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TGF-β Signaling Regulates Neuronal C1q Expression and Developmental Synaptic Refinement · 10.1038/nn.3560

Name: Verified Rigor Record — TGF-β Signaling Regulates Neuronal C1q Expression and Developmental Synaptic Refinement · 10.1038/nn.3560
Item: TGF-β Signaling Regulates Neuronal C1q Expression and Developmental Synaptic Refinement · 10.1038/nn.3560
Rating: 71
Author: Αlpha¹

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Αlpha¹ Editorial

This rigor review was examined and confirmed by Αlpha¹ Editorial · July 6, 2026

Αlpha¹ Rigor Review

Rigor Reviewed

WARN

Warnings to address · Interventional Study

Eight dimensions — click a segment for details

Critical

High-priority

Model-reported confidence: highReviewer agreement: lowEngine: 7.6.0Source: full text

Not verified in this report

No data or code availability links were detected to verify.

This Αlpha¹ Rigor Review uses AI Rigor Reviewers trained on a curated corpus of high-fidelity and retracted papers, with expert supervision and curation. It can still make mistakes; verify each finding against the source before relying on it.

The paper's main methodological strengths are a well-motivated scientific premise, use of complementary genetic and pharmacological approaches, and blinded analysis. Its main weaknesses are the complete absence of data/code sharing, unverified statistical assumptions, missing sex and housing details for the animals, and inadequate resource identification (antibody catalog numbers, mycoplasma testing). A reader should weigh these gaps when assessing the robustness of the conclusions.

All 8 dimensions were applicable and evaluated from the full manuscript text by three independent reviewers. The statistics verification component checked only 3 of many tests, so the mathematical correctness of most reported tests is unverified. The citation check found no retracted or missing references.

12 major claims checked against the paper's own evidence: all adequately supported.

Recomputed 3 tests: 3 consistent, 0 inconsistent, 3 via agent-written checks.

Rigor

Eight Dimensions2 pass · 5 warn · 1 fail

12 major claims12 supported

The introduction and results section extensively cite prior work (e.g., Stevens et al., 2007; Schafer et al., 2012) and clearly state that an astrocyte-derived factor regulates C1q expression. The paper identifies TGF-β as this factor through a systematic in vitro screen and then tests its necessity in vivo using genetic and pharmacological models, directly addressing the limitations of prior studies (unknown identity of the factor).

Sub-criteria

prior work citedADEQUATE

premise rationaleADEQUATE

limitations addressedADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper thoroughly establishes its scientific foundation by citing prior work on complement in synaptic pruning, the role of C1q, and the unknown regulators of C1q expression, and provides a clear rationale linking TGF-β to this process.

Evidence

direct quote[Introduction, paragraphs 1-3]

“C1q −/− and C3 −/− mice exhibit sustained defects in synaptic refinement and elimination... however, the signals regulating complement-mediated pruning during development remain poorly understood.”

direct quote[Introduction, paragraph 5]

“A screen investigating how astrocytes influence neuronal gene expression first identified the C1q genes among the few genes that were highly upregulated in developing RGCs in response to astrocytes.”

direct quote[Introduction, paragraph 6]

“In the current study, we identified transforming growth factor beta (TGF-β) as the factor secreted by astrocytes necessary and sufficient for C1q expression in purified RGCs.”

PASSReviewer 2· 95% conf

The scientific premise is clearly established, with extensive prior work cited and a logical rationale linking background to objectives.

Evidence

direct quote[Introduction, paragraphs 1-4]

direct quote[Introduction, paragraph 4]

“In the current study, we identified transforming growth factor beta (TGF-β) as the factor secreted by astrocytes necessary and sufficient for C1q expression in purified RGCs.”

direct quote[Introduction, paragraph 3]

“Remarkably little is known about the signals controlling C1q expression and function in the brain.”

PASSReviewer 3· 95% conf

The paper extensively cites prior work, establishes a clear rationale, and identifies the gap in knowledge about signals regulating C1q expression.

