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Cited by 5Pancreatic function and diabetesPancreatitis Pathology and TreatmentPancreatic and Hepatic Oncology Research

TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration

Open access
Melzer, Michael Karl, Schirge, Silvia, Gout, Johann, Arnold, Frank, Srinivasan, Dharini, Burtscher, Ingo, Allgöwer, Chantal, Mulaw, Medhanie +11 more·2023·BMC Biology
DOI 10.1186/s12915-023-01553-xPMC PMC10029195
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Abstract

Background: The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. Results: We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. Conclusions: TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

AI snapshot

Written from the paper’s full text.

TL;DR

TBX3 is dynamically expressed in pancreatic mesenchyme and epithelium during development and shifts to stromal cells in adult pancreas, but compartment-specific Tbx3 deletion is largely dispensable for organogenesis. After caerulein-induced acute pancreatitis, epithelial Tbx3 loss prolongs acinar proliferation and increases fibrosis and inflammatory programs, suggesting TBX3 fine-tunes regeneration.

Key findings

  • TBX3 expression is developmentally dynamic: murine scRNA-seq and Tbx3-Venus reporter showed mesenchymal/neural-crest expression at E12.5–E15.5, some acinar precursor expression around E18.5/P7, and adult restriction to stellate and endothelial cells, not exocrine or endocrine cells; human data showed TBX3 in pancreatic endoderm/progenitors and adult stellate cells.
  • Epithelial Tbx3 knockout (Ptf1aCre) and mesenchymal Tbx3 knockout (Nkx3-2Cre) produced no significant changes in pancreatic morphology, exocrine markers, islet size, or insulin/glucagon-positive cell percentages; human iPSC TBX3 knockdown did not significantly alter trilineage potential.
  • In caerulein-induced acute pancreatitis, Tbx3-KO(epi) mice had significantly more KI-67+ acinar cells per field at 72 h (4.3 vs 1.7) and 168 h (3.0 vs 1.2), and significantly more Sirius Red fibrosis at 72 h, without significant differences in ADM, edema, inflammatory infiltration, or apoptosis.
  • RNA-seq at 72 h identified 1152 differentially expressed genes in Tbx3-KO(epi) (762 up, 390 down) but only 1 at 168 h; enriched gene sets included Il6-Jak-Stat3, inflammatory response, E2F targets, G2M checkpoint, and acinar-specific NF-κB, with increased B cells and a trend for neutrophils; Lef1 was upregulated and its promoter had a putative TBX3 binding score of 0.82 (p=0.0009).

Methods

  • Re-analysis of public single-cell and bulk transcriptomic datasets (GSE101099, GSE109774, GSE162547, GSE84133, GSE131817), plus a Tbx3-Venus reporter mouse, conditional Tbx3 knockouts using Ptf1aCre (epithelial) and Nkx3-2Cre (mesenchymal), and a doxycycline-inducible shTBX3 human iPSC pancreatic differentiation model on porcine urinary bladder scaffold.
  • Caerulein-induced acute pancreatitis in adult Tbx3-KO(epi) and control mice, with sacrifice at 24, 72, and 168 h; histology, immunofluorescence, qRT-PCR, and RNA-seq at 72 and 168 h analyzed with DESeq2, GSEA, and putative TBX3-DNA binding analysis.
  • Statistics used Mann-Whitney tests, two-way ANOVA with Sidak post-test, significance at p<0.05 (*) and p<0.01 (), and GSEA significance at FDR<0.25.

Limitations

  • Stated by the paper: the study was underpowered to detect TBX2 compensation during pancreatic development, and a double Tbx2/Tbx3 knockout in both compartments would be necessary but was beyond scope. The paper also states technical limitations of scRNA-seq for weakly expressed genes, especially acinar cells, and notes protease/RNase-rich acinar tissue can impair intact RNA isolation.
  • Evident from the text: the pancreatitis regeneration experiments used only epithelial Tbx3 knockout, so mesenchymal Tbx3 contribution to injury response was not tested; the Lef1–TBX3 interaction is supported by a putative promoter binding score rather than direct functional validation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/s12915-023-01553-x

Cites

90 of 90 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

36 of 44 cited sources checked · 11 supported · 0 flagged · accuracy not yet measured

  • Study design and conductown data

    Specific recombination in epithelial cells of the pancreas was confirmed by immunohistochemical stainings for tdRFP.

