Krüppel Regulates Cell Cycle Exit and Limits Adult Neurogenesis of Mushroom Body Neural Progenitors in Drosophila

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors investigated factors required for neural progenitors to exit the cell cycle before the adult stage. They first show that Kr is turned on in pupal stage MBNBs, and depletion of Kr from pupal stage NBs leads to retention of MBNBs into the adult stage. Then they demonstrate that these retained NBs maintain the expression of Imp, and co-depletion of Imp abolishes the extended neurogenesis. Further, they show that co-depletion of kr-h1 significantly reduces the retained MBNBs caused by loss of kr, suggesting antagonistic genetic interactions between these two. In addition, they demonstrate that over-expressing Kr-h1 leads to the striking phenotype of tumor-like neuroblast overgrowth in adult brains.

Strengths:

(1) The authors leveraged well-controlled, powerful genetic tools (including temporal control of RNAi knockdown using the Gal80ts system), and provided strong evidence that Kr expression in pupal stage MBNBs is required to repress Imp and promote the end of neurogenesis. Similarly, the experimental result of co-depleting Kr-h1 and Kr, and the striking phenotype upon Kr-h1 mis-expression, support the antagonistic roles played by Kr-h1 and Kr in this process.

(2) The sample sizes, quantification methods, and p-values are well documented for all experiments. In most parts, the data presented strongly support their conclusions.

(3) Identification of two transcription factors with opposite roles in controlling cell cycle exit, and their possible interactions with the Imp/Syp axis, is highly significant for the study on how the proliferation of neural progenitors is regulated and limited before the adult stage.

Weaknesses:

(1) The nature of the KrIf-1 allele is not clear. It is mentioned that this allele leads to misexpression of Kr in various tissues. However, it is not clear if Kr is mis-expressed or lost in MBNBs in the KrIf-1 mutant. If Kr is mis-expressed in MBNBs in the KrIf-1 mutant, then it would be difficult to explain why both loss of Kr and mis-expression of Kr in MBNBs lead to the same NB retention phenotype. The authors should examine Kr expression in MBNBs in the KrIf-1 mutant.

(2) Some parts of the regulations and interactions between Kr, Kr-h1, Imp, Syp, and E93 are not well-defined. For example, the data suggest that Kr is turned on in the pupal stage MBNBs, and is required to end neurogenesis through repressing Imp and Kr-h1. To further support this conclusion, the authors can examine if Kr-h1 expression is up-regulated in kr-RNAi. The authors suggested that Kr-h1 may act upstream or in parallel to Imp/Syp, but also suggested that Kr-h1 may repress E93. The expression of Imp, Syp, and E93 can be examined in brains with Kr-h1 mis-expression to determine where Kr-h1 acts. If Imp expression is elevated when Kr-h1 is mis-expressed, then Kr-h1 may act upstream of Imp. If Imp/Syp expression does not change, then Kr-h1 may act on the E93 level.

Reviewer #2 (Public review):

Summary:

In this paper, the authors study the role of Kruppel in regulating the survival of mushroom body neuroblasts. They first confirm that adult wild-type brains have no proliferation and report that Kruppel mutants and Kruppel RNAi in neuroblasts show a few proliferative clones; they show that these proliferative clones are localized in the mushroom body. They then show that Kruppel is expressed mostly during pupal stages and acts by downregulating the expression of Imp, which has been shown to positively regulate neuroblast proliferation and survival. Expectedly, this also affects neuronal diversity in the mushroom body, which is enriched in gamma neurons that are born during the Imp-expression window. Finally, they show that Kr acts antagonistically to Kr-h1, which is expressed predominantly in larval stages.

Strengths:

The main strength of this paper is that it identified a novel regulator of Imp expression in the mushroom body neuroblasts. Imp is a conserved RNA-binding protein that has been shown to regulate neural stem cell proliferation and survival in different animals.

Weaknesses:

(1) The main weakness of the paper is that the authors want to test adult neurogenesis in a system where no adult neurogenesis exists. To achieve this, they force neuroblasts to survive in adulthood by altering the genetic program that prevents them from terminating their proliferation. If this was reminiscing about "adult neurogenesis", the authors should at least show how adult neurons incorporate into the mushroom body even if they are born much later. On the contrary, this more likely resembles a tumorigenic phenotype, when stem cells divide way past their appropriate timing.

(2) Moreover, the figures are, in many cases, hard to understand, and the interpretation of the figures doesn't always match what one sees. The manuscript would benefit from better figures; for example, in Figure 2C, Miranda expression in insc>GFP in Kr-IF-1 is not visible.

(3) The authors describe a targeted genetic screen, but they don't describe which genes were tested, how they were chosen, and why Kruppel was finally selected.

