Chromatin priming and Hunchback recruitment integrate spatial and temporal cues in Drosophila neuroblasts

  1. Ayanthi Bhattacharya
  2. Hemalatha Rao
  3. Sonia Q Sen

  • Curated by eLife

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    eLife assessment

    The study provides an important advance towards understanding how spatial and temporal transcriptional programs are integrated to regulate lineage-specific chromatin and enhancer activation. The functional evidence is currently incomplete, but the current data provide a solid correlative and conceptual foundation. Functional experiments directly linking Gsb occupancy to chromatin state and regulation of some lineage-specific targets would further strengthen the causal interpretation of the model. Clarifying the scope of conclusions and explicitly acknowledging the technical limitations of current chromatin assays would provide a more balanced interpretation of the manuscript.

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Abstract

Neural stem cells generate diverse cell types by integrating spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. In Drosophila neuroblasts (NBs), spatial patterning sets lineage identity, while a temporal transcription factor (TTF) cascade sets birth order. Two proposed mechanisms could integrate these inputs. In direct regulation, spatial transcription factors (STFs) and TTFs co-occupy and activate TS enhancers within NBs. In epigenetic regulation, STFs first prime NB-specific chromatin, creating ‘sites of integration’ (SoIs) that later recruit TTFs.

We test this in two identified NBs — NB5-6 and NB7-4 — and their candidate STFs, Gooseberry (Gsb) and Engrailed (En), together with the first TTF, Hunchback (Hb). In NB5-6, Gsb is expressed transiently, suggesting a chromatin-based memory of its activity. In NB7-4, En expression persists throughout development so integration could either be epigenetic or direct. We used chromatin engagement by the STFs as the discriminator between these models. If integration is epigenetic, the STF must engage less-accessible chromatin to establish NB-specific SoIs; if regulation is direct, the STF need not.

We find that En binds only to pre-accessible loci in NB7-4 and En+Hb co-binding marks the most accessible enhancers. This suggests that NB7-4 likely relies on an unknown priming factor to establish SoIs, with direct En–Hb co-binding mediating enhancer activation.

In NB5-6, Gsb binds both open and less-accessible chromatin and Gsb+Hb co-binding marks the most accessible enhancers. When ectopically expressed, Gsb remodels chromatin globally in the non-cognate NB7-4, and at endogenous NB7-4 SoIs, it specifically reduces accessibility as well as Hb binding. This suggests that in NB5-6 Gsb likely acts together with other NB5-6–specific factors to recognize less-accessible chromatin and to promote Hb recruitment while restricting Hb occupancy to appropriate enhancers. Together these findings support a unified two-step model: NB-specific combinations of TFs — each NB’s “STF code” — first prime chromatin and then recruit and restrict Hb to ensure lineage-specific enhancer activation.

Article activity feed

  1. eLife

    eLife assessment

    The study provides an important advance towards understanding how spatial and temporal transcriptional programs are integrated to regulate lineage-specific chromatin and enhancer activation. The functional evidence is currently incomplete, but the current data provide a solid correlative and conceptual foundation. Functional experiments directly linking Gsb occupancy to chromatin state and regulation of some lineage-specific targets would further strengthen the causal interpretation of the model. Clarifying the scope of conclusions and explicitly acknowledging the technical limitations of current chromatin assays would provide a more balanced interpretation of the manuscript.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    It has long been known that Drosophila embryonic ventral nerve cord neuroblasts incorporate both spatial and temporal transcription factor expression to generate 30 distinct neuroblasts and lineages per hemisegment. This manuscript aims to elucidate the mechanism by which this integration of spatial and temporal transcription factors occurs through "direct regulation" or "epigenetic regulation". Direct regulation is defined as both spatial and temporal factors binding to open chromatin and working together to dictate specific lineages. Epigenetic regulation is defined as a spatial factor priming the chromatin in a neuroblast-specific manner to allow for the integration of temporal factors to generate specific lineages. The authors conclude that there is a two-step model in which a spatial transcription factor code "primes" the chromatin in terms of accessibility and then recruits temporal factors to ensure lineage-specific enhancer activation.

    Strengths:

    The authors tested two models, "direct regulation" vs "epigenetic regulation" in a well-defined pool of neural stem cells during normal development.

