Nutritional control of body size through FoxO-Ultraspiracle mediated ecdysone biosynthesis

Thank you for sending your work entitled “Nutritional Control of Body Size Plasticity through FoxO-Ultraspiracle Mediated Ecdysone Biosynthesis” for consideration at eLife. Your article has been favorably evaluated by Ian Baldwin (Senior editor), a Reviewing editor, and 3 reviewers.

The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Overall, all of the reviewers felt that the manuscript was interesting, largely well done, and addressed the important issue of the achievement of “critical weight”. The proposal that FoxO binds to Usp to form a novel complex to inhibit a peak of ecdysone, thus regulating the critical weight transition is intriguing, and in general the experiments demonstrating FoxO's involvement in CW are convincing. However the reviewers agreed that additional experiments were needed to support the main conclusions of the manuscript.

1) Show that FOXO and Usp interact in Co-IPs in fed wildtype L3 larvae. In Figure 1, it is shown that FoxO binds Usp in Co-IPs of extracts from starved but not fed larvae. For this interaction to be relevant to the regulation of the critical weight transition, the authors need to show that the dFoxO-Usp interaction also occurs in fed early L3 larvae (pre-critical weight). While the GST pulldowns with recombinant proteins in Figures 1 and 5 provide convincing evidence that FoxO and Usp have the potential to bind in vitro, the westerns are less convincing. The westerns show a weak Co-IPed band above the background smear that we are told corresponds to FoxO in . Given that demonstrating this point is so essential for the main conclusion of the paper about a molecular FoxO-Usp interaction, further controls need to be shown. One suggestion is to show that the FoxO band (co-IPed from larval extracts with anti-Usp) present in wild type animals is missing in FoxO null mutants.

2) Demonstrate the effects of feeding ecdysone at precritical weight times. If the model is correct then one would expect that feeding 20E just after ecdysis to L3 will lead to a smaller critical weight and precocious metamorphosis.

3) Concerns were raised about the specificity of the Gal4 drivers used. The P0206 driver used as a driver to express UAS-FoxO in the PG is also expressed in the CA and in oenocytes, which are known to regulate larval growth. The reviewers felt that it was important to use other drivers (Aug21 for the CA and the oenocyte 'specific” driver PromE-GAL4 from Joel Levine) as additional controls. Although the P0206-GAL4 driver has been used in similar studies of critical weight, the authors note that phm-GAL4 driven expression of UAS-FoxO is lethal in FoxO mutants but not in control animals. This observation may indicate that the presence of FoxO in the peripheral tissues of control animals is modifying the phenotypic effects of PG-specific FoxO over-expression. Although the authors suggest that animals expressing P0206-FoxO are viable due to the moderate levels of FoxO expression, these animals could be surviving due to the presence of FoxO in oenocytes. Therefore, the observations described in Figure 4A and Figure 6 could result from FoxO activity in either the oenocytes and not the PG or a synergistic interaction between these two tissues. The authors may address these problems with two simple experiments. Does oenocyte-specific expression of UAS-FoxO in a FoxO mutant delay the onset of pupariation? And, if so, is this delay 20E dependent? Finally, both of these Figures lack the necessary P0206-GAL4 negative control.

4) The authors' interpretations of the in vivo effects of UAS-FoxO NK (Figure 4 onwards) as a way of specifically impeding the FoxO-Usp interaction are not necessarily correct. The experiments have been done very thoroughly (and often sensibly using a FoxO null background) but the main result is that the effects of UAS-FoxO NK upon critical age/weight, pupal weight, gene expression (e74B, dib, phm) and ecdysone tend to be INTERMEDIATE between controls and UAS-FoxO animals. This indicates that, at best, the FoxO-Usp interaction only makes a partial contribution towards the overall FoxO activity relevant to ecdysone gene repression. At worst, if general FoxO functions are compromised in the NK protein then the authors would not be demonstrating any specific in vivo role for Usp-FoxO interactions, simply that partially reducing the general activity of FoxO gives a hypomorphic phenotype. Therefore, the authors should examine the effects of FoxONK overexpression while simultaneously over expressing Usp. If the complex is truly important then coexpression of foxONK together with Usp should look just like foxONK. If however, Usp coexpression still augments the delay to critical weight, the result is more consistent for a role of Usp in CW determination that is independent of complex formation.