    “Specific recombination in epithelial cells of the pancreas was confirmed by immunohistochemical stainings for tdRFP in Ptf1aCre/+ [35] x LSL-tdRFPKI/KI [55, 56] mice (Additional file 1: Fig. S3d) [56].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [35] Kawaguchi Y, Cooper B, Gannon M, Ray M, MacDonald RJ, Wright CVE. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nat Genet. 2002;32(1):128–34. source
    • cites [55] Luche H, Weber O, Rao TN, Blum C, Fehling HJ. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur J Immunol. 2007;37(1):43–53. source

      The source’s ‘Cell type-specific activation and expression of tdRFP in mice’ section reports that activated reporter cells can be monitored histologically, and Figure 8 shows tdRFP detection in spleen and lymph-node sections. It does not report Ptf1aCre crosses or confirm recombination in pancreatic epithelial cells; the citing sentence points to its own supplementary figure for that result. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [56] Walter K, Tiwary K, Trajkovic-Arsic M, Hidalgo-Sastre A, Dierichs L, Liffers ST, et al. MEK inhibition targets cancer stem cells and impedes migration of pancreatic cancer cells in vitro and in vivo. Stem Cells Int. 2019;2019:8475389. source

      Methods §2.1 describes mice carrying Ptf1a-driven Cre and an LSL-tdRFP reporter, and Results §3.3 says Ptf1a-mediated RFP expression reports pancreatic and pancreas-derived cells. Neither passage reports immunohistochemical staining for tdRFP or says that staining confirmed recombination specifically in pancreatic epithelial cells. Page numbers were not available in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Study design and conductown data

    TBX3 antibody specificity was confirmed using a global Tbx3 knockout.

    “By using a global knockout of Tbx3 through the replacement of the first three exons by a Cre-knockin [48], we confirmed TBX3 antibody specificity (Additional file 1: Fig. S3a).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [48] Hoogaars WMH, Engel A, Brons JF, Verkerk AO, De Lange FJ, Wong LYE, et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 2007;21(9):1098–112. source

      The cited paper’s Materials and methods describes a Cre knock-in that produced a Tbx3 null allele, supporting the identification of a Tbx3 knockout. But it says the first three codons in the first exon—not the first three exons—were replaced. The antibody-specificity confirmation is presented as the citing authors’ own result, not a finding attributed to this source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Study design and conductcitation

    The Tbx3-Venus reporter system is validated.

    “To visualize the activity of the murine Tbx3 promoter at the protein level, we used a validated Tbx3-Venus (Tbx3tm1(Venus)Vmc) [11, 42–47] reporter system (Fig. 2a).”
    Quote in the paperSubstantial claim7 of 7 sources checked
    • cites [11] Russell R, Ilg M, Lin Q, Wu G, Lechel A, Bergmann W, et al. A dynamic role of TBX3 in the pluripotency circuitry. Stem Cell Rep. 2015;5:1155–70. source
    • cites [42] Kunasegaran K, Ho V, Chang THT, De Silva D, Bakker ML, Christoffels VM, et al. Transcriptional repressor Tbx3 is required for the hormone-sensing cell lineage in mammary epithelium. PLoS One. 2014;9:e110191. source
    • cites [43] van Eif VWW, Stefanovic S, van Duijvenboden K, Bakker M, Wakker V, de Gier-de Vries C, et al. Transcriptome analysis of mouse and human sinoatrial node cells reveals a conserved genetic program. Development. 2019;146:dev173161. source

      The supplied passages contain bibliographic metadata but no discussion of a Tbx3-Venus reporter. Searches of the indexed copy for Tbx3-Venus, its allele designation, and reporter validation returned no relevant passage, so this copy does not establish whether the system was validated. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [44] Mohan RA, Bosada FM, Weerd JHV, van Duijvenboden K, Wang J, Mommersteeg MTM, et al. T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system. Proc Natl Acad Sci U S A. 2020;117(31):18617–26. source

      The available indexed passages labeled Abstract contain citation metadata and keywords, but no description or validation of a Tbx3-Venus reporter. Searches for “Venus,” “validated reporter system,” and the allele name found no relevant passage, so this copy cannot establish the claim. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
    • cites [45] Mohan RA, Mommersteeg MTM, Domínguez JN, Choquet C, Wakker V, de Gier-de Vries C, et al. Embryonic Tbx3+ cardiomyocytes form the mature cardiac conduction system by progressive fate restriction. Development. 2018;145:dev167361. source

