8) mouse anti-22C10 (1:100, DSHB); (9) mouse anti-Lz (1:100, DSHB); (10) mouse anti-Gl (1:20, DSHB); (11) mouse anti-Pros (1:20, DSHB) (12) mouse anti-β-galactosidase (1:100, Promega); (13) chicken anti-β-galactosidase (1:100, Abcam); (14) guinea pig anti-Sens (1:100, gift of Hugo Bellen); (15) mouse anti-Yan (1:5, DHSB); (16) mouse anti-HA (1:1000, Santa Cruz Biotechnology); (17) mouse anti-Myc (1:1000, Santa Cruz Biotechnology). Secondary antibodies and phalloidin were obtained from Jackson Laboratories and Invitrogen. Imaginal discs and adult flies were prepared as described in Anderson et al., 2012.

Comparison of in vivo Elav and Lz Protein Levels Between Normal and fl(2)d Mutant Cells

Third instar larval eye-antennal discs containing fl(2)d mutant clones were stained with antibodies against Elav and Lz, viewed and photographed on a Zeiss Axioplan II fluorescent compound microscope. The image files were imported into Adobe Photoshop and the rectangular marquee tool was then used to select regions of the fl(2)d loss-of-function clones. The Analysis Tool within Adobe Photoshop was used to determine the mean pixel intensity of Elav staining within the fl(2)d loss-of-function clones and the neighboring wild type tissue. In order to compare the relative level of Elav expression in the clone to that of the surrounding wild type tissue, the mean pixel intensity measurements for the clone was divided by that of the wild type tissue to yield a fold difference ratio. We examined and determined the pixel intensity ratio for clones in multiple discs. In order to determine the average fold difference for a single disc the fold differences for all clones within an individual disc were added and then averaged. In order to determine the average fold difference between fl(2)d clones and wild type tissue for the entire experiment we added and averaged the fold differences for the discs that we had examined. These methods allowed us to eliminate any experimental differences (such as antibody penetration) that may have existed between discs. Similar methods were used to determine the fold difference in Lz levels between fl(2)d clones and wild type tissue.

DNA Constructs

Fl(2)d encodes a protein that is 536 amino acids in length (Penalva et al., 2000). Fl(2)d NT contains amino acids 1–100 (contains the histidine and glutamine stretches) fused to GFP while Fl(2)d CT contains amino acids 101–536 (contains predicted three coiled coil motifs). The So FL, So ΔSD and Optix FL proteins are described in Weasner et al., 2007 and diagramed in Figure 1A (see figure legend for details on nomenclature).

Figure 1. Fl(2)d interacts with Sine Oculis and functions during eye development.

Figure 1

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(A) A schematic drawing depicting the molecules that were tested for physical interactions in yeast two-hybrid and immunoprecipitation assays. So-FL = full-length So protein, So-ΔSD = a variant of So in which the Six domain has been deleted, Optix-FL = full-length Optix protein, Fl(2)d-FL = full-length Fl(2)d protein, Fl(2)d-NT = a variant of Fl(2)d that contains the histidine (blue) and glutamine (green) stretches fused to GFP, FL(2)d-CT = a variant of Fl(2)d that contains the three predicted coiled-coil (orange) domains. (B) Blots of co-immunoprecipitations from Kc167 cells. NF = nuclear fraction, M = mock, IP = immunoprecipiation (pulldown), IB = immunoblot (blot). Names of tested proteins are listed to the right of each row. The nomenclature is same as in panel A with the addition of Myc and HA epitopes. Fl(2)dFL is capable of binding SoFL in (row 1) but the interaction is lost when the Six domain is deleted (row 2). The Fl(2)dNT variant retains the ability to bind So (row 5) but the Fl(2)dCT fails to do so (row 4). (C, D) Fl(2)d protein is distributed throughout the entire eye imaginal disc. (E, F) So and Fl(2)d proteins are co-distributed within developing photoreceptors. (G, H) Reductions in Fl(2)d levels either via loss-of-function clones (G) or expression of an fl(2)d RNAi construct (H) causes a roughening of the external surface of the compound eye. (I–K) Fl(2)d protein is lost in fl(2)d loss-of-function null clones. (L–N) Expression of an fl(2)d RNAi construct with DE-GAL4 eliminates Fl(2)d protein in the dorsal half of the eye. Genotypes and molecules are listed within each panel. Anterior is to this right.

