Specification of the endocrine primordia controlling insect moulting and metamorphosis by the JAK/STAT signalling pathway

  1. Mar García-Ferrés
  2. Carlos Sánchez-Higueras
  3. Jose Manuel Espinosa-Vázquez
  4. James C-G Hombría

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

The corpora allata and the prothoracic glands control moulting and metamorphosis in insects. These endocrine glands are specified in the maxillary and labial segments at positions homologous to those forming the trachea in more posterior segments. Glands and trachea can be homeotically transformed into each other suggesting that all three evolved from a metamerically repeated organ that diverged to form glands in the head and respiratory organs in the trunk. While much is known about tracheal specification, there is limited information about corpora allata and prothorathic gland specification. Here we show that the expression of a key regulator of early gland development, the snail gene, is controlled by the Dfd and Scr Hox genes and by the Hedgehog and Wnt signalling pathways that induce localised transcription of upd , the ligand of the JAK/STAT signalling pathway, which lies at the heart of gland specification. Our results show that the same upstream regulators are required for the early gland and tracheal primordia specification, reinforcing the hypothesis that they originated from a segmentally repeated organ present in an ancient arthropod.

Summary statement

By analysing snail regulation in the CA and PG we describe their specification network, which converges on JAK/STAT pathway activation and reveals shared upstream regulators with the trachea.

Article activity feed

  5. Review Commons

    Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Reviewers 1 and 2 are very positive about our manuscript, while reviewer 3 is surprisingly critical.

    However, except for the first observation, most of reviewer 3´s comments are based on incorrect interpretations of our results.

    We have integrated the useful comment into our revised version, and we will discuss in the following sections why reviewer 3’s remaining criticisms should be disregarded.

    __Reviewer 1: __

    Reviewer 1 has only minor suggestions and is satisfied that we prove convincingly our claims. The reviewer also finds our results reinforce our previously proposed hypothesis that the glands and the trachea evolved from common metamerically repeated ancient primordia.

    We have introduced the following changes to the text to accommodate Reviewer’s 1 minor suggestions.

    Main suggestion: Insert a paragraph in discussion explaining the relevance of new insights to more basal insects that do not form a ring gland.

    RESPONSE:We have introduced at the end of Discussion the following paragraph:

    “Our analysis of snail activation in the CA and PG shows that these glands and the trachea share similar upstream regulators, reinforcing the hypothesis that both diverged from an ancient segmentally repeated organ. In Drosophila melanogaster the CA and the PG primordia experiment a very active migration after which they fuse to the corpora cardiaca forming the ring gland (Sanchez-Higueras and Hombria, 2016). This differs from more basal insects where the CA fuses to the corpora cardiaca but not to the PG, and from the Crustacea where the three equivalent glands are independent of each other (Chang and O'Connor, 1977; Laufer et al., 1987; Nijhout, 1994; Wigglesworth, 1954). As the mechanisms we here describe relate to the early specification of the glandular primordia in Drosophila, it will be interesting to investigate if the equivalent genes are also involved in the endocrine gland specification of more distant arthropods”.

    Additional comment 1: Introduction, pg 3, a paragraph starting with "In comparison to the extensive knowledge we have of ..." - consider omitting or greatly shortening, this text breaks a flow as it is focused on tracheal development. I understand the authors' logic, but this information distracts from the main focus on CA and PG. RESPONSE:We agree that the trachea description paragraph breaks the flow of the introduction to gland development. As suggested by the reviewer, we have deleted most of the descriptive text on trachea development but left all the references so that interested readers can find the information.

    Additional comment 2: *Beginning of discussion, pg 11:

    • change 2nd sentence to: " Our results indicate that the HH and the Wnt pathways act indirectly to negatively regulate the spatial activation ..."
    • the following sentence, starting with "Engrailed activation off hh transcription ...." is way too long and hard to follow, consider breaking into two sentences. RESPONSE:*We have changed both sentences as suggested

    Additional comment 3:* In Fig 4B, mx and lb segments should be labeled so this panel is consistent with labeling in 4A.* RESPONSE:We have changed Fig.4B labels to be consistent with 4A

    Additional comment 4: In Fig 6, reduce a font size for labels on right-hand side (A1, A2, A1+A2 proximal, etc), so that they are visualy distinct from panel labels on left-hand side (A, B, C,..).

    RESPONSE:We have changed Fig.6 Font size as suggested

    __Reviewer 2 __

    The reviewer is positive and agrees that the results we present in “this paper add to our understanding of how the CA and PG primordia are specified and highlights important similarities with the specification of the tracheal primordia”. The reviewer’s comments focus specially on the activation vs. maintenance of sna.

    Specific Comment a): Referring to Fig 1G-J, the reviewer says: It is not clear to me from either this figure or from the text whether the initial pattern of expression of the sna-rg reporter in stage 11 embryos is WT and then disappears at stage 12, or whether it is always defective. In trying to understand the activation process, I think it would be important to know for sure whether rg enhancer activity during the initiation phase in stage 11 is WT or not.

