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RAR - Reviewer Attention Report

AFD-127 24 reviewer-attention items 2 paper-level hypotheses
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Alternative paper-level hypotheses 2

Paper-level alternatives surfaced by HRAN and checked against the broader manuscript context. A reviewer would inspect whether the authors address these hypotheses across the paper.

Alternative paper-level hypothesis

anterior-to-posterior coordination of NSC reactivation

severity S unaddressed

Across the perturbation panels, posterior reactivation may depend on the activation state of anterior source cells and/or descending neuronal output in general, rather than on a specific propagated qNSC-to-qNSC relay mechanism.

Mechanism: shared upstream source-state / broad-output model vs specific propagated relay

Reason to inspect: This paper-level hypothesis synthesizes the convergent causal ambiguity across five perturbation panels. In every case (PTEN, AKT, Kir2.1, TrpA1, neuronal Kir2.1), the manipulation is expected to alter the state of the targeted anterior cells. The posterior phenotype could therefore reflect loss or gain of an upstream source state or descending neuronal output, rather than propagation of a specific signal. The paper does not include any experiment that factorially separates anterior NSC state from anterior neuronal output from posterior outcome. The paper's own conflict note acknowledges this ambiguity. The convergence of multiple panel-level candidates on this single axis elevates it to a high-severity paper-level concern.

Suggested experiment: Factorially separate anterior NSC state, anterior neuronal activity, and posterior outcome. For example: (1) silence descending neurons while independently forcing anterior NSC reactivation via AKT; (2) force anterior NSC reactivation via a non-bioelectric, non-insulin method and test posterior effects; (3) directly activate posterior NSCs while anterior NSCs remain quiescent to test whether the relay is necessary or merely permissive.

Relevant panels: Figure 2B Figure 2C Figure 3B Figure 3C Figure 6D
Alternative paper-level hypothesis

qNSCs / claimed transient neuronal state

severity S unaddressed

The paper's central 'mixed stem cell-neuron' state may instead be a quiescent NSC program that selectively deploys neuronal/synaptic modules without qNSCs actually becoming neuronal.

Mechanism: partial neuronal program vs bona fide neuronal identity

Reason to inspect: The paper defines the 'neuronal' identity of qNSCs primarily through a UCell neuron-score metric based on top neuronal genes from mature neuronal clusters, plus expression of neuronal/synaptic genes. However, selective deployment of neuronal gene modules is well-documented in non-neuronal cells (e.g., synaptic machinery in immune cells, neuronal transcription factors in stem cells). The paper's hard constraints confirm that both quiescent and reactivated NSCs retain stem-cell markers (deadpan, worniu, klumpfuss), meaning the cells never lose NSC identity. The paper does not test broader neuronal identity criteria such as electrophysiological properties characteristic of neurons, axon/dendrite morphology, or functional synaptic transmission from qNSCs. The neuron-score metric is a transcriptomic similarity measure, not a functional identity test. The distinction between 'qNSCs become neuronal' and 'qNSCs deploy neuronal modules while remaining stem cells' is the central novelty claim and remains unresolved.

Suggested experiment: Test whether qNSCs satisfy functional neuronal identity criteria: electrophysiological properties (action potentials, synaptic currents), morphological features (axon/dendrite formation), or functional synaptic transmission. Alternatively, show that the neuronal gene program in qNSCs is qualitatively distinct from known cases of neuronal module deployment in non-neuronal cells.

Relevant panels: Figure 4C Figure 4D Figure 4E Figure 5A Figure 5B Figure 5C

Needs synthesis 2

Claims linked to several panels where neither panel in itself provides sufficient evidence. The useful review task is to inspect the panels together.

Figure 3
Figure 3A Figure 3B
Synthesis claim C9
Author claim

We found that misexpression of Kir2.1 in the brain lobes (Fig. 3A) led to impaired reactivation of VNC qNSCs (Fig. 3B).

Specific point under review

Misexpression of Kir2.1 in the brain lobes led to impaired reactivation of VNC qNSCs.

Questions for Reviewer

  1. What comparison or control would help interpret this point: comparison/control condition without Kir2.1 misexpression?

  2. Is there evidence for brain lobe Kir2.1 misexpression:no Kir2.1 misexpression / control (led to impaired reactivation), considering the panels together or the surrounding figure context?

