Mask is an interacting partner of Notch

In a yeast two-hybrid screen, we have identified Mask as an interactive partner of Notch. The same round of screen yielded multiple positive clones of Su(H), a well-recognized binding partner of Notch-ICD, thereby confirming the validity of our approach. We employed the amino terminus of Notch-ICD (amino acids 1896–2109) as a bait to screen a Drosophila 0–24 h embryonic library containing of 6 X 106 cDNAs. A total of eleven positive clones (His +) were isolated (Fig.S1 A), and it was determined that these clones encodes overlapping mask cDNAs. By analyzing the sequences of overlapping positive clones, it was identified that the second ankyrin repeat domain of Mask (amino acids 1650–2087 of LD04107 clone, Accession number: AY069323 which is corresponding to amino acids 2572–2718 of full-length Mask protein, Accession number: AF425651) was important for binding to Notch-ICD.

Confirmation of the interaction between Notch and Mask was achieved through co-immunoprecipitation experiment (Fig. 1B, C). Protein lysate was prepared from third instar larval tissues co-expressing Notch-ICD and Mask protein by wing specific vestigial-GAL4 (vg-GAL4) driver strain. In co-IP experiment, Notch-ICD was immunoprecipitated using anti-Mask antibody from protein samples extracted from larval wing discs where both proteins were co-expressed (Fig. 1B). To rule out the presence of Notch in our western blot due to non-specific binding with A/G beads, we put a control lane without adding the primary antibody (anti-Mask). Absence of band in this control lane shows the specificity of the interaction. Conversely, we co-expressed both the protein and used anti-Notch antibody (C17.9 C6) to immunoprecipitate its binding partners and Mask was found to be immunoprecipitated with Notch which was detected using anti-Mask antibody in western blot (Fig. 1C). Here also the absence of Mask band at the negative control lane confirms the specificity of the binding. These results confirm that Mask is physically associated with Notch.

To gain some additional insight about domain specific interaction of Mask and Notch, GST-pull-down experiments were done utilizing purified GST-Mask domains (Fig. 1D). Various fusion proteins of GST-Mask, including GST-ANK1 (aa546-aa1043), GST-ANK2 (aa2312-aa2644) and GST-KH (aa3036-aa3100), were expressed in bacteria and isolated using Glutathione Sepharose beads. Following thorough washing, the beads were incubated with extracts from third instar larval salivary glands overexpressing Notch-ICD using sgs-GAL4 driver. Through domain specific analysis of the Mask protein, it was revealed that the second ankyrin domain of Mask is necessary for binding to Notch-ICD (Fig. 1D). These findings suggest that Mask directly interacts with Notch-ICD and Mask binds with Notch through its second ankyrin repeat domain.

Further, we investigated the subcellular localization of these two proteins. Immunocytochemical analysis revealed that endogenous Mask and Notch colocalizes in the same subcellular compartment (Fig. 1E- O). Pearson’s correlation coefficient of the colocalization of Mask and Notch is approximately 0.5 (Fig. 1P).

