A Notch-dependent transcriptional mechanism controls expression of temporal patterning factors in Drosophila medulla

  1. Alokananda Ray
  2. Xin Li

  • Curated by eLife

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    Evaluation Summary:

    In Drosophila neural progenitors (neuroblasts), sequentially-expressed transcription factors (known as temporal transcription factors) ensure the generation of various types of neurons and glia as they divide. However, the mechanisms regulating and finetuning the speed of temporal factor transitions has remained unclear and under-investigated. Here the authors concentrate on a specific temporal transition occurring in medulla neuroblasts and demonstrate that lineage-intrinsic Notch signaling facilitates this transition via at least two identified enhancers. This work provides important insights on the signals and mechanisms that promote temporal transitions in neural progenitors, and therefore regulate cellular diversity in the brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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Abstract

Temporal patterning is an important mechanism for generating a great diversity of neuron subtypes from a seemingly homogenous progenitor pool in both vertebrates and invertebrates. Drosophila neuroblasts are temporally patterned by sequentially expressed Temporal Transcription Factors (TTFs). These TTFs are proposed to form a transcriptional cascade based on mutant phenotypes, although direct transcriptional regulation between TTFs has not been verified in most cases. Furthermore, it is not known how the temporal transitions are coupled with the generation of the appropriate number of neurons at each stage. We use neuroblasts of the Drosophila optic lobe medulla to address these questions and show that the expression of TTFs Sloppy-paired 1/2 (Slp1/2) is directly regulated at the transcriptional level by two other TTFs and the cell-cycle dependent Notch signaling through two cis -regulatory elements. We also show that supplying constitutively active Notch can rescue the delayed transition into the Slp stage in cell cycle arrested neuroblasts. Our findings reveal a novel Notch-pathway dependent mechanism through which the cell cycle progression regulates the timing of a temporal transition within a TTF transcriptional cascade.

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  1. Version published to 10.7554/elife.75879 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Neural stem cells express cascades of transcription factors that are important for generating the diversity of neurons in the brain of flies and mammals. In flies, nothing is known about whether the transcription factor cascades are build from direct gene regulation, e.g. factor A binding to enhancers in gene B to activate its expression. Here, Xin and Ray show that one temporal factor, Slp1/2, is regulated transcriptionally via two molecularly defined enhancers that directly bind two other transcription factors in the cascade as well as integrating Notch signaling. This is a major step forward for the field, and provides a model for subsequent studies on other temporal transcription factor cascades.

    Thanks for the positive comments!

    Reviewer #2 (Public Review):

    The manuscript addresses an important question concerning the mechanisms regulating temporal transitions in Drosophila neural progenitors called neuroblasts. Here, they concentrate on a specific transition between the transcription factors Ey and Slp1/2 that are sequentially expressed within a cascade involving at least 6 temporal transcription factors. Using a combination of new transgenes, bioinformatics and genome-wide profiling of transcription factor biding sites (Dam-ID), they functionally characterize two enhancers of the Slp1/2 genes that are active during this transition. This led to the identification of the Notch pathway as an important facilitator of the transition. They also show that Notch signaling requires cell cycle progression and that Slp1/2 is a direct target of Ey, validating the importance of transcriptional cross-regulatory interactions among the temporal transcription factors to trigger progression.

    In my opinion, the study is very interesting, representing the first careful analysis of enhancers involved in temporal transitions in neural progenitors, and leading to new insights into the mechanisms promoting temporal progression.

    Thanks for the positive comments!

    Reviewer #3 (Public Review):

    In this manuscript, the authors present data to suggest that transcriptional activation of the Slp1/2 temporal factors in the medulla neuroblasts of the developing Drosophila optic lobe is dependent on two enhancer elements. The authors concluded that these two enhancers were able to be activated by Ey and Scro, two other factors identified to be involved in the temporal cascade of the medulla NB. The authors show that cell cycle progression is necessary for Notch signaling, and that Notch signaling activates and sustains the temporal transcription factor cascade. The authors use GFP reporter assays to correlate the enhancer activity to Slp1/2 expression and used DamID to show in-vivo binding of Su(H) and Ey to the enhancer fragments.

