The Drosophila EcR-Hippo component Taiman promotes epithelial cell fitness by control of the Dally-like glypican and Wg gradient

Reviewer #1 (Public Review):

Summary:

Schweibenz et al are investigating how cells with lower levels of Tai are out-competed by neighboring wild-type (WT) cells. They show that clones homozygous for a tai hypomorphic mutation are disadvantaged and are killed by apoptosis. But tai-low clones are partially rescued when generated in a background that is heterozygous for mutations in apoptotic genes, in the Hippo pathway component warts, or for the Wg/Wnt pathway negative regulator Apc. They then follow up in the link between tai LOF and Wg. The story then shifts away from clones and into experiments that have Tai RNAi depletion or Tai over-expression in the posterior compartment of the wing disc, using the anterior compartment as a control. These non-clonal experiments show that depletion of Tai in the posterior compartment of wing discs results in less Wg in this compartment. This is shown to be due to a reduction in the glypican Dally-like protein (Dlp). The fact that long-range Wg is reduced in tai-depleted discs that also show a reduction in Dlp, suggests that Tai somehow positively promotes Wg distribution. There is some data in the supplementary materials suggesting that Tai promotes dlp mRNA expression but this was not compelling. In fact, the compelling data was that Dlp protein in tai mutant clones is not abundantly on the cell surface, but instead somehow retained in the mutant cell. The authors don't further examine Dlp protein in tai clones. The final figure (Figure 8) shows that there is less Wg at the DV margin in wing discs when tai is depleted from wg-producing cells. In sum, the authors have uncovered some interesting results, but the story has some unresolved issues that, if addressed, could boost its impact. Additionally, the preprint seems to have 2 stories, one about tai and cell competition and the other about tai and Wg distribution. It would be helpful to reorder the figures and improve the narrative so that these are better integrated with each other.

Strengths:

The authors are studying competition between tai-low clones and their fitter WT neighbors, and have uncovered an interesting connection to Wg.

Weaknesses:

(1) It would be good to know whether the authors can rescue tai-low clones by over-expression UAS-Dlp.

(2) The data about tai-promoting dlp (Figure S4) is not compelling as there are no biological replicates and no statistical analyses.

(3) The data on Wg distribution seems disjointed from the data about cell competition. The authors could refocus the paper to emphasize the cell competition story. The role of Dlp in Wg distribution is well established, so the authors could remove or condense these results. The story really could be Figsured 1, 2, 3 and 7 and keep the paper focused on cell competition. The authors could then discuss Dlp as needed for Wg signaling transduction, which is already established in the literature.

(4) The model of tai controlling dlp mRNA and Dlp protein distribution is confusing. In fact, the data for the former is weak, while the data for the latter is strong. I suggest that the authors focus on the altered Dlp protein distribution on tai-low clones. It would also be helpful to prove the Wg signaling is impeded in tai clones (see #5 below).

(5) I don't know if the Fz3-RFP reported for Wg signaling works in imaginal discs, but if it does then the authors could make clones in this background to prove that cell-autonomous Wg signaling is reduced in tai-low clones.

Reviewer #2 (Public Review):

The authors investigate the properties of the transcriptional co-activator Taiman in regulating tissue growth. In previously published work they had shown that cells that overexpress Tiaman in the pupal wing can cause the death of thoracic cells adjacent to the wing tip to die and thus allow the wing to invade the thorax. This was mediated by the secretion of Spz ligands. Here, they investigate the properties of cells that are homozygous for a hypomorphic allele of taiman (tai). They show that homozygous mutant clones are much smaller than their wild-type twin spots and that cells in the clones are dying by apoptosis which is inferred from elevated levels of anti-Dcp1 staining (Figure 1).

By generating clones during eye development, the authors screen for dominant modifiers that increase the representation of homozygous tai tissue in the adult eye (Figure 2). They find that reducing the levels of hid, the entire rpr/hid/grim locus and Apc (and/or Apc2) each increase the representation of tai clones. They then show that the survival of tissue to the adult stage correlates with the size of lones in the third-instar larval wing disc (Figure 3). The rest of the study derives from the modification of the phenotype by Apc and investigates the interaction between Wnt signaling and tai clone survival.

