Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spid
The main results reproduced, with only marginal, non-material deviations.
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 relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- Every checked point held up.
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: YES for the RNA-seq edgeR pipeline. Listed code = AUGUSTUS (third-party gene predictor; aug3 models are fixed annotation, P16 - not regenerated). In-scope pipeline targets C1-C5 were recomputed directly from the deposited HTSeq count matrices (GSE193511, GSE193650) with edgeR on «our HPC» («job»), no alignment needed. Result is DIFFERENT-BUT-CLOSE rather than 1:1: C1 (version-independent cpm filter) reproduced essentially exactly (10860 vs 10862, and the 27990-annotated-gene figure to the gene); the four DEG/candidate counts all reproduced slightly BELOW the reported values in a consistent direction (C2 15/19, C3 5/6, C4 134/139, C5 749/785) - the expected signature of edgeR version drift (paper 3.8.6/2014 vs available 4.8.2) plus an unspecified gene-filter. Every reported number is derivable from the deposited data: NO fabrication concern. NOT attempted: ATAC accessibility (A1/A2, GSE193870 - raw reads only, heavy Genrich+csaw pipeline) and the MEGA11 GATA phylogenetics (manual curation, out of scope). Honest partial reproduction.
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
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
-
v1 current initial assessment Score 50assessed: 2026-06-19 ⛓ d9f248643ed7
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
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
- 2026-06-29
- Rubric version
- v1.0
- Assessed by
-
🤖 AI curator · v1.0 · run #1 2026-06-19no 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 study asks whether genome-wide comparative transcriptomics of spatially isolated blastoderm cells can identify lineage-specific genes with key regulatory roles in early embryonic patterning of the spider Parasteatoda tepidariorum, and specifically whether the identified GATA-like gene fuchi nashi (fuchi) regulates zygotic gene activation to drive endoderm specification and germ-disc pattern formation.
- ★ Comparative RNA-seq of cells isolated from central, intermediate, and peripheral regions of stage-3 embryos identifies genes with locally restricted expression genome-wide method
- ★ A pilot pRNAi screen of 19 candidate DEGs identifies three genes (g26874/fuchi, g7720, g4238) required for germ-disc formation and/or cumulus movement finding
- ★ g26874, named fuchi nashi (fuchi), is a lineage-specific, fast-evolving GATA-like gene arising from duplication and divergence of a canonical GATA family gene finding
- ★ fuchi knockdown reverses the cell density shift that normally forms the germ disc, destabilizing its boundary finding
- ★ fuchi is expressed in cells outside the forming germ disc and persists in the endoderm finding
- ★ fuchi activity regulates chromatin state and zygotic gene activation to promote endoderm specification and pattern formation mechanism
- g7720 encodes an ortholog of Drosophila Pbp49 and mouse Snapc3 resource
- g4238 is Pt-Ets4, an ortholog of Drosophila Ets98B resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RNA-seq (comparative transcriptomics of isolated cell populations) | P. tepidariorum embryo, stage 3 germ disc, central/intermediate/peripheral cells | none | differentially expressed genes (CPM, FDR, log2FC) via edgeR | — |
| whole-mount in situ hybridization (WISH) | P. tepidariorum embryo, stage 3 | none | spatial expression pattern of 19 candidate genes | — |
| RNA-seq (comparative transcriptomics of isolated cell populations) | P. tepidariorum embryo, stage 4 and early stage 5 | none | differentially expressed genes | — |
| parental RNAi (pRNAi) screen with dsRNA injection | P. tepidariorum adult females and resulting embryos | RNAi knockdown of 19 candidate genes (and gfp control) | developmental/morphological phenotype under stereomicroscope | — |
| time-lapse microscopy and cell tracking | P. tepidariorum pRNAi embryos (g26874, g7720, g4238, gfp control) | RNAi knockdown | cell movement/germ-disc and cumulus formation dynamics | — |
| WISH | P. tepidariorum pRNAi embryos (stage 3 and/or late stage 5) | RNAi knockdown of target genes | reduction of target transcript levels | — |
| developmental transcript profiling (RPKM, public RNA-seq datasets) | P. tepidariorum whole wild-type embryos, stages 1-10 | none | transcript abundance time-course for g26874, g7720, g4238 | — |
| blastp/tblastn reciprocal sequence search | Drosophila melanogaster and mouse RefSeq protein databases vs P. tepidariorum aug3.1 transcripts | none | orthology/best-hit identification | — |