mask genetically interacts with Notch Pathway components

To explore the functional consequences of the physical interaction between Mask and Notch proteins, we examined potential genetic interactions between mutant alleles of mask and Notch, as well as other components involved in the Notch signaling pathway, in trans-heterozygous combinations. For this analysis, we utilized two distinct strong loss-of-function alleles of mask: mask10.22 and mask5.8,along with a weaker hypomorphic allele mask6.3 (Fig. 2 A2-A4) [80]. The presence of a trans-heterozygous combination of the Notch null allele, N 54 l9 (Fig. 2B1), or a hemizygous Notch hypomorphic allele, Nnd−3(Fig. 2 C1), together with any of the three mask alleles, led to an increased manifestation of the wing-nicking phenotype (Fig. 2 B2-B4 and Fig. 2 C2-C4). This result suggests that the Notch function is further diminished in this scenario. Graphical representation shows the percentage of flies showing wing nicking phenotype with trans-heterozygous combination of mask and Notch alleles (Fig. 2H). In contrast, when we introduced the gain-of-function Notch allele known as Abruptex mutation (NAx−16172) (Fig. 2D1), which causes a shortened longitudinal vein IV (L4) and V (L5), in combination with mask mutations, we observed an elongation of the L4 vein extending to the wing margin (Fig. 2D2-D4). Graphical representation shows the length of L4 in only NAx−16172 allele and in combination with different mask alleles compared to wild type (Fig. 2I). Flies with a hemizygous genotype of dx, a cytoplasmic modulator of Notch signaling, show minor distal vein thickening (Fig. 2 E1). A trans-heterozygous combination of dx and any mask null mutant shows no significant phenotype in the adult wings (data not shown). However, in the case of a hemizygous combination of dx152 with mask hypomorph alleles, the wing phenotype displays distal wing vein thickening (Fig. 2E2) and with mask null allele shows an increase in vein thickening along with wing notching (Fig. 2 E3,E4). Graph shows the percentage of flies showing wing nicking phenotype in only dx152 allele and in combination with different mask alleles (Fig. 2H). C96-GAL4 driven expression of dominant negative Notch (UAS-Notch-DN) displays loss of marginal wing blade (Fig. 2 F1). A trans-heterozygous combination of a mask allele and a dominant negative allele of Notch further enhanced the wing nicking phenotype, indicating a further decrease of Notch function (Fig. 2 F2-F4). Graph shows the average wing area in alone Notch-domainant-negative and in combination with different mask alleles compared to wild type (Fig. 2J). Dominant negative C-terminal Mam truncation display a fully penetrant wing nicking phenotype using C96-GAL4 driver (Fig. 2G1) [37, 79]. Reducing the dose of mask in these individuals elicited an enhanced wing notching phenotype (Fig. 2 G2-G4). We checked the genetic interaction between Notch ligand Delta and mask (Fig.S2 A-C’). Notably, heterozygous Delta mutants (Dl BX6) exhibited vein thickening and extra vein material deposition in adult wing tissue (Fig. S2 B). This phenotype was further exacerbated in a trans-heterozygous combination with the mask loss-of-function allele (mask 6.3), suggesting a potential genetic interplay between mask and Delta in wing development (Fig. S2 C). We also checked the Notch target Cut in heterozygous Dl BX6 flies, results showed that there was a subtle reduction in Cut expression (Fig. S2 B’), the phenotype was further aggravated in trans-heterozygous combination with mask 6.3 allele (Fig. S2 C’). Our genetic interaction results revealed that, mask and Notch pathway components shows very strong genetic interaction which reiterate a functional relationship between mask and Notch consistent with their molecular interaction.

Fig. 2

Genetic interaction of mask and Notch pathway components. (A1-G4) Representative wings from individuals with indicated genotype. Wings from wild type individuals (A1) and mask heterozygous (A2-A4) showed normal wing phenotype. Wings from N54 l9 heterozygotes (B1) showed wing nicking phenotype, which was enhanced in trans-heterozygous combination with mask allele (B2-B4). Wings from male Nnd−3 hemizygotes (C1) showed nicking phenotype which was enhanced in combination with mask allele (C2-C4). Gain-of-function allele of Notch NAX−16172 shows shortened the longitudinal vein L5 and L4 (D1), which in combination with different allele of mask rescued the length of L4 completely (D2-D4). Hemi-zygotes of dx allele dx152 showed vein thickening in the distal region of the wing blade and sporadic wing nicking phenotype (E1), which was enhanced by combination with different mask loss-of-function allele (E2-E4). Dominant negative Notch showed wing nicking phenotype in heterozygous condition (F1), which was enhanced in trans-heterozygous condition with different mask allele (F2-F4). Dominant negative Mastermind, MamH in heterozygous condition shows wing notching phenotype (G1), which was enhanced in trans-heterozygous combination with different mask allele (G2-G4). Scale bar 500 µm (A1-G4). (H) Represents the percentage of flies showing wing nicking phenotype of different mask allele with combination of NN54 l9, NNd−3 and dx152 respectively. (I) Represents the length of L4 in NAX−16172 and combination with different mask allele in male and female. (J) Represents the wing area in C96-GAL4 driven Dominant-negative-Notch with combination of different mask allele. For every set of experiment at least 200 flies were observed and measurement of wing area and vein length was measured for at least 25 flies and the data was employed for statistical analysis. Significance was determined using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-test, with a p-value < 0.05 considered statistically significant (**p < 0.01, ***p < 0.001). Scale bar 500 µm