The authors then investigate interactions between tai and the wingless (wg) pathway. First, they show that increasing tai expression increases the expression of a wg reporter (nkd-lacZ) while reducing tai levels decreases its expression (Figure 4) indicating that wg signaling is likely reduced when tai levels are decreased. This finding is strengthened by examining wg-lacZ expression since the expression of this reporter is normally restricted to the D/V boundary in the wing disc by feedback inhibition via Wg signaling. Expression of the reporter is increased when tai expression is reduced and decreased when tai expression is increased (Figure 5).

The authors then look at Wg protein away from the DV boundary. They find increased levels when tai expression is increased and decreased levels when tai is decreased. They conclude that tai activity increased Wg protein in cells (Figure 6). They suggest that this could be the result of the regulation of expression of Dally-like protein (Dlp). Consistent with this idea, increasing tai expression increases Dlp levels, and decreasing tai decreases Dlp levels (Figure 7). They then show that increasing Dlp levels when tai is reduced increases Wg levels which presumably means that Dlp is epistatic to tai. Puzzlingly, increasing both tai and Dlp decreases Wg.

The authors then examine the effect of reducing Dlp in the cells that secrete Wg. They find that increasing tai results in the diffusion of Wg further from its source while reducing tai reduces its spread (Figure 8). They then show that in clones with reduced tai, there is increased cytoplasmic Dlp (Figure 9). They therefore propose that tai clones fail to survive because they do not secrete enough Dlp which results in reduced capture of the Wg for those cells and hence decreased Wg signaling.

Evaluation

While the authors present good evidence in support of most of their conclusions, there are alternative explanations in many cases that have not been excluded.

From the results in Figure 1 (and Figure 3), the authors conclude that "The data indicate the existence of an extracellular competition mechanism that allows normal tai[wt] cells to kill tai[k15101] neighbors" (line 127). However, the experiments have been done with a single allele, and these experiments do not exclude the possibility that there is another mutation on the same chromosome arm that is responsible for the observed phenotype. Since the authors have a UAS-tai stock, they could strengthen their results using a MARCM experiment where they could test whether the expression of UAS-tai rescues the elimination of tai mutant clones. Alternatively, they could use a second (independent) allele to demonstrate that the phenotype can be attributed to a reduction in tai activity.

By screening for dominant modifiers of a phenotype one would not expect to identify all interacting genes - only those that are haploinsufficient in this situation. The authors have screened a total of 21 chromosomes for modification and have not really explained which alleles are nulls and which are hypomorphs. The nature of each of the alleles screened needs to be explained better. Also, the absence of a dominant modification does not necessarily exclude a function of that gene or pathway in the process. This is especially relevant for the Spz/Toll pathway which the authors have previously implicated in the ability of tai-overexpressing cells to kill wild-type cells. The most important discovery from this screen is the modification by the Apc alleles. This part of the paper would be strengthened by testing for modification by other components of the Wingless pathway. The authors show modification by Apc[MI01007] and the double mutant Apc[Q8] Apc2[N175A]. Without showing the Apc[Q8] and Apc2[N175A] alleles separately, it is hard to know if the effect of the double mutant is due to Apc, Apc2,` or the combination.

RNAi of tai seems to block the formation of the Wg gradient. If so, one might expect a reduction in wing size. Indeed, this could explain why the wings of tai/Df flies are smaller. The authors mention briefly that the posterior compartment size is reduced when tai-RNAi is expressed in that compartment. However, this observation merits more emphasis since it could explain why tai/Df flies are smaller (Are their wings smaller?).

In Figure 7, the authors show the effect of manipulating Tai levels alone or in combination with increasing Dlp levels. However, they do not include images of Wg protein distribution upon increasing Dlp levels alone.

In Figure 8, there is more Wg protein both at the DV boundary and spreading when tai is overexpressed in the source cells using bbg-Gal4. However, in an earlier experiment (Figure 5C) they show that the wg-lacZ reporter is downregulated at the DV boundary when tai is overexpressed using en-Gal4. They therefore conclude that wg is not transcriptionally upregulated but is, instead secreted at higher levels when tai is expressed in the source cells. Wg protein is reduced in the DV stripe with tai is overexpressed using the en-Gal4 driver (Figure 6B') and is increased at the same location when tai is overexpressed with the bbg-Gal4 driver. (Figure 8) I don't know how to reconcile these observations.

