Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No authors-side cause for any deviation
- ✓The central claim held under reproduction
- 🔴A deviation arose in the data or preprocessing
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Described well enough -> 1:1 reproducible. The paper's bulk-RNA-seq DE claim (1588 up / 1480 down in Tbx5-mutant pSHF, >=1.5 FC & 5% FDR) was reproduced by running the documented third-party tool CSBB-v3.0 DifferentialExpression (RUVSeq+edgeR, by co-author Chaturvedi, repo @00ca12d) verbatim on the authors' own deposited GSE75077 count matrix. The up-count reproduces to within 2 genes (1586 vs 1588) and, critically, only the empirical-RUVg normalization variant lands in range (plain upper-quantile overshoots to ~2000-2200), so the reproduction also disambiguates a method the paper left unspecified. The down-count is systematically ~10% low (1322-1393 vs 1480) across all filter parameters -- a directional asymmetry most consistent with edgeR/RUVSeq version drift (2026 packages vs the paper's ~2016 stack), not a data/logic error and no fabrication signal (headline numbers are derivable from shipped data+tool). Every named Fig-1B gene reproduces in direction and significance, including the thesis gene Aldh1a2 (down, FC 0.43) and the whole RA->Hedgehog->Wnt axis. NOT attempted (optional hard 20%): rebuilding the exact 2016-era R/edgeR/RUVSeq versions to close the down-count gap, and re-aligning raw FASTQ from SRP066296 (we reproduce DE from the deposited count matrix, i.e. downstream of alignment); both out of scope per 80/20. Wet-lab assays (ISH, qPCR, mouse/Xenopus genetics) out of scope as non-pipeline.
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
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v1 current initial assessment Score 84assessed: 2026-06-15 ⛓ e634ae09d4d2
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Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-15
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-09-19
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether retinoic acid (RA) signaling is the molecular link between mesodermal Tbx5 activity and endodermal Shh expression, establishing how Tbx5 coordinates a conserved RA-Hedgehog-Wnt gene regulatory network for cardiopulmonary development.
- ★ Tbx5 directly maintains Aldh1a2 expression in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer mechanism
- ★ Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the foregut endoderm through a conserved MACS1 enhancer mechanism
- ★ Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate wnt2/2b expression, which induces pulmonary fate in the foregut endoderm mechanism
- ★ Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior mechanism
- ★ Tbx5-/- mouse CP tissue shows reduced pSHF/pulmonary transcriptional program and gained aSHF/pharyngeal gene expression finding
- ★ Tbx5 loss-of-function in Xenopus (morpholino or CRISPR) phenocopies mouse Tbx5-/- cardiopulmonary defects and reduces aldh1a2 expression, demonstrating conservation finding
- RA signaling negatively regulates Fgf8/Fgf10-positive aSHF fate finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq | mouse E9.5 micro-dissected cardiopulmonary (foregut mesoderm+endoderm) tissue | Tbx5 knockout | differentially expressed genes | — |
| RT-qPCR | mouse E9.5 CP tissue | Tbx5 knockout | Aldh1a2, Fgf8, Fgf10 relative expression | — |
| whole-mount immunostaining/confocal imaging | mouse E9.5 Shh:GFP transgenic embryos | none (WT) | Tbx5, Aldh1a2, Nkx2-1 protein co-expression | confocal microscopy |
| gene set enrichment analysis (GSEA) | mouse CP tissue transcriptome vs single-cell RNA-seq gene sets | computational/none | enrichment of aSHF/pharynx vs pSHF/lung gene sets among Tbx5-/- DEGs | — |
| digital and serial-section in-situ hybridization / 3D reconstruction | mouse E9.5 foregut | none (WT) | Aldh1a2, Tbx5, Tbx1, Shh, Fgf8, Fgf10 expression domains | online Spatial Mouse Atlas |
| in-situ hybridization | Xenopus tropicalis F0 embryos | tbx5 exon5 CRISPR/Cas9 mutation | aldh1a2 and other target gene expression | Cas9 protein (PNA Bio CP01-20) |
| immunofluorescence quantification (3D volume rendering) | Xenopus laevis Tbx5-MO morphants and X. tropicalis tbx5 CRISPR mutants (NF34) | Tbx5 knockdown (MO) or mutation (CRISPR), with/without human TBX5 RNA rescue | Aldh1a2 protein volume pixel intensity in foregut lpm/pSHF | Nikon Elements Analysis AR software |
| transgenic reporter imaging | Xenopus Tg(WntRE:dGFP) Wnt/β-catenin reporter embryos | Tbx5 depletion | Wnt reporter GFP expression in ventral foregut | — |
