CoChat
Sign Up
Cited by 13Congenital heart defects researchDevelopmental Biology and Gene RegulationSingle-cell and spatial transcriptomics

Cell diversity and plasticity during atrioventricular heart valve EMTs

Open access
Lotto, Jeremy, Cullum, Rebecca, Drissler, Sibyl, Arostegui, Martin, Garside, Victoria C., Fuglerud, Bettina M., Clement-Ranney, Makenna, Thakur, Avinash +2 more·2023·Nature Communications
DOI 10.1038/s41467-023-41279-6PMC PMC10492828

Published version of a preprint

Open this paper in CoChat — free accountOpen PDF

Abstract

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9- deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

AI snapshot

Written from the paper’s full text.

TL;DR

Single-cell transcriptomics of >50,000 murine embryonic heart/atrioventricular canal cells maps endocardial and epicardial EMTs during atrioventricular valve formation, revealing epithelial-mesenchymal plasticity (EMP) and distinct progenitor states. Sox9 loss increases EMP and stalls EndMT, while EpiMT proceeds through a Hic1-expressing AV EPDC population.

Key findings

  • From 53,521 QC-passed single cells, 48,822 wildtype cells resolved into 18 populations spanning E7.75–E12.5; EndMT analysis of 10,354 endocardial/endocardial-derived cells identified eight populations and bifurcation into VEC progenitors versus AV mesenchyme.
  • During EndMT, cells in AV endocardial and VEC II clusters co-express endothelial and mesenchymal profiles in domains of high fluid shear stress response, indicating EMP; Piezo1 is the most highly expressed mechanosensitive ion channel in endocardium.
  • In E10.5 Sox9 cKO AVCs, mesenchymal cells are reduced, AV endocardial/VEC II proportions increase, and robust EMP cells increase four-fold; residual cKO mesenchyme shows myofibroblastic gene activation.
  • EpiMT analysis of 4,633 epicardial cells identified five populations including an AV EPDC population marked by Hic1; Hic1 and Itgb5 activation in some epicardial cells by E10.5 suggests EpiMT may initiate earlier than previously understood, and Hic1nLacZ+ AV sulcus cells co-express SOX9 at E12.5/E14.5.

Methods

  • Generated five scRNA-seq libraries from E10.5 and E12.5 AVCs, including Tie2-cre;tdTomato lineage tracing and Sox9fl/fl;Tie2-cre cKO/control libraries, and complemented them with published E7.75, E8.25, and E9.25 heart datasets.
  • Dissociated AVCs, FACS-purified live cells, prepared 10x Chromium 3′ RNA-seq libraries, aligned with Cell Ranger, and processed with scran/scater, PhenoGraph clustering, Harmony batch correction, Palantir trajectory inference, and NicheNet ligand-receptor analysis.
  • Validated findings by immunofluorescence on cryosections and FACS-sorted cells, including Hic1nLacZ/+ LacZ staining combined with SOX9, ERG, TNNT2, WT1, CD31, PDGFRα, and vimentin staining.

Limitations

  • The paper states that epicardial lineage contribution to AV mesenchymal cells could not be confirmed without lineage tracing, and that validation of EndMT/EpiMT convergence would likely require dual lineage tracing with sorting or scATAC-seq.
  • The paper notes that some transcriptional differences across batches persist after Harmony correction, though these genes are predominantly associated with general biological processes and metabolism.
  • The paper states that no statistical method was used to predetermine sample size, experiments were not randomized, researchers were not blinded, and embryo sex was not determined; it also notes EMP is a continuum but was visualized as a binary threshold.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-023-41279-6

Cites

94 of 94 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

60 of 79 cited sources checked · 27 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    AV mesenchyme, the progenitor pool for VICs, is formed early in development through two EMTs from endocardium and epicardium.

