Connecting Hippo Pathway and Cytoophidia in Drosophila Posterior Follicle Cells

CTP synthase (CTPS), the rate-limiting enzyme in the de novo synthesis of CTP, assembles into a filamentous structure termed the cytoophidium. The Hippo pathway regulates cell proliferation and apoptosis. The relationship of the nucleotide metabolism with the Hippo pathway is little known. Here, we study the impact of the Hippo pathway on the cytoophidium in Drosophila melanogaster posterior follicle cells (PFCs). We find that the inactivation of the Hippo pathway correlates with reduced cytoophidium length and number within PFCs. During the overexpression of CTPS, the presence of Hippo mutations also reduces the length of cytoophidia in PFCs. In addition, we observe that knocking down CTPS mitigates hpo (Hippo)-associated over-proliferation. In summary, our results suggest that there is a connection between the Hippo pathway and the nucleotide biosynthesis enzyme CTPS in PFCs.


Introduction
The nucleotide metabolism plays a crucial role in various cellular processes, including the synthesis of DNA and RNA and the generation of cellular energy.The synthesis of purines and pyrimidines follows two primary pathways: the de novo nucleotide synthesis and the salvage pathway.CTP synthase (CTPS) assumes the role of the rate-limiting enzyme in the de novo synthesis pathway for CTP [1,2].CTPS exhibits a dynamic equilibrium among monomers, dimers and tetramers [3].In the presence of a substrate, CTPS presents at a high concentration of the active tetramer type [4].The activity of CTPS is increased in cancer cells, such as those in liver neoplasia and kidney tumors [5].
Cytoophidia are widely distributed during both the larval stage [16] and oogenesis of Drosophila.Within the Drosophila ovaries, cytoophidia are observed in various cell types, including follicle cells, nurse cells and oocytes [17][18][19].Cytoophidia can be categorized into two distinct classes: micro-cytoophidia, measuring approximately 1-4 µm in length, and macro-cytoophidia, which can extend to up to 30-40 µm [6].Functionally, cytoophidia serve as scaffolds [20][21][22] and effectively prolong the half-life of CTPS [23].Considering that CTPS is a rate-limiting enzyme, it is crucial to gain a comprehensive understanding of its regulatory mechanism.
The Hippo pathway was first discovered in Drosophila in 1995 [24,25].The mutations of the Hippo pathway result in an over-proliferative phenotype.In Drosophila, the core of the Hippo pathway coordinates a series of phosphorylation events that ultimately inhibit the transcriptional coactivator Yorkie (Yki).Initially, the Sterile 20-like kinase Hippo (Hpo) [26] associates with the WW domain scaffolding protein Salvador (Sav) [27] to phosphorylate and activate the DBF family kinase Warts (Wts).Subsequently, the activated Wts interacts with Mob as a tumor suppressor (Mats) [28] to phosphorylate and inhibit Yki [29], creating a 14-3-3 binding site.Yki then binds with the 14-3-3 protein, retaining Yki in the cytoplasm [30].
The Hippo pathway is evolutionarily conserved in mammals [31].The Hippo pathway controls the expression of downstream genes, including diap1, bantam and cyclin E [32], to regulate proliferation and apoptosis, thereby maintaining tissue size homeostasis [33].Furthermore, the Hippo pathway is implicated in the regulation of the lipid metabolism and organ regeneration [34].The dysregulation of the Hippo pathway has been identified in various cancers, such as malignant mesothelioma [35] and prostate adenocarcinoma [36].A previous study has found that YAP1 increases the glutamine level and enhances de novo nucleotide synthesis in zebrafish [37,38].The knockdown of YAP/TAZ decreased the expression of CTPS1 in MDA-MB-231 cells [39].However, the mechanism by which the Hippo pathway regulates nucleotide synthesis remains unknown.Therefore, our study aimed to investigate the connection between the Hippo pathway and cytoophidia.
Using the Drosophila egg chamber as a model [40], we observed that the Hippo pathway and cytoophidium assembly interconnect in posterior follicle cells (PFCs).Mutations in the Hippo pathway result in over-proliferation and Notch accumulation in the PFCs.Hippo pathway mutations reduce the length and number of cytoophidia within the PFCs.In addition, Hippo pathway mutations reduce the length of the cytoophidium, even when CTPS is overexpressed.CTPS knockdown suppresses the over-proliferative phenotype in hpo mutant PFCs.Therefore, our study connects the Hippo pathway and cytoophidia in PFCs.