Evidence

direct quote[Introduction, paragraph 5]

“Previous findings suggested that an astrocyte-derived factor triggers neuronal C1q expression”

direct quote[Introduction, paragraph 5]

“the identity of the astrocyte-derived signal(s) that regulate C1q expression has remained unknown”

direct quote[Introduction, last paragraph]

“we identified transforming growth factor beta (TGF-β) as the factor secreted by astrocytes necessary and sufficient for C1q expression in purified RGCs”

The study uses genetic knockout (retina-specific TGFβRII −/−) and pharmacological inhibition with proper controls, including wild-type littermates and vehicle-treated groups. Blinding and randomization are reported for key analyses (e.g., LGN analysis, microglial engulfment), but a formal power analysis is absent. The paper does not report an independent replication of the main findings in a separate cohort, though the in vivo results are corroborated by multiple approaches (genetic and pharmacological).

Sub-criteria

controlsADEQUATE

power analysisNOT REPORTED

blinding levelsADEQUATE

outlier handlingNOT REPORTED

randomization unitNOT REPORTED

inclusion exclusionNOT REPORTED

randomization methodINADEQUATE

replicate distinctionADEQUATE

independent replicationNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

The study design is rigorous for an animal interventional study, with appropriate controls and blinding, but lacks a pre-specified power analysis and does not report a separate independent replication cohort.

Evidence

direct quote[Methods, LGN analysis and Microglial Engulfment Analysis]

“All images were collected and quantified 'blind,' and compared to age-matched littermate controls.”

direct quote[Methods, Statistical Analysis]

“data for each experiment were collected and processed randomly and animals were assigned to various experimental groups randomly as well.”

direct quote[Methods, Statistical Analysis]

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications”

PASSReviewer 2· 90% conf

The study design is rigorous, with clearly described genetic and pharmacological approaches, controls, and blinded analysis; randomization and blinding are partially reported but adequate for the study type.

Evidence

direct quote[Statistical Analysis, Methods]

“Data collection and analysis were performed blind to the conditions of the experiments.”

direct quote[Statistical Analysis, Methods]

“data for each experiment were collected and processed randomly and animals were assigned to various experimental groups randomly as well.”

direct quote[Results, multiple sections]

“To determine if TGF-β signaling regulates C1q expression in vivo during retinogeniculate refinement, we used genetic and pharmacological approaches to block TGF-β signaling and then measured C1q expression in vivo.”

WARNReviewer 3· 70% conf

The study uses appropriate genetic and pharmacological approaches but lacks a priori power analysis, detailed randomization method, and comprehensive blinding descriptions.

Evidence

direct quote[Statistical Analysis, Methods]

“animals were assigned to various experimental groups randomly as well”

direct quote[Statistical Analysis, Methods]

“Data collection and analysis were performed blind to the conditions of the experiments”

direct quote[Statistical Analysis, Methods]

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications”

The paper uses mice but does not state sex in any section. Age (e.g., P5, P10) is given but no body weight or health assessment. Species and strain are reported adequately. No information on housing conditions (light cycle, temperature, enrichment) is provided.

Sub-criteria

demographicsN/A

sex reportedNOT REPORTED

sex justifiedN/A

age weight healthINADEQUATE

housing conditionsNOT REPORTED

species strain sourceADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

The paper reports the species, strain, and source of mice, and provides age information, but does not report sex of the animals, and does not provide weight or health status.

Evidence

direct quote[Methods, Mice]

“Floxed TGFβRII mice (B6.129S6- TGF -β r2 tm1Hlm ) were obtained from the NCI Mouse Repository and crossed to the CHX10-Cre line, Tg(Chx10-EGFP/cre,-ALPP)2Clc/J (Jackson Lab), to generate retina-specific TGFβRII −/− mice.”

direct quote[Methods, Mice and various figure legends]

“Experiments were approved by the institutional animal use and care committee... RGCs acutely isolated from P5 WT (white bar) and retinal TGFβRII −/− (grey bar) retinas”

absence[Methods, Mice]

Sex of mice is not reported.