    “During mouse embryogenesis, AV mesenchyme, which comprises the progenitor pool for VICs, is formed early in development through two epithelial-to-mesenchymal transitions (EMTs) from distinct cellular sources: endocardium and epicardium4–6”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [4] Markwald, R. R., Fitzharris, T. P. & Manasek, F. J. Structural development of endocardial cushions. Am. J. Anat. 148, 85–119 (1977). source
    • cites [5] Wessels, A. et al. Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart. Dev. Biol. 366, 111–124 (2012). source

      The Introduction says early AV-cushion mesenchyme derives from endocardial EMT and later gives rise to valve interstitial cells. The Results say nearly all cushion mesenchymal cells are endocardially derived at ED12, with very limited epicardial contribution; epicardially derived cells populate the lateral cushions later, around ED14–14.5. This contradicts the claim that early AV mesenchyme forms through EMTs from both sources. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [6] Gittenberger-de Groot, A. C., Vrancken Peeters, M. P. F. M., Mentink, M. M. T., Gourdie, R. G. & Poelmann, R. E. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ. Res. 82, 1043–1052 (1998). source

      The Discussion describes transformation of epicardial cells into EPDCs and endocardial cells into AV-cushion mesenchyme; the Results report EPDCs in developing valve cushions. But this chicken–quail chimera study does not establish the claim for mouse embryogenesis or identify the AV mesenchyme as the progenitor pool for valve interstitial cells. Its Figure 8 discussion also places epicardial-cell entry into AV cushions later, not at the earliest stage shown. Page numbers were not available in the indexed passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    By E9.0, fluid shear stress from blood flow and signals from the myocardium including BMPs induce EndMT in a subset of endocardial cells lining the cushions.

    “By E9.0, fluid shear stress from blood flow and signals from the myocardium, including BMPs, induce an endocardial-to-mesenchymal transition (EndMT) in a subset of endocardial cells lining the cushions9–13.”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [9] Jiao, K. et al. An essential role of Bmp4 in the atrioventricular septation of the mouse heart. Genes Dev. 17, 2362–2367 (2003). source

      The paper’s introductory discussion (page unavailable) says myocardial signals contribute to cushion formation and that TGF-β2 can activate EMT in explant assays. Its Results and Discussion detect Bmp4 near the forming cushion at E9.0, but find EMT proceeds when Bmp4 is deleted; other BMPs may compensate. The copy does not establish fluid shear stress as an inducer or the claimed combined mechanism by E9.0. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [10] Ma, L., Lu, M.-F., Schwartz, R. J. & Martin, J. F. Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Development 132, 5601–5611 (2005). source

      The Introduction says a subset of endocardial cells undergoes EMT to form cushions, and the section ‘Bmp2 is a crucial inducer of EMT in the AV cushion’ reports that BMP signaling is required to induce it. Our copy does not establish that fluid shear stress induces this transition or that it occurs by E9.0; its E9.0 passage concerns myocardial Bmp2 expression. Page numbers are unavailable in the indexed copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [11] Granados-Riveron, J. T. & Brook, J. D. The impact of mechanical forces in heart morphogenesis. Circ. Cardiovasc Genet. 5, 132–142 (2012). source

      The Conclusions and Figure 2 describe blood-flow-related shear stress increasing Notch activity in endocardial cushion cells, some of which undergo EMT. But The Notch Connection places the start of mouse cushion EMT at E9.5, not E9.0. Its BMP10 discussion concerns trabecular cardiomyocyte proliferation, not myocardial BMP signals inducing cushion EndMT; no page numbers are available in our copy. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [12] Goddard, L. M. et al. Hemodynamic forces sculpt developing heart valves through a KLF2-WNT9B paracrine signaling axis. Dev. Cell 43, 274–289.e5 (2017). source

      The Introduction (page unavailable) says cushion formation begins at E9.5–10.5 with endocardial-to-mesenchymal transformation, supporting the EndMT component but not the stated E9.0 timing. The Results and Discussion link fluid shear to later cushion remodeling, not to induction of EndMT; the passages found do not identify myocardial BMP signals as an EndMT trigger. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [13] Fukui, H. et al. Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces. Science 374, 351–354 (2021). source
  • Premisecitation

    AV mesenchymal cells are also generated from the epicardium via EpiMT starting around E12.5.

    “AV mesenchymal cells are also generated from the epicardium via the epicardial-to-mesenchymal transition (EpiMT) starting around E12.5 (Fig. 1a).”
    Quote in the paperNot a substantial claim4 of 4 sources checked
    • cites [5] Wessels, A. et al. Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart. Dev. Biol. 366, 111–124 (2012). source
    • cites [6] Gittenberger-de Groot, A. C., Vrancken Peeters, M. P. F. M., Mentink, M. M. T., Gourdie, R. G. & Poelmann, R. E. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ. Res. 82, 1043–1052 (1998). source