Downregulation of Mask results in downregulation of Notch signaling activity

Tissue-specific GAL4 drivers were exploited to study the downregulation effect of mask in third instar larval wing imaginal discs. The Mask-RNAi lines (UAS-Mask-RNAi) was used to investigate the loss-of-function effects of mask on Notch signaling activity as well as the effect on the endogenous Notch protein levels (Fig. 3A- 3P). We utilized one strong and another relatively milder RNAi lines targeting mask. Downregulation of mask by using strong RNAi line and engrailed-GAL4 in the posterior compartment of the wing imaginal disc at 25 °C shows larval lethality and at 18 °C results in a downregulation of Cut (Fig. 3B) and NRE-GFP (Fig. 3F), which is expressed under the influence of Notch Signaling at the dorsoventral boundary of the wing imaginal discs. Similar kind of reduction of Cut was observed when we downregulates Mask using milder RNAi line (Fig. S3 A-S3B’). Similarly, there was a subtle reduction of NRE-GFP expression at the posterior domain when we downregulates Mask using milder RNAi line (Fig. S3D-S3E’). Loss of Cut was observed upon downregulating Mask with strong RNAi at the anterior–posterior boundary using patched-GAL4 at 25 °C but shows pupal lethality (Fig. 3J). However, the downregulation of mask using ptc-GAL4 did not affect the level or localization of the endogenous Notch protein level (Fig. 3N). (Total number of discs examined = 20, for each case). We utilized the antibody designed against NECD (C458.2H) from DSHB to examine the status of the full-length Notch receptor under Mask downregulation conditions. For this, we downregulated Mask at the anterior–posterior (AP) boundary using ptc-GAL4 and assessed full-length Notch receptor expression. Our results indicated no significant differences in the expression pattern or distribution of full-length Notch in the Mask downregulation background (Fig. S3 G-J). We further investigated the impact of Mask downregulation on additional Notch signaling components in wing imaginal disc cells, specifically Delta and Suppressor of Hairless [Su(H)]. To assess this, we downregulated Mask at the dorsal–ventral (DV) boundary of the wing imaginal disc using c96-GAL4 and analyzed Delta and Su(H) expression levels (Fig. S4 A-H). Our results indicate that Mask downregulation does not significantly affect Delta (Fig. S4 C) or Su(H) expression. (Fig. S4 G).

Fig. 3

Reducing Mask level down regulates Notch signaling activity. A-H Representative images from third instar wing imaginal disc show the expression of Cut and NRE-GFP upon reducing the level of Mask using posterior domain specific engrailed-GAL4. Abrogating Mask in the posterior domain using Mask-RNAi results into abolished Cut (A-D) and NRE-GFP (E–H) expression. A and E shows the RFP marked posterior domain where Mask was downregulated. B and F shows the abrogated Cut and NRE-GFP expression upon Mask downregulation, respectively. C and G is the merged images of A,B and E,F respectively. D and H are the merged images of C and G with DAPI respectively. B’ and F’ are the intensity of Cut and NRE-GFP along with DV boundary, respectively. Q represents the relative intensity of Cut and NRE-GFP at the anterior and posterior domain. A total number of disc examined = 20, for each case. A total number of 5 discs were used for quantification in each case followed by unpaired t-test to determine the significance of our findings. (I-P) Downregulation of Mask using patched-GAL4 along with AP boundary shows the abrogated Cut (I-L) expression at the AP-DV junction but no change at the level of Notch protein (M-P). I and M shows the GFP marked AP boundary where Mask was downregulated. J and N show the level of Cut and Notch protein respectively, upon downregulation of Mask at AP boundary. K, O is the merged images of I,J and M, N respectively. L and P are the merged images with DAPI. J’ and N’ show the intensity of Cut and Notch at DV boundary. (R-W) Loss-of-function clones of mask using mask10.22 allele were generated with FLP/FRT system; mask.10.22 clones were marked by the absence of Green Fluorescent Protein (GFP) expression. Cut staining in wing disc of such clones display significant reduction in the expression (S); whereas Notch staining shows no significant change in expression and localization of Notch protein (V). Scale bars, 20 µm (A-P) and (R-W). Unpaired t-test was performed to determine the p-value (**p < 0.01, ***p < 0.001)