    Weaknesses:

    The data in this study cannot clearly substantiate these two models.

    Overall, there are a number of issues that are inconsistent and not supportive of the model proposed in this manuscript. Firstly, there is no evidence of pioneer factor activity in any of the NB lineages described - i.e., any changes in chromatin accessibility being shown over time. The authors must show chromatin conformation changes during the window of spatial transcription factor expression in order to convince the readers of this phenomenon. Secondly, the phenotypic data do not align with the sequencing data - the story would be more cohesive if the sequencing data and phenotypic data were in the same NB subtypes. On one hand, we are shown that Gsb misexpression induces loss of chromatin accessibility in NB 7-4, however in the widespread loss model, we are not shown a phenotype in these NB7-4 - which suggest that the chromatin accessibility at these sites (sites that have already been distinguished as SoIs for that NB subtype) does not play an important role in distinguishing NB 7-4 identity. However, the authors report loss of NB3-5 identity but have no evidence as to how the chromatin has changed (or if it has at all) in that subtype, leaving the readers to wonder how the loss of identity occurred.

  3. eLife

    Reviewer #2 (Public review):

    Summary:

    This article by Bhattacharya et al. investigates how neural stem cells (NSCs, NBs) in Drosophila integrate spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. Prior to this work, it was understood that NSCs utilize spatial transcription factors (STFs) and temporal transcription factors (TTFs) to determine lineage identity and birth order, but the mechanisms of integration were not fully elucidated. The authors employed chromatin profiling techniques to analyze the binding of STFs and TTFs in two specific neuroblast lineages, NB5-6 and NB7-4. They found that Gsb (an STF) binds both accessible and less-accessible chromatin in NB5-6, while En (another STF) binds only to pre-accessible chromatin in NB7-4. The findings support an "STF code" where the combination of pioneer and non-pioneer spatial factors, along with temporal factors, triggers neuroblast-specific enhancer activation and determines lineage identity.

    Strengths:

    The experiments are well-executed, the interpretations are generally sound, and the figures are clear and elegant. However, some conclusions are drawn too broadly without essential functional data. Therefore, additional work is needed to more effectively convey the central message.

    Weaknesses:

    (1) Integration of TaDa and functional data on Gsb for the STF model

    The authors demonstrate that TaDa profiling maps Gsb binding across the genome and identifies candidate chromatin-priming sites in NB5-6. Gsb LOF/GOF experiments reveal effects on NB identity. Combining TaDa data with LOF and GOF analyses indicates that Gsb influences NB5-6 specification by binding to both open and relatively closed chromatin, helping maintain NB5-6 identity while limiting NB3-5 fate.

    However, the study does not establish a direct link between specific LOF/GOF phenotypes and particular genomic targets. For instance, analyzing Gsb occupancy at lineage-specific identity factors or terminal selector genes (such as Lbe, Ap, or Eya for NB5-6; and Ems, etc., for NB3-5) in wild-type and manipulated conditions (Gsb misexpression) would directly connect chromatin binding to the regulation of fate determinants. These investigations would strengthen the mechanistic connection between the correlative TaDa profiles and the observed identity changes, supporting the idea that Gsb functions as a context-dependent chromatin-priming factor within the STF code, rather than as a generic transcription factor.

    (2) Gsb misexpression reveals bidirectional chromatin remodelling

    Experiments with ectopic Gsb expression demonstrate bidirectional chromatin remodeling in NB7-4, showing decreases in accessibility at some binding sites and increases at others. While the authors show that Gsb can disrupt chromatin upon misexpression, interpreting its "pioneer-like" or chromatin-priming activity is complex due to several factors: the misexpression occurs in a non-native lineage, the direct versus indirect effects rely on whole-embryo Dam-Gsb peaks instead of NB7-4-specific binding, and heat-shock-induced chromatin changes are not fully accounted for. These issues make it challenging to definitively determine Gsb's role in chromatin priming.

    A complementary approach would be to perform Gsb knockdown/loss-of-function in its native NB5-6 lineage and profile chromatin accessibility (TaDa or CATaDa). This would allow a cleaner, more physiologically relevant assessment of Gsb's contribution to priming, SoI establishment, and Hb recruitment. Such an experiment would strengthen the causal link between Gsb occupancy and chromatin state and clarify whether Gsb truly acts as a context-dependent pioneer in vivo, rather than producing indirect effects due to ectopic misexpression.