    RESPONSE: As suggested by the reviewer, we have included st11 embryos in Fig. 1 as panels G,J which illustrate that early sna-rg activation occurs normally in snaΔrgR2embryos prior to apoptosis kicking in. To make space for these images, we have taken out the st12 embryos that we had in our previous submitted version. This does not affect the manuscript’s message, as st12 phenotypes are similar to those at st13 which are presented in Fig. 1H,J.

    Moreover, in this revised version, the embryos in Fig. 1G-J have also been double stained with the apoptosis marker DCP1 to highlight the cell death observed in the gland primordia of snaΔrgR2 embryos (Fig. 1G’-J’).

    Specific Comment b) The authors argue that the rg deletion removes the only region driving sna expression in CA/PG. I'm not convinced that necessity necessarily implies sufficiency with respect to the requirements for rescue. While the sna-rg reporter is expressed in a pattern that seems to mimic the endogenous gene, do we know that a rg-sna transgene would fully rescue the rg deletion mutant?

    RESPONSE: In our previous paper (Sanchez-Higueras 2014) we presented evidence that in sna null embryos, a Snail BAC gene lacking the sna-rg CRM can fully rescue the mesoderm phenotypes but not the ring gland ones. This proved that in the BAC transgene there was no shadow CRM capable of rescuing the gland formation in the absence of sna-rg. In the current paper we show that deleting the endogenous sna-rg CRM in the sna locus results in the absence of sna transcription from the gland primordia.

    Making a sna-rg- construct expressing sna to test if this rescues the snaΔrgR2 homozygous mutants could be done, but it will delay this publication without adding much to the paper: we already know that sna-rg is sufficient to drive activation in all the CA and the PG cells (Sanchez-Higueras 2014 Fig 2J-M) and it would be expected to rescue the gland formation in snaΔ*rgR2 *homozygous mutants.

    Having said that, we have changed the wording in the manuscript to one that may be acceptable to the reviewer.

    Instead of:

    “These results prove that snaΔrgR2 deletes the only regulatory region driving sna expression in the CA and PG gland primordia…”

    We now say:

    “These results prove that the snaΔrgR2 deletes mutation inactivates the only regulatory region driving sna expression in the CA and PG gland primordia…”

    Specific Comment c) is Sna required for maintaining sna expression?

    RESPONSE:This experiment is relevant to the maintenance mechanism of sna expression in the ring gland, and not to its activation which is the main focus of this paper.

    The search for the maintenance mechanisms is currently been followed in the laboratory and we prefer not deal with it in this paper. Providing a negative answer to this question would not be satisfactory, as we would need to search for the factors controlling sna’s maintenance.

    Specific comment d)* The authors show that there is an expansion in the number of sna-rg reporter expressing cells along the AP axis when upd is ectopically expressed using a sal-Gal4 driver. Though not mentioned in the text at this juncture, sal is expressed in the PG primordia, while seven-up (svp) is expressed in the CA primordia. I assume that the upd induced expansion is only observed for the PG primorida (LB) and not the CA primordia (Mx)-at least this is what the figure looks like. **(…) *How about svp driven upd-assuming there is a svp-Gal4 driver-does it cause an expansion of Ca but not PG.

    RESPONSE: As the reviewer has noticed, there is a stronger expansion of sna-rg-GFP expression in the labial segment than in the maxillary segment. This is not due to the use of the sal-Gal4 line. We see the same effect with arm-Gal4 which drives similar expression on the maxilla and the labium. To illustrate this point, we have included two new panels (Fig.5D-E) where the ectopic expression of Upd has been induced with arm-Gal4. These embryos have been stained with anti-Sal to label the PG. This experiment shows clearly that the PG has expanded much more than the CA.

    There are several reasons why expansion of the glands could be more efficient in the labium than in the maxilla. One possible reason is the temporal response to Upd activation. Upd induction by the arm-Gal4 and sal-Gal4 lines may occur after the cells in the maxilla are no longer capable of activating sna-rg but still capable of activating it in the labium. This temporal hypothesis is based on our results showing that the CA expresses more transiently the upd gene and that STAT activation lasts for longer in the labium than in the maxilla (Fig. 4A-D)].

    A second possibility, that we favour, is the existence of dorso-ventral repressor genes modulating sna-rg expression intrasegmentally. Some of our results point towards the sna-rg CRM receiving repressor inputs that modulate intrasegmental spatial expression in the dorso-vental axis. When we delete the A2 distal region of the sna-rg enhancer, its expression in the labium expands ventrally (Fig. 6E,G and Sup.Fig. 4D). If a similar repressor was also modulating sna-rg in the maxilla it could be blocking its expansion. However, at this stage we have no solid data to support any of these hypotheses. As explained before for the maintenance mechanisms of sna-rg expression, our ongoing work aims to isolate and characterize further elements controlling the ring gland gene network, including these negative regulators.