      The supplied passages and indexed results show bibliographic information, not a description or validation of the reporter system. Searches for Tbx3-Venus, the allele designation, and Venus reporter validation found no passage addressing the claim in our copy. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [46] van Weerd JH, Mohan RA, van Duijvenboden K, Hooijkaas IB, Wakker V, Boukens BJ, et al. Trait-associated noncoding variant regions affect tbx3 regulation and cardiac conduction. Elife. 2020;9:e56697. source

      The Results section (‘Identification of accessible regulatory elements within the murine Tbx3 locus’; page unavailable) uses Tbx3Venus/+ mice to isolate Tbx3+ cardiomyocytes, supporting that this is a reporter system used for that purpose. The cited passages do not describe a validation of the reporter, so they do not establish the ‘validated’ part of the claim. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [47] van Eif VWW, Stefanovic S, Mohan RA, Christoffels VM. Gradual differentiation and confinement of the cardiac conduction system as indicated by marker gene expression. Biochim Biophys Acta Mol Cell Res. 2020;1867(3):118509. source
  • Findingown data

    TBX2 staining intensity remains similar in control and Tbx3-KO pancreata in both epithelial and mesenchymal knockout models.

    “However, the overall staining intensity remained similar in control (epi) and Tbx3-KO (epi), as well as control (mes) and Tbx3-KO (mes) pancreata, albeit nuclear and cytoplasmatic protein distribution varied across various animals without a relevant genotype-specific trend.”
    Quote in the paperSubstantial claim
  • Findingown data

    TBX3 protein expression, as assayed with the Venus reporter, presents specific and developmentally dynamic expression patterns in both the pancreatic epithelium and the mesenchyme.

    “Thus, TBX3 protein expression, as assayed with the Venus reporter, presents specific and developmentally dynamic expression patterns in both the pancreatic epithelium and the mesenchyme.”
    Quote in the paperSubstantial claim
  • May combine two claims. 11
  • Classified as probably not a substantial claim. 20

  • May combine two claims.

    Findingcitation

    Venus protein expression correlates mostly with RNA findings, except weak acinar expression around E18.5 which appears underestimated in scRNA sequencing data.

    “These patterns correlate mostly with the RNA findings, except the weak but robust acinar expression around E18.5 which appears underestimated in scRNA sequencing data potentially attributed to technical limitations for weakly expressed genes [50–54].”
    Quote in the paperNot a substantial claim2 of 5 sources checked
    • cites [50] Svensson V, Natarajan KN, Ly LH, Miragaia RJ, Labalette C, Macaulay IC, et al. Power analysis of single-cell rnA-sequencing experiments. Nat Methods. 2017;14(4):381–7. source
    • cites [51] Hicks SC, Townes FW, Teng M, Irizarry RA. Missing data and technical variability in single-cell RNA-sequencing experiments. Biostatistics. 2018;19(4):562–78. source
    • cites [52] Zheng Y, Zhong Y, Hu J, Shang X. SCC: an accurate imputation method for scRNA-seq dropouts based on a mixture model. BMC Bioinformatics. 2021;22(1):5. source
    • cites [53] Islam S, Zeisel A, Joost S, La Manno G, Zajac P, Kasper M, et al. Quantitative single-cell RNA-seq with unique molecular identifiers. Nat Methods. 2014;11(2):163–6. source
    • cites [54] Papalexi E, Satija R. Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol. 2018;18:35–45. source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TBX3 is critically involved in pluripotency maintenance and lineage-specific exit from the pluripotency circuitry in mouse embryonic stem cells.

    “Finally, TBX3 is not only critically involved in pluripotency maintenance but also in lineage-specific exit from the pluripotency circuitry in mouse embryonic stem cells [11–17].”
    Quote in the paperNot a substantial claim7 of 7 sources checked
    • cites [11] Russell R, Ilg M, Lin Q, Wu G, Lechel A, Bergmann W, et al. A dynamic role of TBX3 in the pluripotency circuitry. Stem Cell Rep. 2015;5:1155–70. source

      The Results section on lineage commitment reports that removing TBX3 changes differentiation in mouse ESCs, supporting its role in lineage-specific exit. But the Discussion explicitly says TBX3 is dispensable for maintaining pluripotency, contrary to the claim that it is critically required for maintenance. No page numbers are available in our copy. (LLM, conf=0.91)

      Experimental — accuracy not yet measured
    • cites [12] Klingenstein M, Raab S, Achberger K, Kleger A, Liebau S, Linta L. TBX3 knockdown decreases reprogramming efficiency of human cells. Stem Cells Int. 2016;2016:6759343. source

