Cell cycle and temporal transcription factors regulate proliferation and neuronal diversity of dedifferentiation-derived neural stem cells

  1. Kellie Veen
  2. Francesca Froldi
  3. Qian Dong
  4. Edel Alvarez-Ochoa
  5. Phuong-Khanh Nguyen
  6. Kieran F Harvey
  7. John P D McMullen
  8. Owen Marshall
  9. Patricia R Jusuf
  10. Louise Y Cheng

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Abstract

Dedifferentiation is the reversion of differentiated cells to a stem cell like fate, whereby, the gene expression program of mature cells is altered and genes associated with multipotency are expressed. Appropriate terminal differentiation of NSCs is essential for restricting the overall number of neurons produced; in addition, faithful production of neuronal subtypes that populate the brain is important for NSC function. Both characteristics of NSCs are specified through temporal patterning of the NSCs driven by the successive expression of temporal transcription factors (tTFs). In this study, we found that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression fail to undergo timely expression of temporal transcription factors (tTFs), where they express mid-tTF, Sloppy-paired 1 (Slp-1) and fail to express late-tTF Tailless (Tll); consequently generating an excess of Twin of eyeless (Toy) positive neurons at the expense of Reversed polarity (Repo) positive glial cells. In addition to disrupted production of neuronal/glial progeny, Dpn overexpression also resulted in stalled progression through the cell cycle, and a failure to undergo timely terminal differentiation. Mechanistically, DamID studies demonstrated that Dpn directly binds to both Dichaete (D), a Sox-box transcription factor known to repress Slp-1, as well as a number of cell cycle genes. Promoting cell cycle progression or overexpression of D were able to re-trigger the progression of the temporal series in dedifferentiated NBs, restoring both neuronal diversity and timely NB terminal differentiation.

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    Reply to the reviewers

    Reviewer #1 (Evidence, reproducibility and clarity):

    Summary
    Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.
    Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.
    Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.

    Major comments:

    • Are the key conclusions convincing?
      Most conclusions are convincing; however, some issues are pointed out below.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.
    Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.

    While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.

    We will characterise the temporal identity, termination and cellular identity of Lola-Ri induced ectopic neuroblasts. If these parameters are disrupted, we will overexpress D to assess whether this can trigger the progression of the temporal series.

    Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.

    We will include that Dpn is a target of Notch when first mentioned.

    Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.
    Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.

    We will make a more conservative claim and clarify all of our conclusion are medulla neuron-specific.

    Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.
    Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.
    Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.

    We will assess the temporal identity of neuroblasts in nerfin-1 mutant clones.

    Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.

    Ey is shown in Fig-2 (D-D’’) already. Slp is used as a marker of mid tTFs as Ey is expressed also in neurons thus would also be present in deep sections of control clones, whereas Slp is not expressed in neurons. We therefore used Slp as a proxy for mid-temporal identity throughout our study. We will include this text in our revision.

    In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.

    In Figure 2H, we have shown in clones that overexpression of Dpn induced a significant increase of Slp. In Figure S3B-B’’, we have shown that Dpn overexpression causes an upregulation of Slp at 6 hr APF. We can think we have pretty convincingly shown that Dpn overexpression activates Slp.

    For Dichaete, our existing data shows that Dpn overexpression did not significantly alter D expression. To assess if using a stronger driver might allow us to see some changes, we will induced dedifferentiation via Dpn overexpression using the Eyeless-Gal4 driver. In this experiment, we will quantify the amount of D upon Dpn overexpression. Depending on this result, we will revise our conclusion on whether Dpn overexpression represses D.

    Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
    Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.

    • Are the data and the methods presented in such a way that they can be reproduced?
      Yes

    Are the experiments adequately replicated and statistical analysis adequate?
    Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.

    We will repeat Notch experiments to increase the n number for these experiments.

    Minor comments:

    • Specific experimental issues that are easily addressable.
      There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.

