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 cell cycle before the adult stage. They first show that Kr is expressed 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. In the revised manuscript, they provide evidence suggesting that Kr does not regulate Kr-h1 expression, but rather Kr and Kr-h1 may regulate the late temporal gene E93 expression in parallel.

Strengths:

(1) The authors leveraged well-controlled powerful genetic tools (including temporal control of RNAi knock down using the Gal80ts system), and provided strong evidence that Kr is required for the Imp to Syp transition and to promote the end of neurogenesis through E93 in mushroom body neuroblasts. 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) In the revised manuscript, Kr immunostaining was done in KrIf-1 mutant brains using tyramide signal amplification (TSA) to enhance the weak endogenous Kr signal, and they conclude that Kr is ectopically detected in MBNBs and their progeny. However, a wildtype control with TSA amplified Kr immunostaining was not provided, so it is not known whether the elevated signal comes from TSA amplification or the mutant condition. If Kr is indeed mis-expressed in MBNBs in the KrIf-1 mutant, the authors should provide a hypothesis to explain why both loss of Kr and mis-expression of Kr in MBNBs lead to the same NB retention phenotype.

Reviewer #2 (Public review):

In this paper, the authors study the role of Krüppel in regulating the termination and survival of mushroom body neuroblasts. They first confirm that adult wild-type brains have no proliferation and report that Kr mutants and Kr RNAi specifically in neuroblasts prolong MBNB lifespan, enabling continued neurogenesis in the adult brain. They further show that Kr acts during pupal stages to regulate the elimination to the mushroom body neuroblasts, as its depletion leads to neuroblast retention, revealing a previously unrecognised postembryonic function of Kr distinct from its established role in embryonic neurogenesis. Mechanistically, this is achieved by the regulation of the Imp/Syp transition and E93 expression. Finally, they show that Kr acts antagonistically to Kr-h1, which is expressed predominantly in larval stages. Together, these results identify Kr as a mushroom body neuroblast regulator coordinating intrinsic and extrinsic signal to regulate neuroblast termination.

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.

Moreover, the authors provide a series of evidence that this regulator, Kruppel, acts by integrating extrinsic signals (ecdysone) with intrinsic timers (Imp-Syp transition) to regulate neuroblast termination.

Weaknesses:

No significant weaknesses were identified.

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.

Comment on revised version.

The authors have clarified the expression status of Kr in the KrIf-1 mutant, as well as the regulatory interactions among Kr, Kr-h1, and E93. My other concerns have also been satisfactorily addressed. I congratulate the authors on this excellent work and on discovering a novel mechanism that regulates temporal progression and the termination of division in mushroom body neuroblasts.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

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.

We thank Reviewer #1 for their thoughtful and constructive assessment of our manuscript. We are grateful that the reviewer recognised the strength of our genetic approaches, the significance of identifying Kr and Kr-h1 as opposing regulators of MBNB termination, and the value of linking these factors to the Imp/Syp axis. We have revised the manuscript substantially in response to the reviewer’s comments. In particular, we have further characterised Kr expression in the KrIf-1 mutant background, examined the relationship between Kr, Kr-h1 and E93, analysed Kr-h1 expression in MBNBs using a Kr-h1::GFP BAC reporter, revised the proposed model, improved figure presentation and corrected typographical errors throughout the manuscript.

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.

We agree that clarifying the nature of Kr expression in the KrIf-1 mutant background is important. We have therefore performed additional Kr immunostaining in KrIf-1 mutant brains using tyramide signal amplification (TSA) to enhance the weak endogenous Kr signal. These new data show that Kr is ectopically detected in MBNBs and their progeny in KrIf-1 mutant late pupal and adult brains (Fig. S3G, H), and quantification of TSA-enhanced Kr signals further supports Kr misexpression in MBNBs during late pupal stages (Fig. S3I). We have added these data to the revised manuscript. These results indicate that the KrIf-1 phenotype is associated with aberrant Kr expression in MBNBs rather than a simple loss of Kr expression. We agree with the reviewer that it may appear counterintuitive that both Kr depletion and Kr misexpression can lead to MBNB retention. We now discuss this point more explicitly. Our interpretation is that MBNB termination requires tight control of Kr activity during a narrow developmental window. Loss of Kr prevents the normal progression of the Imp-to-Syp transition and E93 activation, whereas ectopic or mistimed Kr expression in KrIf-1 mutants may also interfere with the same termination programme. Thus, both reduced Kr activity and inappropriate Kr expression can disrupt the precisely regulated Kr-dependent transition required for timely MBNB cell-cycle exit and elimination.

We have revised the Results and Discussion to make this interpretation clearer and to avoid describing KrIf-1 simply as equivalent to Kr loss of function.

(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.

We thank the reviewer for this helpful suggestion. We agree that the regulatory relationships between Kr, Kr-h1, Imp/Syp and E93 were not sufficiently defined in the original manuscript. In the revised manuscript, we have therefore clarified which relationships are supported by our data and which remain unresolved.

During revision, we placed particular emphasis on the relationship between Kr, Kr-h1 and E93, because our genetic interaction data showed that co-depletion of Kr-h1 partially suppresses MBNB retention caused by Kr depletion (Fig. 5A). This indicated that Kr and Kr-h1 functionally oppose each other during MBNB termination. We therefore asked whether this functional antagonism could be explained by a simple linear pathway in which Kr promotes termination by suppressing Kr-h1, which in turn antagonises E93.

We first tested whether Kr-h1 regulates E93 in the MBNB lineage. Kr-h1 depletion increased E93 expression in the MBNB lineage, whereas Kr-h1 overexpression strongly reduced E93 expression (Fig. S5B). These data support the conclusion that Kr-h1 antagonises E93 expression in this lineage, consistent with the established antagonistic relationship between Kr-h1 and E93 in other developmental contexts.

We next examined whether Kr regulates this Kr-h1–E93 axis by suppressing Kr-h1 expression. To address this, we used a functional Kr-h1::GFP BAC reporter. Kr-h1::GFP was readily detected in larval MBNBs but was strongly downregulated during pupal development, consistent with the MBNB-lineage-specific RNA-seq dataset of Liu et al. (2015). However, Kr depletion did not significantly increase Kr-h1::GFP levels in late pupal MBNBs (Fig. 5B and Fig. S5C). Moreover, despite the absence of detectable Kr-h1::GFP upregulation, Kr depletion strongly reduced E93 expression in the progeny region of the MBNB lineage, while the weak E93 signal detectable in MBNBs themselves was not substantially altered (Fig. 5B).

These results argue against a simple linear pathway in which Kr promotes MBNB termination by directly repressing Kr-h1 expression. Instead, they suggest that Kr supports E93 expression within the MBNB lineage through a mechanism that does not require detectable upregulation of Kr-h1. Thus, Kr and Kr-h1 functionally oppose each other during MBNB termination, but they are unlikely to act through a simple linear Kr–Kr-h1–E93 pathway. We have revised the model accordingly, proposing that Kr and Kr-h1 act through convergent or parallel mechanisms to regulate the MBNB termination programme.