      The Introduction reports that TBX3 helps maintain the stem-cell state and that its depletion leads to differentiation of murine pluripotent stem cells. It also describes roles in mesendodermal and endodermal differentiation, but does not explicitly establish lineage-specific exit from pluripotency in mouse embryonic stem cells. The paper’s own experiments concern human cells. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [13] Weidgang CE, Russell R, Tata PR, Kühl SJ, Illing A, Müller M, et al. TBX3 directs cell-fate decision toward mesendoderm. Stem Cell Reports. 2013;1:248–65. source
    • cites [14] Quarta C, Fisette A, Xu Y, Colldén G, Legutko B, Tseng YT, et al. Functional identity of hypothalamic melanocortin neurons depends on Tbx3. Nat Metab. 2019;1(2):222–35. source

      The Results section reports that TBX3 loss impairs maturation of hypothalamic neurons derived from human embryonic stem cells; the Discussion also links Tbx3 generally to differentiation and cell-fate commitment. Neither passage addresses pluripotency maintenance or lineage-specific exit from pluripotency circuitry in mouse embryonic stem cells (searched: pluripotency, mouse embryonic stem cells, lineage-specific exit). (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [15] Cioffi M, Vallespinos-Serrano M, Trabulo SM, Fernandez-Marcos PJ, Firment AN, Vazquez BN, et al. MiR-93 controls adiposity via inhibition of Sirt7 and Tbx3. Cell Rep. 2015;12(10):1594–605. source

      The Discussion (page unavailable) says Tbx3 maintains pluripotency in pluripotent stem cells and promotes mesendodermal differentiation during embryogenesis. It does not specifically establish lineage-specific exit from the pluripotency circuitry in mouse embryonic stem cells; the paper's own experiments concern adipocyte precursors. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [16] Kartikasari AER, Zhou JX, Kanji MS, Chan DN, Sinha A, Grapin-Botton A, et al. The histone demethylase Jmjd3 sequentially associates with the transcription factors Tbx3 and Eomes to drive endoderm differentiation. EMBO J. 2013;32(10):1393–408. source

      The section “Tbx3 recruits Jmjd3 to the enhancer region of Eomes locus” reports that Tbx3 binds the Eomes enhancer during differentiation, and “Sequential two-step activation of Eomes locus leads to definitive endoderm differentiation” reports impaired endoderm differentiation after Tbx3 knockdown. These passages support a role in lineage-specific differentiation of mouse embryonic stem cells. They do not establish the separate claim that Tbx3 is critical for maintaining pluripotency. Page numbers were not provided in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [17] Lu R, Yang A, Jin Y. Dual functions of T-box 3 (Tbx3) in the control of self-renewal and extraembryonic endoderm differentiation in mouse embryonic stem cells. J Biol Chem. 2011;286:8425–36. source

  • Classified as probably not a substantial claim.

    Premisecitation

    The pancreatic mesenchyme is essential for proper lineage formation.

    “Given the essential role of the pancreatic mesenchyme for proper lineage formation [49, 57], Tbx3 was also deleted in this compartment.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [49] Landsman L, Nijagal A, Whitchurch TJ, VanderLaan RL, Zimmer WE, MacKenzie TC, et al. Pancreatic mesenchyme regulates epithelial organogenesis throughout development. PLoS Biol. 2011;9(9):e1001143. source

      The Results section, “Pancreatic Mesenchyme Is Required for Precursor Cell Proliferation” (page unavailable), reports that mesenchymal depletion reduced endocrine and exocrine cell masses, supporting a role in their expansion. But it also says every pancreatic epithelial lineage formed, and the Discussion reports no alteration in the main cell types’ differentiation capacity. The source therefore does not support an essential role in lineage formation if that means establishing or differentiating those lineages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [57] Cozzitorto C, Mueller L, Ruzittu S, Mah N, Willnow D, Darrigrand JF, et al. A specialized niche in the pancreatic microenvironment promotes endocrine differentiation. Dev Cell. 2020;55(2):150–62 e6. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Batch effects were corrected using FindIntegrationAnchor() and IntegrateData() in the reciprocal PCA approach of the Seurat workflow.