    (3) En is not a pioneer factor

    The authors conclude that Engrailed (En) is not a pioneer factor, based on the observation that En binding correlates with accessible chromatin and that En is not enriched at NB5-6-specific SOIs. However, this conclusion is not sufficiently supported by the functional data.

    First, the absence of En binding at NB5-6-specific SOIs does not necessarily indicate an inability to engage closed chromatin. These regions were not selected for the presence of En consensus motifs, so their lack of occupancy may simply reflect the absence of En binding motifs rather than a lack of pioneering capacity. A systematic motif analysis at NB5-6-specific SOIs is needed to determine whether En binding sites are present but unoccupied.

    Second, the claim that En lacks pioneer activity relies solely on a single steady-state TaDa/DamID occupancy assay at one developmental stage. Because pioneer factor interactions can be transient, low-affinity, and stage-specific, such binding may not be detected by TaDa, which also depends on local GATC density and methylation kinetics and may yield false negatives. Given these technical limitations, the absence of En binding at less accessible regions does not definitively rule out a priming role.

    In the absence of direct functional assays (En LOF/GOF), the authors should explicitly acknowledge these technical and conceptual limitations and tone down the claim that "En lacks pioneer activity".

    (4) Clarity of STF-code Model and Central Message

    The manuscript begins by presenting two models, direct and epigenetic, but the central takeaway of the paper is not clear. Specifically, the nuanced roles of the spatial factors Gsb and En as chromatin-priming versus stabilizing/effector factors within an STF code, and the resulting division of labor, are not clearly illustrated. The distinction between Gsb as a chromatin-priming factor and En as a cofactor-dependent activator/stabilizer should be explicitly presented in a stepwise model for better clarity. The authors could strengthen this by providing a schematic with two sequential stages illustrating how neuroblast identity factors (STF code) change chromatin states to drive lineage-specific enhancer activation. The schematic can be shown from the neuroectoderm to individual NB lineages to make it more panoramic.

    (5) Identification of Priming Factors in NB7-4

    While the authors suggest that an unknown priming factor might be responsible for establishing sites of integration in NB7-4, they do not identify or explore potential candidates for this role. Further investigation into what factors might be involved in chromatin priming in NB7-4 could provide a more complete understanding of the mechanisms at play.

    (6) Functional Validation of STF Code Components

    The study proposes an STF code for each neuroblast lineage, but the specific components of these codes, beyond Gsb and En, are not fully explored. Identifying and validating additional factors that contribute to the STF code in each lineage could strengthen the conclusions.

  4. eLife

    Author Response:

    eLife assessment:

    The study provides an important advance towards understanding how spatial and temporal transcriptional programs are integrated to regulate lineage-specific chromatin and enhancer activation. The functional evidence is currently incomplete, but the current data provide a solid correlative and conceptual foundation. Functional experiments directly linking Gsb occupancy to chromatin state and regulation of some lineage-specific targets would further strengthen the causal interpretation of the model. Clarifying the scope of conclusions and explicitly acknowledging the technical limitations of current chromatin assays would provide a more balanced interpretation of the manuscript.

    We thank the reviewers and editors for their comments on our manuscript. We address here the concerns raised by them.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    It has long been known that Drosophila embryonic ventral nerve cord neuroblasts incorporate both spatial and temporal transcription factor expression to generate 30 distinct neuroblasts and lineages per hemisegment. This manuscript aims to elucidate the mechanism by which this integration of spatial and temporal transcription factors occurs through "direct regulation" or "epigenetic regulation". Direct regulation is defined as both spatial and temporal factors binding to open chromatin and working together to dictate specific lineages. Epigenetic regulation is defined as a spatial factor priming the chromatin in a neuroblast-specific manner to allow for the integration of temporal factors to generate specific lineages. The authors conclude that there is a two-step model in which a spatial transcription factor code "primes" the chromatin in terms of accessibility and then recruits temporal factors to ensure lineage-specific enhancer activation.

    We thank the reviewer for this clear and succinct summary and for accurately capturing the central idea of the model we propose. In particular, we appreciate that the reviewer highlights the distinction between the previously proposed “direct regulation” and “epigenetic regulation” models, which our work suggests may operate together within neuroblast lineages through a combinatorial spatial transcription factor code.