Hippo Pathway Is Essential for Oocyte Polarity
In Drosophila oogenesis, the establishment of the body axis depends on the polarization of the oocyte [41,42].The asymmetric distribution of mRNA and proteins within the oocyte plays a pivotal role in determining the anterior-posterior (AP) body axis [43].For example, gurken (grk) mRNA localizes adjacent to the oocyte nucleus [44], while bicoid (bcd) mRNA is found at the anterior pole [42], and oskar (osk) mRNA is concentrated at the posterior pole [41].The dysregulation of the Hippo pathway disrupts oocyte migration, leading to the over-proliferation of anterior and posterior follicle cells, as well as the mis-localization of Osk and Staufen (Stau) [45,46].
We used a mosaic analysis with a repressible cell marker (MARCM) technique [47] to generate FRT/FLP mitotic clones of mutated cells with a loss of function (wts x1 ) allele of wts.In control egg chambers, the OSK signal was localized in the crescent at the posterior side (Figure 1A,B), while single-layer follicle cells surrounded the germline cells (Figure 1C).The Notch intracellular domain (NICD) signal was observed at the apical part of the follicle cells (Figure 1A,D).The GFP signal indicated that the follicle cells expressed GFP but had no mutation (Figure 1E).In the wts x1 egg chambers, when the posterior follicle cells were GFP-positive clones (wts x1 clones) (Figure 1F,J), we observed that the OSK mis-localized to the middle of the oocyte (Figure 1G), and we also observed the over-proliferative phenotype (Figure 1H) and the accumulation of the NICD signal (Figure 1I).These results are consistent with those of previous studies [45,46].We found that the signal of GFP in the wts x1 PFCs was lower than other GFP-positive clones (Figure 1J), but the NICD signal accumulated in the wts-mutant PFCs.Hence, in order to mark the wts x1 PFCs, we chose the NICD signal results are consistent with those of previous studies [45,46].We found that the signal of GFP in the wts x1 PFCs was lower than other GFP-positive clones (Figure 1J), but the NICD signal accumulated in the wts-mutant PFCs.Hence, in order to mark the wts x1 PFCs, we chose the NICD signal as a marker (the yellow line in Figure 1I).The over-proliferative phenotype is effectively framed by the yellow line (Figure 1H).

Cytoophidium Length and Number Are Decreased in wts-Mutant PFCs
To investigate whether the wts x1 mutation affects CTPS cytoophidia assembly in PFCs, we used antibodies targeting the NICD and CTPS for the immunostaining of egg chambers.In the control egg chambers, cytoophidia were observed in germline cells and follicle cells (Figure 2A,D).The NICD signal localized at the apical side of the follicle cells (Figure 2B), and a layer of follicle cells surrounded the germline cells (Figure 2C).In the wts x1 egg chambers shown in Figure 2F, the accumulated NICD signal is framed by a yellow line (Figure 2G) to mark the wts x1 PFCs, and the over-proliferative phenotype was

Cytoophidium Length and Number Are Decreased in wts-Mutant PFCs
To investigate whether the wts x1 mutation affects CTPS cytoophidia assembly in PFCs, we used antibodies targeting the NICD and CTPS for the immunostaining of egg chambers.In the control egg chambers, cytoophidia were observed in germline cells and follicle cells (Figure 2A,D).The NICD signal localized at the apical side of the follicle cells (Figure 2B), and a layer of follicle cells surrounded the germline cells (Figure 2C).In the wts x1 egg chambers shown in Figure 2F, the accumulated NICD signal is framed by a yellow line (Figure 2G) to mark the wts x1 PFCs, and the over-proliferative phenotype was observed, as shown with the yellow line (Figure 2H).We compared the length of cytoophidia in the NICD accumulation area with a normal area (Figure 2I).Comparing the GFP-positive clones, which had no accumulated NICD, with the GFP-negative clones, we found that the length of cytoophidia had no difference (data no shown) in the wts x1 egg chambers.An analysis revealed that the length of cytoophidia in the accumulated NICD was shorter compared to the adjacent cells (Figure 2K).By counting the numbers of cytoophidia and follicle cells, we found that the proportion of cytoophidium in the accumulated NICD PFCs was decreased compared to other GFP-positive clones and GFP-negative clones (Figure 2L).
observed, as shown with the yellow line (Figure 2H).We compared the length of cytoophidia in the NICD accumulation area with a normal area (Figure 2I).Comparing the GFP-positive clones, which had no accumulated NICD, with the GFP-negative clones, we found that the length of cytoophidia had no difference (data no shown) in the wts x1 egg chambers.An analysis revealed that the length of cytoophidia in the accumulated NICD was shorter compared to the adjacent cells (Figure 2K).By counting the numbers of cytoophidia and follicle cells, we found that the proportion of cytoophidium in the accumulated NICD PFCs was decreased compared to other GFP-positive clones and GFP-negative clones (Figure 2L).