Yeast 2-Hybrid, Kc167 Immunoprecipitation and Transcriptional Activation Assays

Full-length so, optix and DSix4 cDNAs were cloned into the pDEST32 vector and used to screen a yeast two-hybrid library (Life Technologies ProQuest Two-Hybrid System) that was made with RNA from wandering third instar larvae (spiked with RNA from eye-antennal imaginal discs: Suppl. Tables 1–3). The physical interactions between So and Fl(2)d was confirmed with a directed yeast 2-hybrid assay and immunoprecipitation from Kc167 cells. The Qiagen Effectene Transfection Reagent was used to transfect Kc167 cells with combinations of each of the following plasmids: (1) UAS-SoFL-Myc; (2) UAS-SoΔSD-Myc; (3) UAS-Fl(2)dFL-HA; (4) UAS-Fl(2)dNT-HA; (5) UAS-Fl(2)dCT-HA; (6) UAS-OptixFL-Myc; (7) UAS-ElavFL-HA/Myc; (8) mt-GAL4. 0.4ug of each plasmid was used. Protein induction, purification and immunoprecipitation were performed as described in Anderson et al., 2012. Yeast transcriptional assays to determine the activation strength of Sine Oculis, Fl(2)d and Eyes Absent proteins were performed as described in Anderson et al., 2012.

RT-PCR

Eye-antennal discs were dissected from third instar larvae in DEPC treated PBS. Tissue was lysed in Buffer RLT (Qiagen) with 1% β-mercaptoethanol and homogenized using Qiashredder (Qiagen). Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen). RNA was DNase treated using RQ1 RNase-free DNase (Promega). RT-PCR was performed using the Qiagen OneStep RT-PCR Kit and the following primer pairs (EX2F-EX3R, EX1F-EX3R, EX2F-5999). PCR products were analyzed on agarose gels. PCR using EX2F and EX3R primers yields a 350bp product that corresponds to the LD33076/cDNA-1 coding transcript. A 366bp product corresponding to the RE14370 non-coding transcript is generated from EX1F and EX3R primers and a 374bp product corresponding to the RE58603 non-coding transcript is obtained from the EX2F and EX5R primers. The sequences of the EX2F, 5999, EX3R and EX4R primers are described within Borgeson and Samson, 2005. The sequence of the EX1F primer is: 5′-CGCAGCGGATCTGGTCTC-3′ and the EX5R is 5′-CAGGCGGCTTCTATCAATC-3′. Detection of the RP49/RpL32 transcript served as the positive control in this experiment. The primers RP49F and RP49R are described in Borgeson and Samson, 2005. PCR products were verified by sequencing.

Results

Identification of a So-Fl(2)d Complex

In order to identify SIX protein containing complexes, we screened a yeast two-hybrid library that was made with RNA from third instar larvae for interactions with full-length So, Optix and DSix4 proteins (the library was spiked with RNA from eye-antennal discs: Suppl. Table 1–3). Of the 50 positive clones that were recovered from the So interaction screen 20 corresponded to Fl(2)d. We confirmed the formation of the So-Fl(2)d complex though a directed yeast two-hybrid assay (data not shown) and co-immunoprecipitation from Kc167 cells (Fig. 1A, B, row 1). Of the 30 positive clones that were recovered and analyzed from the Optix interaction screen none corresponded to Fl(2)d. Immunoprecipitation from Kc167 cells also failed to detect interactions between Optix and Fl(2)d (Fig. 1B, row 3). We did not test for interactions with DSix4 (in Kc167 cells) since it is not expressed within the developing eye.

Previous studies have implicated the SIX-domain (SD) in mediating protein-protein interactions with transcriptional co-factors such as Eya and Gro (Pignoni et al., 1997; Kobyashi et al., 2001). Fl(2)d also appears to interact with So via the SD as we were unable to immunoprecipitate Fl(2)d from Kc167 cells with a variant of So lacking the SD (SodeltaSD: Fig. 1A, B, row 2). In order to determine which portion of Fl(2)d interacts with So we generated two variant forms of Fl(2)d (Fig. 1A). One variant contains the histidine and glutamine stretches fused to GFP (Fl(2)dNT-GFP) while the other contains the three predicted coiled-coil domains (Fl(2)dCT). We were able to immunoprecipitate So from Kc167 cells with Fl(2)dNT-GFP (Fig. 1B, row 5) but not Fl(2)dCT (Fig. 1B, row 4). This suggests that the formation of the So-Fl(2)d complex is mediated by interactions between the SD of So and the N-terminal region of Fl(2)d.

