PONE-D-22-19798Imp is required for timely exit from quiescence in Drosophila type II neuroblastsPLOS ONE

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #3: No

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5. Review Comments to the Author

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Reviewer #1: Balancing the proliferation and quiescence is essential for the normal functions of stem cells. In this manuscript, Munroe et al investigated the role of Insulin-like growth factor II mRNA- binding protein (Imp) in proliferation and quiescence of Drosophila type II neuroblasts (TIINBs). They found Imp displayed a high-to-low temporal gradient in TIINBs and the Imp gradient can be effectively shifted to different times in development by ImpRNAi and Imp overexpression. They further suggested that pnt-Gal4 UAS-GFP could be used as a marker to identify proliferating type II neuroblasts. By quantifying the number of proliferating TIINBs, they found that Imp knockdown delayed exit from quiescence, whereas overexpression of Imp did not induce precocious exit from quiescence in TIINBs. Moreover, comparable levels of Imp were observed in quiescent and proliferating TIINBs while its antagonist protein Syp expressed at a significantly lower level.

Overall, this paper suggests a necessary but not sufficient role of Imp in mediating timely exit from quiescence in TIINBs. Experiments were carefully performed, the data presented are of high quality, and interpretations of the results are comparatively justified. I only have several minor suggestions for the authors to consider:

1. For all the Imp/syp immunostaining data, the numbers of TIINBs for statistical analysis were not provided in Methods or Figure legends.

2. Line 133,“Syp levels in quiescent TIINBs were slightly higher than Syp levels in proliferative TIINBs (Figure 4F), showing a correlation between higher Syp levels and neuroblast quiescence.”Whereas the figure legend of Fig4E claimed that “Imp and Syp levels are the same in quiescent and proliferating type II neuroblasts.”Based on the data, the legend needs to be fixed.

Reviewer #2: Munroe et al. report a role for Imp in exit from quiescence of type II neuroblasts in Drosophila. They examine and quantify Imp protein levels in Type II neuroblasts at different larval stages, something that had been missing (although much needed) in the field. They also test whether Imp regulates exit from quiescence based on high to low Imp expression and based on the known role of Imp in regulating neuroblast" decommissioning". While the data presented in this manuscript is solid and well presented, this reviewer has one point that may need to be further addressed:

Authors report that Pnt-Gal4 is only active in proliferating type II neuroblasts. By this logic, using Pnt-Gal4 should only knockdown Imp in proliferating neuroblasts. Is knockdown of Imp delaying the exit from quiescence or suppressing proliferation? or both? Is there data to suggest that Pnt-GAL4/UAS transgene expression perdures in quiescent type II neuroblasts?

Typo:

Line 66, It is the non-mushroom body neuroblasts that require low Notch signaling to be driven out of quiescence. MB neuroblasts do not enter and exit quiescence. Authors should also check the reference for this statement.

Reviewer #3: 

Overall evaluation and significance

Munroe et al., investigate the role of Imp, an important RNA-binding protein (RBP) involved in Drosophila neurogenesis, in neuroblast (NB) quiescence exit. Re-entry of NBs into cell cycle has been previously shown to be regulated by nutrition-dependent glial niche and the Notch signalling pathway, while the role of Imp in this process has not been explored. Here, the authors characterised the kinetics of Type II NB quiescence exit upon modulation of the Imp expression gradient. They observed that reducing the level of Imp, which accelerates Imp protein depletion in Type II NBs, delays exit from quiescence, while the overexpression of Imp did not lead to a noticeable effect. Taken together, the authors suggest a previously unknown role of Imp in promoting re-entry of quiescent NBs into cell cycle. The manuscript is high quality and of value and interest. It should be accepted for publication, once the authors have addressed our concerns, either by revising the text or if they would prefer, providing additional data.

Major comments

The authors first present a detailed comparison of Imp expression kinetics in Imp RNAi and Imp OE experiments. The study then aims to demonstrate that modulating Imp affects Type II NB quiescence exit. However, their choice of PntP1-GAL4 driver needs to be explained / justified, in light of the main conclusion of this work.

1. Throughout the manuscript, the authors utilise PntP1-GAL4 line to identify proliferating NBs and to drive UAS transgenes. However, in Figure 3, the authors describe that PntP1-GAL4 only becomes active in proliferating NBs and not in quiescent cells. Therefore, the Imp RNAi should be inactive in quiescent NBs, which does not support the authors' conclusion that reduced level of Imp leads to delayed quiescence exit. The authors should include more information that justifies their choice of PntP1-GAL4 over more conventionally used wor-GAL4, ase-GAL80.

2. Although the knock down of Imp in Figure 2C-D seems convincing on the population-wide scale, a key piece of data is missing that GFP-negative quiescent NBs are under PntP1-GAL4/Imp-RNAi control (Figure 4A). Can the authors provide an explanation or data that Imp levels are reduced in quiescent NBs in Imp RNAi conditions compared to the wild-type at 24-48 ALH, and also discuss why Imp RNAi is active despite the lack of GFP expression? Is it possible that other cell types (e.g. glia) might be affected by the driver?

3. Are all PntP1>GFP-positive NBs CycE-positive? The population of GFP-positive but CycE-negative cells may suggest that quiescence exit is a multi-step process where Imp plays a role in the initial step.

Minor comments

1. Figure 2A-B: Representative wild-type series of Type II NB images should be provided to match the quantification shown in Figure 2C.

2. Line 213: Please provide replicate information in all figure legends. In particular, what does the symbol represent in Figure 4A and 4C? It should be clear how many brains were quantified per biological replicate.

3. Figure 4E-F: Syed et al., 2017 (doi.org/10.7554/eLife.26287) have shown lack of Syp expression in Type II NBs at stages before 48 ALH and upon disruption of ecysone signalling. Can the authors explain the discrepancy?

4. Line 197: Immunofluorecence quantification method should be explained in more detail. Was the Raw Integrated Density calculated over 3D volume of the NBs or on select 2D planes? Why were nuclear areas and intensities removed from the analysis? Were any background subtraction method used?

5. In Figure 2C, how were the fluorescence intensity signals normalised between biological replicates and different genotypes?

6. Line 168: The role of Syp in entering the embryo-to-larval neuroblast quiescence is not yet established. The text should be revised. Perhaps the authors meant 'decomissioning and cell cycle exit'?

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Imp is required for timely exit from quiescence in Drosophila type II neuroblasts

PONE-D-22-19798R1

Dear Dr. Doe,

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Hongyan Wang, Ph.D.

Academic Editor

PLOS ONE