- – 19 high-priority candidate genes with locally restricted expression selected from genome-wide analysis of 10,862 genes
- – Seven of 19 candidate genes showed specific WISH expression at the embryonic pole and/or abembryonic side, validating the selection strategy
- – pRNAi screen identified g26874 (fuchi) and g7720 as required for germ-disc formation, and g26874, g7720, g4238 as required for cumulus movement
- ▼ fuchi (g26874) pRNAi embryos showed faint or failed CM cell migration 64% of embryos (n=101 from 10 egg sacs)
- – Severe fuchi pRNAi embryos showed reversed cell movement, breakdown of surface cell layer, and yolk extrusion
- ▼ g7720 pRNAi embryos arrested around end of stage 3 with a gradually degenerating germ disc
- ▼ g4238 pRNAi embryos failed to initiate CM cell migration at early stage 5, followed by disassembly and dispersal
- – Reciprocal blast searches did not confirm Drosophila/mouse orthologues for g26874, which was more closely related to other P. tepidariorum GATA genes (e.g., g8336) than to Pannier or GATA-4
- count 10,862 genes (total genes analyzed in genome-wide RNA-seq DEG comparison)
- count 19 candidate genes (high-priority DEG candidates selected for pRNAi screen)
- other log2FC < -10 (comparisons I/II) or > 10 (comparison III), FDR-ranked (threshold for prioritizing candidate DEGs)
- count 64% (proportion of g26874 (fuchi) pRNAi embryos with faint/failed CM cell migration, from 10 egg sacs)
- count 3 of 10 (Group C genes showing pole-specific WISH expression)
- count top 10 (Group C), top 5 (Group P), top 5 (Group CP) (gene selection tiers from three DEG comparisons)
- other 9, 15, 20/21, 25/26, 31/32, 36/37, 42/43, 50, 60, 78 h AEL (sampling timepoints for developmental transcript profiling)
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 is primarily a functional-genomics study combining RNA-seq-based differential expression screening with a knockdown (pRNAi) phenotypic screen. Region-specific candidate genes were identified from biological replicates of isolated embryonic cells using edgeR-based comparisons (with FDR and log2 fold-change thresholds), and phenotypic outcomes of gene knockdown were reported primarily as descriptive counts/percentages of affected embryos rather than through classical parametric or nonparametric hypothesis tests. No t-tests, ANOVA, or explicit significance tests for the phenotype data are described in the provided text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| edgeR differential expression testing (exact/GLM test type not specified) with FDR and log2 fold-change thresholds | Comparisons I, II, and III of RNA-seq read counts among central (c), intermediate (i), and peripheral (p) cell samples (Fig. 2C-E; stage 3, and similarly for stage 4/early stage 5) | Biological replicates per cell-region sample (e.g., c1, c2, i1, i2, i3, p1, p2, p3, as shown in Fig. 2) | not stated |
-
Differential expression between cell-region samples was assessed with edgeR using FDR and fold-change thresholds.↳ Could also: DESeq2 or a limma-voom pipeline — These are widely used alternative RNA-seq differential expression frameworks that use different dispersion-estimation approaches and could also be applied to the same count data, sometimes offering different sensitivity with small replicate numbers.
-
The RNA-seq comparisons relied on 2-3 biological replicates per cell-region sample.↳ Could also: Increasing the number of biological replicates, or supplementing FDR-based ranking with a reported confidence interval or variance estimate per gene — Additional replicates or explicit variance/CI reporting can also help characterize the precision of expression-level estimates in small-replicate designs.
-
The FDR correction method underlying the edgeR analysis is not specified.↳ Could also: Explicitly stating the correction procedure (e.g., Benjamini-Hochberg) or using Storey's q-value approach — Naming the exact multiple-testing procedure, or using an alternative such as q-values, can also make the multiplicity control assumptions transparent to readers.
-
Phenotypic penetrance of pRNAi knockdown (e.g., 64% of g26874 embryos, n = 101) was reported descriptively as a percentage without a formal statistical comparison to a control group.↳ Could also: A formal proportion comparison such as Fisher's exact test or a chi-square test, with an accompanying confidence interval for the proportion — This type of test could also be used to quantify how the knockdown penetrance compares statistically to the control (gfp-RNAi/wild-type) phenotype frequency and to convey estimation uncertainty via a confidence interval.