Cytoophidium Length and Number Are Decreased in Hpo-Mutant PFCs
To confirm that the reduction in the cytoophidium length can indeed be regulated by the Hippo pathway, we generated FLP/FRT mitotic clones in the egg chamber using a truncating (hpo BF33 ) allele of hpo.In the control egg chambers (Figure 3A), the NICD signal (Figure 3B) and cytoophidium length (Figure 3D) had no significant differences between the GFP-positive and GFP-negative clones (Figure 3E).There was a layer of follicle cells surrounding the germline cells (Figure 3C).The GFP-positive clones indicated that the follicle cells expressed GFP but had no mutations.In the hpo BF33 egg chambers (Figure 3F), the accumulated NICD signal is framed by the yellow line in Figure 3G.The over-proliferative phenotype was also observed in the hpo-mutant PFCs (Figure 3H).Furthermore, during the quantification of the cytoophidia length, we observed that the hpo BF33 resulted in a decrease in the cytoophidium length in the NICD accumulation area (Figure 3I,K) compared with the cytoophidia in other follicle cells.Similarly, the proportion of cytoophidium in accumulated NICD PFCs was decreased compared to the other GFP-positive clones and GFP-negative clones (Figure 3L).

Hpo Mutation Decreases Cytoophidium Length Even If CTPS Is Overexpressed
To investigate whether CTPS overexpression can reduce the cytoophidia length in the hpo mutant PFCs, we generated a stock containing the hpo BF33 mutation and UAS-CTPS-mCherry-HA.In the control with the CTPS overexpressed egg chamber (Figure 4A), the yellow lines shown in Figure 4A marked the GFP-positive clones, which overexpressed CTPS.The NICD signal localized at the apical side of the follicle cells (Figure 4B).The follicle cells surrounded the germline cells (Figure 4C).The length and abundance of cytoophidia increased in the GFP-positive clones (Figure 4D).
In hpo BF33 with CTPS overexpressed egg chambers (Figure 4F), the hpo mutant PFCs shown in 4G are framed by a yellow line based on the accumulated NICD signal.The overproliferative phenotype was observed and is shown within the yellow line in Figure 4H.The cytoophidium length increased within the GFP-positive clones (Figure 4F,I).The GFPpositive follicle cells took hpo mutation and overexpressed CTPS (Figure 4J).Additionally, it is worth noting that the length of cytoophidia in the accumulated NICD clones was shorter than the that in the other GFP-positive clones (Figure 4I,K).

CTPS Knockdown Suppresses hpo BF33 -Induced Excessive Proliferation
To investigate whether the CTPS is required for cell proliferation regulated by the Hippo pathway, we generated a stock containing hpo BF33 and UAS-CTPS-RNAi.Here, we chose the hpo BF33 egg chamber as a control (Figure 5A), and the surface matched the accumulated NICD signal (Figure 5A'-A"').In hpo BF33 with CTPS knockdown egg chambers (Figure 5B), we observed that the NICD signal accumulated in the hpo BF33 with CTPS knockdown PFCs (Figure 5B').By comparing the volume of surface, we found that the NICD accumulated volume in the hpo BF33 with CTPS knockdown was smaller than that in the hpo BF33 egg chambers (Figure 5E).This result indicated that CTPS knockdown suppresses the over-proliferation induced by hpo BF33 mutation.

CTPS Overexpression Increases the Volume of NICD in Hpo Mutant PFCs
Although the hpo BF33 mutation still resulted in a reduction in the cytoophidium length in PFCs when the CTPS was overexpressed, we attempted to determine whether CTPS overexpression exacerbates over-proliferation associated with hpo BF33 .In the hpo BF33 with CTPS overexpressed egg chamber (Figure 5C), the volume of NICD accumulation was observed, and it is marked in a light red surface in Figure 5C"-C"'.The quantification showed that CTPS overexpression increased the volume of NICD accumulated in hpo BF33 with CTPS-OE PFCs by comparing with the hpo BF33 egg chamber (Figure 5E).