  3. Is there evidence for VNC qNSC reactivation state:reactivation readout measured in VNC qNSCs (impaired reactivation of VNC qNSCs), considering the panels together or the surrounding figure context?

  4. Can the figure, legend, or methods clarify whether no explicit control image or comparator state is displayed in this graph?

  5. This claim-panel pair has not yet been evaluated by CLEAR. Should it remain in reviewer attention?

Figure 5
Figure 5A Figure 5B Figure 5C Figure 5D Figure 5E Figure 5F
Synthesis claim C30
Author claim

Thus, the synaptic proteins Hig and Hasp are both required for timely NSC reactivation, suggesting that a synaptic mechanism may be involved.

Specific point under review

The synaptic proteins Hig and Hasp are both required for timely NSC reactivation.

Questions for Reviewer

  1. This claim-panel pair has not yet been evaluated by CLEAR. Should it remain in reviewer attention?

Needs experiment 7

Alternative panel explanations or hypotheses, with a suggestion for a discriminating experiment. A reviewer would check if the authors address these hypotheses elsewhere in the paper.

Figure 1
Figure 1D
Experiment claim C3
Author claim

We found that qNSCs reactivate first in the brain lobes (4 h after larval hatching—ALH), followed by qNSCs in the thoracic (8 h ALH) and abdominal (20 h ALH) VNC (Fig. 1C,D).

Alternative to consider: The regional reactivation sequence could arise from anterior-posterior differences in nutrient/insulin access rather than an actively propagated inter-regional signal.

Suggested experiment: Deliver exogenous insulin uniformly across the CNS (e.g., ex vivo culture with saturating insulin) and test whether the anterior-to-posterior reactivation sequence persists.

Questions for Reviewer

  1. Could this alternative explanation be addressed: No experiment directly tests whether equalizing nutrient/insulin delivery across the CNS would abolish the anterior-to-posterior sequence. The gradient model is a parsimonious null that the paper does not formally exclude?

Figure 2
Figure 2B
Experiment claim C6
Author claim

Upon misexpression of an inhibitor of insulin signaling (PTEN) in the brain lobe qNSCs, we found that reactivation of ventral nerve cord qNSCs was severely impaired (Fig. 2B).

Alternative to consider: PTEN misexpression may impair posterior reactivation because it prevents the targeted anterior NSCs from entering the upstream active/reactivated state needed to emit some downstream cue, rather than because insulin-pathway state itself is the propagated coordinating signal.

Suggested experiment: Score reactivation of the targeted brain-lobe NSCs in the PTEN condition. Also test whether a non-insulin manipulation that blocks anterior NSC reactivation (e.g., cell-cycle arrest) produces the same VNC phenotype.

Questions for Reviewer

  1. Could this alternative explanation be addressed: The experiment cannot distinguish signal identity from source-state requirement. This is a fundamental interpretive gap for the relay model?

Figure 2
Figure 2C
Experiment claim C7
Author claim

Remarkably, this was sufficient to induce reactivation of the ventral nerve cord qNSCs (Fig. 2C).

Alternative to consider: Constitutively active AKT may rescue posterior reactivation because it forces anterior NSCs into an upstream active/reactivated state that then emits another cue, rather than because AKT/insulin-pathway activation itself is the propagated signal.

Suggested experiment: Drive anterior NSC reactivation via an orthogonal pathway (e.g., forced cell-cycle entry, CycE overexpression) under starvation and test whether posterior reactivation is also rescued.

Questions for Reviewer

  1. Could this alternative explanation be addressed: Sufficiency of AKT does not identify the transmitted signal. Any manipulation that forces anterior NSC reactivation might produce the same posterior rescue?

Figure 3
Figure 3B
Experiment claim C9
Author claim

We found that misexpression of Kir2.1 in the brain lobes (Fig. 3A) led to impaired reactivation of VNC qNSCs (Fig. 3B).

Alternative to consider: Kir2.1 may impair VNC reactivation indirectly by preventing anterior NSCs from reaching the active/reactivating source state, rather than showing that membrane potential itself is the propagated instructive variable.

Suggested experiment: Score reactivation of the targeted brain-lobe NSCs in the Kir2.1 animals. If they fail to reactivate, the posterior phenotype is explained by source-state loss, not bioelectric signal identity.