    Strengths:

    The authors tested two models, "direct regulation" vs "epigenetic regulation" in a well-defined pool of neural stem cells during normal development.

    We thank the reviewer for recognizing this aspect of the study.

    Weaknesses:

    The data in this study cannot clearly substantiate these two models.

    Overall, there are a number of issues that are inconsistent and not supportive of the model proposed in this manuscript. Firstly, there is no evidence of pioneer factor activity in any of the NB lineages described - i.e., any changes in chromatin accessibility being shown over time. The authors must show chromatin conformation changes during the window of spatial transcription factor expression in order to convince the readers of this phenomenon.

    Thank you for raising this point. In most studies, pioneer or chromatin-priming activity is inferred from a transcription factor’s ability to bind regions of relatively low accessibility and to remodel chromatin upon perturbation, rather than from direct developmental time-course measurements of chromatin accessibility.

    In our study we provide two lines of evidence consistent with such activity. First, TaDa profiling shows that Gsb occupies both accessible loci and regions that are relatively less accessible in NB5-6. Second, ectopic expression of Gsb in the non-cognate NB7-4 lineage results in clear chromatin remodelling, with loci both gaining and losing accessibility (Fig. 6). These perturbation experiments demonstrate that Gsb is sufficient to alter chromatin accessibility in vivo and therefore support a chromatin-priming role for it.

    We agree that a developmental time-course would be very informative. The difficulty is that, in this system, the relevant sequence unfolds extremely rapidly and across two different cellular contexts. Spatial transcription factors such as Gsb are expressed in the neuroectoderm, neuroblasts are then specified and delaminate, and Hb expression begins almost immediately after NB formation — on the order of minutes to tens of minutes. Before delamination there is no neuroblast to target with NB-specific drivers, and once the NB forms the temporal program is already underway. More generally, resolving chromatin accessibility changes across this transition would require temporally precise profiling at very high resolution in vivo, likely with live or near-live methods, and is not feasible with the Dam-based lineage-restricted approaches currently available.

    Secondly, the phenotypic data do not align with the sequencing data - the story would be more cohesive if the sequencing data and phenotypic data were in the same NB subtypes. On one hand, we are shown that Gsb misexpression induces loss of chromatin accessibility in NB 7-4, however in the widespread loss model, we are not shown a phenotype in these NB7-4 - which suggest that the chromatin accessibility at these sites (sites that have already been distinguished as SoIs for that NB subtype) does not play an important role in distinguishing NB 7-4 identity. However, the authors report loss of NB3-5 identity but have no evidence as to how the chromatin has changed (or if it has at all) in that subtype, leaving the readers to wonder how the loss of identity occurred

    Thank you for raising this point regarding the alignment between the chromatin and phenotypic analyses. The reviewer’s comment made us realise that the rationale for these experiments may not have been sufficiently clear in the original manuscript and could therefore be perceived as misaligned. We therefore explain the logic of the experimental design here and will edit the manuscript in the revision to clarify this point for readers.

    The chromatin experiments were designed to test whether Gsb is capable of remodelling chromatin when introduced into a non-cognate lineage. For this purpose, NB7-4 provided a suitable lineage with clean genetic access for TaDa/CATaDa experiments, allowing us to assess whether ectopic Gsb expression can alter chromatin accessibility in vivo.

    The functional role of Gsb, however, was examined within the spatial domain in which it is normally expressed. We knocked-down Gsb broadly and early in development and assayed its effects on NB5-6. Consistent with its established role in row-5/6 patterning, reduction of Gsb disrupted the specification of NB5-6 identity. In the converse experiment, broad misexpression of Gsb led to a partial expansion of NB5-6 markers. Because spatial patterning in the ventral nerve cord is organized into mutually exclusive row identities, changes in NB5-6 specification can be accompanied by reciprocal effects in neighbouring lineages. In our experiments, this is reflected in changes in markers of adjacent identities, particularly NB3-5. For this reason, NB3-5 markers provide a sensitive and informative readout of altered NB5-6 specification in the phenotypic analyses.

    We recognize that this point may not have been clear in the original manuscript. To avoid similar confusion for readers, we will make this reasoning explicitly clear in the revision.