      The Endocardial Cushion Invasion section reports epicardium-derived cells in the AV cushions of chicken–quail chimeras at stages 32–43, and the Discussion describes epicardial cells transforming into a subepicardial cell population. The paper does not establish that AV mesenchymal cells begin to arise by this route around mouse embryonic day E12.5; its E12.5 reference concerns lethality in mouse knockout models, not the timing of AV cushion invasion. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [23] Zhou, B., von Gise, A., Ma, Q., Hu, Y. W. & Pu, W. T. Genetic fate mapping demonstrates contribution of epicardium-derived cells to the annulus fibrosis of the mammalian heart. Dev. Biol. 338, 251–261 (2010). source

      The Results report epicardium-derived cells at the atrioventricular junction with mesenchymal markers and evidence of epicardial-to-mesenchymal transition. The cited paper does not establish that this process starts around E12.5; its reported observations here are at E14.5 and later (page numbers unavailable in our copy). (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [24] Zhou, B. et al. Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart. Nature 454, 109–113 (2008). source

      The passage labeled Abstract (page unavailable) says that some epicardial cells transition to a mesenchymal phenotype, and the References list a study of epicardium-derived cells in atrioventricular cushions. The cited paper does not itself establish that AV mesenchymal cells arise through this transition starting around E12.5. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premiseown data

    Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation.

    “Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [4] Markwald, R. R., Fitzharris, T. P. & Manasek, F. J. Structural development of endocardial cushions. Am. J. Anat. 148, 85–119 (1977). source
    • cites [5] Wessels, A. et al. Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart. Dev. Biol. 366, 111–124 (2012). source
    • cites [6] Gittenberger-de Groot, A. C., Vrancken Peeters, M. P. F. M., Mentink, M. M. T., Gourdie, R. G. & Poelmann, R. E. Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions. Circ. Res. 82, 1043–1052 (1998). source
  • Premisecitation

    Endocardial Gata4 is required for EndMT initiation.

    “Endocardial Gata4 is required for EndMT initiation15”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Rivera-Feliciano, J. et al. Development of heart valves requires Gata4 expression in endothelial-derived cells. Development 133, 3607–3618 (2006). source
  • May combine two claims. 51
  • Classified as probably not a substantial claim. 16
  • The quote only partly states this claim. 1

  • May combine two claims.

    Premisecitation

    VEGF signalling induces NFATc1 expression, which represses EndMT and maintains endothelial identity in non-transitioning endocardium.

    “VEGF signalling is also implicated – inducing NFATc1 expression, which represses EndMT and maintains endothelial identity in non-transitioning endocardium21.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [21] Wu, B. et al. Nfatc1 coordinates valve endocardial cell lineage development required for heart valve formation. Circ. Res. 109, 183–192 (2011). source

      The Results section on EMT and cell-cell contact says Nfatc1 maintains the endocardial phenotype by suppressing Snail2. The Discussion and fate-mapping results also support its role in limiting EMT in valve endocardial cells that remain in the endocardium. Our copy does not establish the attributed upstream step that VEGF signalling induces Nfatc1 expression. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    EndMT is controlled by transcription factors including SNAI2, GATA4, and SOX9, activated in response to cues including TGFβ, WNT, and NOTCH.

    “This transdifferentiation event is controlled by various transcription factors, like SNAI214, GATA415, and SOX916,17, which are activated in response to cues from neighbouring cell types, including the TGFβ superfamily9,10, WNT18, and NOTCH19,20.”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [14] Niessen, K. et al. Slug is a direct Notch target required for initiation of cardiac cushion cellularization. J. Cell Biol. 182, 315–325 (2008). source
    • cites [15] Rivera-Feliciano, J. et al. Development of heart valves requires Gata4 expression in endothelial-derived cells. Development 133, 3607–3618 (2006). source
    • cites [16] Garside, V. C. et al. SOX9 modulates the expression of key transcription factors required for heart valve development. Development 142, 4340–4350 (2015). source
    • cites [17] Akiyama, H. et al. Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa. Proc. Natl Acad. Sci. USA 101, 6502–6507 (2004). source

  • May combine two claims.

    Findingown data

    PhenoGraph clustering identified 18 distinct populations within 48,822 wildtype cells, including emergent mesenchymal, myocardial, and endothelial populations.

    “We used PhenoGraph39 clustering to identify diversity captured within the 48,822 wildtype cells and annotated 18 distinct populations, including emergent mesenchymal, myocardial, and endothelial populations”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [39] Levine, J. H. et al. Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis. Cell 162, 184–197 (2015). source

  • May combine two claims.