    In the text we said: “We found that D overexpression did not significantly increase neuronal production, suggesting that it is likely that cell cycle progression lies upstream or in parallel to the temporal series, to promote the generation of neurons.”

    In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).

    We will add “medulla NBs” to the end of this sentence.

    In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).
    Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.

    We will expand in discussion to discuss the link between cell cycle/tTFs.

    In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?
    In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.
    Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.

    While Dpn is overexpressed, it progresses through the temporal series at a slower pace due to a delay in cell cycle progression, as well as delayed onset of D, these NBs still eventually reach the terminal temporal identity, and are thus about to undergo terminal differentiation. We will include an additional piece of data that shows NBs induced by Dpn overexpression do eventually turn on Tll.

    Are prior studies referenced appropriately ?
    Yes, but in a few places, some references can be added.
    An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.14134

    We beg to disagree with the reviewer in terms of Pros is not required for terminal differentiation of medulla neuroblasts. Li et al., 2013 shows that nuclear Pros is found in the oldest NBs. We do agree that differentiated state of medulla neurons is less stable, possibly owing to absence of Pros, and we will include that in our discussion.

    In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.
    Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.

    We will discuss the study looking at N targets in NB hyperplasia in the discussion of the revised manuscript.

    We will mention that Dpn is a target of Notch in the results section.

    Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.

    We will comment on Brat in the discussion.

    Are the text and figures clear and accurate?
    The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.
    There are 1 or 2 places where figure calling is wrong in the text.
    The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.

    We thank the reviewers for identifying these mistakes, and will correct them.

    Do you have suggestions that would help the authors improve the presentation of their data and conclusions?
    The presentation is okay, in my opinion.

    Reviewer #1 (Significance):

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.

    This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.
    However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.

    • Place the work in the context of the existing literature (provide references, where appropriate).
      Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.
      Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).

    The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.
    Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.

    • State what audience might be interested in and influenced by the reported findings.
      The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
      Drosophila, central nervous system patterning and cell fate determination of neural stem cells.

    Reviewer #2 (Evidence, reproducibility and clarity):

    Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.
    Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.

    This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:

    1. Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?

    We will include an expression analysis of EyOK107-GAL4 and eyR16F10-GAL4. GMR31HI08-GAL4 expression analysis was previously published (Vissers et al., 2018). We will explain in the text the benefits of each driver.

    1. The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.

    We will repeat this experiment and mark the progeny with the neuronal marker Elav, to demonstrate that they are neurons. Additionally, we will add the control to this figure.

    1. How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).
      Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?
      Additional explanations in the methods section is needed to assess the robustness of the data.

    We will expand the methods section and mention that we used similar thresholding in antibody staining between control and uas dpn in all instances, so even if the antibody is weaker (eg hth) it is consistently quantified. Additionally, we can increase the intensity of Ey in Figure 2D-2D’, as it is expressed at low levels.

    1. This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'
      The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.

    We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence.

    1. Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".

    We will split figure 2 into two images. The first image including A-H and the control data. And the second including I-Q and the control data. This will increase the size of the images. Additionally, we will group I and J with corresponding data.

    1. Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.

    Most NBs have terminated by this time point, we will explain this within the text.

    1. Figure 3K-M: Please explain how the Toy signal was measured and quantified.

    We will expand the methods section and explain how Toy quantification is made.

    1. The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."
      More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.
      Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?

    The DamID data do actually show that Dpn binds (i.e. there is a statistically significant peak at FDR<0.01) directly at these loci (see the TaDa supp fig A & B). Whether it’s doing anything functional or not, we can’t say, but our data shows that Dpn directly binds to slp1 and D. We will clarify the sentence to indicate this in our revision.

    1. This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.

    Our data already shows that these NBs express Tll, the terminal temporal transcription factor (Figure 4F). In addition, we show that there is an increase in Tll+ and Repo+ progeny (Figure 4K, L). Together, this suggests that D overexpression promotes the progression of the temporal series. However, it is possible that Mira+ cells can disappear via cell death. We will assess this possibility by staining for cell death marker Dcp1 at 6hr APF.

