Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts

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   eLife

   Jun 2, 2020

   ###This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on May 29, 2020, follows.

   Summary

   In their manuscript entitled "Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts," Kizhedathu and colleagues investigate the developmental regulation of Chk1 activation in larval tracheoblasts of the Dorsal Trunk segment tr2. They find that four Wnt ligands are required to achieve a level of active Chk1 (pChk1) needed to maintain tracheoblasts in G2 arrest. This regulation is achieved by autocrine signaling in which the canonical Wnt pathway is activated by 4 Wnt ligands expressed in the trachea at high levles. Wnt signaling is required for transcription of Chk1. None of the 4 highly expressed Wnts are dispensable. Release from G2 is …

   ###This manuscript is in revision at eLife

   The decision letter after peer review, sent to the authors on May 29, 2020, follows.

   Summary

   In their manuscript entitled "Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in Drosophila tracheoblasts," Kizhedathu and colleagues investigate the developmental regulation of Chk1 activation in larval tracheoblasts of the Dorsal Trunk segment tr2. They find that four Wnt ligands are required to achieve a level of active Chk1 (pChk1) needed to maintain tracheoblasts in G2 arrest. This regulation is achieved by autocrine signaling in which the canonical Wnt pathway is activated by 4 Wnt ligands expressed in the trachea at high levles. Wnt signaling is required for transcription of Chk1. None of the 4 highly expressed Wnts are dispensable. Release from G2 is required for activation of the dpp/tkv/pMad pathway that spurs continuing cell divisions.

   This is a Research Advance manuscript following up on work reported in a prior publication entitled "Negative regulation of G2-M by ATR/Chk1 (Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila." The current work makes several important contributions. Having initially identified a G2 cell cycle arrest dependent on Chk1 and Atr, but not upon DNA damage, the authors now show that the cell cycle arrest is maintained through a canonical Wnt signal that mediates transcription of chk1 mRNA. The authors identify 4 Wnt ligands expressed in the tracheoblasts and show that all 4 are required for G2 maintenance via chk1 transcription, although individually dispensable for fz3 transcription. The authors also show that the dpp pathway signal required to drive tracheoblast cell divisions cannot operate during G2 cell cycle arrest. Lastly, authors note that Wnt5 is thought to signal through a nonconical pathway, but in this instance contributes to canonical signaling.

   Essential Revisions

   1. Additional controls to confirm the requirement for 4 Wnts:

   Is there any chance that there are off target effects from the RNAi that may cause this? Do the Wnt ligands impact each others' expression? Wnt ligand KD followed by qPCR analysis of the Wnt ligands could be useful. This issue is important to discuss, and test.

   Test of second independent RNAi line where classic loss of function alleles are not available.

   Test RNAi of the non-expressed Wnt ligands and addition of a supplemental table documenting the screen (Wnt and other pathways) with RNAi line numbers, drivers, temperatures and results.

   1. Clarify ability of overexpressed Wnts to rescue, and determine the requirement for nonconical Wnt pathway:

   Address whether derailed or doughnut are required in tracheoblasts.

   Authors Wnt threshold model hinges on ability of overexpressed Wnt to compensate for loss of one Wnt ligand. However, this was only reported for one loss of function case (Wnt 6 RNAi), and only with one overexpressed Wnt (Wnt5). Authors should test ability of other Wnts to rescue, and should also address whether a conventional Wnt can substitute, when overexpressed, for Wnt5.

   1. Address inhibition of mitoses during L2:

   The authors show that downregulating the Wnt pathway in L2 stage does lead to the reduction of Chk1 mRNA (figures 3A and 2, respectively), but that this does not result in tracheoblast mitoses. This indicates that in L2, in contrast to L3, there must be some additional control which is lifted after L2/L3 metamorphosis. The authors should discuss this issue and present possible explanations. If they have any more relevant data, this should also be presented.

   1. Move Figure 5S into results:

   P. 14 lines 358-369. Minimally, it is not appropriate to introduce data in the discussion that is not discussed in the main results section. Moreover, the experiments and results are super interesting. I could be wrong because I am not an expert in the cell cycle, but I don't think the idea of a G2 arrested cell continuing to grow physically because it still expresses cell cycle promoting genes while in G2 arrest is really out there in the literature on cell and organ size control (although I think mammalian oocytes arrest in G2 and they get really big, but they also may have bridges to nurse-like cells to supplement growth). As such, I would strongly encourage the authors to make Fig 5-Figure supplement 1 part of the main figures and discuss the basic findings in the results section. Then it could be revisited in the Discussion section to put the result in the bigger picture context.

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2. 

   Version published to 10.1101/2020.06.01.127266 on bioRxiv

   Jun 1, 2020