Regarding the Imp/Syp axis, the original manuscript had already shown that Kr depletion causes persistent Imp expression in adult MBNB lineages and that co-depletion of Imp suppresses MBNB retention caused by Kr depletion. These data supported the conclusion that persistent Imp expression is required for MBNB retention following Kr depletion. In the revised manuscript, we strengthened this conclusion by analysing Imp and Syp expression during late pupal development, when MBNBs normally undergo cell-cycle exit and elimination. We found that Kr-depleted MBNBs maintain higher Imp expression and show reduced Syp expression compared with controls (Fig. 4A, B), supporting the idea that Kr promotes MBNB termination by facilitating the normal Imp-to-Syp transition.

We also considered whether Kr-h1 acts upstream of the Imp/Syp transition. Kr-h1 overexpression caused persistent Imp expression in proliferating adult NBs (Fig. 5E). However, this manipulation produced a severe tumour-like overgrowth phenotype and likely represents a strongly non-physiological state. We therefore do not conclude that Kr-h1 normally acts upstream of Imp/Syp during MBNB termination. Rather, our revised interpretation is that Kr promotes MBNB termination through the Imp-to-Syp transition and by supporting E93 expression, whereas Kr-h1 counteracts termination at least in part by repressing E93. How the Imp/Syp and E93-associated pathways converge during MBNB termination remains an important question for future work.

We have revised the Results, Discussion and Fig. 6D model accordingly, distinguishing experimentally supported relationships from more speculative connections.

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.

We thank Reviewer #2 for their critical assessment of our manuscript and for the specific suggestions regarding manuscript framing, figure clarity, public dataset analysis and textual accuracy. We have revised the manuscript extensively in response to these comments. In particular, we have clarified that our study does not aim to describe physiological adult neurogenesis in Drosophila, but rather investigates how normally terminating neural progenitors can retain or regain neurogenic potential when the developmental termination programme is disrupted. We have also revised the text to avoid overstatement about functional integration of adult-born neurons, improved figure presentation, removed the incomplete targeted-screen framing, added analysis of public MBNB-lineage transcriptomic data, and corrected multiple textual and citation issues.

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.

We appreciate this important point and agree that Drosophila does not normally show substantial physiological neurogenesis in the adult central brain under standard conditions. We have therefore revised the manuscript to clarify the framing of our study. Our aim is not to claim that Kr depletion reveals a normal physiological adult neurogenesis programme, but rather to identify developmental mechanisms that normally terminate MBNB proliferation and restrict latent neurogenic potential in the adult brain.

We note, however, that the original manuscript already provided evidence that the persistent proliferative cells induced by Kr depletion or the KrIf-1 mutation are associated with the MB lineage and generate neuronal progeny in young adult brains. EdU-positive clones were detected predominantly in the dorsoposterior MB cell body region (Fig. 2A, B). These clones generally contained a single Mira- and insc>GFP-positive NB-like cell, including mitotic cells with cortical Mira localisation (Fig. 2C, D), together with surrounding Elav-positive neuronal progeny (Fig. 2E). In addition, persistent mitotic cells in KrIf-1 mutant brains were found in the MB cell body region marked by mb247>GFP and OK107>GFP, and showed Mira localisation consistent with dividing MBNBs (Fig. 2F-H). Thus, although we have not directly demonstrated mature circuit integration, the original data support the conclusion that Kr depletion and the KrIf-1 mutation allow MBNBs to persist beyond their normal termination window and generate neuronal progeny within the MB region.

At the same time, our data do not establish the precise neuronal identity, connectivity or functional incorporation of the newly generated progeny into mature MB circuits, nor do they determine the long-term behaviour of these progeny in older adult brains. We have therefore revised the text and Discussion to avoid overinterpreting these cells as fully integrated adult-born MB neurons, and now state that these issues remain important questions for future work.

We also agree that prolonged progenitor proliferation can become pathological in some contexts. However, we do not think that the Kr-depletion and KrIf-1 phenotypes are equivalent to tumour-like overgrowth, at least under the conditions analysed here. In young adult brains, retained MBNBs remained spatially restricted to the MB cell body region, and each EdU-positive clone generally contained only a single NB-like cell with asymmetric Mira localisation, surrounded by Elav-positive neuronal progeny (Fig. 2C-E, G, H). This pattern is more consistent with persistent asymmetric MBNB divisions than with tumour-like expansion. This contrasts with Kr-h1 overexpression, which caused extensive tumour-like NB overproliferation with impaired neuronal differentiation; we now describe this latter phenotype as tumour-like NB overgrowth (Fig. 5C-E). We have revised the manuscript to distinguish these phenotypes more clearly.

(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.

We thank the reviewer for pointing out the need to improve figure clarity. We have revised the figure presentation throughout the manuscript to improve readability, including increasing label size, improving panel labelling and ensuring that the interpretation in the Results is consistent with the corresponding figure panels and legends.

Regarding the specific concern about Fig. 2C, we agree that the Mira signal was not sufficiently clear in the merged image. We have therefore added a Mira-only single-channel panel to Fig. 2C to allow the relevant signal to be evaluated more directly. We have also revised the text to clarify that retained MBNBs were identified based on combined positional, morphological and marker criteria, rather than by Mira signal alone.

(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.

We agree that the original description of a targeted genetic screen was insufficient. Because the screen was preliminary and incomplete, we concluded that presenting partial screening information would not be sufficiently informative and could overstate the scope of the present study. We have therefore removed the screen-based framing from the revised manuscript. Instead, the revised manuscript presents Kr as a candidate selected for focused functional analysis based on its known role in neuroblast temporal regulation and the strong MBNB retention phenotype observed upon Kr depletion and in the KrIf-1 mutant background.

(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.

We thank the reviewer for this helpful comment. We agree that our original wording could have been clearer. Our intention was not to argue that Kr cannot have a temporally restricted function in MBNBs, but rather that Kr does not behave like a canonical temporal transcription factor whose expression undergoes a strong stage-specific switch in this lineage.

We have revised both the text and the corresponding quantification. In the original version, Kr signal levels in pupal MBNBs appeared to vary modestly across pupal stages. However, after reanalysing cytoplasmic and nuclear Kr signals separately using Welch’s ANOVA followed by Dunnett T3 multiple-comparison correction, no pairwise comparisons reached statistical significance (Fig. 3E, F). Thus, the revised quantification does not support a significant temporal change in Kr protein levels during the pupal stages analysed, and we have avoided describing Kr as showing a clear pupal expression peak.