(4) The authors argue that Kr does not behave as a typical tTF in MBNBs. However, they show no expression in the embryo, limited expression in the larva and early pupa, and a peak around P24-P48. This sounds like a temporally regulated expression of a transcription factor. Importantly, they mentioned that they tested their observations against different datasets (FlyAtlas2, modENCODE, and MBNB-lineage-specific RNA-seq data), but they don't provide the data.

(5) Finally, the contribution of Kr to the neuronal composition of the mushroom body is expected (since Imp is known to regulate neuronal diversity in the MB), but the presentation in the paper is very incomplete.

Unfortunately, based on the above, I am not convinced that the authors can use this framework to infer anything about adult neurogenesis. Therefore, the impact of this work is limited to the role of Kruppel in regulating Imp, which has already been shown to regulate the extent of neuroblast division, as well as the neuronal types that are born at different temporal windows.

Reviewer #3 (Public review):

Summary:

Drosophila neuroblasts (NBs) serve as a well-established model for studying neural stem cell biology. The intrinsic genetic programs that control their mitotic potential throughout development have been described in remarkable detail, highlighting a series of sequentially expressed transcription factors and RNA-binding proteins that together constitute the temporal patterning system.

However, the mechanisms that limit the number of NB divisions remain largely unknown in a specific subset of NBs known as mushroom body neuroblasts (MB NBs). Unlike other NBs, which terminate proliferation before or shortly after the onset of metamorphosis, MB NBs continue dividing until the end of metamorphosis, ceasing only just before adulthood.
In this study, the authors identify the transcription factor Krüppel (Kr), a member of the conserved Krüppel-like family, as temporally regulated in MB NBs. They demonstrate that Kr knockdown during pupal stages maintains expression of the RNA-binding protein Imp and results in prolonged MB NB proliferation into adulthood. Their data suggest that Kr contributes to the timely silencing of Imp during metamorphosis. The authors further identify Kr-h1, a related transcription factor, as a potential antagonist. While Kr-h1 appears dispensable for the timely termination of MB NBs under normal conditions, its overexpression leads to their continued proliferation and tumor-like expansion in adults.

This work provides the first evidence for a transcription factor-driven temporal regulation mechanism in MB NBs, offering new insight into the control of neural stem cell self-renewal. Given the evolutionary conservation of Krüppel-like factors, this study may have broader implications for the neural stem cell field.

Strengths:

(1) The study possibly identifies a new series of temporal transcription factors that are specific for mushroom body neuroblasts.

(2) The mechanism could be conserved in vertebrates.

Weaknesses:

Some proposed regulatory interactions, particularly between Kr, Kr-h1, and other temporal factors like Imp, Chinmo, and E93, have not been thoroughly investigated, which weakens the support for the proposed model. Additional experimental validation is needed to confirm these relationships and strengthen the mechanistic framework.

Author response:

We thank the editors and reviewers for their thoughtful and constructive evaluation of our manuscript, “Krüppel Regulates Cell Cycle Exit and Limits Adult Neurogenesis of Mushroom Body Neural Progenitors in Drosophila.” We are pleased that all reviewers recognised the novelty and significance of identifying Krüppel (Kr) as a key transcription factor promoting timely termination of mushroom body neuroblast (MBNB) proliferation, and the potential antagonistic function of Kr-h1.

We appreciate the helpful suggestions aimed at improving the mechanistic clarity and presentation of our findings. Below, we outline how we plan to address the major points raised in the full revision.

(1) Characterisation of the KrIf-1 allele and Kr expression

We agree that clarifying the nature of the KrIf-1 allele is important. In response to this concern, we will examine Kr expression in KrIf-1 mutant larval, pupal, and adult brains using immunostaining and available reporter lines. These experiments will help determine whether the observed neuroblast retention phenotype correlates with altered Kr expression in MBNBs.

(2) Regulatory relationships between Kr, Kr-h1, Imp, Syp, Chinmo, and E93

We are currently performing additional experiments to clarify the interactions among these temporal factors. For instance, we are testing whether Kr-h1 overexpression alters the expression of Imp, Syp, and E93. We have obtained a published E93 antibody from Dr Chris Doe (Syed et al., 2017) and will include E93 expression analysis in our revised manuscript.

While Chinmo is of interest, its expression is well established to be regulated downstream of Imp/Syp via mRNA stability (Liu et al., 2015; Ren et al., 2017). Given that we currently lack reliable tools to assess Chinmo levels, we will focus primarily on Imp, Syp, and E93 as readouts for Kr/Kr-h1 function. If we succeed in obtaining Chinmo antibodies or reporter lines in time, we will include corresponding data.