Next we utilize FLP-FRT system [93] and different loss-of-function mask alleles to generate mask mutant somatic clones in larval wing imaginal discs. In mask10.22 mutant clones we investigate the expression of Notch target gene, Cut and we observed that Cut protein level was significantly reduced in mask10.22 mutant clones compared with wild type sister cells at the DV boundary (Fig. 3R-T). We also observed that the mask10.22 clones are relatively small in size; respect the DV boundary and very few mutant clones are present at the DV boundary (Fig. 3 R-W). Interestingly, in mask10.22 mutant clones, no significant change in the Notch protein level was observed compared with the surrounding wild-type cells, except there is a reduction of endogenous Notch puncta that represents the vesicular Notch (Fig. 3V). These observations confirmed that mask not only positively regulates Notch signaling but also play vital role in the modulation of Notch downstream target genes (Fig. 3). Thus, downregulation of mask resulted in reduced Notch signaling activity without affecting the level of endogenous Notch receptor.

Yki is a known interactor of Mask thus to assess the involvement of Yki, we overexpressed NLS-tagged Yki (UAS-Yki-NLS-HA) in the Mask downregulation background, with C96-GAL4 and analyzed Cut expression (Fig. S4 J-J’’). The results indicated that Yki-NLS-HA overexpression did not cause any significant changes in Cut expression at the DV boundary (Fig. S4 J) compared to control one (Fig. S4I). Mask is essential for cell survival [80] and plays a role in the nuclear translocation of Yki [77]. We sought to determine whether the downregulation of Notch target gene expression due to Mask loss is dependent on these factors or occurs independently. To explore this, we overexpressed the effector caspase inhibitor p35 in a Mask downregulation background with DV boundary specific C96-GAL4, and examined Cut expression. The results showed that Cut expression remained impaired at the DV boundary (Fig. S4 K). Adult wing phenotype also suggests that there was no significant change in coexpression of Mask-RNAi and p35 (Fig S4 K’) compared to control one (Fig. S4I’’), although overexpression of Yki-NLS-HA in Mask downregulation background results into pharate adult phenotype (Fig. S4 J’’). Graphical representation shows that there was no significant difference in Cut expression in experimental group compared to control group (Fig. S24L).

Mask loss- and gain-of-function resembles wing and eye phenotypes of Notch mutants