    In the revised manuscript we now describe the different effects of Upd ectopic activation on the expression of sna-rg in the maxilla and the labium (underlined text is new to this revised version):

    “To test if generalised Upd expression in the maxilla and labium can activate sna-rg expression independently of other upstream positive or negative inputs, we induced UAS-upd with either the sal-Gal4 or the arm-Gal4 lines. We observe that, these embryos have expanded sna-rg expression along the antero-posterior axis in the maxillary and labial segments (Fig. 5C). Analysis of Sal expression, which labels the PG primordium (Sanchez-Higueras et al., 2014), shows that Upd ectopic expression induces a moderate expansion of the CA primordia while resulting a much larger increase of the PG primordium (Fig. 5D-E). This expansion occurs mostly in the anterior and posterior axis from cells where the Hh and the Wnt pathways are normally blocking sna-rg expression, while expansion is less noticeable in the dorso-ventral axis. This indicates that most of the antero-posterior intrasegmental inputs provided by the segment polarity genes converge on Upd transcription but that the dorso-ventral information is registered downstream of Upd.”

    The differential response of sna-rg to Upd activation in the maxillary and labial segments is also mentioned in Fig. 5 legend. (see Continuation comment d).

    Continuation comment* d) “It looks to me also like the vvl domain is expanding as well. This information should be clarified.”

    RESPONSE: Yes, ectopic upd expression also expands *vvl1+2 *expression. We have previously published that vvl1+2 is a direct target of JAK/STAT signalling in the trachea (Sanchez-Higueras 2019 and Sotillos et al. 2010 Dev.Biol). Although *vvl1+2 *expands dorsally in the Mx, those cells do not activate sna-rg dorsally. The ventral restriction of sna-rg in the maxilla is controlled by Dfd while in the labium its dorsal expression depends on Scr. We explain this in Fig.5’s figure legend where we now say (underlined text is new to this revised version):

    (C) Ectopic Upd expression driven with sal-Gal4 induces ectopic sna-rg and vvl1+2 expression in the gnathal segments, which for sna-rg is more pronounced in the labium than in the maxilla. Note that in the maxillary segment Upd can induce ectopic dorsal vvl1+2 but not sna-rg expression, this is expected as Dfd only induces sna-rg ventrally in the maxilla. (D-E) *sna-rg-GFP *embryos stained with anti-GFP (green) and anti-Sal (red). In control embryos (D) Sal labels the PG primordium but not the CA. In arm-Gal4 embryos ectopically expressing Upd, the PG is more expanded than the CA as shown by number of cells co-expressing Sal and GFP.

    Specific Comment e) The authors note a difference between CA and PG in the requirement for STAT binding sites in the enhancers. Is that related to the fact that svp is expressed in CA and sal is expressed in PG? Would driving svp expression using the sal-Gal4 driver maintain sna-rg expression.

    RESPONSE: During our preliminary ongoing experiments on sna maintenance mechanisms we looked in svp mutants and did not notice a change in sna-rg expression, thus it is unlikely that Svp is responsible for the difference. As said above, we continue looking for genes involved in gland formation. Sal could be involved in the maintenance of sna in the PG, but as Sal is expressed in the maxilla and labial segments before gland formation, it is difficult to disentangle if Sal is required for sna activation or maintenance (or both).

    Specific Comment f) Do svp or sal have a role in initiating sna expression when upd is present or maintaining sna expression after upd disappears? Presumably there is already published data that would answer these questions.

    RESPONSE: As explained above we did not find any effect of svp on activation of sna-rg, however we find that in sal mutants the labium does not express sna-rg. This shows that sal is likely to be another positive input. As in sal mutants both trh and Ubx become ectopically expressed in the Lb (Casanova1989* Roux's archives of developmental biology* 198: 137-140; Castelli-Gair 1998 IJDB42:437-444) we have done the experiment in sal trh double mutants and in sal Ubx,abdA,Abd-B mutants. In both cases we still see a failure of sna activation in the Lb reinforcing the idea that Sal is an additional positive input. However, we prefer not to add the sal experiments as they would complicate the paper which currently focuses on the similar requirement of the Wnt, Hh and JAK/STAT signalling pathways.

    __Reviewer 3 __

    Reviewer is very critical. We accept some of the points raised and have modified the manuscript accordingly. However, as we detail below, the most serious criticisms are incorrect and do not affect the conclusions reached by our work.

    We agree with the following comment:

    “In the Dfd Scr double mutant, both the CA and PG expression of the snail-rg-GFP reporter is still there - admittedly, the gland cells look abnormal at late stages, but this reporter that is supposed to function as a proxy for gland induction is still expressed. That either means that expression of sna-rg-GFP is not a proxy or that the glands are still being specified in the absence of the Hox genes that are proposed to specify these organs. The reporter should not be expressed if these Hox genes are what specify these endocrine organs.”