Additional comments:

Reviewer #1:

1) The presence of ecdysone receptor (EcR) is shown to reduce the Usp-FoxO interaction in vitro (Figure 1F) but there is no follow up of the in vivo functional significance of EcR in this context. This issue is left hanging and so adds little to the paper. At present, the reader is left wondering whether or not EcR directly regulates ecdysone biosynthetic gene expression in the prothoracic gland and whether or not this regulation is antagonized by FoxO. Addressing this may add significant extra mechanism to the model.

2) Is there any functional relevance for a well-fed larva of dFoxO/Usp repression of ecdysone biosynthesis and its release near critical age? Loss of dFoxO activity is known to have no obvious effect on the final size/viability of adult flies from well-fed larvae (MA Junger et al. 2003). Moreover, FoxO overexpression only delays the small L3 ecdysone peak rather than abolishing it.

3) The present manuscript will be almost impenetrable for readers outside the immediate field and, even for insiders, it will be challenging in places. For example, many Results sections finish with a raw result and a figure citation but they would benefit from one sentence of interpretation/conclusion. Also, the figures and legends need clarifying in several places such as, for Figure 4, when (and when not) FoxO null mutant backgrounds are being used in an experiment (better indicated on the figure itself).

4) It will be confusing to readers that phm and dib expression often peak only after critical weight is attained in controls and in some genetic manipulations (e.g. Figure 3). Moreover, the order of the phm expression peaks for the three genotypes in Figure 3D and E doesn't match the order in which their critical weights are attained. How is this observed sequence of events compatible with the model in Figure 7? This finding needs addressing as it casts doubts upon whether increased mRNAs of ecdysone biosynthetic genes are really the trigger for critical weight.

Reviewer #2:

I have a minor comment regarding Figure 2D. The authors state that critical weight in Phm>FoxO, Usp animals is significantly increased compared with Phm>FoxO animals. This is unclear based on the bar graph, even with the 95% confidence intervals. Providing these values in the figure legend would provide a more convincing argument.

Reviewer #3:

It is unclear how the authors determine CW. The plots they provide in Figures 2 and 4 are time to pupation verses time at which starvation is imposed. How are 'weights' determined from this type of plot? It would seem to me that they need to show a standardization plot in which weight gain is linear with respect to developmental time. I assume that they have done this, but the method to determine CW should be better described in either the figure legend or the methods.

The authors play up the fact that they have discovered a key regulator of the small ecdysone peak that is associated with CW. In this regard, I was surprised that the authors never mention a recent paper from the King-Jones lab (Ou Q, Magico A, King-Jones K.PLoS Biol. 2011 Sep;9(9):e1001160.) which showed that PTTH also regulates the small ecdysone peak through effects on the nuclear localization of the NHR HR4. Since HR4 is proposed to negatively regulate biosynthetic enzymes prior to CW just like the FoxO/Usp complex proposed here, it would seem to me that this warrants significant mention. Are these two processes related or coordinated? Does HNR4 associate with FoxO? It would seem that at some level they have to be and this should be discussed.

Lastly, In Manduca and to some extent in Drosophila, there is data suggesting that limiting oxygen is the key to determination of critical weight. Once again there is no discussion of this point.

