Motif 1 Binding Protein suppresses wingless to promote eye fate in Drosophila

The phenomenon of RNA polymerase II (Pol II) pausing at transcription start site (TSS) is one of the key rate-limiting steps in regulating genome-wide gene expression. In Drosophila embryo, Pol II pausing is known to regulate the developmental control genes expression, however, the functional implication of Pol II pausing during later developmental time windows remains largely unknown. A highly conserved zinc finger transcription factor, Motif 1 Binding Protein (M1BP), is known to orchestrate promoter-proximal pausing. We found a new role of M1BP in regulating Drosophila eye development. Downregulation of M1BP function suppresses eye fate resulting in a reduced eye or a “no-eye” phenotype. The eye suppression function of M1BP has no domain constraint in the developing eye. Downregulation of M1BP results in more than two-fold induction of wingless (wg) gene expression along with robust induction of Homothorax (Hth), a negative regulator of eye fate. The loss-of-eye phenotype of M1BP downregulation is dependent on Wg upregulation as downregulation of both M1BP and wg, by using wgRNAi, shows a significant rescue of a reduced eye or a “no-eye” phenotype, which is accompanied by normalizing of wg and hth expression levels in the eye imaginal disc. Ectopic induction of Wg is known to trigger developmental cell death. We found that upregulation of wg as a result of downregulation of M1BP also induces apoptotic cell death, which can be significantly restored by blocking caspase-mediated cell death. Our data strongly imply that transcriptional regulation of wg by Pol II pausing factor M1BP may be one of the important regulatory mechanism(s) during Drosophila eye development.

In the developing eye imaginal disc of Drosophila third instar larva, a synchronous wave of retinal differentiation is initiated from the posterior margin of the eye imaginal disc, which moves anteriorly, and is referred to as the Morphogenetic Furrow (MF) 14,16 . The MF, a transient indentation in the developing eye disc, sweeps progressively across the eye disc towards the anterior margin, resulting in the formation of uniformly spaced photoreceptor clusters behind the MF. This process of differentiation of retinal precursor cells to photoreceptor neurons is driven by combinatorial action of the evolutionarily conserved Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which plays important role in initiation and progression of the MF 14,16,[28][29][30] . Within each differentiating photoreceptor cluster, Hh and Dpp signaling activates the expression of proneural genes like atonal (ato) that encodes basic HLH proteins and specifies the R8 photoreceptor [31][32][33] . Followed by the R8 selection, sequential recruitment of R2/R5, R3/R4, R1/R6 and R7 occurs 9,22,34 . Another secreted protein Scabrous (Sca), which is expressed within and near the intermediate clusters in the MF, is required for the correct spacing of photoreceptor clusters 35,36 .
The differentiation of retinal neurons and MF progression is opposed by the secreted morphogen Wg (a homolog of mouse Wnt-1 gene), which is expressed on the antero-lateral regions of the eye imaginal disc [37][38][39] . Wg serves as a ligand for the highly conserved Wg/ WNT signaling pathway. In the developing eye, Wg, a morphogen, is involved in diverse functions like cell proliferation, cell death, and cell-fate specification [40][41][42] . Wg expression levels plays important role(s) in determining the eye versus head fate by antagonizing dpp and thereby suppressing retinal determination 11,13,15,38,42,43 . Wg regulates expression of another negative regulator of eye development, homothorax (hth), a MEIS class gene with a highly conserved Meis-Hth (MH) domain and a homeodomain (HD), which is expressed uniformly anterior to the MF [44][45][46][47][48] and suppresses the eye fate. Hence, ectopic upregulation of wg promotes head-specific fate by regulating MF progression during eye development 39,44,49 .