Questions for Reviewer

  1. Could this alternative explanation be addressed: The experiment does not distinguish bioelectric signal propagation from upstream source-cell state failure. This is the same causal ambiguity as Figures 2B/2C but in the bioelectric domain?

Figure 3
Figure 3C
Experiment claim C11
Author claim

We expressed the temperature-activated Ca2+ channel dTrpA1 (Pulver et al, 2009) in brain lobe qNSCs and found increased reactivation in the VNC based and found increased reactivation as shown by pH3 labeling and expression of Worniu (Fig. 3C).

Alternative to consider: dTrpA1 may accelerate VNC reactivation because it prematurely pushes anterior NSCs into the upstream active/reactivating state that releases another cue, rather than because depolarization itself is the inter-regional instructive signal.

Suggested experiment: Determine whether TrpA1 accelerates reactivation of the targeted brain-lobe NSCs. Test whether a non-bioelectric method of accelerating anterior NSC reactivation produces the same posterior acceleration.

Questions for Reviewer

  1. Could this alternative explanation be addressed: Sufficiency of depolarization does not identify the transmitted signal. The posterior effect may track anterior source-cell state rather than bioelectric signal identity?

Figure 5
Figure 5F
Experiment claim C28
Author claim

Downregulation of hig in neurons also significantly impairs qNSC reactivation (Figs. 5F and EV3).

Alternative to consider: Pan-neuronal hig knockdown may impair NSC reactivation by broadly disrupting neuronal support or network output, not specifically by disrupting direct neuron-qNSC synaptic signaling.

Suggested experiment: Restrict hig knockdown to the candidate contacting neurons (e.g., using a more specific driver) and compare the phenotype with pan-neuronal knockdown.

Questions for Reviewer

  1. Could this alternative explanation be addressed: The phenotype could reflect broad neuronal dysfunction rather than specific neuron-qNSC synaptic signaling. A restricted knockdown in candidate contacting neurons would be needed?

Figure 6
Figure 6D
Experiment claim C32
Author claim

Blocking neuronal firing drastically delayed the onset of reactivation as marked by Worniu (Fig. 6D) and completely abolished the proliferation of qNSCs in the ventral nerve cord, which was assessed using pH3 staining (Fig. 6D).

Alternative to consider: The Figure 6D phenotype may reflect a general requirement for descending neuronal activity or trophic support in posterior reactivation, rather than a dedicated relay of anterior NSC state.

Suggested experiment: Manipulate descending neuronal activity while independently controlling anterior NSC state (e.g., silence descending neurons while forcing anterior NSC reactivation via AKT) and test whether posterior reactivation follows neuronal output or anterior NSC state.

Questions for Reviewer

  1. Could this alternative explanation be addressed: The experiment cannot distinguish a specific relay of anterior NSC state from a general requirement for descending neuronal activity. This is a central interpretation fork for the paper's relay model?

Evidence question 9

CLEAR could not find sufficient evidence for the claim in the referenced panel. A reviewer should investigate the strength of the claim vs the experimental setup.

Figure 1
Figure 1C
Evidence claim C3
Author claim

We found that qNSCs reactivate first in the brain lobes (4 h after larval hatching—ALH), followed by qNSCs in the thoracic (8 h ALH) and abdominal (20 h ALH) VNC (Fig. 1C,D).

Specific point under review

qNSCs reactivate first in the brain lobes (4 h after larval hatching—ALH), followed by qNSCs in the thoracic (8 h ALH) and abdominal (20 h ALH) VNC.

Questions for Reviewer

  1. Does the claim's causal/mechanistic language exceed what the experimental design can support? Consider what additional experiment would directly test the claimed mechanism rather than the broader pathway.

  2. Is there evidence for the 4 h ALH timepoint (brain-lobe reactivation at 4 h ALH), considering the panels together or the surrounding figure context?

  3. Is there evidence for the 20 h ALH timepoint (abdominal VNC reactivation at 20 h ALH), considering the panels together or the surrounding figure context?

Figure 1
Figure 1D
Evidence claim C5
Author claim

Intriguingly, qNSCs in the abdominal VNC remain quiescent more than fifteen hours longer than qNSCs in the brain lobes, despite the secretion of insulin-like peptides across the entire CNS emanating from the glial-niche (Chell and Brand, 2010).