1) Show that FOXO and Usp interact in Co-IPs in fed wildtype L3 larvae. In Figure 1, it is shown that FoxO binds Usp in Co-IPs of extracts from starved but not fed larvae. For this interaction to be relevant to the regulation of the critical weight transition, the authors need to show that the dFoxO-Usp interaction also occurs in fed early L3 larvae (pre-critical weight). While the GST pulldowns with recombinant proteins in Figures 1 and 5 provide convincing evidence that FoxO and Usp have the potential to bind in vitro, the westerns are less convincing. The westerns show a weak Co-IPed band above the background smear that we are told corresponds to FoxO in . Given that demonstrating this point is so essential for the main conclusion of the paper about a molecular FoxO-Usp interaction, further controls need to be shown. One suggestion is to show that the FoxO band (co-IPed from larval extracts with anti-Usp) present in wild type animals is missing in FoxO null mutants.

We have performed a new Co-IP experiment examining FoxO-Usp interactions in: 1) fed pre-critical weight wild-type larvae (0-5 h AL3E), 2) fed pre-critical weight FoxO null larvae, 2) starved pre-critical weight wild-type larvae and 4) fed, post-critical weight, wild-type larvae. This experiment is now in Figure 2G. This experiment shows that FoxO and Usp do indeed form complexes in fed pre-critical weight larvae as well as in protein-starved, pre-critical weight larvae. In contrast, we do not see FoxO-Usp complexes in FoxO mutant larvae and fed, post-critical weight larvae.

2) Demonstrate the effects of feeding ecdysone at precritical weight times. If the model is correct then one would expect that feeding 20E just after ecdysis to L3 will lead to a smaller critical weight and precocious metamorphosis.

We have created a new Figure (now Figure 1) showing that starving larvae on 20E-supplemented agar eliminated the developmental delays seen in pre-critical weight larvae (Figure 1B). We also found that larvae fed 20E supplemented fly media become smaller in size (Figure 1–figure supplement 1). In addition, we quantified ecdysone concentrations in starved and fed wild-type larvae (Figure 1A). We found that in starved larvae ecdysone concentrations remain low for the first 18 hours after the moult. In fed larvae, ecdysone concentrations peak at 10 hours after the moult. These data demonstrate that the ecdysone peak itself is suppressed in starved pre-critical weight larvae and that feeding ecdysone to pre-critical weight larvae is sufficient to rescue the developmental delays induced by starvation. We hope this new data will make the experiments that follow easier to understand.

3) Concerns were raised about the specificity of the Gal4 drivers used. The P0206 driver used as a driver to express UAS-FoxO in the PG is also expressed in the CA and in oenocytes, which are known to regulate larval growth. The reviewers felt that it was important to use other drivers (Aug21 for the CA and the oenocyte 'specific” driver PromE-GAL4 from Joel Levine) as additional controls. Although the P0206-GAL4 driver has been used in similar studies of critical weight, the authors note that phm-GAL4 driven expression of UAS-FoxO is lethal in FoxO mutants but not in control animals. This observation may indicate that the presence of FoxO in the peripheral tissues of control animals is modifying the phenotypic effects of PG-specific FoxO over-expression. Although the authors suggest that animals expressing P0206-FoxO are viable due to the moderate levels of FoxO expression, these animals could be surviving due to the presence of FoxO in oenocytes. Therefore, the observations described in Figure 4A and Figure 6 could result from FoxO activity in either the oenocytes and not the PG or a synergistic interaction between these two tissues. The authors may address these problems with two simple experiments. Does oenocyte-specific expression of UAS-FoxO in a FoxO mutant delay the onset of pupariation? And, if so, is this delay 20E dependent? Finally, both of these Figures lack the necessary P0206-GAL4 negative control.

We have used both Aug21- and PromE(800)-Gal4 to drive FoxO expression in the CA and oenocytes of FoxO mutant animals (Figure 6–figure supplement 2). We found neither developmental time nor final body size affected in these animals.

We’ve added the P0206>+ control to Figure 6C. In Figure 7, the P0206>+ control is in black.