    “Batch effects were corrected with the FindIntegrationAnchor() and the IntegrateData() function in the reciprocal PCA approach of the Seurat workflow.”
    Quote in the paperNot a substantial claim4 of 4 sources checked
    • cites [76] Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat Biotechnol. 2015;33(5):495–502. source

      The Online Methods section on principal components analysis describes PCA using R’s prcomp function, not reciprocal PCA integration. Searches for FindIntegrationAnchor, IntegrateData, and batch-effect correction found no passage in our copy addressing the claimed workflow. Page numbers were not available in the indexed passages. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [77] Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411–20. source

      The Results section (‘Overview of Seurat alignment workflow’; page unavailable) supports the general point that Seurat integrates datasets affected by batch differences. It describes a CCA-based alignment workflow, however, not reciprocal PCA, and the retrieved passages do not identify FindIntegrationAnchor() or IntegrateData() as its functions. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [78] Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM, et al. Comprehensive integration of single-cell data. Cell. 2019;177(7):1888–902 e21. source

      The STAR Methods (Method details; page unavailable) identify FindIntegrationAnchors as an anchor-identification function and IntegrateData as part of the integration procedure used for batch correction. But the paper describes CCA, not reciprocal PCA, as the initial dimension-reduction method for reference integration. It therefore supports the Seurat functions and batch-correction approach, but not the specified reciprocal-PCA workflow. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [79] Hao Y, Hao S, Andersen-Nissen E, Mauck WM, Zheng S, Butler A, et al. Integrated analysis of multimodal single-cell data. Cell. 2021;184(13):3573–87 e29. source

      The STAR★Methods ‘Sample integration’ section says the authors used an anchor-based reciprocal PCA workflow to integrate samples. Our copy does not name FindIntegrationAnchor() or IntegrateData() as the functions used, so it supports the workflow but not that implementation detail. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TBX3 is a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells.

    “TBX3 is also a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells [18].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Mukherjee S, French DL, Gadue P. Loss of TBX3 enhances pancreatic progenitor generation from human pluripotent stem cells. Stem Cell Reports. 2021;16(11):2617–27. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Tbx3 interacts with certain parts of the Wnt and Hedgehog signaling pathways.

    “Interestingly, Tbx3 interacts with certain parts of the Wnt and Hedgehog signaling pathways [8–10].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [8] Emechebe U, Pavan Kumar P, Rozenberg JM, Moore B, Firment A, Mirshahi T, et al. T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number. Elife. 2016;5:e07897. source

      The Results section reports that Tbx3 interacts with Kif7, a Hedgehog-pathway protein, and that Hedgehog activation changes Tbx3 localization in cilia. The passages found do not establish an interaction with the Wnt signaling pathway (no page numbers are available in our copy), so that part of the claim remains unsupported. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [9] Lüdtke TH, Rudat C, Wojahn I, Weiss AC, Kleppa MJ, Kurz J, et al. Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell. 2016;39(2):239–53. source
    • cites [10] Aydoğdu N, Rudat C, Trowe MO, Kaiser M, Lüdtke TH, Taketo MM, et al. TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme. Development. 2018;145(23):dev171827. source

      The paper title in our indexed copy identifies TBX3 as acting downstream of canonical WNT signaling in mouse ureteric mesenchyme, supporting the WNT part of the claim. The available passages contain no discussion of Hedgehog signaling, so they do not support that part (searched: Hedgehog, SHH, and TBX3). No page numbers are available for these passages. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Augmented Hedgehog, Notch, and Wnt/b-catenin signaling are mandatory for regeneration in mice after experimental induction of pancreatitis.

    “Among others, augmented Hedgehog, Notch, and Wnt/b-catenin signaling were delineated as mandatory for regeneration in mice after experimental induction of pancreatitis [21–24].”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [21] Murtaugh LC, Keefe MD. Regeneration and repair of the exocrine pancreas. Annu Rev Physiol. 2015;77:229–49. source

      In “Developmental signals regulating pancreatic regeneration” (page not provided), the review reports that Hedgehog signaling is required for acinar re-differentiation after caerulein-induced pancreatitis and that Notch promotes it, although the Notch requirement may be indirect. It also reports that β-catenin is important for proliferative expansion of surviving acini, but not for their re-differentiation; whether that effect depends on Wnt ligands is unknown. Thus, calling augmented signaling through all three pathways mandatory for regeneration overstates the source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [22] Fendrich V, Esni F, Garay MVR, Feldmann G, Habbe N, Jensen JN, et al. Hedgehog signaling is required for effective regeneration of exocrine pancreas. Gastroenterology. 2008;135(2):621–31. source
    • cites [23] Siveke JT, Lubeseder-Martellato C, Lee M, Mazur PK, Nakhai H, Radtke F, et al. Notch signaling is required for exocrine regeneration after acute pancreatitis. Gastroenterology. 2008;134(2):544–55. source
    • cites [24] Keefe MD, Wang H, De La OJP, Khan A, Firpo MA, Murtaugh LC. β-catenin is selectively required for the expansion and regeneration of mature pancreatic acinar cells in mice. DMM. Dis Model Mech. 2012;5(4):503–14. source