In Figure 9, the tai-low clones have elevated levels of Dlp. How can this be reconciled with the tai-RNAi knockdown shown in Figure 7C' where reducing tai levels causes a strong reduction in Dlp levels?

Reviewer #3 (Public Review):

Summary:

In this study, Schweibenz et al., identify the transcriptional coactivator, Taiman (Tai), as a factor that determines the fitness level of epithelial cells by regulating Wingless (Wg), which is an important determinant of cellular fitness. Taiman determines cellular fitness level by regulating levels of cell-surface glypican Dally-like protein (Dlp), which regulates extracellular Wingless (Wg) distribution. Thus, by affecting levels of Wg via glypican regulation, Tai participates in determining cellular fitness, and cells with low Tai levels are eliminated as they are deprived of adequate Wg levels.

Strengths:

(1) The authors make a strong case for the effect of tai on Dlp and Wg levels in experiments where a relatively large group of cells have reduced tai levels.
(2) The claim that tai-low clones are competitively eliminated is supported by experiments that show cell death in them, and their elimination at different time points.
(3) The manuscript is well written.

Weaknesses:

(1) The study has relatively weak evidence for the mechanism of cell competition mediated by Dlp and Wg.

(2) More evidence is required to support the claim that dlp transcription or endocytosis is affected in tai clones.

Other comments:

(1) The authors put the study in the context of cell competition, and the first figure indeed is convincing in this regard. However, most of the rest of the study is not in the clonal context, and mainly relies on RNAi KD of tai in the posterior compartment, which is a relatively large group of cells. I understand why the authors chose a different approach to investigate the role of tai in cell competition. However because ubiquitous loss of tai results in smaller organs, it is important to determine to what extent reducing levels of tai in the entire posterior compartment compares with clonal elimination i.e. cell competition. This is important in order to determine to what extent the paradigm of Tai-mediated regulation of Dlp levels and by extension, Wg availability, can be extended as a general mechanism underlying competitive elimination of tai-low clones. If the authors want to make a case for mechanisms involved in the competitive elimination of tai clones, then they need to show that the KD of tai in the posterior compartment shows hallmarks of cell competition. Is there cell death along the A/P boundary? Or is the compartment smaller because those cells are growing slower? Are the levels of Myc/DIAP1, proteins required for fitness, affected in en>tai RNAi cells?

1. The authors do not have direct/strong evidence of changes in dlp mRNA levels or intracellular trafficking. To back these claims, the authors should look for dlp mRNA levels and provide more evidence for Dlp endocytosis like an antibody uptake assay or at the very least, a higher resolution image analysis showing a change in the number of intracellular Dlp positive punctae. Also, do the authors think that loss of tai increases Dlp endocytosis, making it less available on the cell surface for maintaining adequate extracellular Wg levels?

2. The data shown in the last figure is at odds with the model (I think) the authors are trying to establish: When cells have lower Tai levels, this reduces Dlp levels (S2) presumably either by reducing dlp transcription and/or increasing (?) Dlp endocytosis. This in turn reduces Wg (availability) in cells away from source cells (Figure 6). The reduced Wg availability makes them less fit, targeting them for competitive elimination. But in tai clones, I do not see any change in cell-surface Dlp (9B) (I would have expected them to be down based on the proposed model). The authors also see more total Dlp (9A) (which is at odds with S2 assuming data in S2 were done under permeabilizing conditions.).

As a side note, because Dlp is GPI-anchored, the authors should consider the possibility that the 'total' Dlp staining observed in 9A may not be actually total Dlp (and possibly mostly intracellular Dlp, since the permeabilizing membranes with detergent will cause some (most?) Dlp molecules to be lost, and how this might be affecting the interpretation of the data. I think one way to address this would be to process the permeabilized and non-permeabilized samples simultaneously and then image them at the same settings and compare what membrane staining in these two conditions looks like. If membrane staining in the permeabilized condition is decreased compared to non-permeabilized conditions, and the signal intensity of Dlp in permeabilized conditions remains high, then the authors will have evidence to support increased endocytosis in tai clones. Of course, these data will still need to be reconciled with what is shown in S2.