Through the utilization of different tissue specific GAL4 drivers [7], we checked the downregulation and gain-of-function effect of mask in adult tissues. Two different mask-RNAi lines were used to explore the tissue-specific loss-of-function effects of mask (Fig.S5 A-S5T). Downregulation of mask using strong RNAi line with apterous-GAL4 in the dorsal region of the wing imaginal discs, which form the adult thorax and wing, resulted in crumpled wing along with severely reduced scutellar region and loss of scutellar bristles (S5M). The weaker Mask-RNAi shows crumpled and outwardly directed wings in adult flies (Fig. S5L). Downregulation with strong RNAi of mask using en-GAL4 in the posterior domain of the wing discs shows pupal lethality at 25℃; however, the milder RNAi resulted in reduced posterior cross vein, bending of third vein and overlapped wings (Fig. S5D). At 18℃, strong RNAi shows the same phenotype as of mild RNAi at 25℃ (data not shown). ptc-GAL4 driven Strong RNAi of mask also shows pupal lethality at 25℃ but at 18℃ it shows extremely reduced AP boundary region in between L3 and L4 (Fig. S5I). No adult flies were eclosed from downregulation of Mask using strong RNAi line with ptc-GAL4 and en-GAL4 at 25 °C but at 18 °C, 32% and 28% adult flies ecloses form the pupae, respectively (Fig. S5P). Both strong and mild RNAi mediated downregulation of mask at the wing pouch using nubbin-GAL4 shows wing notching phenotype along with severe reduction in wing size (Fig. S5 F and S5H). Next onwards downregulation was done using strong RNAi only. dpp-Gal4 mediated downregulation of mask at the anterior–posterior boundary of the wing, shows wing notching, reduced anterior region of the wing blade and missing anterior cross vein (ACV)(Fig. S5G). Downregulation of mask at the DV boundary using vg-GAL4 and c96-GAL4 results in severely reduced wing size and wing notching phenotype (Fig. S5 C, S5 K and S5 N). Furthermore, loss of mask at the anterior domain of the wing blade using cubitus interruptus-GAL4 resulted in wing notching, reduced anterior portion of the wing blade and extra vein material in the joining of the first and second vein (Fig. S5B). Consequently, the absence of mask in various sections of the wings blade led to the formation of wing notching, irregular vein formation, and the presence of small areas with disorganized tissue or blister formation in wing blades. The elimination of mask in the eye through the utilization of eyeless-GAL4 led to the absence of ommatidia and a severe decrease in the size of the eye (Fig. S5Q to S5S). Downregulation of Mask using Mask RNAi shows extremely reduced eye phenotype (Fig. S5R and S5S) compared to wild type (Fig. S5Q). These mask-downregulated phenotypes closely resembled the phenotypes observed in cases of Notch loss-of-function. The developmental abnormalities and the percentage of affected flies resulting from Mask downregulation are summarized in Supplementary Table 1. It is widely recognized that Notch plays a crucial role in the development of wing margin, veins, and sensory bristles. The absence of Notch function leads to a reduction in the distance between veins and a decrease in eye size, as documented in previous studies [10, 32, 36, 79] and phenotypes associated with mask downregulation mimic these Notch loss-of-function phenotypes.

Additionally, we overexpress mask in different regions of the wing using various tissue-specific GAL4 (Fig. S5U-S5X). Gain-of-function of mask in the posterior region of the wing using en-GAL4 results loss of wing margin and loss of fifth vain (Fig. S5U). Overexpression of mask at the dorsoventral boundary of the wing disc using vestigial-GAL4 transgene, resulted in severe notching at the posterior wing margin (Fig. S5 V). Overexpression using ap-GAL4 in the dorsal region of the wing resulted in a crumpled wing, loss of wing margin and thick and spreaded posterior cross vein (Fig. S5 W). nubbin-GAL4 mediated mask gain-of-function in the pouch of the third instar larval imaginal disc resulted, drastic notching at the wing margin, loss of fifth vein and loss or incomplete anterior cross vein (Fig. S5X).

Mask shows epistatic interaction with Notch

To investigate the epistatic interaction between Mask and Notch, we assessed whether Mask could mitigate Notch-induced phenotypes. Overexpression of Notch-ICD at the wing margin, driven by C96-GAL4, led to abnormal wing margin bristles (Fig. 4E). This phenotype was notably alleviated by reducing mask expression using Mask-RNAi in the same context (Fig. 4O). Additionally, we confirmed this epistatic relationship by evaluating the expression of the Notch downstream target, Cut [70]. Notably, ectopic Cut expression was observed when Notch-ICD was overexpressed at the AP (Fig. 4A) and DV (Fig. 4C) boundary of the wing imaginal disc using the dpp-GAL4 and C96-GAL4 driver, respectively. However, the expression of ectopic Cut due to Notch-ICD overexpression was significantly rescued by lowering mask expression through UAS-Mask RNAi in the same context (Fig. 4K and M).