Fl(2)d is Expressed and Required in the Eye

We stained eye-antennal discs with an antibody against Fl(2)d and found that the protein is distributed throughout the entire epithelium (Fig. 1C, D). Fl(2)d protein is co-distributed with So in a narrow swathe of cells ahead of the morphogenetic furrow and in all developing photoreceptors within both the compound eyes and ocelli (Fig. 1E, F). fl(2)d is required for correct development of the retina as its removal from the entire eye leads to a roughening of the external surface of the compound eye (Fig. 1G). The fl(2)df01270 mutation is a null allele and no Fl(2)d protein is seen in mutant clones (Fig. 1I–K). Similarly, forcible expression of an RNAi line that targets the Fl(2)d mRNA transcript posterior to the furrow using a GMR-GAL4 driver or within the dorsal compartment using a DE-GAL4 driver also leads to a roughening of the adult eye (Fig. 1H; Suppl. Fig. 3C). The RNAi line is an effective tool to greatly reduce/eliminate Fl(2)d as protein levels are reduced to below detectable levels when the RNAi line is expressed in the dorsal compartment of the eye using a DE-GAL4 driver (Fig. 1L–N). Fl(2)d protein is also eliminated from the peripodial membrane when the RNAi line is expressed under the control of DE-GAL4 (Fig. 1M). Co-expression of a full-length wild type fl(2)d transgene is sufficient to suppress the rough eye phenotype thus confirming that the rough eye phenotype results from knocking down fl(2)d and is not due to an off target effect. Sections of adult retinas in which Fl(2)d were reduced via expression of the RNAi construct reveal a variety of defects in photoreceptor numbers, cell fate and rhabdomere formation (Fig. 2A–D). There moderate rough eye phenotype that results from reductions in Fl(2)d protein levels is consistent with the moderate effects on photoreceptor number and positioning. Our overall conclusion from this set of results is that Fl(2)d plays an important role in eye development.

Figure 2. Loss of Fl(2)d affects photoreceptor numbers, cell fate and rhabdomere structure.

Figure 2

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(A, B) Retinal sections of adult wild type retinas at 63X (A) and 100X (B). (C–D) Retinal sections of adult GMR-GAL4, UAS-fl(2)d RNAi retinas at 63X (C) and 100X (D). Many ommatidia have fewer than the normal seven photoreceptors that are seen in the distal sections of the retina (orange arrow). Some ommatida have multiple small-rhabdomere photoreceptors suggesting that outer photoreceptors have been converted to inner photoreceptors (red arrow). Many photoreceptors appear to have defective rhabdomere structure (purple arrows).

What is the nature of Fl(2)d’s role in eye development? Since So plays a key role in eye specification we first investigated the possibility that Fl(2)d participates with So to promote early steps in eye development. Within the developing eye, So binds to enhancer elements within several retinal determination genes (including itself, ey, eya and dac) and regulates their expression (Halder et al., 1998; Pauli et al., 2005; Pappu et al., 2005; Atkins et al., 2013). In order to determine if Fl(2)d contributes to eye specification we examined the expression of these four factors as well as that of one additional retinal determination factor, Teashirt (Tsh), in fl(2)d loss-of-function mutant clones. We observed no discernable differences in the expression of these genes between wild type and mutant tissue (Table 1; Suppl. Fig. 1A–O).

Table 1.

Fl(2)d regulates a small subset of genes in the developing retina

Gene Expression Pattern Affect
eyeless anterior to the furrow none
teashirt anterior to the furrow none
sine oculis anterior to the furrow
all photoreceptors		 none
eyes absent anterior to the furrow
all photoreceptors		 none
dachshund anterior to the furrow
subset of photoreceptors		 none
hedgehog all photoreceptors none
glass all photoeceptors
undifferentiated cells		 none
senseless R8 photoreceptor none
elav all photoreceptor cells upregulated in clone
22C10/futsch all photoreceptor cells none
prospero R1, R6 and R7 none
lozenge undifferentiated cells
cone cells		 upregulated in clone
cut cone cells none
yan undifferentiated cells none
cyclin A dividing cells anterior to the furrow and within the second mitotic wave none
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So is known to regulate the expression of one of its binding partners, Eya, and vice versa (Halder et al., 1998). This auto-regulatory loop is important for maintaining the proper stoichiometric levels of both factors during development. We set out to determine if a similar regulatory relationship exists between So and Fl(2)d. We have already demonstrated that Fl(2)d does not appear to regulate the expression of so (Table 1; Suppl. Fig. 1J–L). We then analyzed the distribution and levels of Fl(2)d in so3 null mutant retinal clones but did not observe any consistent and/or significant reductions in Fl(2)d levels (Suppl. Fig. 1P–U). This is consistent with the results of a microarray analysis of gene expression in so1 and eya2 loss-of-function mutant discs. The expression of so and eya is abolished in both mutants but the expression level of fl(2)d within the retina is not significantly altered when compared to wild type eye-antennal discs (data not shown). We therefore conclude that So and Fl(2)d do not participate in a regulatory loop. From analysis of gene expression within fl(2)d mutant clones, we also conclude that the So-Fl(2)d complex does not participate in eye specification. This latter conclusion is supported by the fact that removal of fl(2)d from the entire eye disc does not eliminate the adult retina but instead results in defects in photoreceptor and rhabdomere development/maintenance (Fig. 1G; 2A–D). In contrast, removal of so from the developing eye leads to a complete elimination of all photoreceptor cells (Cheyette et al., 1994; Serikaku et al., 1994; Pignoni et al., 1997).

