Hippo signaling differentially regulates distal progenitor subpopulations and their transitional states to construct the mammalian lungs.
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
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
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 to reproduce the deterministic pipeline outputs 1:1 / near-1:1. Resolved that the BRIEF's GSE269537 is the E13.5 BULK RNA-seq (not the scRNA data); scRNA lives in GSE319370, multiomics in GSE324638. C1 (bulk E13.5 DEGs): downloaded the shipped DESeq2 table and recovered EXACTLY 919 DEGs = 308 down + 611 up using nominal pvalue<0.05 (padj<0.05 gives only 134, so the paper uses unadjusted p) — exact match, uniquely identified criterion. C2 (scRNA-seq cells analyzed after QC): re-implemented the repo's obj_create_process_fun QC (CreateSeuratObject min.cells=3/min.features=200; nFeature 200-7500, nCount>per-sample-UMI {5800,5107,3700,3400}, percent.mt<5) on the 4 GSE319370 filtered_feature_bc_matrix.h5 files; got 13791/13267/9035/31392 vs reported 13985/13275/8979/31392 — one EXACT (E17.5_Mt), one within 8 cells, all four within 1.4%. Residuals attributable to the paper's additional EmptyDrops (FDR 0.1%) and non-epithelial lineage-cluster removal, which are not in the QC-only repo function. No fabrication signals: every reported number is derivable from the shipped GEO data with documented/inferred parameters. NOT attempted (honest, out of scope): cluster identities/final_clusters/UMAPs (Figs 6-7), per-cluster FindMarkers DEG lists, Monocle3 pseudotime, TF/GRN, and multiomics (GSE324638) — all gated behind interactive manual clustering decisions not reproducible from the shipped code. Partial, honest pass over deterministic outputs; not a completeness claim. Compute on «our HPC» («job», node n158); data + repo on «infra», only small results on «host».
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 91assessed: 2026-06-16 ⛓ e7d055d4d688
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-16
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-16no human curator yet
- Last updated
- 2026-08-05
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 how Hippo signaling (via YAP/TAZ activity) controls the size and differentiation of the distal SOX9+ progenitor pool in the developing lung, and whether a subdomain of these progenitors is sufficient to drive lung outgrowth and generate the distinct cell types of the conducting airways versus the alveolar epithelium.
- ★ A fraction (15-50%) of the distal SOX9+ tip progenitor subdomain is sufficient to direct lung outgrowth through branch bifurcation, providing a mechanism for lung size control finding
- ★ YAP/TAZ levels must be tightly regulated by Hippo signaling to balance proliferation and differentiation of SOX9+ progenitors; both low YAP/TAZ (Yap loss) and high YAP/TAZ (Lats1/2 loss) deplete or disorganize the SOX9+ progenitor pool mechanism
- ★ Lats1/2 function through Yap/Taz to control SOX9+ progenitor number and differentiation, since removing one allele each of Yap and Taz in Lats1/2-deficient lungs partially rescues branching and cell-type defects mechanism
- ★ Elevated YAP/TAZ activity (via Lats1/2 removal) converts SOX9+/SOX2+ cells into a SOX9-SOX2- state that adopts the AT1 fate, disrupting production of conducting airway cell types and AT2 cells finding
- ★ Single-cell transcriptome and chromatin accessibility analyses identify transitional cell states and candidate regulators of lung cell fate method
- Some developmental paths of transitional cell states identified in mouse lungs correspond to those in human lungs finding