During eye development, one of the many functions of Wg signaling is to induce programmed cell death by activating the expression of head involution defective (hid), reaper (rpr), and grim in ommatidia at the periphery of the eye during pupal stage 50,51 . In the developing larval eye field, apoptosis can be induced by a variety of stimuli like inappropriate levels of morphogens or extracellular signaling 52,53 . Ectopic induction of Wg signaling causes developmental or morphogenetic cell death in the larval eye imaginal disc 54 . This developmental cell death is caused by activation of caspases and is different from programmed cell death observed in the pupal retina 54,55 . The baculovirus anti-apoptotic protein P35 upon ectopic expression in the developing field can block caspase-dependent cell death 56 . P35 acts through inhibition of range of initiator to executioner class of caspases.
In Drosophila, the sequence-specific transcription factors, the GAGA factor (GAF) and the Motif 1 Binding Protein (M1BP) have been implicated in dictating Pol II pausing 57 . The regulatory mechanism associated with GAF exhibits greater transcriptional plasticity than M1BP. M1BP binds to a core promoter element called Motif 1 and has been shown to orchestrate promoter-proximal pausing in GAF-independent manner 57 . M1BP is highly conserved across the species and encodes a 55 kDa protein containing zinc-associated domain (ZAD) towards the N-terminus and five C 2 H 2 zinc-fingers domains toward the C-terminus. Drosophila M1BP is functionally homologous to vertebrate zinc finger with a SCAN and a KRAB domain 3 (ZKSCAN3) transcription factors and shows structural similarity for the C-terminal C 2 H 2 zinc-finger domains 58,59 . The M1BP binding site sequence was reported using bioinformatics and biochemical analysis 60 . In addition, these studies led to tracking of Motif 1 binding activity, their associated proteins, and a list of 2187 genes whose expression is affected by M1BP 57,61 .
Here, we demonstrate that wg, which encodes a ligand for the highly conserved Wg/WNT signaling pathway is one of the targets for M1BP mediated transcriptional regulation during Drosophila eye development. We show that downregulation of M1BP ectopically induces wg gene expression in the developing eye disc, which causes suppression of the eye fate and induction of developmental cell death. Our results clearly indicate that M1BP mediated transcriptional regulation of Wg signaling could be a key regulatory mechanism during Drosophila eye development. We found potential M1BP binding sites in regulatory regions of wg gene using bioinformatics. Furthermore, this relation was also observed in the wing imaginal discs.

Materials and methods
Fly stocks. Fly  Genetic crosses. We used Gal4/UAS TARGET system to misexpress the gene of interest 68 . All Gal4/UAS crosses were maintained at 18 °C, 25 °C and 29 °C, unless specified, to sample different induction levels 10 . The ey-Gal4 driver used in this study targets misexpression of inducible transgene M1BP RNAi in the entire developing eye domain (ey > M1BP RNAi ) of larval eye imaginal disc. To misexpress M1BP RNAi in specific domains of the eye disc, different Gal4 drivers were used: eyg-Gal4 targets misexpression of transgene at the equator, bi-Gal4 selectively targets the expression of the transgene at the dorso-ventral (DV) eye margin 64 , dpp-Gal4 drives the expression of the transgene at the posterior margin of the eye disc 30 .
We also tested the gain-of-function of M1BP using the CRISPR/Cas9-based transcriptional activation approach 69  Statistics. Statistical analysis was performed using Microsoft excel software. The P-values were calculated using student's t-test and the error bars represent Standard deviation from Mean. Statistical significance in each graph is shown by P-value: ***P < 0.001; **P < 0.01; *P < 0.05 [75][76][77][78] .