PASSReviewer 2· 95% conf

Biological variables are well reported, including species, strain, age, and sex; housing conditions are not described, but this is a minor gap for an interventional study.

Evidence

direct quote[Methods, Mice; throughout]

“RGCs acutely isolated from P5 WT (white bar) and retinal TGFβRII −/− (grey bar) retinas using immunopanning showed significantly reduced C1q expression.”

direct quote[Methods, Mice]

absence[Methods, Mice]

Sex of animals is not reported.

FAILReviewer 3· 85% conf

Sex of animals is not reported; age is reported but not weight or health status; housing conditions are not described.

Evidence

absence[Methods, Mice]

No sex of mice reported anywhere in the paper.

direct quote[Methods, Mice]

“All mice were sacrificed at P5”

absence[Methods]

Housing conditions not described.

The paper states that experiments were approved by the institutional animal use and care committee and were conducted in accordance with NIH guidelines. This satisfies the criteria for an animal study (iacuc_statement and regulatory_compliance). The committee is not named explicitly but is described as 'the institutional animal use and care committee,' which is adequate.

Sub-criteria

iacuc statementINADEQUATE

informed consentN/A

irb ethics statementN/A

regulatory complianceADEQUATE

Per reviewer (3) — the votes behind the verdict

PASSReviewer 1· 95% conf

The paper reports an ethics statement with a named approving body and compliance with NIH guidelines for animal research.

Evidence

direct quote[Methods, Mice]

“Experiments were approved by the institutional animal use and care committee in accordance with NIH guidelines for the humane treatment of animals.”

PASSReviewer 2· 95% conf

Ethical compliance is adequately reported: IACUC approval is stated with institutional and NIH guideline adherence.

Evidence

direct quote[Methods, Mice]

“Experiments were approved by the institutional animal use and care committee in accordance with NIH guidelines for the humane treatment of animals.”

WARNReviewer 3· 80% conf

The paper states approval by an institutional animal care committee in accordance with NIH guidelines but does not name the specific committee or provide a protocol number.

Evidence

direct quote[Methods, Mice]

“Experiments were approved by the institutional animal use and care committee in accordance with NIH guidelines for the humane treatment of animals”

The paper identifies mouse strains and sources. Software (GraphPad Prism 5, Imaris, ImageJ) is named with versions. Antibodies are listed with vendors and dilutions, but many lack catalog numbers or RRIDs, and validation data are only referenced for some (e.g., C1q validated in Stephan et al., 2013). No cell line authentication or mycoplasma testing is reported (primary cultures were used). Reagents like cholera toxin b are identified.

Sub-criteria

mycoplasma testingNOT REPORTED

reagents identifiedINADEQUATE

organisms identifiedADEQUATE

antibodies identifiedINADEQUATE

cell line authenticationN/A

software tools identifiedADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 80% conf

Organisms and software tools are identified, but some reagents (antibodies) lack catalog numbers or full identification, and no authentication data are provided for cell lines or mycoplasma testing.

Evidence

direct quote[Methods, Immunohistochemistry]

“C1q (undiluted culture supernatant, Epitomics, validated in Stephan et al., 2013), C3 (Cappel,1:300), vglut2 (Millipore,1:2000), TGFβRII (R&D Systems goat antihuman, 1:200)”

direct quote[Methods, Statistical Analysis]

“GraphPad Prism 5 software (La Jolla, CA) was used for all statistical tests.”

absence[Methods, Neuron and Astrocyte Cultures]

Mycoplasma testing is not reported.

PASSReviewer 2· 85% conf

Key resources are well identified, including antibodies, organisms, reagents, and software; cell line authentication and mycoplasma testing are not applicable.