As requested, we have also included the public MBNB-lineage RNA-seq analysis in the revised manuscript as Fig. S5A. This analysis shows that Kr transcript levels remain low across the analysed developmental stages, in contrast to the stronger temporal changes observed for other regulators, including larval Kr-h1 expression followed by pupal downregulation, reciprocal induction of E93, and marked increases in Syp expression. Because FlyAtlas2 and modENCODE have limited spatial resolution for MBNB-specific expression, we now focus this comparison on the MBNB-lineage-specific RNA-seq dataset.

We have therefore revised the manuscript to describe Kr as a postembryonic regulator required for pupal MBNB termination, rather than as a conventional temporally induced tTF in this lineage. In our revised interpretation, Kr expression is low and not detectably strongly temporally induced in MBNBs, whereas Kr function is temporally required during pupal MBNB termination, as supported by the Gal80ts knockdown experiments.

(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.

We agree that, given the known role of Imp/Syp in MB neuronal temporal identity, altered MB neuronal composition is a plausible consequence of disrupted Kr function. However, we also agree that our study does not directly determine the precise neuronal identities, connectivity or functional properties of the progeny produced by persistent MBNBs. We have therefore revised the manuscript to present this aspect more cautiously.

In the original manuscript, we had shown that Kr depletion and the KrIf-1 mutation affect MB morphology. In the revised manuscript, we have clarified the presentation of these data and added new analysis of the severe MB defects caused by Kr-h1 overexpression (Fig. 6C). We now describe these phenotypes as changes in MB morphology and lineage progression, rather than as direct evidence for altered neuronal composition.

In the Discussion, we state that persistent Imp expression in Kr-depleted MBNBs could bias neuronal progeny toward earlier identities or delay their transition to later identities, but we explicitly note that neuronal identity and connectivity were not directly analysed. We also state that future studies will be required to determine whether prolonged MB neurogenesis alters MB circuit function, learning, plasticity or ageing. Thus, we have revised the manuscript to avoid overstating the contribution of Kr to MB neuronal composition.

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.

We understand the reviewer’s concern and have revised the manuscript to avoid implying that our data establish physiological adult neurogenesis in Drosophila. Instead, we now frame the study as identifying mechanisms that normally terminate MBNB proliferation and restrict latent neurogenic potential in the adult brain.

We respectfully note, however, that the impact of this study is not limited to showing that Kr regulates Imp. First, we identify Kr as a postembryonic regulator required for timely MBNB termination during pupal development. Second, we show that both Kr depletion and the KrIf-1 mutation allow MBNBs to persist beyond their normal termination window and generate neuronal progeny in the adult MB region. Third, we show that Kr promotes not only the Imp-to-Syp transition but also proper E93 expression within the MBNB lineage. Finally, we identify Kr-h1 as a functionally opposing KLF-family factor that represses E93 and can drive tumour-like NB overgrowth when misexpressed. We have revised the manuscript to present these conclusions more clearly and cautiously.

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 MBNBs 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.

We thank Reviewer #3 for their positive and constructive assessment of our study. We are grateful that the reviewer recognised the significance of identifying Kr as a regulator of mushroom body neuroblast termination, and the potential broader relevance of KLF-family transcription factors to neural stem cell regulation.

In response to the reviewer’s comments, we have substantially revised the manuscript. In particular, we have added new data on Kr expression in the KrIf-1 mutant background, Kr-h1 expression in MBNBs, and the regulation of E93 by Kr and Kr-h1. We have also incorporated public MBNB-lineage transcriptomic data to place Kr, Kr-h1, E93, Imp and Syp within the known temporal progression of the MB lineage. Finally, we have revised the proposed model to distinguish experimentally supported interactions from open mechanistic questions.

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.

We agree with the reviewer that some regulatory relationships in the original model required further experimental support. In response, we have added new data and revised the model substantially to distinguish experimentally supported interactions from unresolved mechanistic links.

In the revised manuscript, we provide additional analysis of the Imp/Syp axis after Kr depletion, direct evidence for Kr-h1 expression in MBNBs, and new data showing that Kr-h1 antagonises E93 expression in the MBNB lineage. We also found that Kr depletion reduces E93 expression in the progeny region of the MBNB lineage without significantly increasing Kr-h1::GFP levels in MBNBs, arguing against a simple linear pathway in which Kr promotes termination by directly repressing Kr-h1. Together, these results support a revised model in which Kr promotes MBNB termination by facilitating the Imp-to-Syp transition and supporting E93 expression, whereas Kr-h1 functionally opposes termination at least in part through E93 repression.

Recommendations for the authors:

Reviewing Editor Comments:

The majority of the reviewers agreed that a few major important points should be addressed, in particular:

(1) Better characterization of the nature of the KrIf-1 allele.

(2) Better define the regulations and interactions between Kr, Kr-h1, Imp, Syp, chinmo, and E93.

(3) More information about the genetic screen performed.

(4) The timings of expression of Kr in pupal MBNBs and of KrIf-1.

The reviewers also found that a general proofreading of the manuscript would greatly benefit its readability and comprehension and suggested several small corrections and more detailed explanations.

We thank the Reviewing Editor and Senior Editor for their careful assessment of our manuscript and for summarising the major points that required revision. We have addressed these points as follows.

(1) Better characterisation of the nature of the KrIf-1 allele

We agree that clarifying the nature of the KrIf-1 allele was important. We have now examined Kr expression in KrIf-1 mutant brains using TSA-enhanced Kr immunostaining. These new data show ectopic Kr signal in MBNBs and their progeny in KrIf-1 mutant late pupal and adult brains (Fig. S3G–I), supporting the conclusion that the KrIf-1 phenotype is associated with Kr misexpression rather than simple loss of Kr expression. We have revised the Results and Discussion to explain that MBNB termination requires precise control of Kr activity, and that both Kr depletion and inappropriate Kr expression can disrupt this termination programme.

(2) Better definition of the regulatory relationships between Kr, Kr-h1, Imp, Syp, Chinmo and E93

We have strengthened this part of the manuscript by combining the genetic evidence already present in the original manuscript with new expression analyses of temporal and hormone-responsive regulators. The original manuscript already showed that co-depletion of Imp suppresses MBNB retention caused by Kr depletion, supporting Imp as a key downstream effector of Kr. In the revised manuscript, we further show that Kr depletion causes persistent Imp expression and reduced Syp expression in late pupal MBNBs (Fig. 4), supporting the conclusion that Kr promotes MBNB termination by facilitating the Imp-to-Syp transition.

We have also added new data on Kr-h1 and E93. Kr-h1 depletion increases E93 expression in the MBNB lineage, whereas Kr-h1 overexpression reduces E93 expression (Fig. S5B), supporting an antagonistic relationship between Kr-h1 and E93 in this lineage. In addition, Kr depletion reduces E93 expression in the surrounding progeny of the MBNB lineage without detectably increasing Kr-h1::GFP levels in late pupal MBNBs (Fig. 5B). These data argue against a simple linear Kr-Kr-h1-E93 pathway and instead support a model in which Kr and Kr-h1 function through convergent or parallel mechanisms to regulate MBNB termination.