- – 1588 genes upregulated and 1480 genes downregulated in Tbx5-/- CP tissue (≥1.5 fold change, 5% FDR) 1.5-fold
- ▲ 25% (91/366) of aSHF/pharynx-enriched genes overlapped with genes upregulated in Tbx5-/-, versus only 5% (10/213) of pSHF/lung genes p<0.0001
- ▼ 34% (72/213) of pSHF/lung marker genes were downregulated in Tbx5-/- mutants, versus only 6% (21/366) of aSHF+pharynx genes p<0.001
- – GSEA showed overrepresentation of aSHF/pharynx genes among upregulated genes and pSHF/lung genes among downregulated genes in Tbx5-/- CP tissue NES=1.58 (up), NES=-1.99 (down), both p<0.0001
- – RT-qPCR confirmed Aldh1a2 downregulation and Fgf8/Fgf10 upregulation in Tbx5-/- CP tissue p<0.05
- ▼ Aldh1a2 protein in foregut lpm/pSHF reduced in Xenopus Tbx5-MO morphants and tbx5 CRISPR mutants relative to WT ~28% of WT (p=0.0009, morphants), ~33% of WT (p≤0.0001, mutants)
- ▼ Loss of Tbx5 caused downregulation of aldh1a2 in foregut lpm beginning at NF25 but not at NF15
- ▲ Co-injection of human TBX5 RNA rescued aldh1a2 expression and pulmonary development in Tbx5-depleted Xenopus embryos
- count 1588 upregulated / 1480 downregulated genes (Tbx5-/- vs WT mouse CP tissue RNA-seq DEGs)
- fold_change ≥1.5 fold change, 5% FDR (differential expression cutoff for Tbx5-/- CP tissue)
- pvalue p<0.0001 (hypergeometric test, aSHF/pharynx gene overlap with Tbx5-/- upregulated genes (91/366))
- pvalue p<0.001 (hypergeometric test, pSHF/lung gene overlap with Tbx5-/- downregulated genes (72/213))
- other NES=1.58, p<0.0001 (GSEA enrichment of aSHF/pharynx genes among upregulated genes)
- other NES=-1.99, p<0.0001 (GSEA enrichment of pSHF/lung genes among downregulated genes)
- mean ~28% of WT (p=0.0009) (Aldh1a2 immunostaining intensity in Xenopus Tbx5-MO morphant fg lpm/pSHF)
- mean ~33% of WT (p≤0.0001) (Aldh1a2 immunostaining intensity in Xenopus tbx5 CRISPR mutant fg lpm/pSHF)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper combines reanalysis of published bulk RNA-seq from micro-dissected mouse cardiopulmonary tissue with gene-set enrichment and hypergeometric overlap tests to characterize Tbx5-regulated transcriptional networks, then validates key findings by RT-qPCR and quantitative immunofluorescence in both mouse and Xenopus models. Individual gene comparisons use Student's t-tests or parametric paired t-tests, and results are reported as mean ± SD with significance thresholds. The paper text provided is truncated before the full Methods and statistical sections, so additional tests used in later figures may not be captured here.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Differential expression analysis (bulk RNA-seq; ≥1.5-fold change, 5% FDR threshold) | WT vs Tbx5−/− mouse E9.5 cardiopulmonary tissue; Figure 1A–B | n=5 WT, n=2 Tbx5−/− biological replicates (mouse embryos) | not stated |
| Hypergeometric probability test (HGT) | Overlap of Tbx5-regulated genes with aSHF/pharynx vs pSHF/lung gene sets from scRNA-seq; Figure 1A | Gene set sizes: 366 aSHF+pharynx genes, 213 pSHF+lung genes; 1588 up and 1480 down in Tbx5−/− | not stated |
| Gene Set Enrichment Analysis (GSEA) | Tbx5-regulated transcriptome vs aSHF/pharynx and pSHF/CPP/lung gene sets; Figure 1—figure supplement 1A–B | Full ranked transcriptome from RNA-seq (n=5 WT, n=2 mutant) | not stated |
| Student's t-test (two-tailed, unpaired implied) | RT-qPCR validation of Aldh1a2, Fgf8, Fgf10 in E9.5 WT vs Tbx5−/− CP tissue; Figure 1C | not stated | not stated |
| Parametric two-tailed paired t-test | Quantification of Aldh1a2 immunofluorescence volume pixel intensity in Tbx5 morphant and CRISPR mutant vs control Xenopus fg lpm/pSHF; Figure 2—figure supplement 1B–C | N=3 embryos per group; each dot = one fg lpm/pSHF region | not stated |
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The RNA-seq differential expression reanalysis used n=2 biological replicates in the Tbx5−/− group↳ Could also: A larger number of biological replicates (e.g., n≥3 per group) could also be used, and tools such as DESeq2 or edgeR explicitly model dispersion across replicates — With only two mutant samples, variance estimation is highly uncertain; additional replicates would improve dispersion modeling and increase statistical power for identifying differentially expressed genes
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Multiple Student's t-tests were performed across RT-qPCR targets (Aldh1a2, Fgf8, Fgf10) without a stated correction for multiple comparisons↳ Could also: A Bonferroni or Benjamini-Hochberg correction applied across the family of RT-qPCR comparisons could also be used — Applying a multiplicity correction to the set of RT-qPCR comparisons would explicitly control the family-wise error rate or false discovery rate across the tested genes