    RESPONSE: The reviewer has made a good observation. The expression of sna-rg-GFP is not completely absent in Dfd Scr mutant embryos (Fig. 5F in this revised version), which indicates that although the Hox genes are required to activate upd in the maxilla and labium and in their absence the gland primordia become apoptotic, there must be other positive inputs to the enhancer. However, this does not mean the Hox gene input is irrelevant for gland specification. Not only the Hox genes are required to keep normal levels of upd expression in the Mx and Lb primordia and gland viability, but previously we also showed that cephalic Hox genes influence the dorso-ventral position inside the vvl1+2 expressing cells where the sna-rg enhancer is activated: in the maxilla Dfd induces the ventral vvl1+2 expressing cells to activate sna-rg, while in the labium Scr induces the dorsal vvl1+2 cells to activate sna-rg (Sanchez Higueras 2014). The data presented in this paper indicate that the input of both Dfd and Scr over sna-rg CRM activation are indirect.

    As a result of the reviewer’s criticism, we have tested if the additional positive input could be provided by Ci. In our previous submitted version, we showed that the repressor form of Ci blocks sna-rg activation. In this revised version, we have tested what is the effect of expressing the activator form of Ci. In embryos overexpressing the activator CiPKA isoform, we have observed that the expression of sna-rg and upd are expanded, indicating that Ci can provide the additional Hox-independent positive input. In the revised version we present these new results as Fig.3G and Fig. 4I. We have modified accordingly the scheme that appears in panel 3I to include this. In the main text we describe the result in the Hh regulation section where we have added:

    “Although the above results indicate Ci is not absolutely required for sna-rg expression, we observed that overexpression of CiPKA, the active form of Ci, causes a non-fully penetrant expansion of sna-rg expression (Fig. 3G) suggesting the possibility that sna-rg may be responsive to Ci and to a second activator.”

    … and in the “Regulation of Upd ligand expression by the Wg and Hh pathways” section

    where we say:

    “We also found that ectopic expression of the activator Ci protein results in a non-fully penetrant expansion of upd expression in stage 10 embryos (Fig. 4H-I).”

    We have also modified the final scheme in Fig. 7 to mention that Dfd and Scr prevent the apoptosis of the gland primordia, and that there must be an additional positive input controlling upd activation besides the Hox input. However, in the figure we do not define Ci as the activating input as we would like to have additional evidence before making such claim.

    To clarify that the Hox input is not absolutely required we have modified the text in several places. Where we said:

    “Expression of the sna-rg reporter in the maxilla and the labium requires Dfd and Scr function …”

    We now say:

    “Development of the CA and PG and normal expression of the sna-rg reporter in the maxilla and the labium require Dfd and Scr function …”

    We also mention this in Fig. 5 legend where we have added:

    “In Dfd Scr mutant embryos (F), although the gland primordia become apoptotic, residual GFP expression indicates that there must exist Hox independent inputs activating the sna-rg enhancer.”

    As a result of reviewer 3’s comment, we have noticed a further example of similarity between the gland and the trachea specification, which we have commented in the revised discussion where we added the following paragraph:

    “Another interesting similarity between glands and trachea is that, although ectopic Hox gene expression can ectopically induce sna-rg and trh outside their normal domain, the lack of Hox expression does not completely abolish their endogenous expression, indicating that in both cases a second positive input can compensate for the absence of Hox mediated activation. Our results suggest that, in the glands, this redundant input could be provided by the activating Ci form (Figs. 3G and 4I), but further analysis to confirm this possibility and discard alternative sna-rg activators should be performed.”

    We disagree with the following comments:

    The finding that the CA and PGs form in slightly different DV positions from each other and slightly different DV positions from the trachea (based on the vvl1+2 mCherry reporter staining combined with that of the sna-rg-GFP reporter staining in Figure 5A, where staining does not overlap except where the CA cells have started to migrate over the vvl1+2 mCherry expressing cells) argues pretty strongly against the CA and PG being homologous to each other or absolutely homologous to the trachea primordia

    RESPONSE: This erroneous claim was based on Fig. 5A, that showed a double stained embryo where co-expression is difficult to appreciate without separating the channels. Co-expression of these two reporter lines in the ring gland has been previously documented beyond doubt in our 2014 publication, cited throughout the manuscript, where we presented eight different panels of glands clearly co-expressing both markers at various developmental stages (Current Biology 2014 Fig.2B-I). To prevent any readers reaching the same conclusion as the reviewer, we have modified Fig. 5A to show a double stained sna-rg-GFP *vvl1+2-mCherry *embryo alongside with the two separate channels (panels 5A’ and A’’) to make the co-expression evident.