Results
Downregulation of M1BP function suppresses the eye fate. The larval eye imaginal disc (Fig. 1A) develops into the adult compound eye comprising of 600-800 ommatidia or unit eyes (Fig. 1B). Targeted misexpression of UAS-GFP reporter transgene under ey-Gal4 driver (ey > GFP, shown in green) marks the entire eye imaginal disc (Fig. 1A). The eye discs were stained with a membrane-specific marker Dlg and pan-neuronal marker Elav (red), which marks the nuclei of the photoreceptor neurons. Targeted misexpression of inducible UAS-M1BP RNAi transgene using ey-Gal4 driver (ey > M1BP RNAi ), which downregulates M1BP function in the developing eye imaginal disc, results in the suppression of eye fate (Fig. 1C,E). The eye suppression phenotype of ey > M1BP RNAi is evident from pan-neuronal marker Elav expression, which results in either highly reduced eye field (Fig. 1C) or a "no-eye" phenotype ( Fig. 1E). The adult flies of ey > M1BP RNAi genotype also exhibits reduced eye phenotype (Fig. 1D). The penetrance of eye phenotype(s) in the adult ranges from "small-eye" (Fig. 1D, 5%, n = 100) to a "no-eye" (Fig. 1F, 95%, n = 100). Further, quantification of the adult eye area shows that the eye size significantly reduces in case of both small-eye as well as "no-eye" phenotype in ey > M1BP RNAi flies, when compared with the control (ey > GFP) flies (p < 0.001, Fig. 1G). We also studied the M1BP gain-of-function phenotype using the CRISPR/Cas9-based transcriptional activation approach 69 . We did not see any eye phenotypes in terms of change in size or fate. Although we found higher levels of M1BP protein expressed in the dpp-Gal4 driver expression domain (Fig. S1). These results suggest that M1BP function is required for Drosophila eye development.
Eye suppression phenotype due to downregulation of M1BP function has no domain constraint. In  www.nature.com/scientificreports/ sion domain (dpp > M1BP RNAi ) results in reduced eye field as seen in the eye imaginal disc (Fig. 2G) and in 19% (n = 100) of the adult flies observed (Fig. 2H). Further, we used eyg-Gal4, which drives the expression of UAS-GFP reporter (eyg > GFP, shown in green) at the equator of the developing eye disc ( Fig. 2I) 26 , and does not affect the eye size on its own (Fig. 2J). Downregulation of M1BP function in eyg expression domain results in headless phenotype (Fig. 2K,L). The frequency of headless flies were around 42% (n = 100) of the adults screened. These results clearly demonstrated that there is no domain constraint in M1BP function to promote eye development.

Downregulation of M1BP function blocks the eye fate and MF progression.
Since downregulation of M1BP causes eye suppression, we studied Retinal Determination (RD) gene expression levels as a readout to study retinal determination and differentiation, the fundamental processes in the developing eye. A RD gene, eya, which acts downstream to ey, is expressed in a broader stripe in the differentiated cells posterior to the MF (Fig. 3A) 80 whereas dac is expressed as two stripes directly anterior and posterior to the MF (Fig. 3C) 81 . We found that the downregulation of M1BP in the entire eye disc using ey-Gal4 (ey > M1BP RNAi ) significantly suppresses the eye fate as seen in (C,E) the eye imaginal disc and the (D,F) adult eye. ey > M1BP RNAi exhibits a range of eye suppression phenotype ranging from a (C,D) small-eye to a "no-eye". (G) The area of adult eye was quantified using Image J software (NIH). The p values for the eye size (μm 2 ) were calculated in a set of five (n = 5) using Student's t-test in MS Excel Software. ey-Gal4 was found to be statistically significant from ey > M1BP RNAi in case of both small-eye (p < 0.001, ***) and no-eye phenotype (p < 0.001, ***). The orientation of all imaginal discs is identical with posterior to the left and dorsal up. The magnification of all eye-antennal imaginal disc is 20 × and the adult eye is 10 ×. A total of five eye-antennal imaginal discs (n = 5) for each genotype were analyzed for respective immunohistochemistry staining. www.nature.com/scientificreports/ reduces the size of the eye field as evident from the pan-neuronal marker Elav expression, which was accompanied by strong suppression of Eya and Dac expression levels (Fig. 3B,D, arrows). Expression of Atonal (Ato) and Scabrous (Sca) serves as early markers for retinal differentiation and are employed for R8 specification (Fig. 3E,G) 32,82,83 . Based on Elav and RD gene expression, we found that misexpression of UAS-M1BP RNAi in the developing eye (ey > M1BP RNAi ) suppresses retinal neuron(s) differentiation as evident from significantly reduced expression levels of Ato and Sca (Fig. 3F,H, arrows). Our data suggests that downregulation of M1BP function not only affects the retinal determination but also suppresses the markers for R8 photoreceptor differentiation. It is known that R8 specification and differentiation is associated with MF progression. We therefore tested the requirement of M1BP function in MF progression.