CTPS H355A Overexpression Does Not Increase the Volume of NICD in Hpo Mutant PFCs
To investigate whether the cytoophidium structure affects cell proliferation controlled by the Hippo pathway, we generated a stock containing hpo BF33 and UAS-CTPS H355A -mCherry-HA.It is worth noting that the cytoophidium is depolymerized when the His355 is replaced by Alanine.The H355A mutation only affects polymerization, but not the enzymatic activity [22,48,49].In hpo BF33 with CTPS H355A overexpressed egg chambers (Figure 5D), the GFP-positive clones took hpo BF33 mutation and overexpressed CTPS H355A .The quantification showed that the volume of NICD in hpo BF33 with CTPS H355A -OE had no significant difference compared with the hpo BF33 clones (Figure 5E).These results suggest that the cytoophidium structure contributes to the over-proliferative phenotype induced by the Hippo pathway.

Discussion
The Hippo pathway regulates cell proliferation and apoptosis.We found that mutations of wts and hpo decrease the length and number of cytoophidium in PFCs.A previous study found that the overexpression of dMyc promotes cytoophidium assembly and that the knockdown of dMyc reduces cytoophidium formation and the nucleus size in follicle cells [10].The knockdown of dMyc downregulated CTPS expression in Drosophila cells [50].The overexpression of active Ras increases the length and abundance of cytoophidia [11].The overexpression of dMyc induces cell growth, and the overexpression of active Ras induces over-proliferation in Drosophila intestines.The cytoophidium length reduction phenotype induced by Hippo mutations differs from dMyc and Ras.
Cytophidium formation is related to the intracellular CTPS concentration, and the overexpression of CTPS promotes cytoophidium assembly and increases the CTP concentration [51].The role of the Hippo pathway in the regulation of CTPS expression remains unclear.We hypothesize that the mutation of the Hippo pathway does not result in an abnormal expression of CTPS.CTPS cytoophidia inhibit enzyme activity in S. cerevisiae, Escherichia coli and D. melanogaster [9,[51][52][53].During stage 10a-b of oogenesis, the follicle cells grow rapidly and the number of cytoophidia is reduced [10].Hence, we speculate that the CTPS cytoophidia may depolymerize to release more active CTPS for CTP synthesis in Hippo-mutant PFCs.
In Drosophila oogenesis, follicle cells are suitable for the study of cell proliferation.A previous study found that mutations in the Hippo pathway control the polarity of posterior follicle cells, and Dlg could be found all around the cell periphery in Hippomutant PFCs [46].The cell polarity maintains the cytoophidia formation.The cytoophidia distribute in the basolateral side of follicle cells, and the absence of aPKC decreased the percentage of cells with cytoophidia [21].Hence, the reduced proportion of cytoophidia in Hippo-mutant PFCs may be due to a disruption in the cell polarity.
In the future, we can further investigate whether CTPS expression is regulated by the Hippo pathway.Our study showed that over-proliferation was promoted when CTPS was overexpressed.The knockdown of CTPS suppressed the over-proliferation induced by the Hippo pathway mutation, indicating that CTPS plays a role in the regulation of cell proliferation by the Hippo pathway.Therefore, modulating the activity of metabolic enzymes may offer potential treatment options for cancer caused by Hippo mutations.Hence, it is possible to use 6-Diazo-5-oxo-L-norleucine (a CTPS inhibitor) [17,54] to target CTPS to suppress the over-proliferative phenotype induced by Hippo mutations.
In conclusion, our data demonstrate that the Hippo pathway regulates CTPS cytoophidium formation in Drosophila posterior follicle cells.Interestingly, when CTPS is overexpressed, the Hippo pathway still decreases the length of cytoophidia.Additionally, the knockdown of CTPS suppresses the over-proliferation caused by hpo BF33 .Collectively, this study provides a novel insight into the research on Hippo-related cancer by establishing a connection between the Hippo pathway and CTPS cytoophidia.from https://imagej.net/ij/index.html).The number of cytoophidia and cell nuclei were counted using multi-point tool in ImageJ [56].To quantify over-proliferative phenotype, we chose the volume of accumulated NICD signal based on NICD accumulated in Hippomutant PFCs.The volume of accumulated NICD was boxed by the surface tool in Imaris x64 9.0.1.Representative images with maximum intensity projection are shown in all figures.