    Although we are not including it in this manuscript, the reviewer will also be able to find images in the same 2014 Current Biology publication (Fig.3), where the ectopic activation of Dfd in the trunk leads to the activation of the sna-rg-GFP reporter in the vvl1+2 tracheal cells, proving that the glands and the trachea are formed at homologous positions.

    Having made clear that sna-rg activation in both the CA and the PG occurs in vvl1+2 expressing cells, we now refute a second criticism: The reviewer is puzzled that despite the glands being formed at different dorso-ventral positions in the vvl1+2 expressing patch of cells, we claim both groups of cells are homologous to the trachea.

    We are not saying that the CA are formed at homologous positions to those giving rise to the PG. What we say is that both the CA and the PG are formed at positions homologous to those giving rise to the trachea in the trunk segments.

    To make this clear in the revised version, we have changed the wording of a sentence in the Introduction section that might have originated the confusion.

    Instead of saying:

    “First, the CA, the PG and the traqueal primordia are specified in the lateral ectoderm at homologous positions”.

    Now, it reads:

    “First, the CA and the PG are specified in the cephalic lateral ectoderm at homologous positions to those forming the tracheal primordia in more posterior trunk segments.”

    It has been shown that each tracheal primordium (which are labelled by vvl1+2-mCherry) gives rise to different tracheal branches depending on the positions where they are specified: the dorsal cells give rise to the dorsal tracheal branches, the ventral cells to the ganglionic branches, the medial cells to the dorsal trunk etc. (for illustration see Fig.12 in Manning and Krasnow 1993). Each of these tracheal branches have a different shape and migrate to different positions. We believe that a similar positional specification occurs in the vvl1+2 cells in the maxilla and the labium. In the maxilla only the vvl1+2 ventral cells activate sna and svp (among other genes) to give rise to the CA. In the labium vvl1+2 dorsal cells activate sna, sal, phm (among other genes) to give rise to the PG. This regionalization is similar to what happens during tracheal branch specification, with the only difference that the interaction with Dfd and with Scr is what makes the positional outcome in the maxilla and the labium different (see in our Current Biology 2014 publication Fig. 3E-F and H-J). Thus, when the reviewer considers the equivalence between the CA/PG/trachea homology with that of the wing/haltere or that of the thoracic leg1/2/3 saying: “Indeed, the situation with these endocrine glands and the trachea is completely unlike the situation with the wing and haltere, wherein both structures arise from the same DV position in adjacent segments, or with legs 1, 2 and 3, which arise from the same DV position in adjacent segments”

    …the reviewer should think about the coxa and the tarsi in the legs. The coxa in T1 is not homologous to the tarsi in T2 or T3, but when considering the leg structure as a whole, the coxa and the tarsi form part of the same homologous structure in T1, T2 and T3 despite being formed at different positions inside the leg primordia.

    The reviewer also doubts that the activation of upd occurs in the sna-rg primordium when saying: “Likewise, the STAT10X-GFP staining does not overlap with the sna-rg-mCherry staining (I see red cells and I see green cells - there are no yellow cells). If activation of snail is through Upd activation of STAT signaling, we should see that the snail reporter expression is within the domain of STAT10X-GFP expression.”

    RESPONSE: This is due to the fact that upd activation in the CA is extremely transient, leading to the loss of the x10STAT-GFP expression before the sna-rg-mCherry levels are robust enough in the maxilla. This criticism does not apply to the PG where due to upd expression lasting longer, co-expression of sna-rg-mCherry and x10STAT-GFP in panel 4B should be evident to the reviewer.

    To try to sort the CA co-expression problem, we are currently repeating the experiment but instead of analysing sna-rg-mCherry activation with the RFP antibody, we will do an mcherry RNA in situ. We hope that the mcherry transcript will be detectable earlier than the protein and the co-expression will be evident.

    We strongly disagree when the reviewer says: “This paper provides a strong basis for arguing that the CA and PG are induced independently of Jak/Stat signaling, whereas trachea require this signaling pathway.”

    RESPONSE: When making this claim, the reviewer is ignoring a large number of experiments presented in the manuscript. If the CA and the PG are induced independently of JAK/STAT signalling:

    (1) Why sna-rg expression disappears from the glands in mutants lacking the Upd ligands (Fig. 5B and 6K)?

    (2) Why deleting the region containing the putative STAT binding sites in the sna-rg enhancer causes the loss of enhancer expression (Fig. 6C)?

    (3) Why the smaller enhancer mentioned in point (2) recovers gland expression when adding a STAT binding site from an unrelated gene (Fig. 6G)?

    (4) Why the regained expression of the construct mentioned in (3) is lost by the mutation of two bases affecting this single STAT site (Fig. 6H)?

    The reviewer’s conclusion rests on giving an excessive importance to his reservations to CA co-expression in panel 4A while, surprisingly, disregarding the co-expression in the PG shown in panel 4B and all the experiments presented in Fig. 5 and Fig.6.