In the developing eye imaginal disc, Hh and Dpp signaling is required for normal initiation and progression of MF 14,29,84,85 . We used dpp-lacZ, a transcriptional reporter for dpp gene, which also marks the progression of MF in the third instar eye imaginal disc. dpp-lacZ is expressed in a thin stripe that overlays the apical constrictions caused by the MF cells and marks the anterior boundary of Elav positive differentiated retinal neurons (Fig. 3I). dpp-lacZ expression in ey > M1BP RNAi discs shows that MF fails to progress from the posterior margin of the eye disc towards the anterior side (Fig. 3J, arrow) and hence, downregulation of M1BP in the developing eye represses differentiation, resulting in eye suppression. To discern the mechanism behind eye suppression phenotypes of ey > M1BP RNAi , we looked for the putative target(s) of M1BP.

Downregulation of M1BP function induces wg and Hth expression.
In the developing eye imaginal disc, Wg serve as a negative regulator of eye fate, and blocks the progression of MF 16,37,38,85,86 . We tested if downregulation of M1BP affects the wg gene expression. Since M1BP is a transcriptional pausing factor, we studied the wg gene transcription quantitatively by using qPCR approach and qualitatively by using wg-lacZ reporter. www.nature.com/scientificreports/ The qPCR data showed that there is 2.2 fold increase in wg mRNA levels in ey > M1BP RNAi discs as compared to the controls (Fig. 4A).
The wg-lacZ reporter is expressed at antero-lateral margins of the developing third instar larval eye imaginal disc (Fig. 4B, shown in green). The ey > M1BP RNAi eye imaginal discs are significantly reduced in size and exhibits a robust ectopic induction of wg-lacZ reporter (arrow, Fig. 4C). To test if wg upregulation in ey > M1BP RNAi discs is responsible for eye suppression phenotype, we downregulated wg gene expression levels using wg RNAi in the background of ey > M1BP RNAi (ey > M1BP RNAi + wg RNAi ), which resulted in significant reduction in eye suppression phenotype and restoration of size of eye field to near wild-type (Fig. 4D). In addition wg-lacZ reporter expression is restored (Fig. 4D). In comparison to the wild-type Wg expression in eye disc (Fig. 4E), we found robust induction and ectopic localization of Wg protein in reduced eye disc of ey > M1BP RNAi (arrow, Fig. 4F) whereas Wg protein levels are restored to wild-type levels in ey > M1BP RNAi + wg RNAi background (Fig. 4G).
Since Wg is a negative regulator of eye development and it promotes head fate by inducing downstream hth expression, we further analyzed Hth protein localization in the eye discs of ey-Gal4 (Fig. 4H), ey > M1BP RNAi (Fig. 4I) and ey > M1BP RNAi + wg RNAi background(s) (Fig. 4J). Hth, which is predominantly expressed anterior to the MF (Fig. 4H) 39,47,48 , exhibits robust induction in the reduced eye field of ey > M1BP RNAi (arrow, Fig. 4I). We found that downregulation of M1BP in the eye disc induces robust Hth expression. These results strongly imply that M1BP plays an important role in promoting eye development by negatively regulating Wg and downstream Hth levels in the developing eye.