Regarding Chinmo, we agree that it is an important temporal factor. However, Chinmo is best characterised in this context as an early temporal fate regulator acting downstream of the Imp/Syp programme in MB lineages, where Imp and Syp regulate Chinmo translation post-transcriptionally (Liu et al., 2015). Because our study focuses primarily on late pupal MBNB termination, and because we were unable to obtain reliable Chinmo antibodies or reporter lines within the timeframe of this revision, we did not directly analyse Chinmo expression. We have therefore removed Chinmo from the main model and revised the Discussion to focus on the experimentally supported Imp/Syp and Kr-h1/E93 axes.

(3) More information about the genetic screen

We agree that the original screen-based description was not sufficiently informative. Because the targeted screen was preliminary and incomplete, we have removed the screen-based framing from the revised manuscript. Instead, we now present Kr as a candidate selected for focused functional analysis based on its known role in neuroblast temporal regulation and the strong MBNB retention phenotype observed upon Kr depletion and in the KrIf-1 mutant background.

(4) Timing of Kr expression in pupal MBNBs and in KrIf-1

We have clarified the timing of Kr expression in MBNBs. In wild-type brains, weak Kr signals were detected in MBNBs during pupal development using two Kr antibodies and a Kr::GFP BAC reporter, and these signals were reduced by Kr RNAi (Fig. 3E, F and Fig. S3F). We also analysed MBNB-lineage RNA-seq data, which show relatively low but persistent Kr transcript levels during postembryonic development (Fig. S5A). In the KrIf-1 background, TSA-enhanced Kr staining revealed ectopic Kr expression in MBNBs and their progeny during late pupal and adult stages (Fig. S3G–I). We have revised the manuscript to describe these expression patterns more clearly and to avoid overinterpreting weak embryonic or larval Kr signals.

We have also proofread the manuscript, corrected typographical errors, standardised figure references and nomenclature, improved figure legends and revised several figures for clarity.

Reviewer #1 (Recommendations for the authors):

(1) The authors identified Kr from a targeted genetic screen. Information about how many factors and what factors they have screened will also be very helpful to readers.

We thank the reviewer for this suggestion. In the original manuscript, we briefly referred to a targeted genetic screen that led us to focus on Kr. However, because this screen was preliminary and incomplete, we agree that including partial screening information would not be sufficiently informative and might overstate the scope of the present study. We have therefore revised the manuscript to remove the screen-based framing and instead present Kr as a candidate selected for focused functional analysis based on its known role in neuroblast temporal regulation and the strong MBNB retention phenotype observed upon Kr depletion and in the KrIf-1 mutant background.

(2) In Figure 2C wild type brains, what are the Mira+ cells and insc>GFP+ cells? There seems to be one cell that expresses both. Could it be a dormant NB?

We thank the reviewer for pointing this out. We agree that a small number of weakly Mira-positive and insc>GFP-positive cells can occasionally be observed in adult control brains, as well as in KrIf-1 mutant brains. These cells were few in number, were generally located outside the stereotyped MBNB region, and showed much weaker marker signals than the retained MBNBs observed after Kr depletion or in KrIf-1 mutants. Because they did not appear to express other NB markers examined in this study, such as wor>GFP, we did not investigate them further. Their identity therefore remains unclear, and we cannot exclude the possibility that they represent rare dormant or NB-like cells in the adult brain.

We have revised the text and figure legend to clarify that the retained MBNBs analysed in this study were identified based on their stereotyped MB-region position and strong NB-marker expression.

(3) Kr is normally expressed in stage 10 embryonic NBs. Have stage 10 embryos been examined to make sure that there is no Kr expression in MBNBs?

We thank the reviewer for this important point. We agree that Kr expression in early embryonic neuroblasts is well established. Early embryonic MBNBs can in principle be identified using a detailed positional and combinatorial marker map, as shown by Kunz et al. (2012), who traced individual MBNBs from late embryonic stages back to stage 9 using combinations of Dac, Ey, Svp-lacZ and Rx, together with morphological and positional criteria. However, reliable identification of MBNBs at such early stages is technically challenging in our assay, because our analysis depended on simultaneous detection of OK107>GFP, Mira and Kr, and OK107>GFP/Mira-based identification of the MB lineage was more robust at later embryonic stages. We therefore focused our embryonic Kr analysis on stages at which MBNBs could be confidently identified by their position and marker expression.

In these embryos, Kr was broadly detected in surrounding embryonic CNS cells, including thoracic NBs, but was not robustly detected in OK107>GFP- and Mira-positive MBNBs (Fig. S3A, B). We cannot fully exclude the possibility that MBNBs transiently express very low levels of Kr at earlier embryonic stages before they can be reliably identified in our assay. However, our temporally controlled Gal80ts experiments show that Kr depletion during the pupal stage, but not during earlier embryonic or larval stages, is sufficient to cause MBNB retention in adult brains (Fig. 3C, D). Thus, any transient embryonic Kr expression, if present, is unlikely to be the main cause of the adult MBNB retention phenotype described here.

We have revised the text to avoid overinterpreting the absence of embryonic Kr expression and to emphasise that the critical requirement for Kr in this study occurs during pupal MBNB termination.

(4) In Figure S3C, Kr expression seems to be cytoplasmic. Also in S3D, the expression of KrGFP in NBs is not obvious. Overall, Kr expression in pupal MBNBs is much more convincing than the larval expression.

We agree with the reviewer. Kr expression in larval MBNBs is weak and less convincing than the pupal signal. We have therefore revised the manuscript to describe larval Kr expression more cautiously. In the revised text, we state that Kr was weakly detected in larval MBNBs by antibody staining and that the Kr::GFP BAC reporter showed stronger signals in cells adjacent to MBNBs, likely corresponding to GMCs or immature neurons that may have inherited Kr::GFP from MBNBs.

We now base our main conclusion primarily on the pupal-stage data. At this stage, weak Kr signals were detected in MBNBs using two independent Kr antibodies and the Kr::GFP BAC reporter, and Kr depletion reduced the antibody signal (Fig. 3E, F and Fig. S3F). Importantly, this expression pattern is consistent with our functional data showing that pupal-stage-specific Kr depletion is sufficient to induce MBNB retention in adult brains, whereas earlier embryonic or larval depletion did not produce the same phenotype (Fig. 3C, D). Thus, both the expression and temporal knockdown data support a critical role for Kr during pupal MBNB termination.