Fig. 4

mask shows epistatic interaction with Notch and is required for Notch mediated downstream target gene expression. (A-N) Downregulation of Mask at AP and DV boundary using dpp-GAL4 and C96-GAL4 respectively results in significant reduction of Cut expression. The overexpression of Notch-ICD results into excessive Cut expression at AP (A) and DV (C) boundary. This overexpressed Cut is significantly rescued upon downregulation of Mask using Mask RNAi in the same background in AP (K) and DV (M) boundary respectively. F and H shows the effect of downregulation of Mask upon Cut expression pattern at AP (F) and DV (H) boundary. B and D show that NICD overexpression does not affect the expression of Mask in AP and DV boundary. G and L are the expression pattern of Mask upon Mask downregulation and Notch overexpression plus Mask down regulation at AP boundary. I and N are the expression pattern of Mask upon Mask downregulation and Notch overexpression plus Mask down regulation at DV boundary. E represents the effect of NICD overexpression at the DV boundary using C96-GAL4, J represents the Mask downregulation with C96-GAL4. O represents that Mask downregulation results in the reduction of Notch overexpression phenotypes at the wing margin.. A’, F’, K’ and C’, H’, M’ show the intensity profiling of Cut at the AP and DV boundary, respectively. P represents the average intensity of Cut in Notch overexpressed, Mask downregulation and Mask downregulation in Notch overexpressed background at AP and DV boundary. A total 15 discs for each case were observed and the result was consistent. ImageJ software was utilized for intensity profiling by calculating the mean intensity (integrated density divided by the area of the domain). Quantification was done on a total of five discs for each condition. Significance was determined using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post-test, with a p-value < 0.05 considered statistically significant (**p < 0.01, ***p < 0.001). Q shows the percentage of wing nicking phenotype in Notch overexpressed, Mask downregulation and Mask downregulation in Notch overexpressed background. Profile plot analysis was done by FIJI software. Scale bar 20 µm (A-N)

Overexpression of Notch-ICD with dpp-GAL4 or C96-GAL4 does not affect the endogenous Mask expression pattern (Fig. 4B, D). Downregulation of Mask results wing nicking phenotype with C96-GAL4, similar to Notch loss-of-function phenotype and also shows abrogated Cut expression at AP using dpp-GAL4 (Fig. 4F) and at DV boundary using C96-GAL4 (Fig. 4H). Profile plot display the intensity of Cut were significantly reduced at AP (Fig. 4K’) and DV (Fig. 4M’) boundary when Mask is downregulated in Notch overexpression background compared to only Notch overexpression (Fig. 4A’ and C’). Percentage of wing nicking phenotype due to Mask downregulation using C96-GAL4 was significantly reduced when Notch-ICD was coexpressed in the same background (Fig. 4Q). These results show that in the absence of Mask, Notch-ICD unable to show its complete activity and thus reduction of ectopic Cut expression was observed (Fig. 4P).

We extend our study with wing specific vestigial-GAL4 and eye specific eyeless-GAL4 (Fig. S6 A-I). Overexpression of activated Notch (Notch-ICD) results pupal lethality, very few individuals (3%) can eclose from pupae (Fig. S6B). Downreglation of Mask in the same background increases the percentage of eclosion upto 17% (Fig. S6D, S6I). Similarly, with ey-GAL4 mediated Notch-ICD overexpression and Mask downregulation results extremely proliferative and small eye phenotypes, respectively (Fig. S6 F, S6G). Co-expression of Mask-RNAi and Notch-ICD rescues the over proliferation phenotype as compared to only Notch-ICD overexpression (Fig. S6H).

Further, we investigate the effect of Mask downregulation over different Notch loss-of-function component (Fig. S6). Downregulation of Mask at DV boundary using C96-GAL4 results in wing nicking phenotype and reduction of Cut expression (Fig. S6k, S6k’). Dominant negative form of Mastermind (MamH) overexpression shows wing nicking phenotype and irregular expression of Cut at DV boundary (Fig. S6L, S6L’). Coexpression of MamH and Mask RNAi using C96-GAL4 results into further reduction of Cut expression and increased wing nicking phenotype compared to either Mask-RNAi or MamH expression (Fig. S6M, S6M’). Downregulation of Notch using Notch RNAi (NIRM) with C96-GAL4 driver, results in loss of wing margin and decreased expression of Cut at the DV boundary (Fig. S6 N, S6 N’). Reduction of Notch (UAS-NIRM) and Mask in a same organism results into increased loss of wing margin and further reduction of Cut expression (Fig. S6O, S6O’). Overexpression of dominant negative form of Notch with C96-GAL4 results into loss of marginal wing blade and irregular expression of Cut at the DV boundary (Fig. S6P,S6P’). Down-regulation of Mask in the same background affects both wing phenotype and Cut expression negatively (Fig. S6Q, S6Q’). Graphical representation shows the area of wing blade (Fig. S6R) and intensity of Cut (Fig. S6S) in different control and experimental group.