Evidence

direct quote[Methods, Immunohistochemistry]

“C1q (undiluted culture supernatant, Epitomics, validated in Stephan et al., 2013 ), C3 (Cappel,1:300), vglut2 (Millipore,1:2000), TGFβRII (R&D Systems goat antihuman, 1:200), pSmad (Millipore,1:200), Calretinin (Millipore,1:1000), Iba1 (Wako, 1:400), CD68 (Serotec,1:300), TUJ1 (Covance, 1:400)”

direct quote[Methods, Statistical Analysis]

“GraphPad Prism 5 software (La Jolla, CA) was used for all statistical tests.”

direct quote[Methods, various]

“Specific TGFβRI-specific inhibitor (SB431542, Tocris, 30µM)”

WARNReviewer 3· 80% conf

Antibodies are listed with vendors but lack catalog numbers or RRIDs; mycoplasma testing is not reported; reagents are partially identified; software and organisms are adequately identified.

Evidence

direct quote[Methods, Immunohistochemistry]

“C1q (undiluted culture supernatant, Epitomics, validated in Stephan et al., 2013 )”

absence[Methods, Cell culture]

Mycoplasma testing not reported.

direct quote[Methods, Statistical Analysis]

“GraphPad Prism 5 software (La Jolla, CA) was used for all statistical tests”

The paper explicitly names tests (one-way ANOVA, two-way ANOVA, Student's t-test) and provides exact p-values (e.g., p=0.0226, p=0.0395) and post-hoc tests (Bonferroni). However, the data presentation mostly uses bar graphs without individual data points (e.g., Figures 2, 4, 5). The paper states that data distribution was assumed normal but this was not formally tested, which is inadequate for assumptions verification. Effect sizes and confidence intervals are not reported.

Sub-criteria

tests namedADEQUATE

exact p valuesADEQUATE

effect sizes ciNOT REPORTED

data presentationINADEQUATE

software identifiedADEQUATE

assumptions verifiedINADEQUATE

mathematical plausibilityN/A

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Statistical tests are named and exact p-values are generally reported, but data presentation could be improved (e.g., individual data points in bar graphs are not shown in many figures), and assumptions are not formally tested.

Evidence

direct quote[Methods, Statistical Analysis and figure legends]

“Analyses used include one-way ANOVA, two-way ANOVA, and Student’s t-test. For ANOVA analysis, Bonferonni post hoc tests were used.”

direct quote[Methods, Statistical Analysis]

“For all statistical analyses, data distribution was assumed to be normal but this was not formally tested.”

paraphrase[Figures 2-6]

“Bar graphs represent mean + SEM; no individual data points are plotted.”

WARNReviewer 2· 85% conf

Statistical analysis is generally sound, but several sub-criteria are incomplete: assumptions are not formally tested, exact p-values are often reported as thresholds, and effect sizes/confidence intervals are absent.

Evidence

direct quote[Statistical Analysis, Methods]

“For all statistical analyses, data distribution was assumed to be normal but this was not formally tested.”

direct quote[Figure legends, multiple]

“***p<0.001, **p<0.01, *p<0.05”

absence[Results and Methods]

Effect sizes and confidence intervals are not reported.

WARNReviewer 3· 75% conf

Statistical tests are named and exact p-values are often reported, but assumptions are not verified, effect sizes/confidence intervals are absent, and data presentation uses bar-only graphs without individual points.

Evidence

direct quote[Statistical Analysis, Methods]

“For all statistical analyses, data distribution was assumed to be normal but this was not formally tested”

absence[Statistical Analysis, Methods]

Effect sizes and confidence intervals not reported.

direct quote[Figure legends]

“p<0.0001, t(4)=37.48”

There is no data availability statement in the manuscript. No accession numbers or repository links are provided for raw data or custom code. The paper is from 2013, when such practices were less common, but for a methodology rigor analysis, this is a gap.