    *Reviewer 3 **Minor comments: RESPONSE: *Both comments have been taken into account in the revised version.

    In summary, in this revised version we have answered most queries raised by reviewers 1 and 2. Moreover, reviewers 1 and 2 agree that the results presented in this manuscript reinforce the hypothesis that the CA and the PG glands and the trachea derive from the divergent evolution of a metamerically repeated homologous organ.

    Reviewer 3 has made a good point that we have taken into account and has improved the revised submission.

    However, reviewer 3 is wrong when concluding:

    “This paper provides a strong basis for arguing that the CA, PG and trachea are not homologous structures”, and when saying: “the CA and PG are induced independently of Jak/Stat signaling, whereas trachea require this signaling pathway”.

    As we argue above, these conclusions are erroneous because:

    (1) Are based on the incorrect interpretation of Fig 5A and ignore previous published evidence cited throughout the manuscript.

    (2) It does not take into account key experiments presented in this work, while giving too much weigh to a result that can be easily interpreted.

    (3) It misinterprets the arguments justifying the positional homology between the CA/PG glands and trachea primordia.

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This paper focuses on the specification of two endocrine glands that form from head ectoderm, the corpora allata (CA), which forms in the maxillary segment and secretes Juvenile hormone, and the prothoracic glands (PG), which form in the labial segment and secrete Ecdysone. Secretion of both hormones results in a larval molt. Secretion of only Ecdysone induces metamorphosis, the transition of the larvae into the adult forms. Both the CA and PGs form in positions homologous to the tracheal primordia (approximately) and previous reports indicate that ectopic expression of the appropriate Hox genes can result in homeotic transformations of the glands into tracheal primordia and of tracheal primordia into glands. Using a GFP reporter construct for the snail gene as a proxy for gland specification, the authors show that CA and PG formation is regulated by two segment polarity genes: Hh and Wnt, with Hh signaling activating reporter gene expression and Wnt signaling inhibiting reporter gene expression. They also suggest that their endocrine gland GFP reporter is regulated by the two Hox proteins expressed in those segments: Dfd (maxillary) and Scr (labial) (although figure 5D,E argue against this conclusion). They presumably show that reporter gene regulation by Wnt signaling and Hh signaling is indirect and through localized transcriptional activation of the JAK/STAT signaling pathway ligand gene upd (however, the STAT reporter and the snail reporter are expressed in different cells (fig 4B) - so I'm not so convinced of this conclusion). The authors also find that the CA and PG primordia form at slightly different dorsal ventral positions and that DV positional information is controlled downstream of upd JAK/STAT signaling.

    Major comments:

    The paper is well written and makes for a nice story, but the corresponding data are not supportive of most of the conclusions drawn by the authors.

    First, in the Dfd Scr double mutant, both the CA and PG expression of the snail-rg-GFP reporter is still there - admittedly, the gland cells look abnormal at late stages, but this reporter that is supposed to function as a proxy for gland induction is still expressed. That either means that expression of sna-rg-GFP is not a proxy or that the glands are still being specified in the absence of the Hox genes that are proposed to specify these organs. The reporter should not be expressed if these Hox genes are what specify these endocrine organs. This finding might explain why mutating the Hox consensus binding sites had no effect on expression of the smaller snail reporters.

    The finding that the CA and PGs form in slightly different DV positions from each other and slightly different DV positions from the trachea (based on the vvl1+2 mCherry reporter staining combined with that of the sna-rg-GFP reporter staining in Figure 5A, where staining does not overlap except where the CA cells have started to migrate over the vvl1+2 mCherry expressing cells) argues pretty strongly against the CA and PG being homologous to each other or absolutely homologous to the trachea primordia. Likewise, the STAT10X-GFP staining does not overlap with the sna-rg-mCherry staining (I see red cells and I see green cells - there are no yellow cells). If activation of snail is through Upd activation of STAT signaling, we should see that the snail reporter expression is within the domain of STAT10X-GFP expression. This would be consistent with observing a loss of upd mRNA in the maxillary and labial segments with loss of Dfd and Scr, but not seeing a loss of the sna-rg-GFP reporter. This would also argue against the proposed homology between the glands and the trachea. Indeed, the situation with these endocrine glands and the trachea is completely unlike the situation with the wing and haltere, wherein both structures arise from the same DV position in adjacent segments, or with legs 1, 2 and 3, which arise from the same DV position in adjacent segments. This paper provides a strong basis for arguing that the CA, PG and trachea are not homologous structures and that the CA and PG are induced independently of Jak/Stat signaling, whereas trachea require this signaling pathway.

    Minor comments:

    Page 3: tracheal is misspelled in the first paragraph, line 3.

    Page 5, end of first sentence in first full paragraph: "lethal" should be changed to "non-viable". I think the authors mean that homozygous embryos die, not that they cause the death of other life forms.