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A parametric paired t-test was used for immunostaining quantification with N=3 embryos per group↳ Could also: A non-parametric Wilcoxon signed-rank test could also be applied at this sample size — With very small n, normality assumptions underlying parametric tests are difficult to verify; a non-parametric alternative makes no distributional assumption and is often preferred when n<10
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Dispersion for RT-qPCR results is reported as SD↳ Could also: A 95% confidence interval or SEM could also be reported alongside the mean — For small n, 95% CIs convey both the spread and the uncertainty of the mean estimate, facilitating interpretation of biological variability and supporting effect-size reasoning
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GSEA p-values are reported as nominal thresholds (p<0.0001) without explicit statement of the permutation procedure or FDR q-values↳ Could also: Reporting GSEA FDR q-values alongside NES and nominal p-values is also standard practice (e.g., as recommended in Subramanian et al., 2005) — FDR q-values from GSEA account for multiple gene-set testing and are commonly reported to allow readers to assess significance relative to the full collection of tested gene sets
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Gene-set overlap significance was assessed with a hypergeometric test on binary gene lists defined by a fixed fold-change and FDR threshold↳ Could also: A rank-based method such as GSEA or a Fisher's exact test on continuously ranked gene scores could also be used for the same overlap question — Threshold-free rank-based approaches use the full distribution of effect sizes rather than a binary cutoff, which can be more sensitive to moderate but consistent shifts across a gene set
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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ALDH1A2 protein is reduced to ~28% of WT in Tbx5 morphant Xenopus foregut lateral plate mesoderm, demonstrating conserved Tbx5-dependent regulation across vertebratesimaging xenopus laevis foregut lpm down 2021×1papers★ This paper is the founder (earliest)
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ALDH1A2 is downregulated in Tbx5-/- mouse cardiopulmonary tissue at E9.5 as validated by RT-qPCR, establishing TBX5 as a direct driver of retinoic acid biosynthesis; FGF8 and FGF10 are reciprocally upregulatedqPCR mouse cardiopulmonary tissue down 2021×1papers★ This paper is the founder (earliest)
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GSEA shows aSHF/pharynx genes enriched among upregulated transcripts (NES=1.58) and pSHF/lung genes enriched among downregulated transcripts (NES=-1.99) in Tbx5-/- cardiopulmonary tissue, statistically confirming an anterior-posterior identity switchRNA-seq mouse cardiopulmonary tissue mixed 2021×1papers★ This paper is the founder (earliest)
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25% of aSHF/pharyngeal-enriched genes are upregulated in Tbx5-/- mouse cardiopulmonary tissue, indicating TBX5 normally represses anterior second heart field identityRNA-seq mouse cardiopulmonary tissue up 2021×1papers★ This paper is the founder (earliest)
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34% of pSHF/lung marker genes are downregulated in Tbx5-/- mouse cardiopulmonary tissue at E9.5, indicating TBX5 promotes posterior second heart field and pulmonary identityRNA-seq mouse cardiopulmonary tissue down 2021×1papers★ This paper is the founder (earliest)
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TBX5 knockout in mouse cardiopulmonary tissue at E9.5 broadly dysregulates 3068 genes (1588 up, 1480 down at ≥1.5-fold, 5% FDR), demonstrating extensive transcriptional control of cardiopulmonary patterningRNA-seq mouse cardiopulmonary tissue mixed 2021×1papers★ This paper is the founder (earliest)
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
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Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
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What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-34643182
Paper: Rankin, Steimle, Yang et al. Tbx5 drives Aldh1a2 expression to regulate a RA-Hedgehog-Wnt gene regulatory network coordinating cardiopulmonary development. eLife 2021;10:e69288. PMCID PMC8555986.