Author response:

eLife assessment

“…The evidence however is incomplete, since the tai loss-of-clone phenotype is based on one allele and the mechanism involved in cell competition through Dlp and Wg lacks adequate supporting data.”

We agree with the need for a second allele and are adding supporting data from a new tai lof allele we have generated by Crispr.

We also agree that additional functional data would help demonstrate that differences in Dlp levels are required for the mechanism of Tai cell competition. Experiments are ongoing to test whether normalizing Dlp levels across clonal boundaries rescues elimination of Tai-low clones.

Reviewer #1:

Overall Statements:

“There is some data in the supplementary materials suggesting that Tai promotes dlp mRNA expression, but this was not compelling.”

We are currently testing effects on Tai on dlp and dally transcription using qPCR and reporter transgenes. As noted below, the effects of Tai on Dlp trafficking are ‘strong’, so resolving effects on Dlp transcription will complement this localization data.

“The authors don't further examine Dlp protein in tai clones.”

As noted by the Reviewer, we do examine Dlp levels and localization in tai-low clones (see Figure 9), but these experiments are challenging due to their very small size and the hypomorphic nature of the tai allele (tai[k15101]) that was used. Experiments are in progress to examine the effect of our Crispr null allele of tai on Dlp levels and localization in wing clones.

“In sum, the authors have uncovered some interesting results, but the story has some unresolved issues that, if addressed, could boost its impact. Additionally, the preprint seems to have 2 stories, one about tai and cell competition and the other about tai and Wg distribution. It would be helpful to reorder the figures and improve the narrative so that these are better integrated with each other.”

We agree. The results of our modifier screen required that we first understand how Tai regulates the Wg pathway before could apply this to understanding the competitive mechanism. Thus, the paper is composed of three sections: 1. the screen, 2. the Tai-Dlp-Wg connection in the absence of competition, and 3. the contribution of Dlp-Wg to the tai[low] ‘loser’ phenotype. These sections use different techniques (e.g., clonal mosaics with genomic alleles, Gal4/UAS and RNAi to define the effect of Tai loss on Wg and Dlp). Ongoing experiments return to clonal mosaics to test whether elevating Dlp can rescue tai lof clones in the same manner as Apc/Apc2 alleles (see Figs. 2-3), which elevate Wg pathway activity.

Specifics:

“It would be good to know whether the authors can rescue tai-low clones by over-expression UAS-Dlp.”

As noted above, experiments are ongoing to test whether normalizing Dlp levels across clonal boundaries rescues elimination of Tai-low clones.

“The data on Wg distribution seems disjointed from the data about cell competition. The authors could refocus the paper to emphasize the cell competition story. The role of Dlp in Wg distribution is well established, so the authors could remove or condense these results. The story really could be Figs 1, 2, 3 and 7 and keep the paper focused on cell competition. The authors could then discuss Dlp as needed for Wg signaling transduction, which is already established in the literature.”

We appreciate the suggestion to reorganize the figures to focus the first part of the story on competition, and then follow with the role of Tai in controlling Dlp. We will consider this approach pending the results of ongoing experiments.

“The model of tai controlling dlp mRNA and Dlp protein distribution is confusing. In fact, the data for the former is weak, while the data for the latter is strong. I suggest that the authors focus on the altered Dlp protein distribution on tai-low clones. It would also be helpful to prove the Wg signaling is impeded in tai clones (see #5 below).”

We agree but are currently testing how dlp reporters and mRNA respond to Tai in order to rigorously test a Dlp transcriptional mechanism. To complement the ‘strong’ evidence that Tai regulates Dlp distribution, we are testing Dlp in clones of our Tai Crispr null. Since submission, we have also assessed the effect of blocking the endocytic factor shibire/dynamin in Dlp distribution in Tai deficient cells to complement the data on Pentagone that is already in the paper (see Fig. S3).

