Decision letter

  1. Marianne Bronner
    Reviewing Editor; California Institute of Technology, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Functional evolution of a morphogenetic gradient" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Marianne Bronner as the Senior Editor and Reviewing Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Kwan et al. investigate BMP patterning in embryos of the fly Megaselia and compare this with known mechanisms acting in Drosophila. Data support the view that evolutionary changes are responsible for differences in tissue specification found within these two fly species. In Megaselia, serosa and amnion form as two distinct tissues, whereas the aminoserosa forms as one merged tissue in Drosophila. The data, composed mostly of RNAi, ectopic expression, and colocalization of BMP ligands and target genes, makes a compelling case for differences in feedback control of BMP signaling between these two species as causative for these tissue differences. Specifically, the data suggest that the gene networks acting in these two fly species have changed with regard to the regulation of the gene eiger (egr). In Drosophila, prior published studies have demonstrated egr is a positive regulator of BMP signaling, acting downstream of decapentaplegic (dpp) and zerknüllt (zen) genes. Here, the authors show that in Megaselia, egr also acts downstream of dpp but, in contrast to the case in Drosophila, that in Megaselia egr is not regulated by zen (or rather, it has a minor role). Instead, in Megaselia, egr is regulated by the dorsocross genes (doc/doc2). This change in regulation of egr is proposed as the mechanism by which amnion regulation (through doc genes and hnt) was separated from serosa regulation (mediated by zen).

The results are interesting, novel and well controlled and the manuscript is well written. Some additional experiments are required as outlined below.

Essential revisions:

1) Why does Mab-egr RNAi reduce pMad levels uniformly but not the width of the pMad stripe; whereas, Mab-doc genes RNAi reduces pMad levels and the width of the stripe?

Essentially, pMad within the amnion domain of Mab-egr RNAi embryos appears "normal" (stripe is just as wide) whereas in the Mab-doc mutants the pMad stripe is thinner. Could this relate to a role of Mab-doc genes in regulating a Mab-cv-2? Or other target genes besides egr?

2) Is there cv-2 in Megaselia and does it also act together with Mab-egr to support positive feedback? The authors may not want to address cv-2 experimentally, but at least a sentence or two should be added to the text to put the current study in context of what's known in Drosophila.

3) How certain are the authors that Mab-zen RNAi is working? (e.g. Figure 3—figure supplement 1, panels A-C). Is zen expression lost at st 5 and st 6? Can Mab-zen RNAi lead to efficient decrease in levels earlier to support results of Figure 2H, I and Figure 3L, for example?

4) On the flip side, how certain is it that Mab-zen levels are not affected upon Mab-dpp RNAi? Levels in Figure 2I do appear reduced relative to Figure 2H. Can zen levels be quantified? And possibly compared to zen levels upon Mab-zen RNAi.

https://doi.org/10.7554/eLife.20894.016

Author response

Essential revisions:

1) Why does Mab-egr RNAi reduce pMad levels uniformly but not the width of the pMad stripe; whereas, Mab-doc genes RNAi reduces pMad levels and the width of the stripe?

Essentially, pMad within the amnion domain of Mab-egr RNAi embryos appears "normal" (stripe is just as wide) whereas in the Mab-doc mutants the pMad stripe is thinner. Could this relate to a role of Mab-doc genes in regulating a Mab-cv-2? Or other target genes besides egr?

2) Is there cv-2 in Megaselia and does it also act together with Mab-egr to support positive feedback? The authors may not want to address cv-2 experimentally, but at least a sentence or two should be added to the text to put the current study in context of what's known in Drosophila.

We agree that BMP signaling and Mab-doc/doc2 activity together could regulate more than one gene with a role in shaping the BMP gradient and thank the reviewers for pointing this out. We address this issue in a new paragraph (–subsection “Mab-doc-dependent control of Mab-egr expression contributes to a positive feedback circuit that promotes BMP signaling during gastrulation”, fourth paragraph) and added a brief description of cv-2 experiments in Megaselia to the revised manuscript (Figure 4—figure supplements 3, 4 and 5).