      The Results section, ‘β-catenin is required for acinar cell proliferation during regeneration’ (page unavailable), reports reduced proliferation of β-catenin-deficient acinar cells after experimentally induced pancreatitis. The Introduction also reports that Notch1 and the Hedgehog component Smo are required for regeneration in this model. But the Discussion says the role of Wnt proteins themselves remains to be determined, and the paper does not establish that augmented signaling in all three pathways is mandatory for regeneration. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Redundancy between Tbx3 and Tbx2 to compensate for each other has been reported in several organs.

    “Interestingly, redundancy between Tbx3 and Tbx2 to compensate for each other has been reported in several organs [9, 10].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [9] Lüdtke TH, Rudat C, Wojahn I, Weiss AC, Kleppa MJ, Kurz J, et al. Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell. 2016;39(2):239–53. source
    • cites [10] Aydoğdu N, Rudat C, Trowe MO, Kaiser M, Lüdtke TH, Taketo MM, et al. TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme. Development. 2018;145(23):dev171827. source

      The available copy contains bibliographic metadata and a link to the paper, but no passage discussing functional redundancy or compensation between Tbx2 and Tbx3 in any organ. Searches for “redundancy” and “redundant” did not locate evidence for the claim. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    TBX3 and TBX2 are closely entangled in other regulatory programs, such as Wnt and Hedgehog signaling, and both are essential for proper limb, lung, and ureter development.

    “TBX3 and TBX2 are closely entangled in other regulatory programs, such as Wnt and Hedgehog signaling, and both are essential for proper limb [8], lung [9], and ureter development [10].”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [8] Emechebe U, Pavan Kumar P, Rozenberg JM, Moore B, Firment A, Mirshahi T, et al. T-box3 is a ciliary protein and regulates stability of the Gli3 transcription factor to control digit number. Elife. 2016;5:e07897. source

      The Results section ('Tbx3 is required for normal limb bud initiation') reports defective limb initiation in Tbx3 mutants, supporting the TBX3 portion of the limb-development claim. The Results section on the Hand2/Shh pathway lists Tbx2 among transcripts reduced after Tbx3 loss, but does not establish that TBX2 itself is essential for limb development. The separate lung and ureter citations are not judged here. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [9] Lüdtke TH, Rudat C, Wojahn I, Weiss AC, Kleppa MJ, Kurz J, et al. Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell. 2016;39(2):239–53. source
    • cites [10] Aydoğdu N, Rudat C, Trowe MO, Kaiser M, Lüdtke TH, Taketo MM, et al. TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme. Development. 2018;145(23):dev171827. source

      The paper’s title states that TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme. Citation [10] is attached to ureter development, but the available passages do not establish the stronger claim that each gene is essential for proper ureter development. The limb and lung claims have separate citations. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Common mechanisms and similar genetic programs between stem cell induction, organ homeostasis, and regeneration from injury have been identified for the pancreas.

    “Common mechanisms and similar genetic programs between stem cell induction, organ homeostasis, and regeneration from injury have been identified for the pancreas [20, 21].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [20] Arnold F, Mahaddalkar PU, Kraus JM, Zhong X, Bergmann W, Srinivasan D, et al. Functional genomic screening during somatic cell reprogramming identifies DKK3 as a roadblock of organ regeneration. Adv Sci. 2021;8(14):2100626. source
    • cites [21] Murtaugh LC, Keefe MD. Regeneration and repair of the exocrine pancreas. Annu Rev Physiol. 2015;77:229–49. source

      The sections on developmental signals and transcription factors report that regulators involved in pancreatic development or homeostasis also have roles in regeneration after injury (page unavailable in our copy). They do not establish the claimed overlap with stem cell induction, so the three-way comparison is only partly supported. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Whole transcriptome analysis of Tbx3-KO (epi) vs control at 72h after acute pancreatitis shows clear difference in PCA, with 1152 DEGs (762 up, 390 down), while at 168h only 1 DEG.