“I don't know if the Fz3-RFP reported for Wg signaling works in imaginal discs, but if it does then the authors could make clones in this background to prove that cell-autonomous Wg signaling is reduced in tai-low clones.”

We thank the reviewer for this suggestion, which we are now testing.

Reviewer #2

Overall Comments:

“While the authors present good evidence in support of most of their conclusions, there are alternative explanations in many cases that have not been excluded.”

We appreciate this point and are conducting experiments for a revised submission that will help test alternative mechanisms and clarify our conclusions.

Specifics:

“However, the experiments have been done with a single allele, and these experiments do not exclude the possibility that there is another mutation on the same chromosome arm that is responsible for the observed phenotype. Since the authors have a UAS-tai stock, they could strengthen their results using a MARCM experiment where they could test whether the expression of UAS-tai rescues the elimination of tai mutant clones. Alternatively, they could use a second (independent) allele to demonstrate that the phenotype can be attributed to a reduction in tai activity.”

As noted above, we agree with the need for a second allele and are adding supporting data from a new tai lof allele we have generated by Crispr.

The tai[k15101] allele acts as a tai hypomorph and has been shown to produce weaker phenotypes than the 61G1 strong lof in a number of papers (Bai et al, 2000; König et al, 2011, Luo et al, 2019, and Zhang et al, 2015). We agree that rescue of tai[k1501] with a UAS-Tai transgene would help rule out effects of second site mutations. We are currently pursuing the reviewer’s second suggestion of phenocopy with a different allele, our new tai Crispr lof.

“The authors have screened a total of 21 chromosomes for modification and have not really explained which alleles are nulls and which are hypomorphs. The nature of each of the alleles screened needs to be explained better.”

We will update the text to better reflect what type of alleles were chosen. In most cases we preferred amorphs or null alleles over hypomorphs, however when the amorph option was not available, we used hypomorphs.

“Also, the absence of a dominant modification does not necessarily exclude a function of that gene or pathway in the process. This is especially relevant for the Spz/Toll pathway which the authors have previously implicated in the ability of tai-overexpressing cells to kill wild-type cells.”

We thank the reviewer for this completely accurate point. The dominant screen does not rule out effects of other pathways such as Spz/Toll. Indeed, we were surprised by the lack of dominant effects by Spz/Toll alleles on tai[low] competition given our prior work. The reciprocally clear dominant effect of Apc/Apc2 led us to consider that Wg signaling plays a role in this phenomenon, which then became the starting point of this study.

“The most important discovery from this screen is the modification by the Apc alleles. This part of the paper would be strengthened by testing for modification by other components of the Wingless pathway. The authors show modification by Apc[MI01007] and the double mutant Apc[Q8] Apc2[N175A]. Without showing the Apc[Q8] and Apc2[N175A] alleles separately, it is hard to know if the effect of the double mutant is due to Apc, Apc2,` or the combination.”

We agree that testing for modification with other components of the Wg pathway would be helpful to strengthen the connection between Tai low clonal elimination and Wg pathway biology. We also agree that separating Apc [Q8] and Apc2 [N175A] would be a good idea to check if both Apc proteins are equally important for rescuing Tai low cell death, and future experiments for the lab could investigate this distinction.

“RNAi of tai seems to block the formation of the Wg gradient. If so, one might expect a reduction in wing size. Indeed, this could explain why the wings of tai/Df flies are smaller. The authors mention briefly that the posterior compartment size is reduced when tai-RNAi is expressed in that compartment. However, this observation merits more emphasis since it could explain why tai/Df flies are smaller (Are their wings smaller?).”

We agree that this is an exciting possibility. Growth effects of Tai linked to interactions with Yorkie and EcR could be due to a distinct role in promoting Wg activity. Alternatively, Tai may cooperate with Yorkie or EcR to control Wg pathway. These are exciting possibilities that we are pursuing in future work

With regard to the “small size” effect of reducing Tai, we have previously shown that RNAi of Tai using engrailed-Gal4 causes the posterior compartment to shrink (Zhang et al. 2015, Figure 1C-F, H). In this paper, we also showed that tai[k15101]/Df animals are proportionally smaller than wildtype animals and quantified this by measuring 2D wing size (Zhang et al. 2015, Figure 1A and 1B)

“In Figure 7, the authors show the effect of manipulating Tai levels alone or in combination with increasing Dlp levels. However, they do not include images of Wg protein distribution upon increasing Dlp levels alone.”