    Reviewer #2 (Significance):

    These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.
    I think the findings will be of interest to cell, developmental cell and stem cell biologists.

    My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.

    Reviewer #3 (Evidence, reproducibility and clarity):

    The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing.

    Our data relate specifically to gene regulation by Dpn in a dedifferentiated context, and do not seek to understand Dpn regulation in wt neuroblasts. The reviewer is assuming our scope is greater here: we’re not trying to claim that we know what Dpn is doing in wt NBs, and it’s not surprising that ectopic effects in neurons may be different to wt NBs.

    To assess whether the mechanisms described apply to more than Dpn overexpression, we will also assess whether the temporal series progression is affected in Lola RNAi and Nerfin-1 mutant.

    Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.
    The author's state:
    "However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."
    at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.

    We will add this to the abstract.

    Results

    1. The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.

    We will include expression analysis of all three drivers in a supplementary figure and explain in the text the benefit of each driver.

    1. The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.

    We have conducted lineage tracing (G-Trace) analysis of the medulla neuron driver GMR31H08-GAL4 which we utilise in our study, this driver is predominantly expressed within the medulla neurons (real time) except for a few GMCs present in the lineage. Therefore, the Mira positive cells induced via Dpn overexpression are most likely from dedifferentiation (We will include this data in a supplemental figure in our revised manuscript).

    To further support this, we will use GMR31H08-GAL4 with a Gal80ts, to restrict the timing to dedifferentiation induction to 3rd instar, so that the driver is restricted to neurons. Similar strategy to induce dedifferentiation was utilised in DOI: 10.1242/dev.141341 and DOI: 10.1016/j.devcel.2014.01.030.

    1. What is a conclusion of fig 2C-H?

    Fig 2C-H assess the expression of tTFs in UAS-dpn induced ectopic NBs. We will make these conclusions clearer in the text.

    1. "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.

    We will rephrase this sentence and further introduce this concept when talking about tumour cell of origin and competence. Additionally, we will make the connection to the experiments which follow it clearer.

    1. The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.

    We agree with the reviewer that the profile is not specific to medulla NBs. To assess Dpn binding profiles specifically in the medulla NBs, we will use the recently-published NanoDam technique (https://doi.org/10.1016/j.devcel.2022.04.008) for profiling GFP-fusion proteins, with a medulla specific driver (eyR16F10-GAL4) and Dpn-GFP (recombineered locus under endogenous control). This should inform us whether the target genes we have identified are relevant in the medulla.

    We will include the tracks of the other transcription factors.

    1. Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.

    We observed that high levels of Slp-1 is correlated with low levels of D. This suggest to us that the initial stochastic differences accounts for where Slp-1 is high is where D is low, and vice versa.

    1. The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.

    We will discuss these models in the Discussion, and make our claims more conservative, as we do not have direct experimental evidence to prove or disprove the model that Dpn is acting as an activator in this context.

    1. Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.

    We will make our claims more conservative, and discuss this point further in the Discussion.

    1. I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?

    The data points towards that Dpn overexpression has two separate roles in regulating the cell cycle. Ofcourse dedifferentiation requires a commitment of neurons into the cell cycle (this we think is still happening), however, we think once these cells have turned on NB markers, they have limited ability to progress through the cell cycle. We will discuss this point in the Discussion.

    1. Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.

    A direct target does not necessarily infer the same phenotype. To assess whether the mechanisms apply to other dedifferentiation models, we will add Lola-RNAi and Nerfin-1 data to our revised manuscript.

    Minor comments:

    1. Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"
    2. In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include
    3. Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"

    We will correct these typos.

    Reviewer #3 (Significance):

    Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.