(3) Expression of Kr-h1 in MBNBs

We fully agree that direct evidence for Kr-h1 expression in MBNBs is important. To address this, we have obtained the Kr-h1::GFP BAC transgenic line (BDSC #96786) and are currently using it to assess Kr-h1 expression in MBNBs. We also tested an anti–Kr-h1 antibody previously reported by Kang et al. (2017), developed in the context of fat body studies, but it did not yield clear signals in larval MBNBs. However, previous work by Shi et al. (2007) clearly demonstrated Kr-h1 expression in the developing MB, including MBNBs, using a custom antibody developed by their lab. We also contacted the Lee lab to request this antibody, but unfortunately, it is no longer available. We will include the results obtained using the GFP BAC line in the revised manuscript and, if needed, pursue RNA in situ hybridisation to further validate Kr-h1 expression in MBNBs.

(4) Temporal Kr knockdown and MARCM analysis

We appreciate the suggestion to validate our RNAi-based temporal knockdown results using MARCM. We plan to perform MBNB-specific MARCM analysis following the strategy described by Rossi et al. (2020). However, this approach requires additional time due to the logistics of acquiring the necessary fly stocks, generating appropriate genetic combinations, and conducting clonal analyses. While we will make every effort to include these data, we note that RNAi-based knockdown offers the advantage of temporal reversibility and has been essential for assessing stage-specific requirements in our current study.

(5) Details of the targeted genetic screen

Kr was initially identified as part of a broader, ongoing effort to screen for candidate transcription factors and cell cycle regulators involved in neuroblast cell cycle exit and/or quiescence. As this screen is still preliminary and incomplete, we prefer not to include the full dataset at this stage. Instead, we will revise the manuscript to clarify that Kr was prioritised for further investigation based on the striking MBNB-specific phenotype observed upon RNAi-mediated knockdown and in the KrIf-1 mutant, rather than through a completed screening process.

(6) Clarifying the model (Figure 6D) and interactions

We will revise the proposed model to distinguish between experimentally supported interactions and speculative ones. As noted above, we will primarily focus on the Imp/Syp and E93 axis in relation to Kr and Kr-h1 activity. Chinmo will be omitted from the model unless further data become available to support its inclusion.

(7) Clarifications on figures and data presentation

We appreciate the feedback on figure clarity. We will revise figures such as 1B, 2C, and 3A to improve legibility and presentation. We will also correct typographical errors and figure references, and clarify the activity patterns of the GAL4 drivers. Specifically, while UASmCD8::GFP expression driven by OK107-GAL4 is markedly weaker in MBNBs than in their neuronal progeny (as seen, for example, in Figure S3C), the driver remains active and functionally relevant in MBNBs. We believe the weak expression in MBNBs likely explains the absence of a NB retention phenotype in OK107>KrIR adult brains (see main text, Lines 374–376). As suggested by the reviewer, we will clarify this point earlier in the manuscript and can include additional data showing OK107>GFP expression patterns in pupal MB lineages as supplementary material.

(8) Analysis of public datasets

We will include results from our analysis of publicly available datasets such as FlyAtlas2, modENCODE, and a time-course RNA-seq dataset specific to MBNBs (Liu et al., 2015). While the spatial resolution of FlyAtlas2 and modENCODE is limited, the MBNB dataset provides valuable temporal information up to 36 h after puparium formation (APF). From this dataset, we observe that Kr expression remains consistently low throughout development, with only a modest increase at 84 h ALH (mean TPM ~11) and 36 h APF (~7), suggesting it does not undergo strong transcriptional regulation in MBNBs. In contrast, Kr-h1 is highly expressed during early larval stages (24–84 h ALH; mean TPM ~55–60) and shows a marked suppression by 36 h APF (mean TPM ~2), consistent with its proposed role in promoting MBNB proliferation. Importantly, Eip93F (E93) exhibits a reciprocal pattern to Kr-h1—with minimal expression until 84 h ALH (mean TPM ~24), followed by a substantial induction at 36 h APF (mean TPM ~104), aligning with its known role in triggering neuroblast termination. These temporal expression dynamics support our model that Kr-h1 and E93 function in opposition during the transition from proliferative to terminating neuroblast states. We will summarise these findings in the revised manuscript, along with appropriate discussion of dataset limitations.

We hope this provisional response conveys our strong commitment to thoroughly addressing the reviewers’ concerns and improving the manuscript. We are currently carrying out additional experiments and will submit a revised version with new data and enhanced clarity in due course.

References:

Kang et al., 2017. Sci Rep. 7(1):16369. doi: 10.1038/s41598-017-16638-1.

Shi et al., 2007. Dev Neurobiol. 67(11):1614–1626. doi: 10.1002/dneu.20537.

Rossi et al., 2020. eLife. 9:e58880. doi: 10.7554/eLife.58880.

Liu et al., 2015. Science. 350(6258):317–320. doi: 10.1126/science.aad1886.

Ren et al., 2017. Curr Biol. 27(9):1303–1313. doi: 10.1016/j.cub.2017.03.018. Syed et al., 2017. eLife. 6:e26287. doi: 10.7554/eLife.26287.