Overexpression of Mask modulates Notch Signaling activity

Downregulation and loss-of-function studies of Mask showed that Mask is a positive regulator of Notch signaling activity. We have also investigated the impact of ectopic expression of Mask on Notch downstream target expression. To achieve this, we overexpressed Mask specifically in the posterior domain of the wing disc, utilizing the en-GAL4 driver (Fig. 5A- L). We investigated the level of Notch signaling activity by checking the expression of Notch downstream targets, Cut, Dpn and Vg, which expresses at the DV boundary of the wing discs. Notch signaling is required for the cell-autonomous activation of Cut [63]. Similarly, activated Notch promotes the expression of Dpn by binding to the Notch-responsive enhancer located in the regulatory region of Dpn [73]. Vg is directly regulated by Notch in the wing pouch [44, 46, 91]. Overexpression of Mask results in a diminish expression of Cut (Fig. 5B), Dpn (Fig. 5F) and Vg (Fig. 5J) at the posterior domain marked with GFP (Fig. 5 A, E, I). Intensity profile plot displays the reduction of the Cut (Fig, 5B’), Dpn (Fig. 5F’) and Vg (Fig. 5J’) at the posterior domain compared to the anterior internal control domain. Statistical analysis shows that the reduction is significant (Fig. 5M). Interestingly, overexpression of Mask with different GAL4 shows loss of partial wing blade phenotype (Fig. S5U-S5X). Posterior region of the adult wing blade is prone to show the phenotype. Upon analyzing the adult wing phenotype, we hypothesized that extensive cell death may be a primary contributing factor to this phenotype.

Fig. 5

Effect of Mask overexpression on Notch signalling activity. (A-L) engrailed-GAL4 driven Mask overexpression at the posterior domain (marked with GFP A, E, I) of third inster wing imaginal disc resulted in the reduction at the expression of Notch target Cut (B), Dpn (F) and Vg (J). C, G and K represents the merged images of AB, EF and IJ respectively. D, H and L represents the merged images with DAPI. B’, F’ and J’ shows the intensity profiling of Cut, Dpn and Vg respectively. (M) compares the average intensity of Cut, Dpn, and Vg per unit area between the anterior and posterior domains when Mask is overexpressed in the posterior domain. A total of 15 discs was examine for each case, and five out of them are used to quantify the intensity. Intensity data shows that increased dosage of Mask significantly reduces the level of Notch target gene expression at the posterior domain. Unpaired t-test was performed to determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001)

Caspase inhibition does not mitigate Mask overexpression-mediated loss of Notch targets

Loss of wing blade and loss of Notch target gene expression due to Mask overexpression can take place due to cell death, and/or perturbation of Notch signaling, and/or due to activation of other signaling cascades that negatively impact Notch signaling.

To check these options, first we block the effector caspase by the expression of p35. p35 inhibits the effector caspases and subsequently stops the cell death. Co-expression of Mask and p35 was driven in the posterior domain using the en-GAL4 driver line to examine the expression of various Notch target genes at the DV boundary. Blocking effector caspase activity resulted in a partial improvement in the expression of the Notch target genes Cut (Fig. 6B), Dpn (Fig. 6F), and Vg (Fig. 6J). Intensity profile plot shows that intensity of Cut (Fig. 6B’), Dpn (Fig. 6F’) and Vg (Fig. 6J’) is significantly low compared to the internal control anterior domain. These results suggest that cell death is not solely responsible for the phenomena.