    Reviewer #2 (Public review):

    Summary:

    This article by Bhattacharya et al. investigates how neural stem cells (NSCs, NBs) in Drosophila integrate spatial and temporal cues to activate neuron-specific terminal selector (TS) genes. Prior to this work, it was understood that NSCs utilize spatial transcription factors (STFs) and temporal transcription factors (TTFs) to determine lineage identity and birth order, but the mechanisms of integration were not fully elucidated. The authors employed chromatin profiling techniques to analyze the binding of STFs and TTFs in two specific neuroblast lineages, NB5-6 and NB7-4. They found that Gsb (an STF) binds both accessible and less-accessible chromatin in NB5-6, while En (another STF) binds only to pre-accessible chromatin in NB7-4. The findings support an "STF code" where the combination of pioneer and non-pioneer spatial factors, along with temporal factors, triggers neuroblast-specific enhancer activation and determines lineage identity.

    We appreciate the reviewer’s careful summary of our findings and their clear articulation of the STF-code framework that emerges from the work.

    Strengths:

    The experiments are well-executed, the interpretations are generally sound, and the figures are clear and elegant. However, some conclusions are drawn too broadly without essential functional data. Therefore, additional work is needed to more effectively convey the central message.

    We thank the reviewer for their positive assessment of the experiments, interpretation, and figures, and we respond to their specific concerns below.

    Weaknesses:

    (1) Integration of TaDa and functional data on Gsb for the STF model

    The authors demonstrate that TaDa profiling maps Gsb binding across the genome and identifies candidate chromatin-priming sites in NB5-6. Gsb LOF/GOF experiments reveal effects on NB identity. Combining TaDa data with LOF and GOF analyses indicates that Gsb influences NB5-6 specification by binding to both open and relatively closed chromatin, helping maintain NB5-6 identity while limiting NB3-5 fate.

    However, the study does not establish a direct link between specific LOF/GOF phenotypes and particular genomic targets. For instance, analyzing Gsb occupancy at lineage-specific identity factors or terminal selector genes (such as Lbe, Ap, or Eya for NB5-6; and Ems, etc., for NB3-5) in wild-type and manipulated conditions (Gsb misexpression) would directly connect chromatin binding to the regulation of fate determinants. These investigations would strengthen the mechanistic connection between the correlative TaDa profiles and the observed identity changes, supporting the idea that Gsb functions as a context-dependent chromatin-priming factor within the STF code, rather than as a generic transcription factor.

    We thank the reviewer for this very helpful suggestion. We agree that illustrating how the TaDa binding profiles relate to known lineage determinants will help connect the genome-wide chromatin data to the developmental phenotypes. In the revision therefore, we will examine Gsb occupancy at several genes associated with NB5-6 and NB3-5 identity (including Lbe, Ap, Eya, and Ems).

    (2) Gsb misexpression reveals bidirectional chromatin remodelling

    Experiments with ectopic Gsb expression demonstrate bidirectional chromatin remodeling in NB7-4, showing decreases in accessibility at some binding sites and increases at others. While the authors show that Gsb can disrupt chromatin upon misexpression, interpreting its "pioneer-like" or chromatin-priming activity is complex due to several factors: the misexpression occurs in a non-native lineage, the direct versus indirect effects rely on whole-embryo Dam-Gsb peaks instead of NB7-4-specific binding, and heat-shock-induced chromatin changes are not fully accounted for. These issues make it challenging to definitively determine Gsb's role in chromatin priming.

    A complementary approach would be to perform Gsb knockdown/loss-of-function in its native NB5-6 lineage and profile chromatin accessibility (TaDa or CATaDa). This would allow a cleaner, more physiologically relevant assessment of Gsb's contribution to priming, SoI establishment, and Hb recruitment. Such an experiment would strengthen the causal link between Gsb occupancy and chromatin state and clarify whether Gsb truly acts as a context-dependent pioneer in vivo, rather than producing indirect effects due to ectopic misexpression.