    Significance

    Nature of significance of advance:

    I think the significant finding is that the CA, PG, and trachea are not homologous structures. But that is not what the authors are concluding. The only findings consistent with the data provided are that Wg signaling represses expression of the snail reporter and Hh signaling activates its expression (Figures 1 - 3). Most of the other conclusions do not seem to be sufficiently supported by the data.

    Context of the work:

    These authors have published that the CA and PG are structures specified in homologous positions to the trachea. It has already been published that CA, PG and trachea primordia express the Vvl transcription factor - although I did not go back to see how that was determined. It has already been published that ectopic expression of specific Hox genes can transform the gland primordia into trachea and vice versa (these experiments may also warrant a closer look). So, idea that CA, PG and TR arose from divergent evolution of a segmentally repeated ancient structure has been proposed.

    Best target audience:

    With the findings that are consistent with the story line (figures 1 - 3), Drosophila embryologists working on the formation of these glands would be interested.

    My field of expertise:

    Drosophila development.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript from Garcia-Ferres et al., describes studies aimed at understanding the specification of precursor cells to two ring gland organs, the corpora allata (CA) and the prothoracic gland (PG). These two glands are specified in the lateral ectoderm at positions that match the tracheae in more posterior segments, and like tracheal cells, the CA/PG primordia express ventral veinless (Vvl). The specification of CA/PG primordia requires the Hox genes Deformed (Dfd) and Sex combs reduced (Scr), while the trachea dependent on BX-C complex genes. CA/PG precursor cells also differ from tracheal precursors in that the former requires the expression of "mesodermal" gene snail (sna), while tracheal cells require trachealess. The sna gene has a complex regulatory region, with distinct enhancers for expression in the mesoderm and in the CA/PG primordia. Garcia-Ferres et al have used a reporter carrying the sna CA/PG enhancer, rg, to studying the mechanisms of CA/PG specification.

    In the first set of experiments, using a Crispr deletion the authors showed that the rg enhancer in the endogenous Sna gene is essential for CA/PG specification. The next used the sna-rg to examine the effects of mutations in (potentially) upstream signaling pathways on CA/PG specification. The CA/PG primordia are located outside of the wingless (wg) (parasegmental) expression domain; however, the authors found that two sets of lateral ectodermal cells express sna-rg in wg mutants. Conversely ectopic expression of wg or armS10 in the maxillia and labium eliminates sna-rg expression indicating that wg is a negative regulator of sna. Unlike wg, mutations in hedgehog (hh) and engrailed (en) eliminate sna-rg expression, indicate that both of these genes are required to promote CA/PG specification. The failure to express sna-rg is due at least in part to repressive activities of Cubitus interruptus (Ci) as sna-rg expression is restored in an en, ci double mutant. Sna-rg expression also depends upon JAK/STAT signaling and is in a deficiency that deletes the three upd genes. A upd dependent reporter is activated in CA/PG primordia in stage10/11 embryo, while upd itself appears to be express in the same cells. Consistent with the idea that the JAK/STAT maybe controlling sna-rg expression in response to wg and hh signaling, the authors finding that expression of upd expands in wg mutants, while it disappears in hh mutants. upd expression appears to be controlled by inputs not only from wg and hh, but also by Dfd and Scr as Dfd Scr mutant embryos lack upd expression. In contrast, the pattern of wg and en expression in the Dfd Scr mutant is normal.

    To confirm that JAK/STAT signaling is required for activating the sna-rg reporter, the authors undertook a functional dissection of the rg enhancers. When they split a truncated rg enhancer, rgR2, into two fragments, A1 and A2, there was no expression of the reporter. As fragment A2 has three STAT binding sites, the authors tested whether expression could be rescued by adding a generic STAT site to A1-it could be. On the other hand, when they mutated the three STAT sites in A2, they found that only PG expression is lost. This result suggests that there is a Upd responsive element in A1+A2; however, activation is likely indirect since the activity of this element is tissue specific. They also putative Hox-Exd-Hth bindings sites in the rgR2 enhancer; however. these sites do not appear to be required for reporter expression.

    This paper adds to our understanding of how the CA and PG primordia are specified and highlights important similarities with the specification of the tracheal primordia. There are some questions that should be addressed.

    Specific Comments:

    There are two phases to sna expression in CA/PG primordia. In the first phase (stage 11) hh signaling to neighboring cells prevents of the Ci repressor protein which would in turn activate upd expression in these cells. However, wg expression anterior in anterior cells blocks Upd expression so that it is turned on only a single set of cells posterior to the hh expressing cells. Upd in turn activates the sna via the STAT binding sites in the rg enhancer. upd expression also depends on the Hox genes Dfd and Scr, and when they are mutant upd is not expressed and sna is not turned on. Upd expression is only transient, and so after it disappears a maintenance mechanism ensures that that sna is expressed until at least stage 16. The authors don't really address the maintenance mechanism so it isn't clear what elements or factors are needed to keep the rg enhancer active after upd expression disappears.