We thank the reviewer for this reminder and have already generated these control images to include in a revised submission paper.

“In Figure 8, there is more Wg protein both at the DV boundary and spreading when tai is overexpressed in the source cells using bbg-Gal4. However, in an earlier experiment (Figure 5C) they show that the wg-lacZ reporter is downregulated at the DV boundary when tai is overexpressed using en-Gal4. They therefore conclude that wg is not transcriptionally upregulated but is, instead secreted at higher levels when tai is expressed in the source cells. Wg protein is reduced in the DV stripe with tai is overexpressed using the en-Gal4 driver (Figure 6B') and is increased at the same location when tai is overexpressed with the bbg-Gal4 driver. (Figure 8) I don't know how to reconcile these observations.”

We thank the reviewer for pressing us to develop an overall model explaining our results and how we envision Tai regulating Dlp and Wg. We are preparing a graphic abstract that illustrates this model and will be included in our revision.

Briefly, we favor a model in which Tai controls the rate of Wg spread via Dlp, without a significant effect on wg transcription. For example, the induction of Dlp across the ‘engrailed’ domain of en>Tai discs (Fig 7B-B”) allows Wg to spread rapidly across the flanks and moderately depletes it from the DV margin (Fig 6B-B”) as noted by the reviewer. Adding a UAS-Dlp transgene in the en>Tai background dramatically accelerates Wg spread and causes it to be depleted from the DV margin and build up at the far end of the gradient adjacent to the dorsal and ventral hinge. Significantly blocking endocytosis of Wg in en>Tai discs with a dominant negative shibire transgene also causes Wg to build up in the same location (new data to be added in a revision) consistent with enhanced spreading. The difference in the bbg-Gal4 experiment is that Tai is only overexpressed in DV margin cells, which constrains and concentrates Wg within this restricted domain; we are in the process of testing whether this effect on Wg is blocked by RNAi of Dlp in bbg>Tai discs.

“In Figure 9, the tai-low clones have elevated levels of Dlp. How can this be reconciled with the tai-RNAi knockdown shown in Figure 7C' where reducing tai levels causes a strong reduction in Dlp levels?”

We apologize for not explaining this data well enough. First, the tai[k15101] allele is a weak, viable hypomorph (as shown in our Zhang et al, 2015 paper) whereas the Tai RNAi line is lethal with most drivers (including en-Gal4) and thus a stronger lof. Second, Tai RNAi lower Dlp levels (Fig 7C) while tai[k15101] causes Dlp to accumulate intracellularly (see Fig. 9A-C). These data indicate that reduced Tai leads to a defect in Dlp intracellular trafficking while its loss reduces Dlp overall levels; these data can be explained by a single role for Tai in Dlp traffic to or from the cell membrane, or two roles, one in trafficking and one Dlp expression. As noted, we are investigating both possibilities using dlp reporter lines and our new tai null Crispr allele.

Reviewer #3:

Overall Weaknesses:

“The study has relatively weak evidence for the mechanism of cell competition mediated by Dlp and Wg.”

The screen and middle section of the paper provide genetic evidence that elevating Wg pathway activity rescues Tai[low} loser cells and that Tai controls levels/localization of Dlp and distribution of Wg in the developing wing disc. Our current work is focused on linking these two finding together in Tai “loser” clones.

“More evidence is required to support the claim that dlp transcription or endocytosis is affected in tai clones.”

As noted above, we are testing whether normalizing Dlp levels across clonal boundaries rescues tai[low] loser clones and assessing effects of Tai on dlp transcription and Dlp trafficking.