    We thank the reviewer for this thoughtful comment. We agree that the ectopic Gsb misexpression experiment in NB7-4 should be interpreted as a test of chromatin-remodelling capacity rather than as a fully physiological assay of Gsb function in its native NB5-6 context. At the same time, we note that ectopic expression in a non-native lineage is a standard approach used to assess pioneering or chromatin-remodelling capacity, precisely because it tests whether a factor can alter chromatin outside its endogenous setting. In the revision, we will explicitly discuss this distinction.

    We also agree that NB7-4-specific Gsb occupancy under misexpression would provide a cleaner distinction between direct and indirect effects. In the current manuscript, we infer likely direct effects from overlap with whole-embryo Gsb Dam profiles: loci that lose accessibility upon Gsb misexpression overlap whole-embryo Gsb binding, whereas loci that gain accessibility generally do not. We interpret this as support for the idea that decreased accessibility is more likely to reflect direct Gsb action, whereas increased accessibility is more likely to be indirect. We will clarify this logic in the revision.

    Regarding the reviewer’s suggestion of profiling chromatin accessibility after Gsb loss in native NB5-6, we completely agree that this would be an important complementary experiment. However, this experiment is not currently possible in our system. Gsb is required before NB specification/delamination, whereas available NB5-6 Gal4 drivers turn on only after this stage, precluding the use of RNAi. Early mutant analysis is also technically difficult because homozygous mutant embryos cannot be readily identified at the required stage, and the TaDa/CATaDa approach in this system requires large amounts of input material collected during the very short Hb window. We also tested an early CRISPR-based strategy using maternally contributed Cas9, but in this context the NB5-6 driver is lost, preventing TaDa/CATaDa profiling. We will therefore revise the manuscript to acknowledge that the current misexpression data support chromatin-remodelling capacity and are consistent with context-dependent priming, while not definitively establishing endogenous priming activity in NB5-6.

    (3) En is not a pioneer factor

    The authors conclude that Engrailed (En) is not a pioneer factor, based on the observation that En binding correlates with accessible chromatin and that En is not enriched at NB5-6-specific SOIs. However, this conclusion is not sufficiently supported by the functional data.

    We thank the reviewer for raising this point. We agree that, in several places, our wording was stronger than warranted by the data. For example, we stated that this pattern “argues against a pioneer role for En” and that the results “indicate that En does not act as a pioneer factor.” We agree that these statements are too definitive given the current evidence. Below, we address each of the reviewer’s specific concerns and explain the reasoning behind our original interpretation.

    First, the absence of En binding at NB5-6-specific SOIs does not necessarily indicate an inability to engage closed chromatin. These regions were not selected for the presence of En consensus motifs, so their lack of occupancy may simply reflect the absence of En binding motifs rather than a lack of pioneering capacity. A systematic motif analysis at NB5-6-specific SOIs is needed to determine whether En binding sites are present but unoccupied.

    We agree that the absence of En binding at NB5-6-specific SOIs alone would not be sufficient to infer a lack of pioneering activity, particularly if these loci do not contain En consensus motifs. That observation was only the starting point for our interpretation. Our reasoning was based on several additional lines of evidence from the genome-wide analysis:

    (1) When we examined En binding genome-wide, we consistently found that En occupancy in NB7-4 is restricted to regions of accessible chromatin.

    (2) Loci that are less accessible in NB7-4 show no detectable En occupancy.

    (3) Accessibility is strongly predictive of En binding: chromatin accessibility is markedly higher at En-bound loci than at En-unbound loci.

    Taken together, these patterns suggested to us that En binding in this lineage occurs primarily at pre-accessible chromatin rather than at less accessible regions that would require priming.

    Our interpretation was also guided by the broader literature. To our knowledge, neither Drosophila Engrailed nor its vertebrate homologues (EN1/EN2) have been reported to bind nucleosome-occluded DNA or initiate chromatin opening, which further informed our original interpretation.

    That said, we agree with the reviewer that these observations are suggestive rather than definitive. We will therefore temper the language throughout the manuscript so that we do not make categorical claims about En lacking pioneer activity. We will also perform the suggested motif analysis at NB5-6-specific SOIs to determine whether En binding motifs are present at these loci, which should help clarify whether the lack of En occupancy reflects motif availability or chromatin state.

    Second, the claim that En lacks pioneer activity relies solely on a single steady-state TaDa/DamID occupancy assay at one developmental stage. Because pioneer factor interactions can be transient, low-affinity, and stage-specific, such binding may not be detected by TaDa, which also depends on local GATC density and methylation kinetics and may yield false negatives. Given these technical limitations, the absence of En binding at less accessible regions does not definitively rule out a priming role.