    Premisecitation

    The processes underlying AV EpiMT remain poorly characterized, as few studies assessing EpiMT focus on chamber epicardium.

    “However, the processes underlying AV EpiMT remain poorly characterized, as the few studies assessing EpiMT focus on chamber epicardium25–29.”
    Quote in the paperSubstantial claim5 of 5 sources checked
    • cites [25] Smith, C. L., Baek, S. T., Sung, C. Y. & Tallquist, M. D. Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling. Circ. Res. 108, e15–e26 (2011). source
    • cites [26] Pennisi, D. J. & Mikawa, T. FGFR-1 is required by epicardium-derived cells for myocardial invasion and correct coronary vascular lineage differentiation. Dev. Biol. 328, 148–159 (2009). source
    • cites [27] Quijada, P. et al. Coordination of endothelial cell positioning and fate specification by the epicardium. Nat. Commun. 12, 1–18 (2021). source
    • cites [28] Gise, Avon et al. WT1 regulates epicardial epithelial to mesenchymal transition through β-catenin and retinoic acid signaling pathways. Dev. Biol. 356, 421 (2011). source
    • cites [29] Compton, L. A., Potash, D. A., Mundell, N. A. & Barnett, J. V. Transforming growth factor-β induces loss of epithelial character and smooth muscle cell differentiation in epicardial cells. Dev. Dyn. 235, 82–93 (2006). source

      The Introduction (page unavailable) says that factors regulating epicardial EMT remain largely unidentified, supporting the claim that its processes were poorly characterized. The Experimental Procedures describe explants grown from chick hearts placed ventral side down, but do not explicitly identify the sampled cells as chamber rather than AV epicardium. The paper also does not establish that the few EpiMT studies focused on chamber epicardium. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    VICs are specialized cardiac fibroblasts, generally quiescent under homeostatic conditions, and their activation is associated with valve disease including calcification and regurgitation.

    “VICs, which are specialized cardiac fibroblasts, are generally quiescent under homoeostatic conditions, and their activation is intimately associated with valve disease states, including calcification and regurgitation2,3.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [2] Cheek, J. D., Wirrig, E. E., Alfieri, C. M., James, J. F. & Yutzey, K. E. Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease. J. Mol. Cell Cardiol. 52, 689–700 (2012). source

      The Results section (“VIC activation, cell proliferation, and cellularity”; page unavailable) reports little to no proliferation in normal adult valves and describes VIC activation in adult human calcific aortic valve disease. The Discussion links VIC activation to valve disease and a previously described calcification process. Our copy does not characterize VICs as specialized cardiac fibroblasts or link their activation specifically to regurgitation; it also finds no myofibroblast activation in the mouse models at the stages analyzed. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [3] Tao, G., Kotick, J. D. & Lincoln, J. Heart valve development, maintenance, and disease. Curr. Top. Dev. Biol. 100, 203–232 (2012). source

  • May combine two claims.

    Premisecitation

    Sox9fl/fl;Tie2-cre embryos die around E12.5 with AV developmental defects characterized by severe cushion hypoplasia.

    “These embryos die around E12.5 with AV developmental defects, characterized by severe cushion hypoplasia17.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] Akiyama, H. et al. Essential role of Sox9 in the pathway that controls formation of cardiac valves and septa. Proc. Natl Acad. Sci. USA 101, 6502–6507 (2004). source

      The Results report severely underdeveloped AV endocardial cushions in Sox9-null embryos, but say those embryos died between 11.5 and 12.0 dpc, rather than around E12.5. The Materials and Methods describe Zp3-Cre, Prm1-Cre, and Wnt1-Cre crosses, not Sox9fl/fl;Tie2-Cre embryos. The cited paper therefore supports the cushion defect in a different mutant, not the full genotype-specific claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingcitation

    At E10.5, NFATc1 co-expression is observed on the flow-side of the cushions with ERG and within the VECs lining the primitive valves at E11.5.

    “At E10.5, we observe NFATc1 co-expression on the flow-side of the cushions with ERG and within the VECs lining the primitive valves at E11.521,48”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [21] Wu, B. et al. Nfatc1 coordinates valve endocardial cell lineage development required for heart valve formation. Circ. Res. 109, 183–192 (2011). source

      The Results section reports Nfatc1-high cells in the cushion endocardium at E10.5 and continued expression in valve endocardial cells from E11.5; the Discussion likewise describes high Nfatc1 expression during valve elongation. Our copy does not establish co-expression with ERG or locate those cells specifically on the flow-side of the cushions. Page numbers are unavailable in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [48] Chang, C. P. et al. A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis. Cell 118, 649–663 (2004). source

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

    Premisecitation

    Hic1 is a tumour suppressor and biomarker of adult mesenchymal progenitors.