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    Referee #3

    Evidence, reproducibility and clarity

    The discoveries that the author describe in this manuscript are very specific to dedifferentiated neuroblasts created by UAS-dpn transgene overexpression. Dpn is endogenously expressed in optic lobe neuroblast throughout larval stage, which makes understanding how Dpn regulates gene expression based on the authors results (suppression of cell-cycle genes, and promotion of a specific temporal state) confusing. Therefore, this manuscript does not advance our understanding of regulation of temporal identity and cell cycle progression in optic lobe neuroblasts during normal neurogenesis.

    The author's state:

    "However, beyond the fact that misexpression of these factors and pathways caused the formation of ectopic NBs, whether these dedifferentiated NBs faithfully produce the correct number and types of neurons or glial cells, or undergo timely terminal differentiation, has not been assessed. These characteristics are key determinants of overall CNS size and function, thus are important parameters when considering whether dedifferentiation leads to tumourigenesis or can be appropriately utilized for regenerative purposes."
    at the end of introduction. If this is a true primary goal of this study, the authors should describe it in abstract. Otherwise, readers will lose enthusiasm to read this manuscript in abstract and no longer read the following sections.

    Results

    1. The authors should describe the expression pattern of all three of the Gal4 drivers used. While there are dotted outlines in the supplemental figure, there should be a description in the main text for the expression pattern of these lines which described with temporal state of NBs these lines are expressed in, and whether they are also expressed in the neurons or not.
    2. The authors claim that overexpression of Dpn in the medulla region causes "dedifferentiation." The data provided however is not sufficient to conclude that dedifferentiation is occurring. The GAL4s used all drive in the NBs, and so it is unclear if the ectopic NBs ever became mature neurons. In addition, the lack of ectopic NBs in the clonal analysis 16hrs AHS does not prove that ectopic NBs at 24hrs AHS must have come from "mature neurons." To demonstrate dedifferentiation, the authors should use a driver system that is specific to mature neurons, and then overexpress dpn and look for mira+ cells. Currently, the authors data does not prove that mature neurons dedifferentiatiate into ectopic NBs upon Dpn OE.
    3. What is a conclusion of fig 2C-H?
    4. "As the tumor cell-of-origin can define the competence of tumor NBs to undergo malignancy identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from". This sentence is confusing. What are the authors investigating in the following experiment? Do they want to see ectopic NBs keep their early identity like Chinmo in ventral cord tumor NB? Or tll-positive NB's progenies can dedifferentiate to ectopic NB, but this ectopic neuroblast is not able to keep proliferation in pupal stage? It is hard to understand the connection of this sentence and the following experiment.
    5. The DamID experiment described used wor-gal4 as a driver, which means the Dpn binding profile generated is coming from not only optic lobe NBs, but central brain NBs and VNC NBs as well. In Magadi et al. (2020), the authors profiled Dpn binding in CNS hyperplasia, and found that dpn strongly bound Nerfin-1 and gcm. However, it does not bind cell cycle genes in this context. How do the authors know that the region that they claim are bound by dpn are bound in medulla NBs? The authors should also include tracks to show dpn binding at Nerfin-1, as well as the other tTFs (hth, ey, tll, and gcm). Providing this data will help to understand if Dpn binding is specific to the mid-temporal genes, as Dpn expression is known to be expressed in all medulla NBs regardless of temporal state.
    6. Currently, the DamID data does not help to interpret the Dpn overexpression phenotype at all. Inside of flip-out clone, some cells show Slp-1 expression while others showed D expression. The authors explain that Slp-1 and D suppress their expression to each other. But the DamID data indicate that both Slp-1 and D are Dpn target genes. If this is true, why did they observe the mosaic expression pattern inside of the same clone.
    7. The authors hypothesized if Dpn activated Slp-1directly. Does this mean that Dpn directly activate transcription of Slp-1? It is well known that Dpn is transcriptional repressor. Hes family proteins form a homodimer or heterodimer with another Hes protein and interacts Gro, which recruits a Histon deacetylase protein. The author's claim does not fit to the model what we currently believe. In addition, the authors claimed that Dpn inhibits cell cycle gene transcription directly. This is inconsistent to their claim that Dpn directly activate Slp-1 expression. If the authors want to claim that Dpn has two different functions in this context, the authors must demonstrate it by experimental results.
    8. Related to the above question, I wondered if the authors guess Dpn activate or repress D transcription by binding to D promoter region because they claimed that Dpn activate Slp-1, while suppress cell cycle genes.
    9. I am confused to the claim that Dpn suppress cell cycle genes expression. Dpn overexpression induces dedifferentiation of neuron into NB and re-entry into the cell cycle. If Dpn suppress cell cycle genes how can the dedifferentiated cell re-enter into the cell cycle?
    10. Figure 6 looked redundant because we know Dpn is a direct target of Notch. It is obvious that an upstream factor overexpression can induce the identical phenotype to the phenotype induced by overexpression of a downstream factor.