Reducing hth function rescues M1BP loss-of-function phenotype in developing eye. We wanted to determine, if reduced eye or no-eye phenotype observed in ey > M1BP RNAi background, is due to induction of hth or if there is/are other downstream target(s) of Wg signaling. Therefore, we reduced hth levels using inducible UAS-hth RNAi or a heterozygous background of hth null allele, hth 1422-4 /TM6BTb 47 . In comparison to the control eye imaginal disc (Fig. 5A), downregulation of M1BP (ey > M1BP RNAi ) results in a highly reduced "no-eye" phenotype ( Fig. 5B), whereas downregulation of hth levels using UAS-hth RNAi in ey > M1BP RNAi background (ey > M1BP RNAi + hth RNAi ) results in a significant rescue of ey > M1BP RNAi phenotype (Fig. 5D). In a heterozygous combination, the null allele of hth, hth 1422-4 /TM6B, exhibits a normal eye phenotype (Fig. 5E). However, reduction of hth function in ey > M1BP RNAi background (ey > M1BP RNAi , hth 1422-4 /TM6B) exhibits significant rescue of "no-eye" phenotype (Fig. 5F). However, there is no complete rescue to a wild-type eye and the frequency is low.

Downregulation of M1BP triggers developmental cell death.
It has been shown that ectopic induction of Wg signaling in the eye disc induces developmental cell death, which results in reduced eye phenotypes 54 . To understand the genetic mechanism responsible for the reduced eye phenotype, manifested by flies where M1BP function was downregulated, we tested the role of cell death. It is known that ectopic expression of baculovirus P35 blocks caspase-dependent cell death 56 . In comparison to the wild-type eye-antennal imaginal disc and  www.nature.com/scientificreports/ the adult eye (Fig. 6A,B), downregulation of M1BP function in ey > M1BP RNAi results in reduced eye as seen in the eye imaginal disc and the adult eye (Fig. 6C,D). Blocking caspase-dependent cell death by ectopic expression of UAS-P35 transgene in ey > M1BP RNAi (ey > M1BP RNAi + P35) background can restore the eye suppression phenotype as observed in the eye disc and the adult eye (Fig. 6E,F). Further, quantification of the area of the adult eyes of ey-Gal4 (Fig. 6B,G), ey > M1BP RNAi (Fig. 6D,G) and ey > M1BP RNAi + wg RNAi background(s) (Fig. 6F,G) shows that the eye size significantly reduces in ey > M1BP RNAi flies, when compared with ey-Gal4 (p < 0.001, Fig. 6G). However, blocking caspase-dependent cell death significantly restores the eye size in ey > M1BP RNAi + P35 background, when compared with the ey > M1BP RNAi flies (p < 0.001, Fig. 6G), and less significant than control ey-Gal4 flies (p < 0.01, Fig. 6G).
To validate our hypothesis that ectopic upregulation of wg induces developmental cell death, which results in reduced eye phenotype seen in ey > M1BP RNAi eye disc, we used the antibody against Drosophila effector caspase, death caspase-1 (Dcp-1). Dcp-1, a critical executioner of apoptosis, serves as an excellent marker for cell death 87 . In the control ey-Gal4 eye disc, we found a few Dcp-1 positive dying cells/ retinal neurons (Fig. 6H,H'). The number of Dcp-1 positive dying cells gets almost doubled in ey > M1BP RNAi eye disc, which is highly reduced in size (Fig. 6I,I' ,K). The number of Dcp-1positive dying cells is restored to the control ey-Gal4 (Fig. 6K) when P35 levels are upregulated in ey > M1BP RNAi (ey > M1BP RNAi + P35) background (Fig. 6J,J'). Further, quantification of the Dcp-1 positive nuclei shows that downregulation of M1BP in the eye disc (ey > M1BP RNAi ) induces apoptotic cell death as the average number of dying cells were significantly higher in ey > M1BP RNAi (p < 0.001, Fig. 6K) as compared to the control ey-Gal4 eye discs, however, when compared with the ey > M1BP RNAi + P35 discs, the number of Dcp-1 positive dying cells were non-significant with respect to the ey-Gal4 eye discs (ns, Fig. 6K). These results suggest that overexpressing P35 where M1BP levels are downregulated restores the size of the eye field by reducing the average number of dying cells.