    “like Hic1, a tumour suppressor62 and biomarker of adult mesenchymal progenitors63,76.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [62] Wales, M. M. et al. p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3. Nat. Med. 1, 570–577 (1995). source
    • cites [63] Scott, R. W., Arostegui, M., Schweitzer, R., Rossi, F. M. V. & Underhill, T. M. Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration. Cell Stem Cell 25, 797–813.e9 (2019). source
    • cites [76] Soliman, H. et al. Pathogenic potential of Hic1-expressing cardiac stromal progenitors. Cell Stem Cell 26, 205–220.e8 (2020). source

  • May combine two claims.

    Findingown data

    AV EPDCs express factors associated with EMT, along with unique genes like Hic1.

    “AV EPDCs express a variety of factors associated with EMT, along with some unique genes, like Hic1, a tumour suppressor62 and biomarker of adult mesenchymal progenitors63,76.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [62] Wales, M. M. et al. p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3. Nat. Med. 1, 570–577 (1995). source
    • cites [63] Scott, R. W., Arostegui, M., Schweitzer, R., Rossi, F. M. V. & Underhill, T. M. Hic1 defines quiescent mesenchymal progenitor subpopulations with distinct functions and fates in skeletal muscle regeneration. Cell Stem Cell 25, 797–813.e9 (2019). source
    • cites [76] Soliman, H. et al. Pathogenic potential of Hic1-expressing cardiac stromal progenitors. Cell Stem Cell 26, 205–220.e8 (2020). source

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

    Findingcitation

    Candidate signals from the endocardium, like TGFβ1 and WNT ligands, are identified as known mediators of valve mesenchymal proliferation.

    “We identify candidate signals from the endocardium, like TGFβ1 and WNT ligands, which are known mediators of valve mesenchymal proliferation46,75.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [46] Alfieri, C. M., Cheek, J., Chakraborty, S. & Yutzey, K. E. Wnt signaling in heart valve development and osteogenic gene induction. Dev. Biol. 338, 127–135 (2010). source

      The Discussion reports that Wnt signaling promotes mesenchymal cell proliferation in atrioventricular cushions, and the Results identify Wnt4 and Wnt9b in the endocardial endothelial cells overlying those cushions. But the Discussion says the roles of those endocardial ligands are unknown, and the cited passages do not establish TGFβ1 as a mediator. No page numbers are available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [75] Garside, V. C., Chang, A. C., Karsan, A. & Hoodless, P. A. Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development. Cell Mol. Life Sci. 70, 2899–2917 (2013). source

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    PCA dimensionality reduction and mutual-nearest neighbours or Harmony batch correction was performed to correct for non-biological confounders between replicates and timepoints.

    “PCA dimensionality reduction and mutual-nearest neighbours84 or Harmony34 batch correction was performed to correct for non-biological confounders between replicates and timepoints.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [84] Haghverdi, L., Lun, A. T. L., Morgan, M. D. & Marioni, J. C. Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors. Nat. Biotechnol. 36, 421–427 (2018). source
    • cites [34] Nowotschin, S. et al. The emergent landscape of the mouse gut endoderm at single-cell resolution. Nature 569, 361–367 (2019). source

  • May combine two claims.

    Findingcitation

    Reduced expression of integrins essential for FN1 adhesion in Sox9 cKO and accumulation of stratified endocardium within Fn1 KO echo the phenotype observed in Sox9 cKO.

    “The latter is supported by the reduced expression of integrins essential for FN1 adhesion in the Sox9 cKO, along with the accumulation of stratified endocardium within the Fn1 KO, which echoes the phenotype we observe in the Sox9 cKO57.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [57] Astrof, S., Crowley, D. & Hynes, R. O. Multiple cardiovascular defects caused by the absence of alternatively spliced segments of fibronectin. Dev. Biol. 311, 11–24 (2007). source

      The “Heart defects in the EIIIA/EIIIB double-null embryos” section reports multilayered endothelium in the outflow tract or atrioventricular cushions, supporting the cited comparison to stratified endocardium. But these embryos lacked two alternatively spliced FN segments, not FN1 itself: the Abstract says FN synthesis and deposition were unaffected. The Sox9 cKO observations are the citing authors’ own comparison, not findings attributed to this source. No page numbers were supplied. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation

    JAMs are essential drivers of EMT in cancer metastasis, modulating morphology, migration, and cell polarity.