Figure 1 .
Figure 1.Wts is required for oocyte polarity.(A-E) The Oskar (OSK, red in (A), white in (B)) protein localizes at the posterior crescent of the stage-9 FRT82B control oocyte.The Notch intercellular domain (NICD, white in (A,D) labels the Notch signal and is located in the membrane of the follicle cells and germline cells.Recombinant cells are marked with GFP, and the GFP-positive clones express GFP but have no mutation (green in (A), white in (E)).(F-J) When wts mutates in posterior follicle cells, OSK (red in (F), white in (G)) relocates to the middle of the stage-9 oocyte.The wts mutant posterior follicle cells show an over-proliferative phenotype and an accumulation of NICD (white in (F,I)).DNA is stained with Hoechst 33342 (blue in (F), white in (H)).Recombinant cells are marked with GFP, and GFP-positive clones take wts x1 mutation (green in (F), white in (J)).Scale bars, 20 µm.

Figure 1 .
Figure 1.Wts is required for oocyte polarity.(A-E) The Oskar (OSK, red in (A), white in (B)) protein localizes at the posterior crescent of the stage-9 FRT82B control oocyte.The Notch intercellular domain (NICD, white in (A,D) labels the Notch signal and is located in the membrane of the follicle cells and germline cells.Recombinant cells are marked with GFP, and the GFP-positive clones express GFP but have no mutation (green in (A), white in (E)).(F-J) When wts mutates in posterior follicle cells, OSK (red in (F), white in (G)) relocates to the middle of the stage-9 oocyte.The wts mutant posterior follicle cells show an over-proliferative phenotype and an accumulation of NICD (white in (F,I)).DNA is stained with Hoechst 33342 (blue in (F), white in (H)).Recombinant cells are marked with GFP, and GFP-positive clones take wts x1 mutation (green in (F), white in (J)).Scale bars, 20 µm.

Figure 2 .
Figure 2. Wts x1 decreases cytoophidium length and number in wts mutant PFCs.(A-E) In the FRT82B control egg chamber, there is no difference in the NICD signal (red in (A), white in (B)) or

Figure 2 .
Figure 2. Wts x1 decreases cytoophidium length and number in wts mutant PFCs.(A-E) In the FRT82B control egg chamber, there is no difference in the NICD signal (red in (A), white in (B)) or the nuclear size (blue in (A), white in (C)) of follicle cells between GFP-positive (green in (A), white in (E)) and GFP-negative clones.Cytoophidia labeled with CTPS (white in (A,D)) exist in follicle cells and germline cells.There is no difference in cytoophidium length between GFP-positive and GFP-negative clones.Recombinant cells are marked with GFP and have no mutation.(F-J) In wts x1 egg chamber, NICD (red in (F), white in (G)) accumulates in posterior follicle cells (yellow line in (G)).After wts x1 mutation, the length of cytoophidia (white in (F,I)) in posterior follicle cells is decreased, while the number of cells increases.DNA is stained with Hoechst 33342 (blue in (F), white in (H)).Recombinant cells are marked with GFP (green in (F), white in (J)) and take wts x1 mutation.Scale bars, 20 µm.(K) Quantification shows that wts x1 decreases the cytoophidium length in posterior follicle cells.**** p < 0.0001, ns = not significant, mean shown with SED (Student's t test).(L) Quantification shows that wts x1 decreases the number of cytoophidium in posterior follicle cells.**** p < 0.0001, ns = not significant, mean shown with SED (Student's t-test).

15 Figure 3 .Figure 3 .
Figure 3. Hpo BF33 decreases cytoophidium length and number in hpo mutant PFCs.(A-E) In the FRT42D control egg chamber, there is no difference in the NICD signal (red in (A), white in (B)) and the nuclear size (blue in (A), white in (C)) of follicle cells between GFP-positive and GFP-negative clones.Cytoophidia labeled with CTPS (white in (A,D)) exist in follicle cells and germline cells.There is no difference in cytoophidium length between GFP-positive and GFP-negative clones.Recombinant cells are marked with GFP and have no mutation (green in (A), white in (E)).(F-J) In the hpo BF33 egg chamber, NICD (red in (F), white in (G)) accumulates in posterior follicle cells (yellow line in (G)).After hpo BF33 mutation, the length of cytoophidia in posterior follicle cells is decreased, while the number of cells increases.DNA is stained with Hoechst 33342 (blue in (F), white in (H)).Recombinant cells are marked with GFP (green in (F), white in (J)) and take hpo BF33 mutation.Scale bars, 20 µm.(K) Quantification shows that the hpo BF33 decreases the cytoophidium length in posteriorFigure 3. Hpo BF33 decreases cytoophidium length and number in hpo mutant PFCs.(A-E) In the FRT42D control egg chamber, there is no difference in the NICD signal (red in (A), white in (B)) and the nuclear size (blue in (A), white in (C)) of follicle cells between GFP-positive and GFP-negative clones.Cytoophidia labeled with CTPS (white in (A,D)) exist in follicle cells and germline cells.There follicle cells.**** p < 0.0001, ns = not significant, mean shown with SED (Student's t test).(L) Quantification shows that hpo BF33 decreases the number of cytoophidium in posterior follicle cells.*** p < 0.001, ns = not significant, mean shown with SED (Student's t-test).