We also discuss the observation that Kr signals in pupal MBNBs appear partly cytoplasmic, raising the possibility that Kr may not act solely as a canonical nuclear transcription factor in this context, or that its localisation may be dynamically regulated during MBNB termination. Thus, we agree that the pupal expression data provide the strongest support for Kr expression in MBNBs, and we have revised the text accordingly.

(5) A thorough proofreading is needed to correct the typos.

We apologise for the typographical and formatting errors in the original manuscript. We have thoroughly proofread the revised manuscript, corrected typographical errors, standardised figure references and gene/protein nomenclature, and revised multiple sections for clarity and readability. We have also revised the figure legends and improved figure presentation where possible.

Reviewer #2 (Recommendations for the authors):

(1) The authors should proofread for typos; there are quite a few ("survivial", "fount", "mitoic", "persistenc").

We apologise for these typographical errors. We have thoroughly proofread the revised manuscript, corrected typographical errors and formatting problems, and revised multiple sections for clarity and readability.

(2) In line 59, "adult eclosion" is incorrect. MBNBs also cease dividing before adult eclosion.

We thank the reviewer for pointing this out. We have revised the wording to avoid the incorrect phrase “adult eclosion” and now state that MBNBs normally terminate during late pupal development, before adulthood.

(3) In line 70, this progression of transcription factors is seen in the mammalian retina, not the brain.

We thank the reviewer for this correction. We have revised the relevant sentence to more accurately describe temporal patterning in mammalian neural progenitors, including the well-characterised example of retinal progenitor temporal progression and broader examples of temporal competence changes in cortical development.

(4) In lines 73-75, I don't think that any of the cited papers show that Cas triggers cell cycle exit. Ref 11 shows that, in UAS-Cas, NBs persist (as the authors also say in lines 79-81).

We agree that the original wording was too strong and potentially misleading. We have revised this section to state more generally that temporal transcription factors can influence NB proliferation and NB lifespan, rather than claiming that Cas directly triggers cell-cycle exit. We have also adjusted the discussion of Cas and Svp to better reflect the cited literature.

(5) In lines 133-134, it should be Figure 2A, B instead of Figure 3A, B.

We thank the reviewer for identifying this error. We have corrected the figure reference.

(6) Figures 1B and 1C are impossible to read. The x-axis should be reorganized and grouped into positive and negative CCRs to make a better argument. Moreover, the purpose of this figure is unclear. We already knew that neurons are postmitotic and all adult clusters are neurons. IF there were any NBs in the adult, they would be so few that they would be invisible with this resolution.

We thank the reviewer for this comment. We agree that the dot plots are dense, because they summarise the expression of 112 selected cell-cycle regulator (CCR) genes across multiple adult and larval brain cell clusters. In preparing the revised manuscript, we have ensured that the figure legends explain the meaning of colour intensity and dot size, and that the submitted PDF preserves sufficient resolution for the gene and cluster labels to be read upon enlargement.

In the original figures, CCR genes were arranged according to broad functional categories. We have not forced all CCR genes into a simple positive-versus-negative classification, because the function of many CCRs is context-dependent. For example, APC/C components can contribute to either mitotic progression or G1 maintenance depending on co-activator context, and mitotic regulators such as Polo and Aurora kinases regulate multiple aspects of mitosis rather than acting simply as positive cell-cycle drivers. We therefore present the genes according to broad functional categories and discuss representative examples of positive and inhibitory regulators in the Results and figure legends, rather than implying that every CCR can be assigned unambiguously to one of two categories.

Regarding the purpose of these analyses, our aim was not simply to restate that adult neurons are postmitotic, nor to claim that scRNA-seq can definitively exclude extremely rare adult progenitors. Rather, we used published scRNA-seq datasets to compare CCR expression programmes across adult and larval brain cell populations in an unbiased manner. The analysis shows that no adult brain cluster displays a coordinated larval NB-like positive CCR expression programme, whereas such a programme is readily observed in larval NB clusters. We have clarified this point in the text and acknowledge the limitation that very rare or non-canonical progenitor-like cells may not be detected by this approach.

(7) The survival assays are confusing. Do the authors believe that the presence or absence of 2-3 clones in the adult brain of a few cells would lead to such a limited survival?

We thank the reviewer for raising this point. We agree that the survival assay required clearer explanation and should be interpreted cautiously. In this experiment, E2F1-Dp and Cdk2-CycE were expressed using TH-Gal4 to test whether forced activation of positive cell-cycle regulators can drive postmitotic neurons into aberrant cell-cycle re-entry. Although only a small number of pH3-positive cells may be detected at any single time point, this represents only a snapshot of an ongoing process and may underestimate the cumulative number of affected cells over time.

TH-Gal4-positive dopaminergic neurons are relatively limited in number, but they include functionally important neuronal populations involved in motor behaviour, arousal, sleep/wake regulation and ageing-associated behavioural decline. Therefore, broad induction of aberrant cell-cycle re-entry and apoptosis within these neurons could plausibly affect organismal survival. However, we agree that the survival phenotype should not be overinterpreted. We cannot exclude the possibility that TH-Gal4 expression outside dopaminergic neurons, developmental effects of the manipulation, or other indirect consequences contribute to the reduced survival. Moreover, the precise cause of death was not determined in this study. We have therefore revised the manuscript to present the survival assay as supporting evidence that inappropriate activation of positive cell-cycle regulators is deleterious in postmitotic neurons, rather than as a direct mechanistic explanation for organismal lethality.

(8) In Figure 2, how many of the proliferative clones are found in the mushroom body. Is it all of them?

We thank the reviewer for this question. In Fig. 2, EdU-positive proliferative clones were scored based on their stereotyped position in the dorsoposterior MB cell body region. Under the conditions analysed, the scored clones were consistently detected within this MB region, and their number did not exceed four per brain hemisphere, consistent with the expected number of MBNBs (Fig. 2A, B). In addition, the identity of these clones as retained MBNB lineages was supported by NB marker expression, Mira localisation, insc>GFP expression where applicable, and their association with MB lineage markers such as mb247>GFP and OK107>GFP in KrIf-1 mutant brains (Fig. 2C-H). We have clarified this point in the revised text and figure legend.

(9) In Figure 2C, why are there so many Miranda-positive cells?

We thank the reviewer for this question. We agree that several Mira-positive cells can be observed in Fig. 2C, including in control brains. The identity of these additional Mira-positive cells remains unclear. They did not consistently co-express the canonical NB markers examined in this study, such as insc>GFP or wor>GFP, and therefore do not appear to represent canonical proliferative NBs. However, we cannot exclude the possibility that some weak Mira-positive cells represent rare dormant or NB-like cells in the adult brain. We have revised the text and figure legend to clarify this point.

(10) In Figure 3C, the authors argue that Kr has an opposing function in NB regulation during larval stages, however, the Kr mutant (which is in both larval and pupal stages) has the highest number of EdU-positive clones. Moreover, conditions 2 and 9 have a comparable (low) number of EdU positive clones, although condition 2 has Kruppel downregulated in all stages.