- Yap-deficient lung epithelium (Yap f/f; Shh-Cre or Sox9-Cre) forms distal lung cysts lined by SOX9+ cells lacking SOX2+ cells, indicating failure to produce SOX2+ progeny finding
- ★ Hippo signaling induces distinct cell fates at different SOX9+ subdomains to produce the conducting airways versus the alveolar epithelium mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Whole-mount imaging and immunostaining | mouse lung, Yap f/f;Shh-Cre and Yap f/f;Sox9-Cre embryos | Yap knockout (epithelial) | SOX9+/SOX2+ cell presence, lung cyst formation | — |
| Whole-mount imaging and immunostaining | mouse lung, Lats1 f/f;Lats2 f/f;Shh-Cre embryos | Lats1/2 knockout (broad epithelial) | SOX9+ domain size, lung wet weight | — |
| Whole-mount imaging, immunostaining, lung wet weight measurement | mouse lung, Lats1 f/f;Lats2 f/f;Sftpc-Cre (Lats1/2-mosaic) with ROSA26-mTmG reporter | mosaic Lats1/2 knockout | lung weight, branching pattern, saccule formation, SCGB1A1/Ac-tub/SFTPC/HOPX/PDGFRA/PDGFRB marker expression | — |
| Whole-mount imaging and immunostaining, cell quantification | mouse lung, Lats1 f/f;Lats2 f/f;Yap f/+;Taz f/+;Shh-Cre or Sftpc-Cre (rescue) embryos | Lats1/2 knockout with heterozygous Yap/Taz removal | branching pattern rescue, percentage of SOX2+, SCGB1A1+, Ac-tub+, SFTPC+, HOPX+ cells | — |
| Immunofluorescence for phospho-YAP (pYAP) and E-cadherin/β-catenin | mouse lung sections, control and Lats1/2-mosaic embryos at 14.5 dpc | mosaic Lats1/2 knockout | relative pYAP intensity in proximal vs distal SOX9+ domains | — |
| Single-cell transcriptome analysis | mouse lung epithelium (developmental stages) | none/comparative across genotypes | identification of transitional cell states and fate-determining candidate genes | — |
| Chromatin accessibility assay | mouse lung epithelial cells | none/comparative | regulatory elements associated with transitional states | — |
| Comparative single-cell analysis | human lung tissue | none | correspondence of transitional cell developmental paths between mouse and human | — |
- ▼ Wet weight of Lats1/2-mosaic (Sftpc-Cre) lungs only slightly less than control at 18.5 dpc
- ▼ Wet weight of Lats1/2 Shh-Cre-deficient lungs was a small fraction of control lungs
- ▼ Number of SCGB1A1+ (club) and Ac-tub+ (ciliated) cells greatly decreased in Lats1/2-mosaic lungs vs controls
- ▼ Saccule formation failed and few/no SFTPC+ (AT2) cells present in Lats1/2-mosaic lungs; disorganized epithelium predominantly HOPX+ (AT1)
- ▲ Removal of one allele each of Yap and Taz partially rescued branching defects and cell-type production in Lats1/2-mosaic lungs
- – pYAP (and thus Lats1/2 activity) preserved in 15-50% of the distal SOX9+ subdomain in mosaic lungs, while nearly absent proximally 15-50%
- – Loss of one, two, or three alleles of Lats1/2 (partial dosage) did not cause apparent lung defects
- count n=4 pairs (lung wet weight comparison, control vs Lats1 f/f;Lats2 f/f;Sftpc-Cre at 14.5/18.5 dpc)
- count n=3 pairs (lung wet weight comparison, control vs Lats1 f/f;Lats2 f/f;Shh-Cre at 14.5/18.5 dpc)
- other 15-50% (estimated preservation of pYAP/distal SOX9+ subdomain in Lats1/2-mosaic lungs)
- count n=3 pairs (quantification of relative pYAP intensity in proximal/distal lung branch domains, 14.5 dpc)
- count n=3 per genotype (quantification of SOX2+, SCGB1A1+, Ac-tub+, SFTPC+, HOPX+ cell percentages across control, Lats1/2-mosaic, and rescued mice at 18.5 dpc)
- pvalue p<0.05; p<0.001 (significance markers for Fig. 1 comparisons (two-tailed Student's t-test))
- pvalue p<0.05; p<0.01; p<0.001 (significance markers for Fig. 2 comparisons (two-way ANOVA))
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.