    I agree with the authors that it is important to define the mechanisms by which Notch, cell cycle control and these temporal transcription factors function through their cis-regulatory elements to establish this self-propagating cascade to generate diverse cell types during neurogenesis. However, the findings in this study offer limited new insights toward reaching this goal for a myriad of reasons. First, studies in invertebrate and vertebrate neurogenesis have agreed on the conceptual framework that transcriptional control plays a key role in regulating the generation of diverse cell types. The data showing the patterns of slp1/2 transcript simply reaffirm the proposed model as well as recently published single-cell transcriptomic analyses of fly optic lobe neuroblasts. Second, it remains unclear how physiologically relevant the enhancer analyses presented in this study are to the regulation of Slp1/2 expression, as the data can only suggest that they act redundantly to each other. It is also troubling to see that mutating binding sites of a single transcription factor appears to completely abolish enhancer activity while Slp1/2 protein expression is delayed in mutant clonal analyses. Third, the authors do not offer any explanation for how Notch signaling contributing to the timing of Slp1/2 expression, considering that Notch signaling should be active during the entire life of the neuroblast based on canonical Notch target gene expression. What action do Ey and Scro play in this timely enhancer activation as both appear to be necessary to activate the enhancers along with Notch. Fourth, many studies including the Okamoto et al., 2016 study cited in this study have contributed to our appreciation of the role of proper cell cycle control in promoting generation of diverse neurons in vertebrate neurogenesis. It is unclear to me if findings from the current study contribute to significant advancement on this regulatory link.

    Thanks for raising these concerns. Here are our responses:

    First, we agree that there have been great advances in this field including classical studies in the ventral nerve cord, recent studies on type II lineages and medulla including our own scRNA-seq study of medulla neuroblasts. These studies have revealed the sequential expression of transcription factors in neuroblasts of different ages, and proposed that these transcription factors form a transcriptional cascade based on the cross-regulations among them. However, these cross-regulations were based on mutant phenotypes, and in most cases, the cis-regulatory elements of these TTFs have not been characterized, and it hasn’t been studied whether these cross-regulations are direct or not. Little is known about exactly how the timing of the transition is regulated and coordinated with cell-cycle control. We have addressed these questions and identified two enhancer elements for slp1/2, and demonstrated that the previous TTF Ey, another TTF Scro, and Notch signaling directly regulate slp expression. Further we demonstrated that Notch signaling is dependent on cell cycle progression in neuroblasts, and supplying Notch signaling rescues the delay in Slp expression in cell cycle mutants. We believe this study has provided important insights in this field and is another step forward.

    Second, now we provide evidence that deletion of both enhancers specifically abolished Slp1 and Slp2 expression in medulla neuroblasts.

    Regarding the concerns about binding site mutation:

    1. Ey: With loss of Ey, Slp is completely lost. The Ey binding site mutation phenotype is consistent with loss of Ey phenotype.

    2. Su(H): For the u8772 250bp enhancer, mutating all four predicted Su(H) binding sites did abolish the reporter expression. During the revision, we generated another construct, in which we mutated the two predicted Su(H) binding sites which are perfect matches to the consensus, and found that this dramatically reduced the reporter expression. For the d5778 850bp enhancer, mutation of Su(H) binding sites caused strong glial expression which prevented us to precisely assess the neuroblast expression. Thanks to the excellent advice from review #3, we used repo-Gal4 and GFP-RNAi to remove the glial expression. This approach turned out very informative. We found that mutation of four or six out of six predicted Su(H) binding sites actually did not decrease the reporter expression in neuroblasts, suggesting that Notch signaling does not active the d5778 850bp enhancer through these binding sites. However, we think this is the explanation why this enhancer drives a delayed expression comparing to the 220bp enhancer and the endogenous Slp. In addition, this also explains why with loss of Notch signaling, endogenous Slp expression is only delayed but not completely lost. This is because although the 220bp enhancer driven expression is completely lost, the d5778 850 bp enhancer still directs a delayed expression of Slp and this expression is not dependent on Notch signaling.

    3. Scro: Mutation of Scro binding sites caused a decreased expression level of the reporter, consistent with the scro RNAi phenotype on Slp, which is a decreased expression level.

    Third, regarding how Notch signaling which is active in the entire neuroblast life, can act to activate Slp expression in a specific time We tested genetic interactions between Ey, Scro, and Notch in the regulation of Slp expression. We found that with loss of Ey, supplying constitutive active Notch or Scro is not sufficient to rescue Slp expression. Thus Ey as the previous TTF, may be required to prime the slp locus, so that Notch signaling and Scro can act to further activate Slp expression. Therefore, Notch signaling requires Ey to specifically further activate Slp at the correct time. We have added these experimental results and discussion.

    Fourth, the Okamoto et al., 2016 study actually concluded that cell cycle progression is not required for the temporal progression. In their experimental setup, they supply Notch to maintain the un-differentiated status of cortical neural progenitors when they block cell cycle progression. The observed that temporal transition still happened, and they concluded that cell cycle progression is not required for temporal transitions. However, they didn’t consider the possibility that Notch signaling, which is itself dependent on cell cycle progression, actually rescued the possible phenotype caused by arrest of cell cycle progression. Our result demonstrated that in Drosophila medulla, supplying Notch signaling can rescue the delay in the transition to the Slp stage in cell-cycle arrested neuroblasts, and further showed that the mechanism is by direct transcriptional regulation. We believe that publication of our results will be informative to the vertebrate study, promoting vertebrate researchers to re-consider the role of cell cycle progression and Notch signaling in temporal progression.