Code artifact: CSBB-v3.0 — Computational Suite For Bioinformaticians and Biologists
(github.com/praneet1988/Computational-Suite-For-Bioinformaticians-and-Biologists).
A third-party bioinformatics toolkit authored by Praneet Chaturvedi (a co-author of
the paper). Per BRIEF rule 2 (P16), applying this published tool to the paper's own data
is a fully valid reproduction. The relevant module is DifferentialExpression
(Perl wrapper → Modules/RUVseq.r / Modules/RUVseq_with_empirical.r): RUVSeq
upper-quantile normalization + edgeR GLM-LRT.
Data: GEO GSE75077, supplementary GSE75077_Transcript_ReadCount.txt.gz
(272 KB). Gene-level read-count matrix, 23,419 genes × 7 samples — 5 wild-type
(WT_CPP_1..5) and 2 Tbx5-mutant (Tbx5_Mut_1,2) microdissected posterior second
heart field (pSHF) at mouse E9.5. Columns already in CSBB-required order (controls
first). Public, no restriction.
In scope (pipeline-derived, attempted)
| id | reported result | location | pipeline |
|---|---|---|---|
| C1 | 1588 up- and 1480 down-regulated genes in the absence of Tbx5 (≥1.5 fold change, 5% FDR) | Results / Fig 1 text | CSBB DifferentialExpression (RUVSeq UQ + edgeR GLM-LRT) on GSE75077 |
| C2 | Direction/identity of key network genes (Aldh1a2, Wnt2/Wnt2b, Shh, Osr1, Hand1, Fgf8 …) in the DE table / Fig 1B heat map | Fig 1B | same DE table as C1 |
Out of scope (not attempted; reasons)
- Wet-lab: in-situ hybridization, qPCR, mouse genetics, RNAscope, Xenopus/explant assays, ChIP-qPCR validations — manual/experimental, not pipeline-derived.
- The biological GRN model (RA-Hedgehog-Wnt) — interpretive, not a single computed value.
- No raw FASTQ realignment: the authors deposited the count matrix (GSE75077 suppl); we reproduce DE from that matrix, as the pipeline downstream of alignment. Re-running RSEM/Bowtie2 from SRP066296 is the optional hard 20% and is not attempted (80/20).
Key ambiguity (recorded honestly)
The paper text states ≥1.5 FC and 5% FDR but does not state the CSBB filter
parameters (Counts threshold, min samples) or which normalization variant
(UQ vs UQ+Empirical/RUVg) produced the 1588/1480 split. We therefore sweep a small
grid of both and report the closest configuration, flagging the rest as parameter
under-specification rather than asserting a single ground truth.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
Using the authors' own deposited GSE75077 count matrix and their documented CSBB-v3.0 DifferentialExpression tool verbatim, the up-regulated count reproduces to within 2 genes (1586 vs 1588) and every Fig-1B gene — including the thesis gene Aldh1a2 (FC 0.43, FDR 4e-6) and the full RA→Hedgehog→Wnt axis — moves as reported, so the central conclusion fully holds. The only deviation is a systematic ~11% shortfall in the down-count (1322 vs 1480) that persists across the entire filter grid, sitting in the edgeR/RUVSeq computation and most consistent with package version drift (2026 vs ~2016 stack), not an authors' or data defect. A minor methodology gap exists on our/paper side — the normalization variant was unspecified and had to be pinned from the numbers — but there is no fabrication signal: headline values are derivable from shared data+tool. Overall a solid, mostly 1:1 reproduction with one small, explainable discrepancy → yellow.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.