    “We could identify 1152 differentially expressed genes (DEG) at 72h (762 up, 390 down in Tbx3-KO (epi)), while only 1 DEG was detected at 168h (Fig. 5b).”
    • Also stated in: § Loss of Tbx3 results in increased susceptibility of acinar cells to fibroinflammatory stimuli · boosted
      “we noticed a clear difference in the principal component analysis (PCA) of whole transcriptomes from Tbx3-KO (epi) mice compared to their respective controls at 72h, while the differences at 168h were virtually absent (Fig. 5a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Neutrophil granulocyte quantification revealed only a trend, not significant.

    “Neutrophil granulocyte quantification revealed only a trend (Fig. 5g,i).”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • May combine two claims.

    Findingown data

    Among Tbx genes at 72h, Tbx18 is significantly downregulated and Tbx21 is overexpressed in Tbx3-KO (epi) mice.

    “Interestingly, most Tbx genes remained unaltered, with Tbx18 being significantly downregulated and Tbx21 overexpressed in the Tbx3-KO (epi) mice.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • May combine two claims.

    Interpretationown data

    Tbx3 has a role in tissue homeostasis and regeneration.

    “indicating that Tbx3 has a role in tissue homeostasis and regeneration.”
    • Also stated in: § Discussion · hedged
      “our data show a relevant but subtle contribution of this factor to adult pancreatic organ homeostasis and development.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • May combine two claims.

    Interpretationreasoning

    Tbx3 is involved in proper regeneration from organ injury, but its absence is well compensated by other genes.

    “Thus, despite the fact that regeneration was achieved in Tbx3-KO (epi) mice, prolonged proliferation in acinar cells and accumulation of fibrosis suggest that Tbx3 is involved in proper regeneration from organ injury, but its absence is well compensated by other genes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All mouse work was performed according to acknowledged ethical guidelines (GV-SOLAS and FELASA) to ensure careful, consistent, and ethical handling of mice.

    “All mouse work aspects were performed according to acknowledged guidelines of the Society of Laboratory Animals (GV-SOLAS) and of the Federation of Laboratory Animal Science Associations (FELASA).”
    • Also stated in: § Mice · plain
      “All the aspects of the mouse work were carried out following strict guidelines to insure careful, consistent, and ethical handling of mice.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The anti-GFP antibody was licensed to capture the Venus protein in Tbx3-positive cells of the seminal vesicle serving as a positive control.

    “Similarly, the anti-GFP antibody was licensed to capture the Venus protein in Tbx3-positive cells of the seminal vesicle serving as a positive control (Additional file 1: Fig. S3b).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All animal care and procedures complied with German legal regulations and were approved by the governmental review board of Baden-Württemberg (or Bavaria).

    “All animal care and procedure were conducted in compliance with the German legal regulations and were previously approved by the local governmental review board of the state of Baden-Württemberg (Permission no. 1477, O.195-4, O.195-6, O.195-10, and O.195-12) or conducted in compliance with the local government of Bavaria.”
    • Also stated in: § Mice · hedged
      “All animal care and procedures followed German legal regulations and if applicable were previously approved by the governmental review board of the state of Baden-Württemberg.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    TBX3 directly binds to the Lef1 promoter and most likely triggers its transcriptional repression.

    “indicating direct TBX3 binding to the Lef1 promoter, most likely triggering its transcriptional repression (Fig. 5l).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cluster identity was annotated by specific marker genes.

    “Cluster identity was annotated by specific marker genes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For human datasets, cells expressing either less than 300 genes or more than 4000 genes, and genes expressed in fewer than 10 cells, were filtered out.

    “For human datasets, cells expressing either less than 300 genes or more than 4000 genes and genes expressed in fewer than 10 cells were filtered out.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    For the murine datasets, cells with less than 1000 expressed genes, more than 4000 genes for E12.5 and E14.5, more than 5000 genes for E17.5, and genes expressed in fewer than 3 cells were filtered out before analysis.

    “For the murine datasets, cells with less than 1000 expressed genes, more than 4000 genes for E12.5, E14.5, and more than 5000 genes for E17.5 and genes expressed in fewer than 3 cells were filtered out before analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For GSE109774, cells expressing more than 5% ERCC RNA spike-ins were additionally removed.

    “For GSE109774, cells expressing more than 5% ERCC RNA spike-ins were additionally removed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    TBX3 is not a master regulator of pancreatic development and organ regeneration, but has a relevant but subtle contribution to adult pancreatic organ homeostasis and development.