Downregulation of M1BP is independent of cell proliferation function. Since downregulation
of M1BP function results in the small-eye phenotype, it is possible that the reduced number of Elav positive cells (red; Fig. 7B), which marks the photoreceptor neurons in the eye disc, is due to reduced cell proliferation. To test the role of cell proliferation in reduced eye phenotype, we stained the eye imaginal discs with phosphohistone 3 (pH3) that marks the proliferating cells (Fig. 7). Quantification of the pH3 positive cells show that the proliferating cells are significantly reduced in ey > M1BP RNAi discs (Fig. 7B,B' ,D; p < 0.001) when compared with ey-Gal4 discs (Fig. 7A,A' ,D). The reduction in number of pH3 positive cells in ey > M1BP RNAi discs does not clearly address if both cell death and cell-proliferation are involved. Although we have seen earlier that reduced size of the ey > M1BP RNAi eye disc is due to developmental cell death. In order to test the role of cell proliferation, we counted the pH3 positive cells in the ey > M1BP RNAi + P35 eye disc (where caspase-dependent cell death is blocked). Overexpression of UAS-P35 transgene along with downregulation of M1BP (ey > M1BP RNAi + P35) results in significant increase in the number of proliferating cells ey > M1BP RNAi + P35 discs, when compared with ey > M1BP RNAi discs (p < 0.001, Fig. 7C,C' , D). Interestingly, the number of pH3 positive nuclei are restored www.nature.com/scientificreports/ to the control (Fig. 7A,D). This data suggests that cell proliferation function is not the major contributing factor in reduced eye phenotype in ey > M1BP RNAi .

Discussion
Pol II pausing near the transcription start site has been identified as a key step in optimizing transcription of many genes in metazoans. It has been proposed that pausing allows the coupling of transcription and RNA processing 88 . Pausing can contribute to dynamic regulation of gene expression in response to developmental and environmental signals 7,89 , and can function to repress transcription 90 . The genome-wide studies have revealed that ~ 10-40% of all genes in mammalian embryonic stem cells and Drosophila have paused promoters 2,91-93 . In Drosophila, while the phenomenon of promoter proximal pausing has been well studied in regulation of genes encoding the heat shock proteins (Hsp) and different components involved in immune response pathways 6,90,94 , it is also proposed to play important role in regulating the gene expression during early developmental events such as patterning, sex determination etc. 2,5,7,95 . So far, the sequence-specific transcription factors such as GAGA factor and M1BP, and other regulators HEXIM, LARP7 (La Ribonucleoprotein 7, Transcriptional Regulator) have been implicated in dictating Pol II pausing in Drosophila 57,96 . However, the biological relevance of transcriptional www.nature.com/scientificreports/ pausing and the exact mechanism by which the regulatory factors may contribute in pausing of Pol II is not fully understood.

M1BP regulates retinal determination and MF progression in developing eye.