    “they are essential drivers of EMT in the context of cancer metastasis, modulating morphology, migration, and cell polarity74.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [74] Lauko, A., Mu, Z., Gutmann, D. H., Naik, U. P. & Lathia, J. D. Junctional adhesion molecules in cancer: a paradigm for the diverse functions of cell-cell interactions in tumor progression. Cancer Res. 80, 4878–4885 (2020). source

      The JAM-A structure and function section says JAM-A induces EMT in some cancers, and the metastasis section links JAM-A to increased EMT in nasopharyngeal cancer. The paper also describes roles for JAMs in migration and polarity, but it does not establish that JAMs are essential drivers of EMT across cancer metastasis or specifically link them to the claimed morphological changes. Its Conclusion emphasizes that JAM-A can have both pro- and anti-tumorigenic roles. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    By E12.5, most cells have segregated into the endocardial and mesenchymal lineages as AV EndMT is complete.

    “By E12.5, most cells have segregated into the endocardial and mesenchymal lineages as AV EndMT is complete.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The three AV mesenchymal populations all express regulators of mesenchymal identity such as Sox9, Msx1, and Tbx20, but differ in timepoint composition or proliferative status.

    “The three AV mesenchymal populations all express regulators of mesenchymal identity, such as Sox916, Msx140, and Tbx2041, but differ in timepoint composition or proliferative status”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two distinct VEC populations emerge in the AVC by E12.5: VECs I expresses Ednrb, Irx5, and Palmd; VECs II is enriched for Wnt4, Nfatc1, and Edn1.

    “Two distinct VEC populations emerge in the AVC by E12.5, including VECs I, which expresses Ednrb, Irx5, and Palmd, and VECs II, which is enriched for Wnt4, Nfatc1, and Edn1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the AV mesenchymal lineage, expression of Gata4 is retained, but endothelial biomarkers are repressed.

    “In the AV mesenchymal lineage, expression of Gata4 is retained, but endothelial biomarkers are repressed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Differentiation potential is high among the early endothelial cluster, but highest where endocardial and mesenchymal lineages begin to bifurcate.

    “Differentiation potential is high among the early endothelial cluster, but highest where endocardial and mesenchymal lineages begin to bifurcate”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mesenchymal cells in Sox9 cKO display an increased mesenchymal gene profile, in part due to increased expression of EMT transcription factors Snai1, Twist1, and Zeb21.

    “Contradictorily, they display an increased mesenchymal gene profile, in part due to increased expression of EMT transcription factors, like Snai1, Twist1, and Zeb21”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Flow cytometry and immunostaining of sorted single-cells from individual E10.5 hearts support increased proportions of endothelium (CD31+ PDGFRα-) and cells exhibiting EMP (CD31+ PDGFRα+) in Sox9 cKO.

    “Flow cytometry and immunostaining of sorted single-cells from individual E10.5 hearts further support increased proportions of endothelium (CD31+ PDGFRα-) and cells exhibiting EMP (CD31+ PDGFRα+) in the cKO”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AV EPDCs retain expression of some epicardial transcription factors, including low Wt1 and Tbx18, and robust Tcf21.

    “While these cells retain expression of some epicardial transcription factors, including low Wt1 and Tbx18, and robust Tcf21”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    During EpiMT, mesenchymal factors are further activated and epicardial factors further reduced as cells transdifferentiate to AV mesenchyme via an AV EPDC intermediate.

    “These mesenchymal factors are further activated, and epicardial factors are further reduced, as cells transdifferentiate to AV mesenchyme via an AV EPDC intermediate during EpiMT.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Comparing AV and chamber EPDC populations reveals transcriptional differences likely related to the fate of the transdifferentiating populations, including enriched Tagln, Acta2, and Twist1 within AV EPDCs and Igf2, Apela, and Upk3b within chamber EPDCs.

    “Comparing AV and chamber EPDC populations reveal transcriptional differences likely related to the fate of the transdifferentiating populations, including enriched Tagln, Acta2, and Twist1 within AV EPDCs and Igf2, Apela, and Upk3b within chamber EPDCs (Supplementary Fig. 5h, i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VEC progenitors retain endothelial biomarkers and activate genes associated with AV and VEC identity including Gata4, Irx5, Nfatc1, Wnt4, Ednrb, and Palmd.