    We take the reviewer’s point that our data cannot definitively rule out En as a pioneer. At the same time, it may be useful to clarify that TaDa is not a snapshot assay. Because Dam-mediated methylation accumulates over time while the fusion protein is expressed, even weak or transient interactions can leave a detectable signal when averaged across many cells and across the duration of the expression window.

    This cumulative nature of the assay is why our consistent observation of strong enrichment of En at accessible loci, and no detectable enrichment at less accessible regions across the genome, led us to infer that En binding in NB7-4 is strongly conditioned on chromatin accessibility. We nevertheless agree that this does not definitively exclude rare or transient interactions below the detection threshold of the assay, and we will temper the language in the manuscript accordingly.

    In the absence of direct functional assays (En LOF/GOF), the authors should explicitly acknowledge these technical and conceptual limitations and tone down the claim that "En lacks pioneer activity".

    Yes, we will do that!

    (4) Clarity of STF-code Model and Central Message

    The manuscript begins by presenting two models, direct and epigenetic, but the central takeaway of the paper is not clear. Specifically, the nuanced roles of the spatial factors Gsb and En as chromatin-priming versus stabilizing/effector factors within an STF code, and the resulting division of labor, are not clearly illustrated. The distinction between Gsb as a chromatin-priming factor and En as a cofactor-dependent activator/stabilizer should be explicitly presented in a stepwise model for better clarity. The authors could strengthen this by providing a schematic with two sequential stages illustrating how neuroblast identity factors (STF code) change chromatin states to drive lineage-specific enhancer activation. The schematic can be shown from the neuroectoderm to individual NB lineages to make it more panoramic.

    We thank the reviewer for this suggestion and for clearly articulating the conceptual point. As the reviewer points out, the literature has generally framed spatial–temporal integration as two alternative models—direct regulation at pre-accessible enhancers versus epigenetic priming by spatial factors. Our results suggest that elements of both mechanisms may operate within a lineage through a combinatorial STF code, with different spatial factors playing distinct roles (for example, Gsb contributing to chromatin priming, while En acts primarily at pre-accessible enhancers together with Hb). We agree that this central idea would benefit from being illustrated more explicitly. In the revision we will add a schematic summarizing this proposed two-step model and clarify the relevant parts of the text.

    (5) Identification of Priming Factors in NB7-4

    While the authors suggest that an unknown priming factor might be responsible for establishing sites of integration in NB7-4, they do not identify or explore potential candidates for this role. Further investigation into what factors might be involved in chromatin priming in NB7-4 could provide a more complete understanding of the mechanisms at play.

    We agree that identifying the factor responsible for establishing sites of integration in NB7-4 would be very informative. However, doing so would require substantial additional experiments to systematically test candidate spatial factors and assess their effects on chromatin accessibility in this lineage. Our goal in the present study was to establish how spatial and temporal cues are integrated at lineage-specific enhancers rather than to fully dissect all components of the STF code in each lineage. Identifying the priming factor in NB7-4 is therefore an important next step that we intend to pursue in future work, and we will clarify this point in the Discussion.

    (6) Functional Validation of STF Code Components

    The study proposes an STF code for each neuroblast lineage, but the specific components of these codes, beyond Gsb and En, are not fully explored. Identifying and validating additional factors that contribute to the STF code in each lineage could strengthen the conclusions.

    We agree that identifying additional components of the STF codes operating in each lineage would be very informative. Our goal in this study was not to comprehensively define all spatial factors involved in each lineage, but rather to understand how spatial and temporal inputs are integrated at lineage-specific enhancers. By examining two well-characterized spatial factors with distinct properties -- Gsb in NB5-6 and En in NB7-4 -- we aimed to illustrate how different members of an STF code can play distinct roles in shaping chromatin accessibility and enhancer activation. Identifying additional factors that contribute to these lineage-specific codes will be an important direction for future work.

  5. Version published to 10.7554/elife.110150.1 on eLife
  6. Version published to 10.7554/elife.110150 on eLife
  7. Version published to 10.1101/2025.11.25.690435 on bioRxiv