    “Altogether, while Tbx3 is not a master regulator of pancreatic development and organ regeneration, our data show a relevant but subtle contribution of this factor to adult pancreatic organ homeostasis and development.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Developmental programs can become re-activated during regeneration from injury.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Immune cells were excluded from the quantification of KI67-positive cells.

    “Immune cells were excluded from the quantification.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Gene expression was normalized to ribosomal protein S18.

    “Gene expression was normalized to ribosomal protein S18.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    KI67-positive cells within only clearly identifiable acinar structures were considered.

    “KI67-positive cells within only clearly identifiable acinar structures were considered.”
    Quote in the paperSubstantial claim
27 of 27

  • The paper relies on this without support.

    Developmental programs can become re-activated during regeneration from injury.

    “Driven by the fact that developmental programs can become re-activated during regeneration from injury, we investigated the relevance of Tbx3 for regeneration after caerulein-induced acute pancreatitis.”
1 of 1

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 4 assigns panel f to two different measurements

    Minor

    The caption labels the inflammatory infiltration score as panel f and also labels representative KI-67 staining as panel f.

  • Figure 2 reports both one and at least two mice for panel f

    Minor

    The Figure 2 caption says panel f used one mouse, but its closing statement says there were at least two per group.

  • Adult RNA results cite a nonexistent Figure 1e panel

    Minor

    The text cites Figure 1e for adult RNA data, while the Figure 1 caption identifies panels a-d and assigns adult pancreata to panel d.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Wickham H. Package ‘ggplot2’: elegant graphics for data analysis. New York: Springer-Verlag; 2016.
  2. Blighe K, Rana S, Lewis M. EnhancedVolcano: Publication-ready volcano plots with enhanced colouring and labelin. R-Package; 2019.

Cited by

Sorted by citations

Related

Authors
Melzer, M. K., Schirge, S., Gout, J., Arnold, F., Srinivasan, D., Burtscher, I., Allgöwer, C., Mulaw, M., Zengerling, F., Günes, C., Lickert, H., Christoffels, V. M., Liebau, S., Wagner, M., Seufferlein, T., Bolenz, C., Moon, A. M., Perkhofer, L., & Kleger, A.
Affiliations
  • Universität Ulm (DE)
  • Helmholtz Munich (DE)
  • TUM Klinikum (DE)
  • Amsterdam University Medical Centers (NL)
  • University of Tübingen (DE)
  • Geisinger Medical Center (US)
Corresponding author
Kleger, Alexander
ORCID
Venue
BMC Biology
Publisher
Springer Science and Business Media LLC
Published
2023-03-20
Volume
21
Issue
1
Pages
55-55
ISSN
1741-7007 (ISSN-L)
DOI
10.1186/s12915-023-01553-x
PMC
PMC10029195
OpenAlex
W4328049263
Semantic Scholar
000d2e5f56346e772e3e19a75cb22e34c76b87d9
Cited by
5
References
90
Citations by year
2026: 2 · 2025: 1 · 2024: 1 · 2023: 1
FWCI
1.21
Type
journal article
Language
en
Open access
Licence
Locations
  • BMC Biology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Pure Amsterdam UMC (acceptedVersion, other-oa)
  • PubMed Central (submittedVersion, cc-by)
  • Data Archiving and Networked Services (DANS) (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • mediaTUM – the media and publications repository of the Technical University Munich (Technical University Munich) (submittedVersion, other-oa)
  • OPen Access Repositorium der Universität Ulm (OPARU) (Ulm University) (submittedVersion, cc-by)
  • PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion, cc-by)
Funders
  • DFG (KL2544/7-1, KL2544/6-1, PE 3337/1-1)
  • Universität Ulm (Clinician-Scientist-Program)
  • Deutsche Krebshilfe (AK70114761)
  • Else Kröner-Fresenius-Stiftung (Clinician-Scientist-Program, Research School for Physicians)
  • Helmholtz-Gemeinschaft
Topics
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
  • Health Sciences › Medicine › Surgery › Pancreatitis Pathology and Treatment
  • Health Sciences › Medicine › Oncology › Pancreatic and Hepatic Oncology Research
Keywords
TBX3, pancreatic development, pancreatic regeneration, acute pancreatitis, T-box transcription factor, pancreatic epithelium, fibrosis, inflammation, tissue homeostasis
MeSH terms
Acute Disease, Adult, Animals, Humans, Pancreas, Pancreatitis, T-Box Domain Proteins, Organogenesis, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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