4) The authors' interpretations of the in vivo effects of UAS-FoxO NK (Figure 4 onwards) as a way of specifically impeding the FoxO-Usp interaction are not necessarily correct. The experiments have been done very thoroughly (and often sensibly using a FoxO null background) but the main result is that the effects of UAS-FoxO NK upon critical age/weight, pupal weight, gene expression (e74B, dib, phm) and ecdysone tend to be INTERMEDIATE between controls and UAS-FoxO animals. This indicates that, at best, the FoxO-Usp interaction only makes a partial contribution towards the overall FoxO activity relevant to ecdysone gene repression. At worst, if general FoxO functions are compromised in the NK protein then the authors would not be demonstrating any specific in vivo role for Usp-FoxO interactions, simply that partially reducing the general activity of FoxO gives a hypomorphic phenotype. Therefore, the authors should examine the effects of FoxONK overexpression while simultaneously over expressing Usp. If the complex is truly important then coexpression of foxONK together with Usp should look just like foxONK. If however, Usp coexpression still augments the delay to critical weight, the result is more consistent for a role of Usp in CW determination that is independent of complex formation.

We agree with the reviewers that if FoxO NK is a weak hypomorph of FoxO, this would explain the intermediate phenotype. In the first version of this manuscript, we addressed whether FoxO NK may simply be a hypomorph of FoxO through several means. First, we found that FoxO NK induced the expression of known FoxO target, InR and 4E-BP, to the same levels as FoxO. Secondly, we explored whether FoxO NK overexpression reduced the size of tissues to the same extent as FoxO. FoxO NK significantly reduced the size of wings, and eyes, although not as much as FoxO. Importantly, FoxO NK and FoxO both reduced the size of the prothoracic gland to the same extent.

Further, because overexpressing Usp in the prothoracic gland on its own did not significantly change either CW or critical age, we concluded that Usp did not play an independent role in CW. However, overexpressing both FoxO NK and Usp would provide better evidence that Usp is not acting on its own to regulate CW. We found that co-overexpressing Usp and FoxO NK did not affect the size and the age at critical weight (Figure 6—figure supplement 2C). Furthermore, we did not see significant difference in body size between P0206>FoxO NK animals and P0206>FoxO NK, Usp animals (Figure 6—figure supplement 2D). Therefore, we concluded that FoxO NK bears proper Usp-independent transcriptional activity with strongly reduced FoxO-Usp affinity.

Additional comments: Reviewer #1: 1) The presence of ecdysone receptor (EcR) is shown to reduce the Usp-FoxO interaction in vitro (Figure 1F) but there is no follow up of the in vivo functional significance of EcR in this context. This issue is left hanging and so adds little to the paper. At present, the reader is left wondering whether or not EcR directly regulates ecdysone biosynthetic gene expression in the prothoracic gland and whether or not this regulation is antagonized by FoxO. Addressing this may add significant extra mechanism to the model.

In the in vitro method (GST-pulldown), we used standardized quantities of FoxO, Usp and EcR protein that are not adjusted to the physiological levels. Our in vitro results show that FoxO and EcR can compete for Usp binding in principle. However in vivo, our co-IP results show that this is unlikely to be the case. We see high levels of Usp in the co-IP overall, and in starved larvae FoxO-Usp binding does not qualitatively diminish the amount of EcR-Usp binding (Figure 2G).

2) Is there any functional relevance for a well-fed larva of dFoxO/Usp repression of ecdysone biosynthesis and its release near critical age? Loss of dFoxO activity is known to have no obvious effect on the final size/viability of adult flies from well-fed larvae (MA Junger et al. 2003). Moreover, FoxO overexpression only delays the small L3 ecdysone peak rather than abolishing it.

Our new co-IP experiment shows that FoxO and Usp make complexes in fed, pre-critical weight larvae (Figure 2G), suggesting that even in fed larvae the complex regulates the timing of the ecdysone pulse. Starving larvae does not abolish critical weight, but rather delays it. Thus this peak is sensitive to, but not dependent on, nutrition. In this way, nutrition, via FoxO/Usp, can tune the timing of the ecdysone peak at critical weight, thereby ensuring optimal growth for the available environment.