We thank the reviewer for this comment. We agree that this part of the temporal knockdown experiment required more careful interpretation, and we have revised the manuscript to avoid overinterpreting small quantitative differences among individual temperature-shift conditions.

First, Fig. 3C does not include a Kr mutant condition. We assume that the reviewer is referring to the constitutive insc>KrIR condition grown at 29°C throughout development (condition 1). This condition does not contain tub-Gal80ts and may therefore produce stronger and more continuous Kr RNAi induction than the Gal80ts-based temporal knockdown conditions. In addition, for practical reasons, flies in all conditions were initially kept at 25°C during egg laying to obtain sufficient embryos before being shifted to the indicated temperature regime, including condition 2. These differences may explain why condition 1 produced the highest number of EdU-positive clones. For this reason, we do not interpret condition 1 as a temporal-control condition and do not use it for direct quantitative comparison with the Gal80ts-based conditions.

Second, regarding the comparison between conditions 2 and 9, we agree that these conditions should not be used to infer a simple relationship between the total duration of Kr knockdown and the number of retained MBNBs. We therefore avoid using these differences to infer an additional larval function of Kr. Instead, we now interpret this temporal knockdown experiment more conservatively: Kr depletion during the pupal period is sufficient to induce MBNB retention in adult brains, whereas depletion restricted to earlier embryonic/larval stages or later adult stages has little or no effect.

Thus, the key conclusion supported by these data is the pupal-stage requirement for Kr in MBNB termination, rather than detailed comparisons among all temperature-shift conditions. We have revised the text accordingly.

Overall, we have revised the manuscript to address the reviewer’s concerns by narrowing the framing of adult neurogenesis, clarifying the distinction between persistent MBNBs and tumour-like overgrowth, improving figure presentation, removing the incomplete screen description, adding public dataset analysis, and correcting textual and citation errors. We believe these revisions make the scope and conclusions of the study more precise.

Reviewer #3 (Recommendations for the authors):

Major Points

(1) Targeted screen: The authors mention performing a targeted genetic screen. Please specify which transcription factors were tested.

We thank the reviewer for this suggestion. In the original manuscript, we briefly referred to a targeted genetic screen. However, this screen was preliminary, incomplete, and included a broader set of candidate regulators rather than a systematically completed transcription-factor collection. We therefore agree that listing a partial set of tested factors would not be sufficiently informative and could overstate the scope of the present study.

We have therefore removed the screen-based framing from the revised manuscript. Instead, the revised manuscript presents Kr as a candidate selected for focused functional analysis based on its established role in neuroblast temporal patterning and the strong MBNB retention phenotype observed upon Kr depletion and in the KrIf-1 mutant background. We also no longer use the preliminary screen as part of the evidence supporting our conclusions.

(2) KrIf-1 allele : The authors report that KrIf-1 mutants retain MB NBs in adults, a phenotype resembling Kr loss of function. They propose that this results from Kr deregulation. However, KrIf-1 is a neomorphic allele known to cause ectopic expression in imaginal discs, raising the possibility that Kr is upregulated, not downregulated, in MB NBs. The authors should present experimental data on Kr expression in MB NBs in the KrIf-1 background to clarify this point.

We agree that clarification of Kr expression in the KrIf-1 background is important. We have therefore performed additional Kr immunostaining in KrIf-1 mutant brains using tyramide signal amplification (TSA) to enhance the weak endogenous Kr signal. These new data show that Kr is ectopically detected in MBNBs and their progeny in KrIf-1 mutant late pupal and adult brains (Fig. S3G, H), and quantification of TSA-enhanced Kr signals further supports Kr misexpression in MBNBs during late pupal stages (Fig. S3I).

These results indicate that KrIf-1 is associated with ectopic or aberrant Kr expression in MBNBs, rather than simple loss or downregulation of Kr expression. We have revised the manuscript to clarify this point and no longer interpret KrIf-1 as a simple Kr loss-of-function condition. Although both Kr depletion and KrIf-1 lead to MBNB retention, our revised interpretation is that MBNB termination requires precise temporal and spatial control of Kr activity. Kr depletion disrupts the normal Imp-to-Syp transition and reduces E93 expression in the MBNB lineage, whereas ectopic or mistimed Kr expression in KrIf-1 mutants may also interfere with the termination programme. Thus, both reduced Kr activity and inappropriate Kr expression can perturb the tightly regulated transition required for timely MBNB cell-cycle exit and elimination.

(3) Line 258 - OK107>GFP subset (Fig. 2G): Please clarify why only a subset of EdU-positive clones co-express OK107>GFP. Is this due to EdU labeling other NB lineages not related to the mushroom body?

We thank the reviewer for raising this point. We do not think that the OK107-negative EdU-positive clones represent unrelated NB lineages. The EdU-positive clones analysed in Fig. 2G were located in the dorsoposterior MB cell body region and were associated with Mira-positive NB-like cells, supporting their identification as persistent MBNB-lineage clones.

The reason that only a subset of EdU-positive clones clearly co-express OK107>GFP is likely that, while OK107-Gal4 strongly labels MB neurons and lineage cells, its activity in MBNBs and very young progeny appears to be weaker or more variable at the stages analysed. Thus, the absence of strong OK107>GFP signal in some EdU-positive cells does not necessarily indicate that these clones belong to non-MB lineages.

We have revised the text and figure legend to clarify this point.

(4) Line 261 - Early adulthood mitotic activity: The statement that NBs retain mitotic activity during early adulthood (citing Technau, 2007; Li and Hidalgo, 2020) is misleading. To my knowledge, this mitotic activity has only been reported in glial cells, not NBs. Please revise accordingly.

We thank the reviewer for pointing this out. We agree that the original wording was misleading, because it could be read as implying that normal NBs retain mitotic activity during early adulthood. We have revised the text accordingly. The revised manuscript now refers more cautiously to the previously described early-adult period during which rare residual cell-cycle or proliferative activity has been reported in the adult Drosophila brain, without attributing this activity to normal persistent NBs.

(5) Line 313 - Public dataset analysis: The manuscript mentions analysis of publicly available data, but no results are presented. Please include these findings.

We agree and have now included the public dataset analysis in the revised manuscript. We analysed a published MBNB-lineage-specific RNA-seq dataset and present the expression profiles of selected temporal regulators in Fig. S5A.

This analysis shows that Imp and chinmo transcripts are high during larval stages and decrease during pupal development, whereas Syp is strongly upregulated during the same period. Kr transcript levels remain relatively low throughout the analysed larval and pupal stages and do not show strong temporal induction. In contrast, Kr-h1 is highly expressed during larval stages and sharply downregulated after the larval-to-pupal transition, whereas E93 is strongly induced during pupal development.