    Minor comments:

    1. Typo in main text: "GMR31HI08-GAL4" should be "GMR31H08-GAL4"
    2. In figure 1E-H the dotted line regions indicated the clones are not shown in the merge image. Please include
    3. Typo in discussion paragraph 2: "temporal series was no sufficient to rescue cycle cycle progression"

    Significance

    Insights into the developmental capacity of dedifferentiated stem cells will likely lead to novel strategy to replenish cells lost due to aging, injury and diseases in regenerative medicine.

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    Referee #2

    Evidence, reproducibility and clarity

    Stem cells can divide asymmetrically to self-renew the stem cell while generating differentiating sibling cells. To restrict the number and type of differentiating sibling cells, stem cells often undergo terminal differentiation. Terminally differentiated cells can dedifferentiate and revert to a stem cell like fate. However, the underlying molecular mechanisms are incompletely understood in vivo.
    Here, Veen et al., use Drosophila neural stem cells (called neuroblasts) to investigate how terminal differentiation is regulated. Neuroblasts faithfully produce the correct number and type of neuronal cells through temporal patterning and regulated terminal differentiation. The authors show that misexpression of the bHLH transcription factor Deadpan (Dpn) induces ectopic neuroblasts, which predominantly express mid-temporal transcription factors at the expense of late-temporal transcription factors. As a consequence, these ectopic neuroblasts also fail to produce Repo positive glial cells and are stalled in their cell cycle progression. The authors provide evidence that promoting cell cycle progression and overexpression of the transcription factor Dichaete (D) is sufficient to restore the temporal transcription factor series, neuronal diversity and timely neuroblast differentiation.

    This is an interesting study that will be of interest to the stem cell field. However, I encourage the authors to consider the following critiques:

    1. Explain the rationale for the three different neuronal/NB drivers (GMR31HI08-GAL4, eyOK107-GAL4, eyR16F10-GAL4. How are they expressed?
    2. The rationale for the Edu experiment (Figure S1I) is not clear. Why is this a measure for the production of neuronal progeny? For the correct interpretation of these results, the authors should also provide control clones or Edu experiments of regular neuroblasts.
    3. How was % of Mira (Figure 1K and below) or the % of tTFs (Figure 2H onward) quantified? For instance, Figure 2C-G often shows clonal signal that is not highlighted with the dashed lines and the corresponding tTF intensity does not match the intensity in the outlined clone (eg. Figure 2D-D'; a large optic lobe clone is negative for Ey. Figure 2E-E'; an unmarked clone is negative for Slp).
      Similarly, the Hth signal is very weak to begin with so it is unclear how this was quantified. How was determined what constitutes real signal vs. background noise?
      Additional explanations in the methods section is needed to assess the robustness of the data.
    4. This sentence should be rephrased: 'As the tumour cell-of-origin can define the competence of tumour NBs to undergo malignancy (Farnsworth et al., 2015; Narbonne-Reveau et al., 2016), we next tested whether the temporal identity of the dedifferentiated NBs were conferred by the age of the neurons they were derived from.'
      The connection between tumorigenicity and temporal identity is not really clear and should be briefly reintroduced for this paragraph.
    5. Figure 2I-N: The experimental outline in I and J should be grouped with the corresponding images to clarify what is compared. Also, there are no images for the control clones, which make a comparison difficult. The images are also too small. I cannot really see the Hth, or Slp signal in the small clones shown in Figure 2K-L".
    6. Figure 3H: It is not clear why there are only a small group of Nbs that are positive for Mira. Please explain.
    7. Figure 3K-M: Please explain how the Toy signal was measured and quantified.
    8. The TaDa data set is very interesting but the following might be an overstatement: "We found that Dpn directly binds to slp1 as well as the Sox-family TF dichaete (D) which is expressed in medulla NBs after slp1 (Li et al., 2013) (Figure S6 A-B)."
      More direct binding assays might be needed to show that Dpn directly binds to slp1 and D. If this is already shown, clarify the sentence to indicate what is published and what is extracted from the data shown here.
      Also, what is the rationale for this statement: "Consistent with the model that D represses Slp-1..."?
    9. This might be an overinterpretation: D overexpression in UAS-Dpn NBs promoted their pre-mature cell cycle exit at 6 hrs APF using eyR16F10-GAL4. The data shows loss of Mira signal, which could occur through different mechanisms.

    Significance

    These appear to be novel and significant findings that will enhance our understanding of the temporal progression and terminal differentiation program of neural stem cells in vivo.
    I think the findings will be of interest to cell, developmental cell and stem cell biologists.

    My primary expertise is in the cell biology of fly neural stem cells and asymmetric cell division of neuroblasts. Although I am not intimately familiar with the differentiation and differentiation literature, I consider the findings reported here relevant and impactful.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Authors show that overexpression of bHLH transcription factor Dpn in the medullary neurons of the Drosophila optic lobe results in the dedifferentiation of these neurons back into the NBs. These dedifferentiated NBs acquire and maintain mid-temporal identity, express Ey and Slp, and show delayed onset of tTF Tailless (Tll), leading to an excess of neurons of mid-temporal fate at the expense of late temporal fate neurons and glial cells. The dedifferentiated NBs are stalled in the cell cycle and fail to undergo terminal differentiation. Over expression of tTF Dicheate (D) or promoting G1/S transition pushed these NBs to late stages of the temporal series, partly rescuing the neuronal diversity and causing their terminal differentiation. They also show that the dedifferentiation of NBs by Notch hyper-activation also exhibited stalled temporal progression, which is restored by D overexpression.
    Authors suggest that cell cycle regulation and tTF are primary to the proliferation and termination profile of dedifferentiated NBs.
    Using these conclusions, the authors emphasize the need to recreate the right temporal profile and ensure appropriate cell cycle progression to use dedifferentiated NSC for regenerative purposes or prevent tumorigenesis originating from differentiated cell types.

    Major comments:

    • Are the key conclusions convincing?

    Most conclusions are convincing; however, some issues are pointed out below.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    The authors have overexpressed Dpn and shown that medulla neurons dedifferentiate to NBs, similar to the loss of function phenotype seen for the Nerfin-1 of which Dpn is a target. They also show that temporal series progression defect is also seen in the case of dedifferentiated NB generated by Notch over-activation.
    Using these two examples, the authors suggest that for dedifferentiated NSC, which are to be used for the regenerative purpose, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately. Authors also claim that to prevent tumorigenesis originating from differentiated cell types, one needs to recreate the right temporal profile and ensure cell cycle progression occurs appropriately.