  3. Version published to 10.1101/2021.10.29.466469v4 on bioRxiv
  4. eLife

    Evaluation Summary:

    In Drosophila neural progenitors (neuroblasts), sequentially-expressed transcription factors (known as temporal transcription factors) ensure the generation of various types of neurons and glia as they divide. However, the mechanisms regulating and finetuning the speed of temporal factor transitions has remained unclear and under-investigated. Here the authors concentrate on a specific temporal transition occurring in medulla neuroblasts and demonstrate that lineage-intrinsic Notch signaling facilitates this transition via at least two identified enhancers. This work provides important insights on the signals and mechanisms that promote temporal transitions in neural progenitors, and therefore regulate cellular diversity in the brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

  5. eLife

    Reviewer #1 (Public Review):

    Neural stem cells express cascades of transcription factors that are important for generating the diversity of neurons in the brain of flies and mammals. In flies, nothing is known about whether the transcription factor cascades are build from direct gene regulation, e.g. factor A binding to enhancers in gene B to activate its expression. Here, Xin and Ray show that one temporal factor, Slp1/2, is regulated transcriptionally via two molecularly defined enhancers that directly bind two other transcription factors in the cascade as well as integrating Notch signaling. This is a major step forward for the field, and provides a model for subsequent studies on other temporal transcription factor cascades.

  6. eLife

    Reviewer #2 (Public Review):

    The manuscript addresses an important question concerning the mechanisms regulating temporal transitions in Drosophila neural progenitors called neuroblasts. Here, they concentrate on a specific transition between the transcription factors Ey and Slp1/2 that are sequentially expressed within a cascade involving at least 6 temporal transcription factors. Using a combination of new transgenes, bioinformatics and genome-wide profiling of transcription factor biding sites (Dam-ID), they functionally characterize two enhancers of the Slp1/2 genes that are active during this transition. This led to the identification of the Notch pathway as an important facilitator of the transition. They also show that Notch signaling requires cell cycle progression and that Slp1/2 is a direct target of Ey, validating the importance of transcriptional cross-regulatory interactions among the temporal transcription factors to trigger progression.

    In my opinion, the study is very interesting, representing the first careful analysis of enhancers involved in temporal transitions in neural progenitors, and leading to new insights into the mechanisms promoting temporal progression.

  7. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors present data to suggest that transcriptional activation of the Slp1/2 temporal factors in the medulla neuroblasts of the developing Drosophila optic lobe is dependent on two enhancer elements. The authors concluded that these two enhancers were able to be activated by Ey and Scro, two other factors identified to be involved in the temporal cascade of the medulla NB. The authors show that cell cycle progression is necessary for Notch signaling, and that Notch signaling activates and sustains the temporal transcription factor cascade. The authors use GFP reporter assays to correlate the enhancer activity to Slp1/2 expression and used DamID to show in-vivo binding of Su(H) and Ey to the enhancer fragments.

    I agree with the authors that it is important to define the mechanisms by which Notch, cell cycle control and these temporal transcription factors function through their cis-regulatory elements to establish this self-propagating cascade to generate diverse cell types during neurogenesis. Further work will be needed to offer new insights toward reaching this goal.

    First, studies in invertebrate and vertebrate neurogenesis have agreed on the conceptual framework that transcriptional control plays a key role in regulating the generation of diverse cell types. The data showing the patterns of slp1/2 transcript reaffirm the proposed model as well as recently published single-cell transcriptomic analyses of fly optic lobe neuroblasts.

    Second, it remains unclear how physiologically relevant the enhancer analyses presented in this study are to the regulation of Slp1/2 expression, as the data can only suggest that they act redundantly to each other. It is also concerning to see that mutating binding sites of a single transcription factor appears to completely abolish enhancer activity while Slp1/2 protein expression is delayed in mutant clonal analyses.

    Third, the authors do not explain how Notch signaling contributes to the timing of Slp1/2 expression, considering that Notch signaling should be active during the entire life of the neuroblast based on canonical Notch target gene expression. What action do Ey and Scro play in this timely enhancer activation as both appear to be necessary to activate the enhancers along with Notch?

    Fourth, many studies including the Okamoto et al., 2016 study cited in this study have contributed to our appreciation of the role of proper cell cycle control in promoting generation of diverse neurons in vertebrate neurogenesis. It is unclear to me how the findings from the current study contribute to significant advancement on this regulatory link.

  8. Version published to 10.1101/2021.10.29.466469v3 on bioRxiv
  9. Version published to 10.1101/2021.10.29.466469v2 on bioRxiv
  10. Version published to 10.1101/2021.10.29.466469v1 on bioRxiv