These expression profiles support the idea that MBNBs undergo a major temporal transition during pupal development, including the Imp/Syp transition and reciprocal changes in Kr-h1 and E93 expression. They are also consistent with our experimental finding that Kr-h1 antagonises E93 expression in the MBNB lineage. We have added this analysis to the Results and incorporated it into the revised model.

(6) Figure 3A - Kr localization: Kr is detected predominantly in the cytoplasm, which contrasts with its function as a transcription factor. Is cytoplasmic localization typical for Kr? Please discuss.

We thank the reviewer for this important observation. We agree that the weak Kr signal in pupal MBNBs appears partly enriched in the cytoplasm, which is unexpected for a canonical nuclear transcription factor. We have revised the Discussion to address this point.

Because this signal was reduced upon Kr RNAi, we think that it is likely to represent a Kr-dependent signal rather than nonspecific background. However, we do not yet know whether the apparent cytoplasmic enrichment reflects dynamic regulation of Kr localisation, a non-canonical aspect of Kr function in postembryonic MBNBs, or technical limitations associated with detecting very low endogenous Kr levels. We therefore avoid overinterpreting the cytoplasmic signal, but note that Kr localisation and activity in pupal MBNBs may be more complex than expected for a canonical nuclear transcription factor. Future studies will be required to determine how Kr activity and localisation are regulated in pupal MBNBs.

(7) Temporal Kr knockdown experiment: The result that Kr knockdown during pupal stages - but not earlier - leads to MB NB persistence is interesting but counterintuitive. Given the limitations of RNAi (e.g., leaky expression, incomplete knockdown), MARCM clones for Kr mutants would provide stronger evidence and improve confidence in this conclusion.

We agree that MARCM analysis of Kr mutant MBNB clones would provide a valuable independent test of the temporal RNAi results. We attempted to establish this approach during revision, but it proved technically difficult within the available timeframe.

A major practical limitation is that MBNBs are very few in number, with only four per brain hemisphere. Conventional MARCM or flip-out approaches therefore generate stochastic clones that are inefficient for reliably recovering and analysing MBNB clones. We also explored the possibility of using MB-lineage MARCM reagents reported in a previous study (Rossi & Desplan, 2020), but the available stocks were not directly compatible with the FRT chromosome required for our Kr mutant analysis. Generating the necessary compatible stocks and recovering sufficient MBNB clones would require substantial additional crossing and screening.

For this reason, we were not able to include Kr mutant MARCM data in the revised manuscript. Instead, we base our conclusion on several complementary lines of evidence, including temporal Gal80ts-based Kr RNAi, two independent Kr RNAi lines, the reduction of Kr signal upon Kr RNAi newly added in this revision, and the independent KrIf-1 phenotype, which supports the idea that precise regulation of Kr activity is required for MBNB termination.

(8) OK107-GAL4 activity: Please indicate early in the manuscript that OK107-GAL4 is not active in MB NBs but is active in their progeny. This would clarify the rationale for using the pan-NB driver insc-GAL4 for manipulating MB NBs.

We thank the reviewer for this suggestion. We have revised the manuscript to clarify the use of OK107-Gal4 and insc-Gal4 earlier in the Results and in the relevant figure legends.

Although OK107-Gal4 activity is not completely absent from MBNBs, its activity in MBNBs is stage-dependent and weaker or more variable than in MB neurons and their progeny. We therefore used OK107-Gal4 mainly to visualise MB structures and MB lineage regions, whereas insc-Gal4 was used for robust NB-specific manipulation. This clarification should help explain why insc-Gal4, rather than OK107-Gal4, was used for MBNB-targeted knockdown experiments.

(9) Figure 5A - Mira+ cell numbers: There appears to be an excess of Mira+ cells compared to the expected number of MB NBs. Please explain this observation.

We thank the reviewer for pointing this out. In Fig. 5A, we quantified the number of EdU-positive clones, not the total number of Mira-positive cells. As shown in Fig. 2, proliferative cells retained in Kr-depleted or KrIf-1 mutant adult brains were identified as persistent MBNBs based on combined criteria, including EdU incorporation, NB marker expression such as Mira and insc>GFP, NB-like morphology, and stereotyped location in the dorsoposterior MB region.

We agree that additional Mira-positive cells can be observed in adult brains, including in control and Kr-depleted brains. However, these cells were not associated with EdU-positive proliferative clones and did not consistently express canonical NB markers examined in this study. Their identity therefore remains unclear, and they were not scored as retained MBNBs.

(10) Line 430 - Kr-h1 over-expression phenotype: The amplification phenotype caused by Kr-h1 overexpression is reminiscent of that caused by chinmo overexpression (Narbonne-Reveau et al., 2016), suggesting that Kr-h1 may promote chinmo and Imp expression. Also, what is the expression pattern of Kr in the supernumerary adult MB NBs following Kr-h1 overexpression? Is Kr silenced? Investigating this would help clarify the regulatory links between Kr, Kr-h1, and Imp/chinmo.

We agree that the Kr-h1 overexpression phenotype is reminiscent of NB amplification phenotypes associated with forced or aberrant activation of early temporal growth programmes, including those linked to Chinmo. Narbonne-Reveau et al. showed that Chinmo can promote NB amplification and tumour growth, and that Chinmo, Imp and Lin-28 form a growth-promoting module in neural tumours. Consistent with this idea, our revised manuscript shows that Kr-h1 overexpression causes extensive tumour-like NB overgrowth, impaired neuronal differentiation and persistent Imp expression (Fig. 5C–E).

Although we did not analyse Chinmo expression in this revision, we did examine Imp expression following Kr-h1 overexpression and found persistent Imp expression in tumour-like NB cells (Fig. 5E). These data are consistent with the idea that excessive Kr-h1 activity can reinforce a progenitor-like state. However, we have interpreted this result cautiously, because Kr-h1 overexpression produces a severe tumour-like phenotype with impaired asymmetric division and neuronal differentiation. Thus, persistent Imp expression in this context could reflect either a more direct effect of Kr-h1 on early temporal programmes or an indirect consequence of the strongly perturbed progenitor-like state.

We also did not directly analyse Kr expression in supernumerary NB-like cells following Kr-h1 overexpression. This is an interesting question, but the severe tissue disruption caused by Kr-h1 overexpression would make it difficult to distinguish a physiological regulatory effect on Kr from an indirect consequence of tumour-like overgrowth. In addition, endogenous Kr signals in MBNBs are weak, making it technically challenging to reliably quantify changes in Kr expression in this strongly perturbed condition.