Fig. 6

Blocking caspase activity to inhibit cell death does not alleviate the loss of Notch target gene expression induced by Mask overexpression. (A-L) Represents the third inster wing imaginal disc overexpressing both Mask and p35 the caspase blocker using posterior domain specific en-GAL4 tagged with GFP (A,E,I). (B, F, J) Co expression of Mask and p35 minorly rescued the abolished Notch target genes but not completely. (C,G,K) represents the merged images from AB, EF and IJ respectively. (D, H, L) Shows the merged image with DAPI. B’, F’, J’ represents the intensity profiling of Cut, Dpn and Vg respectively. (M) compares the average intensity of Cut, Dpn, and Vg per unit area between the anterior and posterior domains when Mask and p35 is overexpressed in the posterior domain. Total 20 (n = 20) imaginal discs were examine out of them five were used to quantify the intensity. An unpaired t-test was subsequently conducted to assess the significance of the results (**p < 0.01, ***p < 0.001). (N) adult wing image shows improvement in loss of wing blade area but not completely rescued, where both Mask and p35 was overexpressed using en-GAL4 driver. (O) Blocking JNK signalling by dominant negative form of basket (UAS-Basket-DN) rescued the wing phenotype completely. (P)Graph showing the percentage of wing area among different genotype mentioned. (Q-S) Overexpression of Mask at posterior domain (marked with GFP) (Q), trigger JNK activation, reported by TRE-JNK reporter line(R), (S) is the merged image of Q and R. (T-V) Overexpression of Basket-DN rescues the loss of Cut (U) due to Mask overexpression. (T) GFP marked the domain and (V) shows the merged image of T and U. (R’, U’) Graph shows the intensity profiling of TRE JNK in Mask overexpression condition and profiling of Cut upon Basket-DN expression in Mask overexpression background. (R’’, U’’) Graph shows the Comparison between anterior and posterior domain specific expression of TRE-JNK and Cut, respectively. A total 15 imaginal discs were examined and out of them total five discs were used to quantify the intensity for R’’, and for U’’ total 10 imaginal discs were examined and 5 out of them were used to quantify the intensity. Statistical analyses were carried out to determine the significance of the data by performing unpaired t-test and determine the p-value (*p < 0.05, **p < 0.01, ***p < 0.001). Scale bar 20 µm (A-L) and (Q–V)

Here we explored wheather Mask overexpression mediated loss of Notch signaling activity is only for Notch targets or Notch ligand also. At this end, we checked the Notch ligand Delta in Mask overexpression background. Overexpression of Mask in the posterior domain using en-GAL4 leads to the downregulation of Delta expression in the third instar larval wing imaginal disc (Fig. S7 A and A’). As Mask overexpression induces cell death, we co-expressed p35 in the Mask overexpression background to inhibit apoptosis. Notably, inhibition of apoptosis rescued Delta expression completely (Fig. S7 C and C’), indicating that the observed downregulation of Delta was primarily due to cell death rather than a direct effect of Mask overexpression on Notch Signaling. These results also suggest that apoptosis inhibition completely rescued the Notch ligand but not the Notch target gene expression.

We also observed that Mask overexpression is enough to trigger JNK activation, observed by the expression of TRE-JNK at the posterior domain (Fig. 6R). The transcriptional response element (TRE) is a regulatory sequence that serves as a binding site for the transcription factors Fos and Jun, commonly utilized to evaluate the activation of JNK signaling [13]. Intensity profile plot shows the expression of TRE-JNK is very strong at the posterior domain as compared to anterior domain (Fig. 6R’). Moreover, we observed that increased Mask level significantly increases acridine orange positive cells at the posterior domain of the disc, where Mask was overexpressed with en-GAL4 (Fig. S7 E). Earlier it was shown that Mask is associated with MAPK pathway [80]. At this point we were interested to check whether JNK is involved in this case or not, because there are plenty of information which showed that JNK and Notch acts antagonistically [84, 89]. To, check the notion we block Basket (the Drosophila JNK) by overexpressing dominant negative form of Basket in Mask overexpression background. Strikingly, our results showed that blocking Basket not only recovers the diminished Cut level but also the expression of Cut expanded (Fig. 6U, 6U’’). These results show that Mask overexpression results into activation of JNK signaling and subsequently downregulates Notch signaling.

Mask expression levels are crucial for proper Notch activity

Our results demonstrated that Mask downregulation correlated with reduced Notch target gene expression (Fig. 3A- 3L). Intriguingly, when we overexpress Mask within the posterior domain of the wing disc using the en-GAL4 driver, a similar kind of reduction in the target gene levels at the DV boundary was observed (Fig. 5A- L). Further analysis of Mask protein levels in this overexpression context revealed an elevated signal within the designated