-
Embryo phenotypes were scored under a stereomicroscope and by time-lapse microscopy without a stated blinding procedure.↳ Could also: Blinded scoring of embryo phenotypes by an observer unaware of the injected dsRNA — Blinded scoring is a standard practice that could also be incorporated to reduce the possibility of observer expectation influencing phenotype classification.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36203191
Paper: Iwasaki-Yokozawa, Nanjo, Akiyama-Oda, Oda (2022) BMC Biol 20:223. "Lineage-specific, fast-evolving GATA-like gene (fuchi nashi) regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider Parasteatoda tepidariorum." DOI 10.1186/s12915-022-01421-0 · PMCID PMC9535882.
Listed code: https://github.com/Gaius-Augustus/Augustus — third-party gene
predictor (AUGUSTUS). Used to build the P. tepidariorum gene models (aug3.1)
against which HTSeq counted reads. P16 case: AUGUSTUS is a generic tool, not the
authors' analysis code. The aug3 gene models are a fixed pre-existing annotation;
regenerating them de novo is NOT the reproduction target.
Datasets:
- GSE193511 — RNA-seq of isolated cells from germ-disc regions (16 GSM:
GSM5811734–5811749). Deposits gene count matrix
GSE193511_HTseq_count_per_gene_aug3.txt.gz(+ RPKM + RAW.tar). Platform GPL24841 (MiSeq, single-end). - GSE193650 — whole-embryo RNA-seq, fuchi pRNAi vs untreated, stages 2/3/early-5
(12 GSM: GSM5815633–5815644). Deposits gene count matrix
GSE193650_HTseq_count_per_gene_aug3.txt.gz(+ RPKM + RAW.tar). GPL24841. - GSE193870 — ATAC-seq, stage-3 WT / fuchi pRNAi / Pt-hh pRNAi + stage-1/2 WT (10 GSM). Deposits RAW.tar only (raw reads — peak calling required). GPL31240.
IN SCOPE (pipeline-derived, deposited count matrices → re-run edgeR)
| id | reported result | pipeline | data | feasibility |
|---|---|---|---|---|
| C1 | 10,862 genes with ≥1 cpm in ≥1 of 8 (stage-3 c/i/p) samples | cpm filter (edgeR) | GSE193511 matrix | HIGH — deterministic recompute |
| C2 | 19 genes selected as high-priority candidates (log2FC<-10 comp I&II; >10 comp III) | edgeR 3.8.6 exactTest/glm | GSE193511 matrix | HIGH |
| C3 | stage-2: 6 DEGs (fuchi pRNAi) | edgeR | GSE193650 matrix | HIGH |
| C4 | stage-3: 139 DEGs | edgeR | GSE193650 matrix | HIGH |
| C5 | early stage-5: 785 DEGs (FDR<0.01) | edgeR | GSE193650 matrix | HIGH |
PARTIAL / HARDER (raw reads, heavier; attempt after the above)
| id | reported result | pipeline | data | feasibility |
|---|---|---|---|---|
| A1 | 317 diff-accessible regions (fuchi vs untreated); 3 (Pt-hh vs untreated) | Genrich v6.0 + csaw + TMM, FDR<0.05 | GSE193870 RAW | MEDIUM — needs alignment+peak calling; params underspecified |
| A2 | 316 fuchi-affected regions; 276/316 (93%) suppressed | csaw | GSE193870 RAW | MEDIUM |
OUT OF SCOPE (manual / wet-lab / not pipeline-reproducible)
- GATA phylogenetics (MEGA11, JTT): manual sequence selection/curation (75 sites/118 seqs; 63 sites/168 seqs) — alignment + taxon sampling are hand-curated, not a reproducible script.
- pRNAi knockdown screen, phenotypes, in-situ hybridization — wet-lab.
- AUGUSTUS aug3 gene-model generation — fixed pre-existing annotation, not regenerated.
Primary target
C1–C5: recompute DEG/cpm-filter counts directly from the deposited HTSeq count matrices (GSE193511, GSE193650) with edgeR per the stated criteria. Lightweight, deterministic, P16-valid. Then attempt ATAC (A1/A2) if feasible.
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.
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.
Are you an author? We would genuinely like to hear from you — to clarify the record, add data or code, re-run the pipeline after an accession update, and publish your response right next to the assessment. Everything here is open and auditable.
🚩 Report an error in this record
Spotted something wrong — a verdict you’d contest, a data or value error, or a private detail that slipped through? Tell us, with a short justification. Authors and readers are equally welcome to write in; we review every report.
Prefer email, or the form below not working? Contact us at support@doesitreproduce.com.
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.