We have therefore revised the Discussion to clarify that the Kr-h1 overexpression phenotype should be interpreted mainly as evidence that excessive Kr-h1 activity can oppose MBNB termination and maintain Imp-positive progenitor-like cells, rather than as evidence for a defined normal regulatory pathway from Kr-h1 to Chinmo, Imp or Kr. In parallel, our revised loss-of-function and expression analyses show that Kr depletion causes persistent Imp and reduced Syp expression, that Kr supports E93 expression in the MBNB lineage (Fig. 5B), and that Kr-h1 antagonises E93 expression (Fig. S5B). Because Kr depletion did not significantly increase Kr-h1::GFP expression (Fig. 5B), we no longer present a simple linear model in which Kr promotes MBNB termination by repressing Kr-h1 expression.

(11) Kr-h1 expression in MB NBs: Please provide direct evidence that Kr-h1 is expressed in MB NBs during development.

We agree that direct evidence for Kr-h1 expression in MBNBs is important. We have therefore analysed Kr-h1 expression using a functional Kr-h1::GFP BAC reporter. Kr-h1::GFP was clearly detected in MBNBs during third-instar larval stages, with nuclear enrichment, but became barely detectable in late pupal MBNBs (Fig. 5B and Fig. S5C).

This developmental downregulation is consistent with the published MBNB-lineage RNA-seq dataset, which shows high Kr-h1 transcript levels during larval stages and strong reduction after the larval-to-pupal transition (Fig. S5A). Together, these data provide direct evidence that Kr-h1 is expressed in MBNBs during development and is downregulated before MBNB termination.

(12) Figure 6D - Model validation: The model in Figure 6D is appealing, but many of the proposed interactions remain untested. For example:

- It is not established that Kr-h1 antagonizes E93 or vice versa in MB NBs.

- The antagonistic relationship between Kr and Kr-h1 has not been experimentally confirmed.

- The temporal expression of Kr-h1 in MB NBs is unknown.

- The repression of E93 by Kr-h1 is unlikely to fully explain the MB NB amplification phenotype.

We thank the reviewer for this helpful critique. We have substantially revised Fig. 6D and the accompanying text to distinguish experimentally supported interactions from unresolved regulatory links.

First, we have now tested the relationship between Kr-h1 and E93 in the MBNB lineage. Kr-h1 depletion increased E93 expression, whereas Kr-h1 overexpression reduced E93 expression (Fig. S5B), supporting the conclusion that Kr-h1 antagonises E93 expression in this lineage. Reciprocal antagonism between Kr-h1 and E93 has been reported in other insect developmental contexts. However, because we did not directly test whether E93 represses Kr-h1 in MBNBs, we now present this possible E93-to-Kr-h1 regulation as an unresolved or putative interaction rather than as an experimentally established relationship in our system.

Second, we have clarified the relationship between Kr and Kr-h1. Our genetic data show that Kr-h1 knockdown partially suppresses MBNB retention caused by Kr depletion (Fig. 5A), indicating that Kr and Kr-h1 have opposing functions during MBNB termination. However, although Kr depletion reduced E93 expression in the MBNB lineage, it did not detectably increase Kr-h1::GFP levels in late pupal MBNBs (Fig. 5B). We therefore no longer present Kr as a direct upstream repressor of Kr-h1. Instead, the revised model proposes that Kr promotes MBNB termination by supporting the Imp-to-Syp transition and E93 expression, whereas Kr-h1 opposes termination at least in part by repressing E93.

Third, we now provide direct evidence for Kr-h1 expression in MBNBs using a functional Kr-h1::GFP BAC reporter. Kr-h1::GFP was detected in larval MBNBs and became strongly reduced during pupal development (Fig. 5B and Fig. S5C). This temporal pattern is also consistent with the published MBNB-lineage RNA-seq dataset, which shows high Kr-h1 transcript levels during larval stages and downregulation after the larval-to-pupal transition (Fig. S5A).

Finally, we agree that repression of E93 by Kr-h1 is unlikely to fully explain the strong tumour-like NB overgrowth caused by Kr-h1 overexpression. In the revised manuscript, we show that supernumerary tumour-like NB cells induced by Kr-h1 overexpression retain high Imp expression (Fig. 5E). This suggests that excessive Kr-h1 activity can oppose MBNB termination and maintain an Imp-positive progenitor-like state.

However, we interpret the Kr-h1 overexpression phenotype cautiously, because this strong gain-of-function condition causes severe tumour-like overgrowth and may not accurately reflect the physiological regulatory relationship between Kr-h1 and the normal MBNB termination programme. We have therefore revised the Discussion to state that Chinmo, an early temporal factor linked to the Imp/Syp programme, may be relevant to the persistent progenitor-like state observed after Kr depletion or Kr-h1 overexpression. However, because we did not directly analyse Chinmo expression in this study, we do not include Chinmo in the main model and leave this question for future work.

Minor Points

(1) OK107-GAL4 activity: Please check and indicate early on in the manuscript that OK107-GAL4 is not active in MB NBs (although being active in their progeny). It would clarify why the authors keep using the pan NB insc-GAL4 to manipulate MB NBs.

As described in our response to Major Point 8, we have clarified earlier in the Results that OK107-Gal4 strongly labels MB neurons and the MB lineage, but is not a robust or consistent driver in MBNBs at the stages relevant to our functional assays. We therefore used OK107-Gal4 primarily for visualising MB structures and MB lineage regions, whereas insc-Gal4 was used for robust NB-specific manipulation.

(2) CCR list: Please clarify how the list of candidate cell cycle regulators (CCRs) was compiled.

We have revised the Materials and Methods to clarify how the list of candidate cell-cycle regulators was compiled. Briefly, the CCR list was manually curated to include core cell-cycle regulators, cyclins and CDKs, DNA replication factors, checkpoint regulators, mitotic regulators, APC/C and ubiquitin-proteasome components, and non-canonical CDKs with known roles in transcriptional regulation.

(3) Line 77-78 - Cas and Svp: The statement that Cas and Svp regulate neurogenesis termination by directly regulating cell cycle regulators needs references. As far as I know, the direct targets of Cas and Svp remain unidentified.

We agree that the original wording was too strong. We have revised the text to state more generally that temporal transcription factors, including Cas and Svp, influence NB proliferation and lifespan, without implying that they directly regulate known cell-cycle target genes.

(4) Line 410: Please add references to support this statement.

We have added the appropriate reference to the original genetic modifier screen by Abrell et al., 2000, in which Kr-h1 mutations were identified as enhancers of the KrIf-1 eye phenotype. We have also revised the wording to state that this finding suggests a functional interaction between Kr and Kr-h1, rather than using it alone as evidence for a direct regulatory relationship.

(5) Typos and minor edits:

- Line 38: "capable" (spelling)

- Line 102: Replace "unexplored" with "explored"

- Line 106: "potential" (spelling)

- Line 243: "mitotic" (spelling)

- Line 260: "KrIf-1" (correct gene formatting)

We have corrected the typographical errors and formatting issues indicated by the reviewer, including spelling errors and KrIf-1 gene formatting. We have also proofread the manuscript throughout.