This mouse genetic study used Cre-mediated mosaic inactivation of Lats1/2 and Yap/Taz to interrogate Hippo signaling in distal SOX9+ lung progenitors, combining immunofluorescence-based cell quantification with single-cell transcriptomic and chromatin accessibility assays. Pairwise quantitative comparisons between control and mutant genotypes used two-tailed Student's t-tests, while multi-genotype, multi-cell-type quantifications used two-way ANOVA; significance was indicated by threshold asterisks rather than exact p-values. All quantitative values are reported as mean ± SEM. The provided text is a partial transcript; additional statistical methods for the single-cell analyses may appear in sections not included here.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| two-tailed Student's t-test | Lung wet weight comparisons between control and Lats1/2-mosaic (Sftpc Cre) and control and Lats1/2 (Shh Cre) mice (Fig. 1c); relative pYAP intensity in proximal vs. distal domains of control and Lats1/2-mosaic lungs (Fig. 3i) | n = 4 pairs (Sftpc Cre) and n = 3 pairs (Shh Cre) for wet weight; n = 3 pairs for pYAP intensity | not stated |
| two-way ANOVA | Quantification of percentage of SOX2+, SCGB1A1+, Ac-tub+, SFTPC+, HOPX+, and SFTPC+HOPX+ cells across control, Lats1/2-mosaic, and rescued genotypes (Fig. 2j) | n = 3 per genotype | not stated |
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Lung wet weight and pYAP intensity were compared between two groups with n = 3–4 biological pairs using a Student's t-test↳ Could also: A Mann-Whitney U (Wilcoxon rank-sum) or Wilcoxon signed-rank (if paired) test could also be used — With n as small as 3–4, the normality assumption underlying the t-test cannot be empirically verified; non-parametric alternatives make no distributional assumption and are equally standard at this sample size
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For Fig. 2j, a two-way ANOVA is applied across three genotypes and six cell-type metrics without a named post-hoc correction↳ Could also: A named post-hoc test such as Tukey HSD (all pairwise) or Dunnett's test (vs. control) could also be applied following the omnibus ANOVA — Specifying the post-hoc procedure clarifies exactly which contrasts were tested and at what adjusted error rate, making the analysis fully reproducible and the multiplicity control explicit
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Dispersion is reported as mean ± SEM throughout↳ Could also: Mean ± SD, or showing individual data points overlaid on a summary bar, would also be standard ways to convey spread — SEM shrinks with larger n and reflects precision of the mean estimate rather than biological variability; SD or individual-point plots communicate the actual spread across replicates, which some reporting guidelines prefer for small n
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P-values are reported only as threshold bands (* <0.05, ** <0.01, *** <0.001)↳ Could also: Reporting exact p-values (e.g., p = 0.018) and standardized effect sizes (e.g., Cohen's d) with 95% CIs would also be standard — Exact p-values convey the actual evidence strength rather than a categorical bin; effect sizes with CIs communicate biological magnitude and support future meta-analyses
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Randomization of sample processing order and investigator blinding during cell counting are not described↳ Could also: Blinded scoring of immunofluorescence images and documented randomization of sample order are also standard practice in mouse phenotyping studies — Documenting blinding and randomization allows readers to assess potential ascertainment bias in subjective fluorescence-based cell counting, particularly relevant when the investigator knows the genotype
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Statistical software is not named anywhere in the provided text↳ Could also: Citing the specific software package and version (e.g., GraphPad Prism 10, R 4.4, Python/scipy 1.13) is also standard in methods sections — Software version documentation supports reproducibility, since default algorithms, rounding, and tie-handling can differ across packages and versions
Citation network
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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-41932885
Paper: Zhang K, Basak M, Zaher Y, Yao E, Wang SA, Aung T, Chuang PT. Hippo signaling differentially regulates distal progenitor subpopulations and their transitional states to construct the mammalian lungs. Nat Commun 2026. PMID 41932885 · PMCID PMC13219436 · DOI 10.1038/s41467-026-71253-x.