We then asked if the So-Fl(2)d complex could be involved in the specification and/or maintenance of photoreceptor cells and/or the non-neuronal accessory cells that comprise each unit eye or ommatidium. To test this hypothesis we generated fl(2)d null mutant clones and analyzed the expression of a suite of genes that are known to regulate each step in ommatidial assembly (Table 1). We tested genes that are expressed in photoreceptor neurons, non-neuronal cone cells and the pool of undifferentiated cells that surround individual photoreceptor clusters in the eye disc that eventually are specified in the pupal stage as pigment cells and members of the bristle complex. The expression level and pattern of nearly every factor that we tested was unaffected by the loss of fl(2)d (Table 1; Suppl. Fig. 2A–X). The two exceptions that we uncovered are elav and lz. In both cases, the levels of the encoded proteins appear to be elevated within fl(2)d clones (Fig. 3A–C, F–H). It should be noted that the expression patterns of either gene is not altered in the mutant tissue. We measured and compared the fluorescence levels between normal and fl(2)d null mutant tissue (see methods) and find that there is 1.55 fold increase in Elav levels and a 1.42 fold increase in Lz levels within fl(2)d mutant clones as compared to wild type tissue (Fig. 3K, L). The loss of Fl(2)d gives a rough eye phenotype that is equal in severity to the over-expression of Lz (Suppl. Fig. 3A–B). These results suggest that Fl(2)d contributes to eye development, in part, through the regulation of elav and lz. Since the increases in Elav and Lz protein levels are restricted to the clonal tissue it appears that Fl(2)d functions autonomously to regulate these two genes. Using high resolution microscopy we have been able to co-localize Fl(2)d with Elav within developing photoreceptor neurons (Fig. 3D, E) and with Lz in the undifferentiated cells that surround each ommatidial cluster (Fig. 3I, J). The co-localization of Fl(2)d with both Elav and Lz supports the contention that the regulation of Fl(2)d of these factors occurs autonomously.

Figure 3. Fl(2)d regulates Elav and Lozenge protein levels in the eye disc.

Figure 3

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(A–J) Confocal images of third instar larval eye discs. (A–C) Elav protein levels are elevated in fl(2)d loss-of-function clones. (D, E) Fl(2)d and Elav protein are both present in developing photoreceptors. (F–H) Lz protein levels are elevated in fl(2)d loss-of-function clones. (I–J) Fl(2)d protein is present in the undifferentiated cells (between developing clusters) and is co-expressed with lz. (K, L) Graphs depicting the fold difference in Elav and Lz protein levels between fl(2)d loss-of-function clones and wild type tissue (see methods). Genotypes and molecules are listed within each panel. Anterior is to the right.

Fl(2)d does not regulate the transcription of either elav or lz loci

In order to understand the mechanism underlying the Fl(2)d dependent regulation of Elav and Lz protein levels we first focused on determining if Fl(2)d plays a role in regulating the transcription of either/both target genes. The presence of histidine and glutamine rich regions within the N-terminal region raised the possibility that Fl(2)d could function as a transcriptional activator either within the context of a So-Fl(2)d complex or through interactions with a yet to be identified transcription factor. Such a complex could activate the expression of a transcriptional repressor, which in turn would dampen the expression levels of elav and lz. This scenario is consistent with the increase that we observe in the expression levels of both genes. We used a yeast activation assay to determine if Fl(2)d has the intrinsic potential to activate expression of a reporter construct. Full-length Fl(2)d was fused to the GAL4 DNA binding domain and the chimeric protein was assayed for its ability to activate three different transcriptional reporters (GAL1-HIS3, SPAL10-URA3 and GAL1-lacZ: only GAL1-lacZ is shown here). We first determined that So, on its own, is a relatively weak transcriptional activator of GAL1-lacZ (Fig. 4A, top; Anderson et al., 2012). By comparison, the transcriptional co-activator Eya is capable of activating the GAL1-lacZ reporter at significantly higher levels than So (Fig. 4A, middle). The activation strength of Eya is stronger than that of Ey which itself contains a potent transcriptional activation domain within its C-terminal (Weasner et al., 2009). Fl(2)d failed to activate transcription of any of the three reporters (Fig. 4A, bottom) suggesting that it does not function as a transcriptional co-activator.