    While I agree with this, I think this is an overreaching conclusion based on just these two examples. If they could show the same for one more method of dedifferentiation (For, e.g. Lola) happening in medulla neurons which happens by a mechanism independent of Nerfin-1, Dpn, Notch axis, the argument will become more convincing and broad.
    Also when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019), they do so in the discussion, but mentioning it here gives a broader context to the reader.

    Another important point that needs a mentioned here is that conclusions are based on dedifferentiation happening in the medulla neurons, which are considered less stable since they lack Prospero. Therefore whether this conclusion can be generalized for all the tumors arising from dedifferentiation in the CNS (eg, those arising from NICD activation in the central brain or thoracic region of the VNC) is another concern. Maybe authors can consider making a more conservative claim.
    Generalizing this conclusion to Prospero expressing NBs lies outside the scope of the current study and cannot be addressed here because central brain Type-I NBs use a different set of tTFs.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Experiments with Lola knockdown/mutants in medulla neurons can be done quickly, in my opinion, and will substantiate this claim.
    Another obvious question that comes to mind is if medulla neurons dedifferentiate on overexpression of Dpn, does the same happen in nerfin-1 mutant clones as well? And if yes, why has the author not done similar experiments for nerfin-1 mutants.
    Please show Ey staining in Fig-2 if possible, it will also help to add a line on why Slp was used as marker for mid tTFs instead of Ey.
    In Model shown in last figure Dpn is shown to repress D and activate Slp. Can authors show that Dpn overexpression represses D and activate Slp either by antibody staining or by RT PCR.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Experiments with Lola and nerfin-1 mutants can be done in a few months. I cannot comment on the cost involved.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Replication and statistical analysis are fine. The activated Notch experiments show only three data points in all the experiments. It will be good to increase this number.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    There is a problem with Fig-5F (both 5E and 5F have % EdU in clone/ % Mira in the clone as y-axis), I do not understand how the Fig-5F let them conclude that D overexpression increases the rate of neuronal production.

    In one place, the authors conclude, "Together, this data suggests that it is likely that cell cycle progression lies upstream of the temporal series, to promote the generation of neurons". Authors should consider adding "medulla NBs" at the end of the sentence since cell cycle progression being upstream of temporal series is already known in Type-I NBs, as pointed out by authors as well (Ameele and Brand 2019).

    In the discussion authors says that "Our data support the possible links between cell cycle progression and the expression of temporal regulators controlling NB proliferation and cellular diversity". This is new information, as the 2019 study did not show how cell diversity changes with a changed tTF profile. I think the authors should elaborate on this point to highlight how this is different from what is already known from the 2019 study (done in the context of Type-I NBs).
    Maybe they need to highlight that the cell cycle directs/regulates the progression of temporal series compared to the earlier observation where temporal series was shown to be downstream of the cell cycle.

    In fig-3J in clones even after 24 AHS, Dpn continues to be overexpressed but these cells undergo terminal differentiation, can authors comment why is it so?
    In one place authors say, "To better assess the cumulative effect of the neurons made throughout development, EyOK107-GAL4 was used to drive the expression of Dpn" maybe some background on why use this specific GAL4.
    Also a line about why GMR31HI08-GAL4 eyOK107-GAL4 and and eyR16F10-GAL4 were used.

    • Are prior studies referenced appropriately ?

    Yes, but in a few places, some references can be added.
    An important point that needs to be mentioned for the context is the medulla neurons do not use Prospero for terminal differentiation and are thus considered less stable (DOI: 10.1242/dev.141341).
    In discussion, the authors say that "It would be interesting to explore whether N similarly acts on these target genes to specify cell fate and proliferation profiles of dedifferentiated NBs." There is a study looking at Notch targets in NB hyperplasia (DOI: 10.1242/dev.126326); whether that study shows if any of the cell cycle genes are downstream of activated Notch, needs a mention here.
    Also, when authors mention N mediated dedifferentiation, they need to inform that Dpn is a direct target of Notch in NBs (Doi. 10.1016/j.ydbio.2011.01.019). They do so in the discussion, but mentioning it in the introduction or results will give a broader context to the reader.
    Another gene that needs a mention is "Brat", which regulates both Dpn and Notch, and causes dedifferentiation and tumors in CNS, I think this gene and its interaction with Dpn and Nerfin and Notch needs to be discussed either in the introduction or discussion.