Sub-criteria

code sharingNOT REPORTED

accession numbersNOT REPORTED

repository depositNOT REPORTED

data availability statementNOT REPORTED

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 90% conf

The paper does not provide a data availability statement, and no data or code have been deposited in a public repository.

Evidence

absence[Entire manuscript]

No data availability statement.

WARNReviewer 2· 90% conf

No data or code availability statement is present; no repository deposit or accession numbers are provided; no code sharing is mentioned.

Evidence

absence[Entire manuscript]

No data availability statement found.

absence[Entire manuscript]

No code repository mentioned.

FAILReviewer 3· 90% conf

No data availability statement, repository deposit, or code sharing is provided.

Evidence

absence[Entire paper]

No data availability statement found.

absence[Entire paper]

No repository deposit mentioned.

absence[Entire paper]

No code sharing mentioned.

Methods are sufficiently detailed for replication, covering cultures, immunohistochemistry, qPCR, and imaging. Limitations are discussed (e.g., focus on retina, generalizability). Conclusions are generally proportional. However, no reporting guideline is referenced (ARRIVE would apply to animal studies). The paper does not pre-register the study or specify all outcomes a priori. Funding and competing interests are acknowledged.

Sub-criteria

funding coiADEQUATE

trial registrationN/A

reporting guidelineNOT REPORTED

methods completenessADEQUATE

all outcomes reportedNOT REPORTED

limitations discussedADEQUATE

conclusions proportionalADEQUATE

Per reviewer (3) — the votes behind the verdict

WARNReviewer 1· 85% conf

Methods are fairly detailed, limitations are discussed, and funding/COI are provided, but the study is not registered (not applicable), no reporting guideline is mentioned, and not all outcomes are explicitly pre-specified.

Evidence

direct quote[Acknowledgments and Author Contributions]

“This work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.), the Ellison Foundation (B.S.), John Merck Scholars Program (B.S.), NINDS (RO1-NS-07100801; B.S.), NIDA (RO1-DA-15043; B.A.B.), NIH (P30-HD-18655; MRDDRC Imaging Core).”

direct quote[Discussion, paragraphs 4 and 5]

“Whether and how neuronal activity regulates complement expression and function are open areas of investigation; however, based on our data, we envision a model in which C1q is secreted locally from RGC terminals in an activity-dependent manner”

absence[Methods]

No reporting guideline is mentioned.

WARNReviewer 2· 85% conf

Reporting is generally transparent, but the paper lacks trial registration and a reporting guideline; methods are complete and limitations are discussed.

Evidence

direct quote[Methods, multiple subsections]

“Retinal ganglion cells were cultured from P8 Sprague-Dawley rats after serial immunopanning steps to yield >99.5% purity as described.”

absence[Entire manuscript]

No reporting guideline mentioned.

direct quote[Acknowledgments]

PASSReviewer 3· 80% conf

Methods are detailed, all outcomes are reported, limitations are discussed, and conclusions are proportional, but no reporting guideline is referenced and no conflict-of-interest statement is provided.

Evidence

direct quote[Methods sections]

“Retinal ganglion cells were cultured from P8 Sprague-Dawley rats after serial immunopanning steps to yield >99.5% purity”

direct quote[Discussion]

“We have not quantified synapses in the dLGN of retinal TGFβRII −/− mice, it is unlikely that defective synaptogenesis could explain our phenotype”

direct quote[Acknowledgments]

“This work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.)...”

11 copyedit issues flagged: mostly consistency, typo, clarity.

Checked 45 references: 45 verified.

Αlpha¹ Rigor Action ItemsFinal pass before journal submission

As a post-publication audit, this paper shows meaningful rigor gaps that an informed reader should weigh. The most consequential gaps are the complete lack of data availability (raw data cannot be independently examined) and the limited statistical verification (assumptions untested, effect sizes absent). These would warrant a data-sharing correction or, ideally, deposition of raw data in a repository. The copyedit issues are minor and do not affect scientific conclusions.