    • a) In Fig. 1, G-J the authors show sna-rg reporter expression in WT and in their sna-rg deletion mutant. The images in G and I are of stage 12 embryos. At this point there is clearly little if any rg reporter expression in the deletion mutant, while high levels are observed in the WT. However, this is after sna expression is supposed to be activated by Upd, which is in stage 11. Moreover, upd expression is turned off in stage 12. (Assuming I didn't miss something) It is not clear to me from either this figure or from the text whether the initial pattern of expression of the sna-rg reporter in stage 11 embryos is WT and then disappears at stage 12, or whether it is always defective. In trying to understand the activation process, I think it would be important to know for sure whether rg enhancer activity during the initiation phase in stage 11 is WT or not. The expectation-at least from what is written in the manuscript-is that the initial expression of the sna-rg reporter will be the same as WT in the sna-rg deletion mutant.
    • b) The authors argue that the rg deletion removes the only region driving sna expression in CA/PG. I'm not convinced that necessity necessarily implies sufficiency with respect to the requirements for rescue. While the sna-rg reporter is expressed in a pattern that seems to mimic the endogenous gene, do we know that a rg-sna transgene would fully rescue the rg deletion mutant?
    • c) If sna protein is not required for initiating (see a) sna expression, is it required for maintaining sna expression?
    • d) The authors show that there is an expansion in the number of sna-rg reporter expressing cells along the AP axis when upd is ectopically expressed using a sal-Gal4 driver. Though not mentioned in the text at this juncture, sal is expressed in the PG primordia, while seven-up (svp) is expressed in the CA primordia. I assume that the upd induced expansion is only observed for the PG primorida (LB) and not the CA primordia (Mx)-at least this is what the figure looks like.
      It looks to me also like the vvl domain is expanding as well. This information should be clarified. How about svp driven upd-assuming there is a svp-Gal4 driver-does it cause an expansion of Ca but not PG.
    • e) The authors note a difference between CA and PG in the requirement for STAT binding sites in the enhancers. Is that related to the fact that svp is expressed in CA and sal is expressed in PG? Would driving svp expression using the sal-Gal4 driver maintain sna-rg expression
    • f) Do svp or sal have a role in initiating sna expression when upd is present or maintaining sna expression after upd disappears? Presumably there is already published data that would answer these questions.

    Significance

    This manuscript will be of interest to scientists seeking to understand fate specification-and how the same pathways/interactions can generate completely different organs-- in this case ring glands as opposed to trachea. The paper does however leave many questions unanswered. This is to be expected given that not all of the key players have been identified, and for those that have, the functions are not fully understood.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study, the authors focus on understanding the regulation of development of corpora allata (CA) and prothoracic gland (PG) in Drosophila. Through a series of well designed experiments they convincingly show that interactions between Scr, Dfd, and JAK/STAT pathways induce localized expression of unpaired (upd) gene, which in turn controls snail (a key regulator of early gland development). Overall, this is an excellent study that extends authors' previous work on evolution and divergence of glands (in head) and respiratory organs (in trunk) from common, metamerically repeated primordia. Specifically, this work provides new information regarding CA and PG specification and as such will be of interest to a broad range of developmental biologists. Text is very well written, and figures are well organized and convey results in a clear way. I have several small comments (detailed below), but other than that this manuscript is ready for publication.

    Aleksandar Popadić

    Main suggestion:

    As the formation of the ring gland is an exclusively dipteran trait, it would be helpful to insert a paragraph in discussion explaining the relevance of new insights to other, more basal insects. In a sense, studies of a ring gland present a tail end of evolution, what do results obtain tell us about the regulation of the PG and CA development in other insects?

    Additional comments:

    1. Introduction, pg 3, a paragraph starting with "In comparison to the extensive knowledge we have of ..." - consider omitting or greatly shortening, this text breaks a flow as it is focused on tracheal development. I understand the authors' logic, but this information distracts from the main focus on CA and PG.
    2. Beginning of discussion, pg 11:
      • change 2nd sentence to: " Our results indicate that the HH and the Wnt pathways act indirectly to negatively regulate the spatial activation ..."
      • the following sentence, starting with "Engrailed activation off hh transcription ...." is way too long and hard to follow, consider breaking into two sentences.
    3. In Fig 4B, mx and lb segments should be labeled so this panel is consistent with labeling in 4A.
    4. In Fig 6, reduce a font size for labels on right-hand side (A1, A2, A1+A2 proximal, etc), so that they are visualy distinct from panel labels on left-hand side (A, B, C,..).

    Significance

    While this is a strictly Drosophila study, it does provide a significant new insight into development of corpora allot and prothoracic gland (which are critical organs for insect growth and development). As such this work will be of interest to a wide audience of biologists (please see my comments below for details).

  9. Version published to 10.1101/2022.07.27.501760v1 on bioRxiv