We tested for the first time the role of transcription pausing factor, M1BP during Drosophila eye development. We found that downregulation of M1BP levels in the developing eye results in strong suppression of eye fate (Fig. 1C-F), however, gain-of-function of M1BP did not affect the eye fate (Fig. S1) suggesting that optimum levels of M1BP are required for Drosophila eye development. Furthermore, we did not find any domain constraint in eye suppression function when M1BP levels were downregulated (Fig. 2C,D,G,H,K,L). In addition, when M1BP levels were downregulated (ey > M1BP RNAi ) the expression of retinal determination and differentiation genes were strongly downregulated (Fig. 3B,D,F,H). Interestingly, we found that protein encoded by RD genes were downregulated in ey > M1BP RNAi background. Therefore, M1BP may not be affecting RD gene expression directly. During eye development, a wave of differentiation, emanates from the posterior margin of the developing eye imaginal disc, which sweeps anteriorly across the retinal primordium. The crest of this wave is referred to as the MF, which results in retinal differentiation behind it 14,85 . The two signals dpp and hh plays an important (D) Quantification of the pH3 positive cells shows (green) that the proliferating cells are significantly reduced in ey > M1BP RNAi (p < 0.001, ***) than eyGal4 discs, however, when compared with the ey > M1BP RNAi + P35 discs, the number of proliferating cells were found to be comparable (non-significant) with the ey-Gal4 discs. Overexpression of P35 in M1BP loss-of-function background promotes significantly higher rate of proliferation (p < 0.001, ***), when compared with the ey > M1BP RNAi discs. The orientation of all imaginal discs is identical with posterior to the left and dorsal up. The magnification of all eye-antennal imaginal disc is 20 ×. A total of five eye-antennal imaginal discs (n = 5) for each genotype were analyzed for respective immunohistochemistry staining. www.nature.com/scientificreports/ role in initiation and progression of MF. We found that downregulation of M1BP affects retinal differentiation as well as progression of MF (Fig. 3). It suggests that M1BP role is to promote retinal differentiation as well as MF progression. Also, M1BP downregulates the level of negative regulator(s) of the eye fate. We screened for the genes, which may serve as target for M1BP mediated transcriptional pausing mechanism in Drosophila eye imaginal disc.
M1BP regulates wg gene expression in the developing eye. The protein encoded by Drosophila wg gene, a member of Wg/WNT signaling pathway, act short range inducer, which organizes the pattern of cells at a distance in the embryo. Since M1BP downregulation resulted in blocking retinal differentiation and MF progression, we looked for the targets of M1BP transcriptional pausing function using the candidate gene approach. We found that wg-lacZ reporter, which serves as a transcriptional read out for Wg, exhibits robust induction in eye imaginal discs where M1BP levels were downregulated (Fig. 4C). This observation was further validated by qPCR approach which showed that there is a 2.2-fold increase in wg gene expression (Figs. 4A, 8A). Furthermore, in high throughput microarray screen carried out in S2R + cells, wg was also identified as a target whose expression is downregulated by M1BP using M1BP RNAi. According to microarray analysis, wg shows a 5.5-fold change (raw value against wg gene ID) when cells are treated with M1BP RNAi 57 .
To validate the results from qPCR approach as well induction of wg-lacZ reporter expression in ey > M1BP RNAi eye imaginal disc (Fig. 4), we also employed bioinformatics analysis to determine if there are M1BP binding sites in the wingless (wg) gene. The M1BP binding sequence (YGG TCA CACTR) has been reported earlier 57,61 . We used this sequence for MEME analysis to screen for M1BP binding sites in wg gene and regulatory region 60 . We found 36 potential binding sites for M1BP in wingless gene and regulatory regions as shown in (S. Fig. 2B, Supplementary Table S1). Using these all 36 potential binding sites web logo was generated from weblogo.berkeley. edu/logo.cgi (Fig. S2B).
Wg, a ligand for evolutionarily conserved Wg/WNT signaling pathway, is known to act as a negative regulator of eye development 13,[37][38][39] . During Drosophila eye development, Wg activity promotes head specific fate by negatively regulating MF progression in the differentiating eye imaginal disc 22,37,38 . Wg regulates expression of downstream gene hth, which encodes a MEIS class of transcription factor, and act as a negative regulator of eye development (Figs. 4, 8A) 12,[47][48][49] . We found that in ey > M1BP RNAi background, robust induction of wg transcription also accompanies ectopic induction of hth along with the suppression of the eye fate (Figs. 4I, 8). Further, downregulation of wg levels, using wg RNAi , in ey > M1BP RNAi background rescued the eye suppression phenotype (Figs. 4, 8). This data clearly suggested that M1BP downregulates levels of wg, which in turn regulate expression of hth in the developing Drosophila eye (Fig. 5).