    • Are the text and figures clear and accurate?

    The main figures are not labeled. Therefore, it was very annoying to deduce the specific figure numbers.
    There are 1 or 2 places where figure calling is wrong in the text.
    The Image Fig-5I shows cycD and CDK4 at the G2-M transition; while the text says it supports G1/S, which is indeed the case, the figure needs modification.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    The presentation is okay, in my opinion.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The factors leading to dedifferentiation of the neurons have been identified previously by groups of Chris Doe (mldc, DOI: 10.1242/dev.093781), Andrea brand (10.1016/j.devcel.2014.01.030.) as well as the authors of this paper (10.1101/gad.250282.114, 10.1016/j.celrep.2018.10.038.). However, many questions remained unaddressed regarding such NB generated from neuronal dedifferentiation. For example, whether these cells contribute to native cell diversity of the CNS, undergo timely differentiation or their progeny cells incorporated into appropriate circuits is not well understood. Successful execution of these phenomena is critical for generating functional CNS and such insights are crucial for understanding the origin of tumorigenesis in CNS or employing dedifferentiated NSC for regenerative purposes.

    This study is an overexpression-based study, however, some of the results give significant conceptual insights into the tumors arising out of the dedifferentiation of the neurons. It also gives insights into the fact that the dedifferentiated cells need to be carefully examined for the temporal factor profile before they can be employed for regeneration or any therapy targeting them.
    However, in my opinion, they need to test this idea at least in one more system of neuronal dedifferentiation, preferably independent of the nerfin-1/Notch/Dpn axis to generalize this claim.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    Cerdic Maurange's group had looked at the role of temporal factors and identified the early phase of malignant susceptibility in Drosophila in 2016 (doi: 10.7554/eLife.13463). Andrea Brand's group has shown in a 2019 paper that cell cycle progression is essential for temporal transition in NBs (doi: 10.7554/eLife.47887). Both these studies were in the context of Type-I NBs, which express Prospero, which is crucial for the differentiation of the neurons.
    Previously the authors have studied type-I NBs and shown by Targeted DamID that Dpn is Nerfin-1 target. They also show that Nerfin-1 mutants show dedifferentiation of neurons. They follow up on this observation in medulla neurons, where they find that Dpn overexpression results in their dedifferentiation into medulla NBs. Medulla NBs differ from Type-I NBs in using a separate set of tTFs. Also, Type-I NB and neurons arising from them use Prospero for terminal differentiation, while medulla neurons do not express Prospero and are therefore considered less stable (DOI: 10.1242/dev.141341).

    The importance of the study lies in the results that show that the NB arising out of dedifferentiation of medulla neurons takes up mid-temporal fate. These NBs are stalled in Slp expressing mid-temporal stage unless the cell cycle is promoted by overexpression of cell cycle genes regulating G1/S transition.
    Authors also show that overexpression of D promotes the progression of temporal series in these dedifferentiated NBs, which could partly rescue neuronal diversity and result in terminal differentiation. Thus D plays an important role in determining the type of neurons these NBs generated. This suggests that knowing the tTF profile of these types of dedifferentiated NBs is vital if these cells were to be used for regenerative purposes. Authors further claimed that cell cycle regulation and tTFs are critical determinants of the proliferation and termination profile of dedifferentiated NBs.

    • State what audience might be interested in and influenced by the reported findings.

    The study will be of broader interest to researchers interested in central nervous system patterning, regeneration, and cancer biology.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Drosophila, central nervous system patterning and cell fate determination of neural stem cells.

  5. Version published to 10.1101/2022.07.24.501087 on bioRxiv