Figure 4 .
Figure 4.Even if CTPS is overexpressed, the hpo BF33 decreases cytoophidium length in PFCs.(A-E) In the FRT42D with CTPS overexpression egg chamber, there is no difference in the NICD signal

Figure 4 .
Figure 4.Even if CTPS is overexpressed, the hpo BF33 decreases cytoophidium length in PFCs.(A-E) In the FRT42D with CTPS overexpression egg chamber, there is no difference in the NICD signal (white in (A,B)) of follicle cells between GFP-positive (green in (A), white in I) and GFP-negative clones.The length and number of cytoophidia labeled with CTPS (red in (A), white in (D)) increase in the GFPpositive follicle cells.DNA is stained with Hoechst 33342 (blue in (A), white in (C)).The GFP-positive clones are marked by yellow line.(F-J) In the hpo mutant with CTPS overexpression, NICD (white in (F,G)) accumulates in posterior follicle cells (yellow box in (F)).After hpo BF33 mutated, the length of cytoophidium in posterior follicle cells (yellow box in (I)) is shorter than those in other GFP-positive regions.DNA is stained with Hoechst 33342 (blue in (F), white in (H)).Recombinant cells are marked with GFP (green in (F), white in (J)) and take hpo BF33 mutation and CTPS overexpression.Scale bars, 25 µm.(K) Quantification shows that hpo BF33 mutation decreased the cytoophidium length in posterior follicle cells, even if CTPS was overexpressed.**** p < 0.0001, mean shown with SED (Student's t-test).

Figure 5 .
Figure 5. CTPS plays a role in the over-proliferative phenotype in hpo BF33 mutant PFCs.(A-A''') In the 3D view of hpo BF33 mutant egg chamber, NICD (red in (A,A')) accumulates in posterior follicle cells (light red box in (A'',A''')).The merged image shows the surface within the accumulated NICD signal.(B-B''') In the 3D view of hpo BF33 with CTPS knockdown, NICD (red in (B,B')) accumulates in posterior follicle cells (light red in (B'',B''')).The merged image shows the surface within the accumulated NICD signal.(C-C''') In the 3D view of hpo BF33 with CTPS overexpression, NICD (red in (C,C')) accumulates in posterior follicle cells (light red in (C'',C''')).The merged image shows the surface within the accumulated NICD signal.(D-D''') In the 3D view of hpo BF33 with CTPS H355A

Figure 5 .
Figure 5. CTPS plays a role in the over-proliferative phenotype in hpo BF33 mutant PFCs.(A-A"') In the 3D view of hpo BF33 mutant egg chamber, NICD (red in (A,A')) accumulates in posterior follicle cells (light red box in (A",A"')).The merged image shows the surface the accumulated NICD signal.(B-B"') In the 3D view of hpo BF33 with CTPS knockdown, NICD (red in (B,B')) accumulates in posterior follicle cells (light red in (B",B"')).The merged image shows the surface within the accumulated NICD signal.(C-C"') In the 3D view of hpo BF33 with CTPS overexpression, NICD (red in (C,C')) accumulates in posterior follicle cells (light red in (C",C"')).The merged image shows the surface within the accumulated NICD signal.(D-D"') In the 3D view of hpo BF33 with CTPS H355A overexpression, NICD (red in (D,D')) accumulates in posterior follicle cells (light red in (D",D"')).The merged image shows the surface within the accumulated NICD signal.Scale bars, 20 µm.(E) Quantification shows that CTPS overexpression promotes the volume of over-proliferative phenotype in hpo BF33 PFCs.CTPS knockdown inhibits the volume of over-proliferative phenotype in hpo BF33 PFCs.CTPS H355A overexpression has no difference with hpo BF33 egg chambers in the volume of accumulated NICD signal.** p < 0.01, *** p < 0.001, ns = not significant, mean shown with SED (Student's t-test).