Code: https://github.com/yzaher/HippoSignaling (commit d28c4171350409fefb1d1febd8a6a445b7b9d901). R scripts only — Seurat/Monocle3 scRNA-seq + multiomics analysis. No bulk RNA-seq DEG script shipped.
Data (3 GEO series, clarified from the Data-availability statement):
- GSE269537 — E13.5 bulk RNA-seq (6 libs: Control rep1-3, Lats1/2-deficient rep1-3,
BGISEQ-500). Supplementary
GSE269537_Processed_data_Lats1-2_SpcCre_13.5dpc.csv.gzis a DESeq2-style table (Gene, log2FoldChange, pvalue, padj, 6 per-sample counts; 23,420 genes). - GSE319370 — scRNA-seq, 4 samples: GSM9516767 E14.5 control, GSM9516768 E14.5 mutant,
GSM9516769 E17.5 control, GSM9516770 E17.5 mutant. Each ships a CellRanger v3
filtered_feature_bc_matrix.h5. - GSE324638 — multiomics (snRNA + snATAC) E14.5/E17.5. Not attempted here.
In scope (pipeline-derived, attempted)
| ID | Result | Reported | Pipeline | Tractability |
|---|---|---|---|---|
| C1 | E13.5 bulk DEG count | 919 total (308 down, 611 up), Fig.1k | threshold on processed DESeq2 table | deterministic — high |
| C2 | scRNA-seq cells analyzed after QC | E14.5: 13,985 Ct / 13,275 Mt; E17.5: 8,979 Ct / 31,392 Mt | CellRanger→Seurat QC (repo obj_create_process_fun) |
deterministic given per-sample UMI thresholds — medium |
Out of scope / not attempted (and why)
- Cluster identities,
final_clusters, UMAP layouts (Figs 6–7): the repo derives these through interactive, manual steps — visualFeaturePlotinspection, hand-codedcase_whencluster→label maps, per-step resolution choices. Not reproducible without the authors' manual judgement; non-deterministic. - Per-cluster / per-sample DEG lists (
FindMarkers, VolcanoPlots scripts): depend entirely on the manual clustering above; only meaningful once C2's clusters are matched. - Monocle3 pseudotime, TF/GRN (
TF_GRN_function.R): downstream of manual clustering. - Multiomics (GSE324638): separate modality, large; not attempted in this pass.
- Wet-lab measurements (qPCR, immunostaining, branch counts): not computational.
- The paper's reanalysis of published external scRNA-seq (E12.5–E18.5) for comparison: out of scope (external data + manual integration).
QC method (from Methods, Seurat v5.2.1; matches repo obj_create_process_fun)
CellRanger 8.0.1, mm10 (refdata-gex-mm10-2020-A). CreateSeuratObject(min.cells=3, min.features=200); subset nFeature_RNA 200–7500 & nCount_RNA > UMI(per-sample) & percent.mt < 5 (pattern ^mt-|^MT-); nfeatures=2000; PCs explaining >90% cumulative
variance; Louvain FindClusters; UMAP. Per-sample UMI thresholds from the repo:
E14.5_Ct 5800, E14.5_Mt 5107, E17.5_Ct 3700, E17.5_Mt 3400. (Paper also mentions EmptyDrops
FDR 0.1% and removal of non-epithelial lineage clusters — see AUDIT for impact on C2.)
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a clean, near-1:1 reproduction. C1 (E13.5 bulk DEGs, Fig.1k) reproduced exactly (919 = 308 down + 611 up) from the shipped GSE269537 DESeq2 table, with the useful finding that the paper used nominal p<0.05 (padj gives only 134). The four scRNA post-QC cell counts reproduced within ≤1.4% (one exact, one within 8 cells); the small residuals lie on our methodology side — the QC-only repo function omits the paper's EmptyDrops and lineage-cluster-removal steps. No fabrication signals, no significance/direction issues; deviations are negligible and fully explained. The deeper biological conclusions were honestly out of scope (gated behind manual clustering), so q7 reflects the tested claims only.
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.