Specifics:

“Most of the rest of the study is not in the clonal context, and mainly relies on RNAi KD of tai in the posterior compartment, which is a relatively large group of cells. I understand why the authors chose a different approach to investigate the role of tai in cell competition. However because ubiquitous loss of tai results in smaller organs, it is important to determine to what extent reducing levels of tai in the entire posterior compartment compares with clonal elimination i.e. cell competition. This is important in order to determine to what extent the paradigm of Tai-mediated regulation of Dlp levels and by extension, Wg availability, can be extended as a general mechanism underlying competitive elimination of tai-low clones. If the authors want to make a case for mechanisms involved in the competitive elimination of tai clones, then they need to show that the KD of tai in the posterior compartment shows hallmarks of cell competition. Is there cell death along the A/P boundary? Or is the compartment smaller because those cells are growing slower?”

Based on data that cell competition does not occur over compartment boundaries (e.g., see review by L.A. Johnston, Science, 2009), we chose not to use UAS-Gal4 to assess competition, but rather to investigate underlying biology occurring between Tai, Wg, and Dlp.

“Are the levels of Myc/DIAP1, proteins required for fitness, affected in en>tai RNAi cells?”

This is, of course, an interesting question given that Myc is a well-studied competition factor and is proposed to be downstream of the Tai-interacting protein Yki. We are not currently focused on Myc, but plan to test its role in the Tai-Dlp-Wg pathway in future work.

“The authors do not have direct/strong evidence of changes in dlp mRNA levels or intracellular trafficking. To back these claims, the authors should look for dlp mRNA levels and provide more evidence for Dlp endocytosis like an antibody uptake assay or at the very least, a higher resolution image analysis showing a change in the number of intracellular Dlp positive punctae. Also, do the authors think that loss of tai increases Dlp endocytosis, making it less available on the cell surface for maintaining adequate extracellular Wg levels?”

As noted above, have added experiments using a dominant-negative shibire/dynamin allele to test whether Tai controls Dlp endocytosis. These data will be added to a revised manuscript. We have also gathered reagents to test effects of Tai gain/loss on Dlp secretion.

“The data shown in the last figure is at odds with the model (I think) the authors are trying to establish: When cells have lower Tai levels, this reduces Dlp levels (S2) presumably either by reducing dlp transcription and/or increasing (?) Dlp endocytosis. This in turn reduces Wg (availability) in cells away from source cells (Figure 6). The reduced Wg availability makes them less fit, targeting them for competitive elimination. But in tai clones, I do not see any change in cell-surface Dlp (9B) (I would have expected them to be down based on the proposed model). The authors also see more total Dlp (9A) (which is at odds with S2 assuming data in S2 were done under permeabilizing conditions.).”

As noted above (under Rev #2 comments), we apologize for not explaining this data well enough. First, the tai[k15101] allele is a weak, viable hypomorph (as shown in our Zhang et al, 2015 paper) whereas the Tai RNAi line is lethal with most drivers (including en-Gal4) and thus a stronger lof. Second, Tai RNAi lower Dlp levels (Fig 7C) while tai[k15101] causes Dlp to accumulate intracellularly (see Fig. 9A-C). These data indicate that reduced Tai leads to a defect in Dlp intracellular trafficking while its loss reduces Dlp overall levels; these data can be explained by a single role for Tai in Dlp traffic to or from the cell membrane, or two roles, one in trafficking and one Dlp expression. We are investigating both possibilities using dlp reporter lines and our new tai null Crispr allele.

“As a side note, because Dlp is GPI-anchored, the authors should consider the possibility that the 'total' Dlp staining observed in 9A may not be actually total Dlp (and possibly mostly intracellular Dlp, since the permeabilizing membranes with detergent will cause some (most?) Dlp molecules to be lost, and how this might be affecting the interpretation of the data. I think one way to address this would be to process the permeabilized and non-permeabilized samples simultaneously and then image them at the same settings and compare what membrane staining in these two conditions looks like. If membrane staining in the permeabilized condition is decreased compared to non-permeabilized conditions, and the signal intensity of Dlp in permeabilized conditions remains high, then the authors will have evidence to support increased endocytosis in tai clones. Of course, these data will still need to be reconciled with what is shown in S2.

We thank the reviewer for this excellent suggestion and are generating mosaic discs to test the proposed approach of synchronous analysis of total vs. intracellular Dlp.