Specific point under review

qNSCs in the abdominal VNC remain quiescent more than fifteen hours longer than qNSCs in the brain lobes, despite the secretion of insulin-like peptides across the entire CNS emanating from the glial-niche.

Questions for Reviewer

  1. Is there evidence for insulin-like peptide secretion across entire CNS from glial niche (despite the secretion of insulin-like peptides across the entire CNS emanating from the glial-niche), considering the panels together or the surrounding figure context?

  2. Could this alternative explanation be addressed: No experiment directly tests whether equalizing nutrient/insulin delivery across the CNS would abolish the anterior-to-posterior sequence. The gradient model is a parsimonious null that the paper does not formally exclude?

  3. Could this alternative explanation be addressed: The paper does not show that Wor induction kinetics are equivalent across regions. An independent early readout (e.g., cell-size increase, EdU incorporation, or a distinct transcriptional reporter) would be needed to confirm the sequence is not marker-specific?

Figure 2
Figure 2C
Evidence claim C8
Author claim

Therefore, to coordinate reactivation between the brain lobes and the VNC, qNSCs appear to be able to propagate a signal along the anterior–posterior axis of the CNS.

Specific point under review

To coordinate reactivation between the brain lobes and the VNC, qNSCs appear to be able to propagate a signal along the anterior–posterior axis of the CNS.

Questions for Reviewer

  1. Does the claim's causal/mechanistic language exceed what the experimental design can support? Consider what additional experiment would directly test the claimed mechanism rather than the broader pathway.

  2. Is there evidence for posterior target explicitly resolved as part of an anterior-to-posterior axis comparison (along the anterior–posterior axis of the CNS), considering the panels together or the surrounding figure context?

  3. Could this alternative explanation be addressed: Sufficiency of AKT does not identify the transmitted signal. Any manipulation that forces anterior NSC reactivation might produce the same posterior rescue?

Figure 4
Figure 4B
Evidence claim C19
Author claim

GO term analysis of qNSC gene expression revealed an enrichment for neuronal genes involved in neurotransmitter release, synaptic assembly and synaptic activity (Fig. 4B).

Specific point under review

GO term analysis of qNSC gene expression revealed an enrichment for neuronal genes involved in neurotransmitter release, synaptic assembly and synaptic activity.

Questions for Reviewer

  1. Is there evidence for GO-term enrichment output (revealed an enrichment), considering the panels together or the surrounding figure context?

  2. Is there evidence for neurotransmitter release (enrichment for neuronal genes involved in neurotransmitter release), considering the panels together or the surrounding figure context?

  3. Is there evidence for synaptic assembly (enrichment for neuronal genes involved in synaptic assembly), considering the panels together or the surrounding figure context?

  4. Is there evidence for synaptic activity (enrichment for neuronal genes involved in synaptic activity), considering the panels together or the surrounding figure context?

Figure 4
Figure 4B
Evidence claim C20
Author claim

In contrast, reactivated NSCs expressed genes for transcription and translation (Fig. 4B).

Specific point under review

Reactivated NSCs expressed genes for transcription and translation.

Questions for Reviewer

  1. Is there evidence for transcription (expressed genes for transcription), considering the panels together or the surrounding figure context?

  2. Is there evidence for translation (expressed genes for translation), considering the panels together or the surrounding figure context?

Figure 4
Figure 4E
Evidence claim C23
Author claim

Therefore, qNSCs transiently become neuronal while maintaining expression of stem cell genes.

Specific point under review

qNSCs transiently become neuronal while maintaining expression of stem cell genes.

Questions for Reviewer

  1. Is there evidence for stem cell gene expression measurement in qNSCs (while maintaining expression of stem cell genes), considering the panels together or the surrounding figure context?

Figure 6
Figure 6A
Evidence claim C31
Author claim

When fully extended, the termini of qNSC projections can be seen close to axonal tracts in the CNS (Fig. 6A,B).

Specific point under review

The termini of qNSC projections can be seen close to axonal tracts in the CNS.

Questions for Reviewer

  1. Can the figure, legend, or methods clarify whether no explicit comparative condition or baseline group is shown?

Figure 6
Figure 6B
Evidence claim C31
Author claim

When fully extended, the termini of qNSC projections can be seen close to axonal tracts in the CNS (Fig. 6A,B).

Specific point under review

The termini of qNSC projections can be seen close to axonal tracts in the CNS.