Figure 4. Fl(2)d does not participate in the regulation of elav or lz transcription.

Figure 4

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(A) Yeast transcription assay. The presence of a blue precipitate indicates that the candidate molecule is a transcriptional activator. Based on the presence/absence of the precipitate it appears that Eya is a strong activator while So is weak activator. Fl(2)d does not appear to function as an activator in this assay. (B–F) SEM images of adult compound eyes. (B) Over-expression of so in developing photoreceptors leads to a rough eye phenotype. (C) Over-expression of eya alone has no effect on the structure of the eye. (D) Simultaneous expression of both so and eya leads to a synergistic effect on eye structure. (E) Over-expression of fl(2)d on its own does not affect the structure of the eye. (F) Simultaneous expression of so and fl(2)d increases the severity of the rough eye slightly but does not qualify as a synergistic effect. (G–L) Confocal images of third instar larval eye discs. (G–I) Expression of an elav-lacZ transcriptional reporter is unaffected in fl(2)d clones. (J–L) Expression of an fl(2)d RNAi construct does not affect expression of a lz-lacZ transcriptional reporter. Bracket in J shows reduction in Fl(2)d protein with the eye disc. The peripodial cells retain Fl(2)d expression since the DE-GAL4 driver is not expressed in this non-retinal tissue. Genotypes are listed either within or below each panel. Molecules are listed within each panel. Anterior is to the right.

An in vivo genetic interaction assay yielded similar results to those observed in the yeast transcription assay. The rationale behind this assay is that if the So-Fl(2)d complex functions as a transcriptional activator then forcibly increasing Fl(2)d levels might augment the activation potential of So and synergistically enhance the rough eye phenotype that results from over-expression of So. This is the case for the So-Eya complex. Expression of so behind the morphogenetic furrow using the GMR-GAL4 driver leads to a severe roughening of the adult eye. The defects are most severe in the posterior third of the eye field where it appears as if the adult eye is devoid of ommatidia (Fig. 4B). This phenotype is dramatically enhanced with the entire eye having a flattened appearance if So and its binding partner Eya are co-expressed (Fig. 4D). Since the expression of eya, on its own, does not affect eye development (Fig. 4C), the enhancement of the rough eye represents a bona fide augmentation of So activation potential by Eya. Like Eya, expression of fl(2)d by itself also does not affect the structure of the eye (Fig. 4E). However, unlike eya, the co-expression of fl(2)d with so does not enhance the rough eye phenotype (Fig. 4F). This results also suggests that the So-Fl(2)d complex is unlikely to function as a transcriptional activator.

We also analyzed the ability of Fl(2)d to regulate the expression of elav and lz as well as several genes that are known to lie upstream of these factors in the eye disc itself. We first analyzed the expression levels of lz-lacZ and elav-GAL4, UAS-lacZ transcriptional reporters in cells in which Fl(2)d levels have been reduced and find that both reporters were unaffected by the reductions in Fl(2)d (Fig. 4G–L). We then analyzed the expression of the so, glass (gl), anterior open (aop/yan) and tramtrack (ttk) genes since these genes encode transcription factors that directly bind to an enhancer element within the lz locus and regulate itsexpression (Behan et al., 2002; Yan et al., 2003; Protzer et al., 2008; Siddall et al., 2009). Our analysis of fl(2)d mutant clones indicates that the expression patterns or levels of these genes are not altered in the mutant tissue either (Table 1; Suppl. Figs. 1J–L, 2A–C, P–R). We were unable to conduct a similar analysis of upstream regulators of elav since these factors are yet to be identified. Nonetheless, in sum the results from the yeast transcriptional assays, the genetic synergism assay and the analysis of fl(2)d clones all indicate that Fl(2)d is unlikely to regulate the transcription of elav, lz or any of the known upstream regulators of these two genes.