3) The present manuscript will be almost impenetrable for readers outside the immediate field and, even for insiders, it will be challenging in places. For example, many Results sections finish with a raw result and a figure citation but they would benefit from one sentence of interpretation/conclusion. Also, the figures and legends need clarifying in several places such as, for Figure 4, when (and when not) FoxO null mutant backgrounds are being used in an experiment (better indicated on the figure itself).

Thank you for your feedback, we’ve worked hard to clarify the text and make the results and figure legends more accessible to a broader audience.

4) It will be confusing to readers that phm and dib expression often peak only after critical weight is attained in controls and in some genetic manipulations (e.g. Figure 3). Moreover, the order of the phm expression peaks for the three genotypes in Figure 3D and E doesn't match the order in which their critical weights are attained. How is this observed sequence of events compatible with the model in Figure 7? This finding needs addressing as it casts doubts upon whether increased mRNAs of ecdysone biosynthetic genes are really the trigger for critical weight.

At 0 h AL3E, phm and dib are already high in the phm>dsFoxO, dsUsp. We presume that this early expression is sufficient to drive ecdysone synthesis and cause premature CW transition. Thus the peaks match the order in which CW is attained in the three genotypes. The increases in phm and dib that occur later in this genotype is presumably the Sgs peak. We have clarified this in the text.

Reviewer #2: I have a minor comment regarding Figure 2D. The authors state that critical weight in Phm>FoxO, Usp animals is significantly increased compared with Phm>FoxO animals. This is unclear based on the bar graph, even with the 95% confidence intervals. Providing these values in the figure legend would provide a more convincing argument.

The error bars for this graph were incorrect. We’ve changed this and also provided a supplementary table with the values for age and size at weight for all genotypes (Supplementary file 3).

Reviewer #3: It is unclear how the authors determine CW. The plots they provide in Figures 2 and 4 are time to pupation verses time at which starvation is imposed. How are 'weights' determined from this type of plot? It would seem to me that they need to show a standardization plot in which weight gain is linear with respect to developmental time. I assume that they have done this, but the method to determine CW should be better described in either the figure legend or the methods.

You are absolutely right, we omitted both a script in the Dryad folder and a clear explanation of how we determine size at CW. To clarify how CW is determined, we have included a complete description of the methodology stating that we first construct growth rate plots for each genotype (weight over age) and use linear regression to calculate the size at CW from the age at which larvae reach CW. We have included a new figure supplement for Figure 1 with the growth curve for wild type (w¹¹¹⁸) larvae (Figure 1–figure supplement 1B), and the growth data and scripts for all remaining genotypes in Dryad.

The authors play up the fact that they have discovered a key regulator of the small ecdysone peak that is associated with CW. In this regard, I was surprised that the authors never mention a recent paper from the King-Jones lab (Ou Q, Magico A, King-Jones K.PLoS Biol. 2011 Sep;9(9):e1001160.) which showed that PTTH also regulates the small ecdysone peak through effects on the nuclear localization of the NHR HR4. Since HR4 is proposed to negatively regulate biosynthetic enzymes prior to CW just like the FoxO/Usp complex proposed here, it would seem to me that this warrants significant mention. Are these two processes related or coordinated? Does HNR4 associate with FoxO? It would seem that at some level they have to be and this should be discussed.

Yes, you are right, this was an oversight. We are focusing on how environmental cues regulate the CW pulse, but should not neglect the lovely work from the O’Connor and King-Jones labs on this subject.

We have added these references in the introduction, and expanded our discussion to discuss the relative roles of insulin/TOR versus PTTH (via DHR4).

Lastly, In Manduca and to some extent in Drosophila, there is data suggesting that limiting oxygen is the key to determination of critical weight. Once again there is no discussion of this point.

We have added this to our text.