    “VEC progenitors retain expression of endothelial biomarkers and further activate expression of a variety of genes associated with AV and VEC identity, including Gata415, Irx545, Nfatc121, and Wnt446, along with genes with uncharacterized roles in AV development, like Ednrb and Palmd”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sox9 cKO has increased proportions of AV endocardial and VEC II cells and reduced numbers of AV mesenchymal cells.

    “This includes increased proportions of AV endocardial and VEC II cells and reduced numbers of AV mesenchymal cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    WT mesenchyme is enriched for transcription factors Klf1, Scx, and Sox5, signalling components Fgfr2, Notch2, Tgfbi, and Pdgfra, and ECM components type II and IX collagens, Fbln2, and Matn4.

    “In WT mesenchyme we see enrichment of transcription factors, including Klf1, Scx, and Sox5, signalling components, such as Fgfr2, Notch2, Tgfbi, and Pdgfra, and ECM components, like type II and IX collagens, Fbln2, and Matn4”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Appreciable overlap of endocardial and mesenchymal factors occurs during EndMT, including expression of Pecam1, Tek, Twist2, and Msx1 and co-localization of SOX9, ERG, and NFATc1.

    “Remarkably, we observe appreciable overlap of endocardial and mesenchymal factors during EndMT, including expression of Pecam1, Tek, Twist2, and Msx1 in our scRNA-seq data (Fig. 2d) and co-localization of SOX9, ERG, and NFATc1 by histology”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Differentiation potential is high among the STM populations, but is highest at the interface between the epicardial and AV EPDC clusters during EpiMT.

    “Differentiation potential is high among the STM populations, but is highest at the interface between the epicardial and AV EPDC clusters (arrows, Fig. 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PDGFA and FGF9 are predicted to signal via PDGFRα/β and FGFR1/2, respectively, during EpiMT.

    “PDGFA and FGF9 are predicted to signal via PDGFRα/β and FGFR1/2, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Some Hic1nLacZ-expressing cells co-express SOX9, indicating induction of AV mesenchymal identity, and do not co-express endothelial ERG or myocardial TNNT2.

    “We find that some of these cells co-express SOX9 (arrowheads, Fig. 6c, d), indicating induction of AV mesenchymal identity, and we do not observe co-expression with endothelial ERG or myocardial TNNT2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AV EPDCs maintain low WT1 and activate SOX9 as they transdifferentiate towards an AV mesenchymal cell state.

    “These cells maintain low WT1 and activate SOX9 as they transdifferentiate towards an AV mesenchymal cell state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Active signalling through TGFβ1 and TGFβ3 is predicted for EpiMT, which are not expected to influence EndMT.

    “However, we further predict active signalling through TGFβ1 and TGFβ3, which are not expected to influence EndMT (Fig. 7c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Dissociation with dispase/collagenase successfully captured endothelial- and epicardial-derived cells but myocardial cells are absent.

    “We found that while dissociation with dispase/collagenase successfully captured endothelial- and epicardial-derived cells, myocardial cells are absent.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Adoption of EMP during EndMT is closely associated with activation of mechanoresponsive genes and high differentiation potential, indicating a role for hemodynamic forces in regulating cell fate identity.

    “The adoption of EMP during EndMT is closely associated with the activation of mechanoresponsive genes and high differentiation potential, indicating a role for hemodynamic forces in regulating cell fate identity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Developmental arrest of endocardial cells that exhibit EMP within the AVC occurs when Sox9 is conditionally ablated, along with mesenchymal gene dysregulation and transdifferentiation to myofibroblasts.

    “We further observe the developmental arrest of endocardial cells that exhibit EMP within the AVC when Sox9 is conditionally ablated along with mesenchymal gene dysregulation and transdifferentiation to myofibroblasts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    These data define distinct AV endocardium, VEC, and mesenchymal populations, along with a population of AV EPDCs.

    “This includes distinct AV endocardium, VEC, and mesenchymal populations, along with a population of AV EPDCs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Immunostaining of Hic1nLacZ/+ AV canals for SOX9, ERG, and TNNT2 at E12.5 and E14.5 shows co-expression of mesenchymal SOX9 and AV EPDC Hic1nLacZ within AV sulcus, suggesting transition to mesenchyme.