Fl(2)d does not stabilize or promote the alternate splicing of elav and lz transcripts

Since Fl(2)d is implicated in the alternate splicing of Sxl, tra and Ubx pre-mRNA transcripts and since WTAP binds to and stabilizes cyclin A2 mRNA transcripts (Granadino et al., 1990; 1992; 1996; Burnette et al., 1999; Ortega et al., 2003; Horiuchi et al., 2006), we set out to determine if Fl(2)d plays similar roles in stabilizing or splicing of elav and/or lz transcripts. Since the loss of fl(2)d leads to an increase (not a decrease) in both Elav and Lz protein levels (Fig. 3A–C, F–H, K, L), it is unlikely that Fl(2)d functions to stabilize either transcript. However, Fl(2)d could still play a role in mRNA stability if its physiological function was to promote rather than inhibit mRNA degradation. One of the mechanisms by which elav transcript are stabilized is through the binding of ELAV protein to the 3′ UTR of elav transcripts via three RNA Recognition Motifs (RRM: Samson et al., 1998). This mechanism appears to be conserved as HuR, a vertebrate homolog of Elav, is also involved in the binding and stabilization of mRNAs (reviewed in Brennan and Steitz, 2001). One possible mechanism that would account for the increase in Elav protein in fl(2)d mutant tissue is if Fl(2)d interacts with Elav and prevents it from binding to and stabilizing the elav transcript. To test this hypothesis we attempted to first determine if a genetic interaction between the fl(2)d and elav loci exists. The over-expression of elav in all cells behind the morphogenetic furrow leads to a severe roughening of the adult eye (Fig. 5A). If Fl(2)d forms a complex with Elav and sequesters it away from mRNA transcripts then co-expression of Fl(2)d would be expected to partially suppress the rough eye phenotype. However, the simultaneous expression of both elav and fl(2)d looks nearly indistinguishable from over-expression of elav alone (Fig. 5B). Experiments in which we reduced Fl(2)d levels either through the expression of RNAi constructs or through the use of loss-of-function mutations also failed to enhance the rough eye phenotype (Fig. 5C, D). The results form these genetic interaction assays suggest that Fl(2)d does not interact with Elav. This conclusion was confirmed by the failure to immunoprecipitate an Elav-Fl(2)d complex from Kc167 cells (Fig. 5E, bottom row). Similarly, Elav and So also do not appear to physically interact (Fig. 5E, top row). The data from these studies suggest that the So-Fl(2)d complex does not contain Elav and that Fl(2)d is unlikely to function to regulate the stability of elav and mRNA transcripts.

Figure 5. Fl(2)d does not cooperate with Elav to regulate elav mRNA transcripts.

Figure 5

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(A–D) SEM images of adult compound eyes. (A) Over-expression of elav in developing photoreceptors leads to a severe roughening of the adult eye. (B–D) Neither elevating nor reducing Fl(2)d levels is sufficient to modify the rough eye phenotype. Genotypes are listed above each panel. Anterior is to the right. (E) Blots of co-immunoprecipitations from Kc167 cells. Nomenclature for proteins is similar to Figure 1. So and Fl(2)d do not appear to physically interact with Elav in this assay. NF = nuclear fraction, M = mock, IP = immunoprecipiation, IB = immunoblot. Names of tested proteins are listed to below each row. (F) RT-PCR of elav coding (LD33076 - lane 1) and non-coding (RE58603 - lane 2, RE14370 - lane 3) transcripts and RP49 control (lane 4) from both wild type retinas and eyes lacking Fl(2)d protein.

Drosophila Fl(2)d interacts with members of the spliceosome and regulates the alternate splicing of three different pre-mRNA transcripts (Sxl: Granadino et al., 1990; 1992; Tra: Granadino et al., 1996; Ubx: Burnette et al., 1999). In each of these cases Fl(2)d has been implicated in regulating a different splicing step, thus its exact molecular role in splicing is not completely understood. One potential role for Fl(2)d in the eye could be to participate in the alternate splicing of the elav and lz pre-mRNA transcripts. The lz locus encodes two different transcripts that differ in the use of exon 5 (Daga et al., 1996; Behan et al., 2005). The larger protein isoform contains an Ets interaction domain which allows it to form a biochemical complex with the Ets transcription factor Pointed (Pnt: Behan et al., 2005). This interaction domain is missing from the shorter protein isoform. The antibody that we have used to detect Lz in the eye disc recognizes an epitope located within the C-terminal region of both Lz protein isoforms (Gupta et al., 1998). Thus, alternate splicing of the differing lz coding transcripts cannot account for the increase in Lz protein levels since the antibody that we are using detects a region of the protein that is found in both isoforms. A shift in the abundance of one transcript for the other would not be predicted to result in increased protein levels (at least with the antibody that we are using).