Questions for Reviewer

  1. Can the figure, legend, or methods clarify whether no explicit control or baseline condition is shown for this graph?

Figure 6
Figure 6D
Evidence claim C32
Author claim

Blocking neuronal firing drastically delayed the onset of reactivation as marked by Worniu (Fig. 6D) and completely abolished the proliferation of qNSCs in the ventral nerve cord, which was assessed using pH3 staining (Fig. 6D).

Specific point under review

Blocking neuronal firing drastically delayed the onset of reactivation as marked by Worniu.

Questions for Reviewer

  1. Is there evidence for an onset-resolving time course or at least early and later timepoints (delayed onset of reactivation), considering the panels together or the surrounding figure context?

  2. Could this alternative explanation be addressed: The experiment cannot distinguish a specific relay of anterior NSC state from a general requirement for descending neuronal activity. This is a central interpretation fork for the paper's relay model?

Citation refinement 3

The specific evidence appears elsewhere in the same figure. The useful reviewer task is to check that the claim points readers to the relevant panel(s).

Figure 4
Figure 4C
Citation claim C15
Author claim

As expected, both quiescent and reactivated NSCs expressed neural stem cell genes such as deadpan, worniu, and klumpfuss.

Specific point under review

Both quiescent and reactivated NSCs expressed neural stem cell genes such as deadpan, worniu, and klumpfuss.

Figure-level evidence path

The specific evidence appears in Figure 4D. The claim should also reference Figure 4D when making this statement.

Figure 4
Figure 4C
Citation claim C21
Author claim

Quiescent, but not reactivated, neural stem cells expressed neuronal genes involved in electrochemical processes, including GABAergic (Gad1, Rdl), cholinergic (nAChRalpha6, mAChR-A) and glutamatergic neurotransmission (VGlut; Fig. 4C).

Specific point under review

Quiescent, but not reactivated, neural stem cells expressed neuronal genes involved in electrochemical processes, including GABAergic (Gad1, Rdl), cholinergic (nAChRalpha6, mAChR-A) and glutamatergic neurotransmission (VGlut).

Figure-level evidence path

The specific evidence appears in Figure 4D. The claim should also reference Figure 4D when making this statement.

Figure 4
Figure 4E
Citation claim C15
Author claim

As expected, both quiescent and reactivated NSCs expressed neural stem cell genes such as deadpan, worniu, and klumpfuss.

Specific point under review

Both quiescent and reactivated NSCs expressed neural stem cell genes such as deadpan, worniu, and klumpfuss.

Figure-level evidence path

The specific evidence appears in Figure 4D. The claim should also reference Figure 4D when making this statement.

Figure text check 1

The panel structure is usable, but the figure text, labels, or legend leave a local ambiguity that should be checked before the claim is interpreted.

Figure 5
Figure 5D
Figure text claim C26
Author claim

In qNSCs, Hig is found along the projection and in the neuropil (Fig. 5D).

Specific point under review

In qNSCs, Hig is found along the projection and in the neuropil.

Questions for Reviewer

  1. Can the figure, legend, or methods clarify whether single-state representative image; no comparative baseline is available within the panel?

Unclear baseline 2

The referenced panel needs a clear baseline or reference condition before the claim can be reviewed against it.

Figure 1
Figure 1B
Baseline claim C2
Author claim

We found that the 3:1 ratio of G2/G0 arrested qNSCs is maintained in the brain lobes and the thoracic VNC, whereas the abdominal NSCs are equally split between G2 and G0 arrest (Fig. 1B).

Specific point under review

The 3:1 ratio of G2/G0 arrested qNSCs is maintained in the brain lobes and the thoracic VNC, whereas the abdominal NSCs are equally split between G2 and G0 arrest.

Questions for Reviewer

  1. Which baseline or reference condition should be used to interpret this claim in Figure 1B?

Figure 5
Figure 5A
Baseline claim C24
Author claim

Among the neuronal genes upregulated in qNSCs, we found several cholinergic receptors: nAChRalpha6, nAChRalpha5, nAChRalpha1, mAChR-A (Fig. 5A).

Specific point under review

Among the neuronal genes upregulated in qNSCs, several cholinergic receptors (nAChRalpha6, nAChRalpha5, nAChRalpha1, mAChR-A) were found.

Questions for Reviewer

  1. Which baseline or reference condition should be used to interpret this claim in Figure 5A?