For Drosophila, Gavin-Smyth et al. (2013) found that the reduction of BMP signaling was less severe in egr deficient embryos than in Medea deficient embryos that completely lack BMP-dependent positive feedback, suggesting that egr is not the only target of BMP signaling involved in the feedback process. Gavin-Smyth et al. (2013) also presented an analysis of the cv-2 phenotype. In Drosophila, the initial expression of cv-2 is not under BMP control but under the control of zen, which is expressed independently of BMP signaling in stage 5 embryos; therefore Gavin-Smyth et al. did not consider it part of the feedback circuitry in Drosophila. They also reported that loss of cv-2 causes an elevation of BMP signaling, indicating it does not simply act as a component in a positive feedback circuit. In the new paragraph of our manuscript, we explicitly acknowledge the possibility of additional BMP/doc target genes acting as feedback components in Megaselia and quote the Gavin–Smyth paper for a potential parallel with Drosophila where BMP signaling seems to control more than one feedback component.

In Megaselia, Mab-cv-2 expression is widely expressed from stage 6 onwards, with slightly higher expression dorsally, and was not grossly perturbed by Mab-doc/doc2 knockdown, suggesting that, like in Drosophila, it is not a component of the feedback process. Interestingly, we find that knockdown of Mab-cv-2 can perturb amnion specification at the beginning of gastrulation, suggesting that Mab-cv-2 might promote BMP signaling at the beginning of gastrulation, unlike in Drosophila where it attenuates BMP signaling. This difference between Megaselia and Drosophila is not completely surprising because Cv-2 has been shown to act in a context and dose dependent manner to either promote or inhibit BMP signaling. In conclusion, while the only confirmed Mab-doc/doc2 target remains Mab-egr, we have modified the manuscript to take into account the likelihood that there are other target genes.

3) How certain are the authors that Mab-zen RNAi is working? (e.g. Figure 3—figure supplement 1, panels A-C). Is zen expression lost at st 5 and st 6? Can Mab-zen RNAi lead to efficient decrease in levels earlier to support results of Figure 2H, I and Figure 3L, for example?

As we have shown previously, Mab-zen RNAi is effective within less than 10 minutes after injection of dsRNA (supplemental Figure 1A in Rafiqi et al. 2010). To clarify this issue, we replaced panels of the original Figure 3—figure supplement 1A-C (showing embryos at stage 8 stained for Mab-zen, Mab-doc and Mab-hnt following Mab-zen RNAi) with four new panels showing representative Mab-zen expression patterns at stage 6 in a control-injected embryo (A) and in a Mab-zen RNAi embryo (B), in addition to stage-matched Mab-zen RNAi embryos stained with Mab-doc and Mab-hnt probes (C, D). In Mab-zen RNAi embryos, only residual punctate Mab-zen expression was observed, consistent with high transcriptional activity in the nuclei during gastrulation. In the cytoplasm, Mab-zen transcript did not accumulate above a background level, indicating that Mab-zen expression is post-transcriptionally knocked down in Mab-zen RNAi embryos.

4) On the flip side, how certain is it that Mab-zen levels are not affected upon Mab-dpp RNAi? Levels in Figure 2I do appear reduced relative to Figure 2H. Can zen levels be quantified? And possibly compared to zen levels upon Mab-zen RNAi.

In the original figure, contrast enhancement differed between the images shown in panels 2H and I. When original pictures of late Mab-dpp RNAi embryos were processed in exactly the same way as the controls, Mab-zen expression levels were similar between controls and late Mab-dpp RNAi embryos (n=6). However, we noticed that the green background caused by the Mab-eve probe varied slightly between embryos. In the revised figure, the image shown in 2I was replaced with an image that was subjected to same contrast enhancement that the image shown in 2I.

https://doi.org/10.7554/eLife.20894.017

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  1. Chun Wai Kwan
  2. Jackie Gavin-Smyth
  3. Edwin L Ferguson
  4. Urs Schmidt-Ott
(2016)
Functional evolution of a morphogenetic gradient
eLife 5:e20894.
https://doi.org/10.7554/eLife.20894

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https://doi.org/10.7554/eLife.20894