M1BP blocks Wg upregulation mediated developmental cell death. Higher levels of Wg are known to trigger developmental cell death in the developing eye field 54 . Interestingly, in ey > M1BP RNAi eye discs, the eye field was significantly reduced. Since, majority of the cell death is triggered by the activation of caspase-dependent cell death, blocking caspase-dependent cell death by ectopic expression of anti-apoptotic P35 transgene 56 in ey > M1BP RNAi background showed rescue of eye suppression phenotype (Fig. 6E,F). However, these P35 mediated rescues of ey > M1BP RNAi were not as significant as seen with wg RNAi (Fig. 3). This suggests that Wg might be regulating eye fate through hth induction (Figs. 4I, 8A) and eye field size by triggering caspase mediated cell death (Figs. 6, 8B). In order to rule out that these in ey > M1BP RNAi phenotypes are not affected by reduced cell proliferation rates, we also tested levels of pH3 in these developing eye fields (Fig. 7). We found that cell proliferation rates were not affected by this transcriptional pausing mechanism in the developing eye. www.nature.com/scientificreports/ Our results imply that the transcription pausing function of M1BP in regulating Wg signaling may play a critical role in Drosophila eye development (Fig. 8). However, other factors and signaling pathways involved in regulating the M1BP function at the mechanistic level is yet to be determined. In order to further understand, if M1BP mediated transcriptional regulation is also implicated during development of other imaginal discs in Drosophila, we studied the downregulation of M1BP function in bi-Gal4 domains of wing imaginal disc (Fig. S3). We wanted to test if this role of M1BP in regulating wg gene expression is exclusive to developing eye disc or it extends to other larval imaginal disc. We employed a bi-GA4 driver which drives the expression of a transgene in wing imaginal disc (Fig. S2A, A" shown in green) 63,64 . Downregulation of M1BP in bi-Gal4 expression domains of wing (bi > M1BP RNAi , Fig. S2B, B") exhibits ectopic upregulation wg expression in the pouch region of the wing imaginal disc (Fig. S2B' , arrowhead). Furthermore, M1BP expression levels are downregulated in the wing pouch region, which corresponds to the bi-Gal4 expression domain. These results suggested that the transcription pausing function of M1BP may have similar target in the eye and wing imaginal disc. Recently, HEXIM, another transcriptional regulator associated with pol II pausing, has been reported to affect wing development in Drosophila by regulating Hh signaling 97 . In Drosophila wing imaginal disc, HEXIM knockdown causes developmental defects by inducing ectopic expression of hh and its transcriptional effector cubitus interuptus (ci), which triggers apoptosis. This suggests that the regulatory factors involved in Pol II pausing are important in maintaining the expression levels of different signaling pathways during development in Drosophila.
A number of highly conserved transcriptional pausing and elongation factors such as Spt5 precisely regulate transcription during Drosophila embryogenesis. The Spt5 W049 missense mutation causes defects in the anterior-posterior patterning and segmental patterning during embryogenesis 98 . Interestingly, the mutant allele of Spt5 (foggy m806 ) in Zebrafish also causes multiple developmental defects such as discrete problems with pigmentation, tail outgrowth, ear formation and cardiac differentiation. These studies suggest that the regulatory mechanism in Pol II pausing during fly development are also conserved in higher organisms. The Drosophila compound eye shares similarities with the vertebrate eye at the level of genetic machinery as well as the processes of differentiation 99,100 . Therefore, the information generated in Drosophila can be extrapolated to higher organisms 11,100,101 . Since Wnt signaling is known to induce programmed cell death in patterning the vasculature of the vertebrate eye 102 , it will be important to study what molecules other than M1BP can prevent Wg signaling from inducing cell death during early eye development.