1.
HIGHdata code
Add a data availability statement (Methods, Data Availability) and deposit raw data (microscopy, qPCR) in a public repository such as Figshare or Dryad with a DOI.
The paper currently has no mechanism for readers to access the underlying data, which is a critical reproducibility gap for a data-driven published study.
2.
HIGHstatistics
Report exact p-values (e.g., p = 0.023) instead of threshold asterisks throughout figure legends, or state that exact p-values are provided in a supplementary table.
Threshold symbols (***, **, *) under-report precision and are insufficient for readers to evaluate statistical evidence.
3.
HIGHstatistics
Test and report normality and equal-variance assumptions for each parametric test, or state that non-parametric tests were used if assumptions were violated (Methods, Statistical Analysis).
The paper states normality was assumed but not tested; this is a gap that undermines confidence in parametric test results.
4.
HIGHstatistics
Report effect sizes (e.g., Cohen's d, η²) and 95% confidence intervals for all key pairwise and ANOVA comparisons (Results, figure legends).
Effect sizes and confidence intervals are needed for readers to assess the magnitude and precision of findings beyond p-values.
5.
HIGHrigor
State the sex of all animals used and provide a scientific justification if only one sex was used (Methods, Mice).
Sex is a fundamental biological variable; its complete absence prevents assessment of potential sex differences in the findings.
6.
HIGHreporting
Add an explicit conflict-of-interest statement (e.g., before Acknowledgments) listing any competing interests or stating 'none declared.'
Funding sources alone do not satisfy standard disclosure requirements; a dedicated COI section is expected.
7.
HIGHreporting
Cite and follow the ARRIVE guidelines in the Methods, adding a sentence such as 'This study adheres to the ARRIVE guidelines for reporting animal research.'
ARRIVE is the standard reporting guideline for preclinical animal studies; its absence is a transparency gap.
8.
MEDIUMrigor
Add an a priori power analysis (Methods, Statistical Analysis) to justify reported sample sizes, specifying expected effect size, alpha (0.05), and target power (0.80).
The paper states sample sizes were based on prior publications; a formal power analysis would strengthen the design's evidentiary value.
9.
MEDIUMrigor
Add catalog numbers or RRIDs for all antibodies (Methods, Immunohistochemistry) and vendor catalog numbers for key reagents.
Antibody and reagent identifiers are essential for reproducibility; current descriptions lack unambiguous identifiers.
10.
MEDIUMrigor
Report mycoplasma testing status for all cell cultures used (Methods, Neuron and Astrocyte Cultures).
Mycoplasma contamination can alter cellular responses; its absence is a quality-control gap for the in vitro experiments.
11.
MEDIUMrigor
Describe the randomization method (e.g., random number generator) and unit (individual animal vs. litter) used for group assignment (Methods, Statistical Analysis).
The method of randomization is not specified, which limits the reader's ability to evaluate potential allocation bias.
12.
MEDIUMrigor
Define pre-specified inclusion/exclusion criteria for animals and data points (e.g., based on CTB fill quality) and report how many animals were excluded (Methods).
Without explicit criteria, selective exclusion cannot be excluded as a source of bias.
13.
LOWrigor
Show individual data points in figures (e.g., scatter plots with means ± SEM) rather than bar-only graphs (Figures 2-6).
Individual data points allow readers to assess data distribution, variability, and potential outliers.
14.
LOWrigor
Report housing and husbandry conditions (light cycle, temperature, cage enrichment) for the animals (Methods, Mice).
Housing conditions can affect physiological outcomes and are expected in animal study reports.
15.
LOWcopyedit
Fix the typo 'immunohistochemisy' to 'immunohistochemistry' (Results, paragraph 16) and correct the inconsistent spacing in 'TGF -β r2 tm1Hlm' to 'Tgfbr2tm1Hlm' (Methods, Mice).
Minor typos do not affect scientific conclusions but detract from manuscript professionalism.

Full text (69,463 chars)Analyzed: Jul 6, 2026

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