The elav locus encodes five different mRNA transcripts: one coding and four non-coding (Borgeson and Samson, 2005). A shift between non-coding and coding isoforms is an attractive model to explain the increased levels of Elav protein in fl(2)d clones. Elav itself is known to participate in the alternate splicing of several mRNAs including erect wing (ewg: Koushika et al., 2000; Soller and White, 2003; 2005), armadillo (arm: Koushika et al., 2000) and neuroglian (nrg: Lisbin et al., 2001). The inability of Elav to co-immunoprecipitate with Fl(2)d suggests that a complex containing both proteins does not mediate the alternate splicing of the elav pre-mRNA transcripts. However, we sought to investigate the possibility that Fl(2)d, on its own, is involved in the alternate splicing of these pre-mRNA transcripts and maintains a balance between the levels of coding and non-coding transcripts. Using RT-PCR we first determined if the non-coding isoforms are present in the wild type eye-antennal disc. Since we observe increases in Elav protein levels within the third instar eye-antennal disc we focused on the two non-coding transcripts (RE14370 and RE58603) that are expressed during the larval stages of development. The other two non-coding transcripts (cDNA-16h and cDNA3h) are only expressed in the adult head (Borgeson and Samson, 2005) and were not considered here. We were able to detect the coding transcript and one of the non-coding transcripts (RE14370) but failed to detect the second non-coding transcript (RE58603) in wild type discs (Fig. 5F, lanes 1–3 top row). We were able to detect all three transcripts in discs completely lacking Fl(2)d protein behind the morphogenetic furrow (GMR-GAL4, UAS-fl(2)d RNAi, UAS-Dicer2: Fig. 5F, lanes 1–3 bottom row). We note that the non-coding transcripts are expressed at very low levels and our inability to detect RE58603 in wild type is likely due to the low abundance of the transcript. We failed to detect a shift between amounts of the coding transcripts as compared to wild type. These data seem to suggest that Fl(2)d is not mediating the alternate splicing of the primary elav mRNA transcript.

The so and fl(2)d genes interact genetically within the developing eye

Since both So and Fl(2)d co-localize in the retina (Fig. 1E, F) and since the loss of fl(2)d has a negative impact on eye development (Figs. 1G, H; 3A–C, F–H) we were interested in determining if the So-Fl(2)d complex functions in the eye and is responsible for regulating the levels of both Elav and Lz. Unfortunately, the role that so plays early in eye specification cannot be experimentally separated from potential roles behind the furrow. Eye development is completely blocked in discs or clones that are mutant for so (Cheyette et al., 1994; Serikaku and O’Tousa, 1994; Pignoni et al., 1997). Similarly, over-expression of so severely inhibits eye development (Fig. 4G; Weasner et al., 2007; Anderson et al., 2012). In both contexts, the analysis of Elav and/or Lz levels is complicated by the actual loss of photoreceptors and undifferentiated cells, thus it is not possible to compare Elav and Lz levels between so and fl(2)d loss of function mutants. To overcome these technical problems we attempted to determine if so and fl(2)d interact genetically in the eye by reducing the levels of Fl(2)d in animals that simultaneously over-express So. We do see a partial restoration of eye development, particularly in the posterior third of the retina, when fl(2)d levels are reduced via expression of an RNAi construct (Fig. 6A bracket, 6B arrow). A greater than 50% reduction in fl(2)d expression levels appears to be necessary as the loss of one copy of fl(2)d was insufficient to suppress the rough eye phenotype (Fig. 6C, bracket). We conclude from these genetic interactions that a So-Fl(2)d complex is functioning in cells behind the morphogenetic furrow during normal eye development.

Figure 6. Partial suppression of the So induced rough eye phenotype by reductions in fl(2)d.

Figure 6

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(A–C) SEM images of adult compound eyes. (A, C) The green brackets mark the posterior region of the adult eye that is devoid of ommatidia. Note that the loss of one copy of fl(2)d does not suppress the rough eye phenotype. (B) The green arrow indicates the partial suppression of the rough eye phenotype when an fl(2)d RNAi construct is expressed in cells behind the furrow. Ommatidia are now present in the posterior margin of the eye. Genotypes are listed above each panel. Anterior is to the right.

Discussion

In this report we describe a novel developmental role for Fl(2)d, the Drosophila homolog of Wilms’ Tumor 1-Associating Protein (WTAP). Removal of fl(2)d from the fly retina results in photoreceptor defects and a roughening of the external surface of the compound eye (Fig 1, G, H; Fig. 2A–D; 3A–C, F–H; Suppl. Fig. 3C). Our expression analysis indicates that Fl(2)d protein regulates the levels of at least two proteins: Lz, a RUNX class transcription factor, and ELAV, a pan-neuronal RNA binding protein (Fig. 3A–C, F–H). To our knowledge this is the first report of a role for any WTAP homolog in eye development. In contrast, Wilms’ tumor suppressor (WT1) protein, the binding partner for WTAP is a well-known regulator of eye development in mammals. Reductions in murine WT1 lead to a dramatic loss of retinal ganglion cells (RGCs), an increase in cell death and a disruption in the growth of the optic nerve (Wagner et al., 2002a). These phenotypes are due to the loss of Pou4f2/Brn-3b a homolog of the Drosophila atonal basic helix-loop-helix (bHLH) transcription factor. Consistent with the loss of-function phenotype, over-expression of WT1 is sufficient to induce Pou4f2 expression in cultured cells and to activate a Pou4f2 enhancer element in the mouse retina (Wagner et al., 2002a; 2003). A role in eye development for WT1 appears to be evolutionarily conserved as the Drosophila homolog, klumpfuss, is an important regulator of cell fate specification and programmed cell death in the fly retina (Rusconi et al., 2004; Wildonger et al., 2005). As WT1 and WTAP are obligate binding partners in many contexts it is likely that WTAP also functions within the mammalian eye.

Fl(2)d was identified in a yeast two-hybrid assay for proteins that interact with So, the founding member of the Six family of homeobox transcription factors (Suppl. Table 1). This interaction (and the domains that mediate it) was confirmed with direct yeast two-hybrid assays and immunoprecipitations from Kc167 (Fig. 1A–B). So and its mammalian homologs are expressed in and regulate the development of several tissues including the retina. But while the So/Six family members are widely expressed within both undifferentiated and differentiating cells of the retina, most reports have focused on their roles in early tissue determination and cell proliferation. In contrast, even though So is distributed within differentiating photoreceptor neurons, its role in their specification and maintenance is poorly understood. Furthermore, despite the identification of over two-dozen different binding partners of So, the overwhelming majority of studies have focused on the part that two specific complexes (So-Eya and So-Gro) play in development and disease. Very little information on other So containing biochemical complexes, beyond their existence, is available.

There is some evidence to suggest that the So-Fl(2)d complex functions in the retina. Both so and fl(2)d are co-expressed within many differentiating cells and reductions in fl(2)d levels is sufficient to partially suppress the effects that over-expression of so has on the structure of the compound eye. However, since eye development is completely blocked in so null mutants, it is, at present, difficult to be certain that the phenotypes associated with fl(2)d mutants are due to disruptions of the So-Fl(2)d complex. Nonetheless, the co-expression of both genes within the developing eye and the genetic interaction between the two factors all suggest that So-Fl(2)d complex may function within the retina. We have explored the possibility that Fl(2)d regulates the expression of elav and lz or functions in the alternate splicing or stabilization of the mRNA transcripts. Our experimental results suggest that the regulation of Elav and Lz protein levels is not due to either of these mechanisms. It leaves open the possibility that Fl(2)d (and by association So) may have novel biochemical activities.

Interestingly, expression of both Six1 and Eya1, the mammalian homologs of so and eya, are elevated in Wilms’ Tumor cells (Li et al., 2002; Sehic et al., 2012). The association of WT1 and Six1 in the eye and kidney of patients with Aniridia and Wilms’ Tumor suggest that there is a conserved connection between WT1/WTAP and members of the SIX family of transcription factors. Finally, our identification of the So-Fl(2)d complex adds to our growing knowledge of how SIX proteins regulate development (Fig. 7).

Figure 7. So-Fl(2)d cooperates with other So-dependent complexes to regulate retinal development in Drosophila.

Figure 7

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This model describes the known So-containing biochemical complexes and the roles that they play in the development of the compound eye. This model includes spatial information, which is based on the expression patterns of binding partners and on the location of loss-of-function phenotypes. X denotes putative transcriptional targets of the So-Sbp complex. R = transcriptional repressors (such as Gro and CtBP) that are bound to So and mediate repression of non-retinal gene regulatory networks.

Supplementary Material

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Highlights.

  • So and Fl(2)d form a biochemical complex.

  • Fl(2)d is expressed within the retina.

  • Loss of fl(2)d leads to defects in eye development.

In fl(2)d mutants, Elav and Lz levels are elevated.

Acknowledgments

We would like to thank Georg Halder, Gakuta Toba, Utpal Banerjee, the Vienna Drosophila RNAi Center, the Bloomington Drosophila Stock Center, Exelixis, Ilaria Rebay and the Developmental Studies Hybridoma Bank for fly stocks and antibodies as well as Rudi Turner for assistance with the adult retinal sections. This work is supported by a grant (R01 EY014863) from the National Eye Institute to Justin P. Kumar.

Footnotes

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01
NIHMS588578-supplement-01.doc (65.5KB, doc)
02
NIHMS588578-supplement-02.tif (1.7MB, tif)
03
NIHMS588578-supplement-03.tif (1.7MB, tif)
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NIHMS588578-supplement-04.tif (1.5MB, tif)

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