    “Immunostaining of Hic1nLacZ/+ AV canals for SOX9, ERG, and TNNT2 at (c) E12.5 (n = 9) and (d) E14.5 (n = 6) shows co-expression of mesenchymal SOX9 and AV EPDC Hic1nLacZ within AV sulcus, suggesting transition to mesenchyme (arrowheads).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    WT1hi Hic1nLacZ lo epicardial cells exist in a single-layered epithelium at E12.5 and E14.5, while Hic1nLacZ hi and WT1lo AV EPDCs stratify and appear to adopt a more mesenchymal phenotype as they transdifferentiate.

    “We further observe that while WT1hi Hic1nLacZ lo epicardial cells exist in a single-layered epithelium at E12.5 and E14.5 (yellow arrowheads, Supplementary Fig. 6c, d), Hic1nLacZ hi and WT1lo AV EPDCs stratify and appear to adopt a more mesenchymal phenotype as they transdifferentiate, in line with our gene profile analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TGFβ1 is not predicted to influence EndMT; BMPs along with INHBA and GDF6 are identified as active drivers of mesenchymal identity.

    “Interestingly, TGFβ1 is not predicted to influence EndMT; however, a variety of BMPs along with INHBA and GDF6 are identified as active drivers of mesenchymal identity (Fig. 7a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    TGFβ1 is the most influential factor in promoting EpiMT and may activate regulators associated with AV EPDC or AV mesenchymal identity, including Hic1, Sox9, Snai2, Postn, Tagln, integrins, and type III and V collagens.

    “Our analysis suggests that TGFβ1 is the most influential factor in promoting EpiMT and may activate a variety of regulators associated with AV EPDC or AV mesenchymal identity, including Hic1, Sox9, Snai2, Postn, Tagln, integrins, and type III and V collagens (Supplementary Fig. 7d).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Around E8.5, a constriction forms at the junction between the atria and ventricles, where myocardial hyaluronan and fibronectin accumulate, establishing the primitive valves called AV cushions.

    “Around E8.5, a constriction forms at the junction between the atria and ventricles, where myocardial hyaluronan and fibronectin accumulate, establishing the primitive valves, called the AV cushions4,7.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Markwald, R. R., Fitzharris, T. P. & Manasek, F. J. Structural development of endocardial cushions. Am. J. Anat. 148, 85–119 (1977). source
    • cites [7] Mjaatvedt, C. H., Lepera, R. C. & Markwald, R. R. Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin. Dev. Biol. 119, 59–67 (1987). source

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

    Study design and conductown data

    Some transcriptional differences across batches persist after Harmony correction, though these genes are predominantly associated with general biological processes and metabolism.

    “Some transcriptional differences across batches persist after correction, though these genes are predominantly associated with general biological processes and metabolism”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    A total of 53,521 single-cells from all datasets passed stringent quality control checks, with a median of 4657 genes detected per cell.

    “A total of 53,521 single-cells from all datasets passed stringent quality control checks, with a median of 4657 genes detected per cell”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Proliferation is not spatially coordinated but is activated ubiquitously in the emerging mesenchyme at E12.5.

    “suggesting proliferation is not spatially coordinated, but is activated ubiquitously in the emerging mesenchyme at E12.5”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    EpiMT may initiate by E10.5, which is significantly earlier than previously understood.

    “This suggests that EpiMT may initiate by E10.5, which is significantly earlier than previously understood.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    EndMT stalls without Sox9, leading to developmental arrest of endocardium exhibiting EMP, while the few cells that transdifferentiate to mesenchyme adopt a more myofibroblastic phenotype.

    “Overall, our data suggest that EndMT stalls without Sox9, leading to the developmental arrest of endocardium exhibiting EMP, while the few cells that do transdifferentiate to mesenchyme adopt a more myofibroblastic phenotype.”
    Quote in the paperSubstantial claim

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

    Interpretationown data

    Differences in gene expression in Sox9 cKO suggest that cues involving BMPs, FGFs, PDGFs or integrin-FN1 interactions could be responsible.

    “Differences in gene expression in the Sox9 cKO suggest that cues involving BMPs, FGFs, PDGFs or integrin-FN1 interactions could be responsible.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Cell signalling analyses suggest TGFβ superfamily, PDGF, FGF, and mechanical signalling may play a role in activation of AV EPDCs.

    “Our cell signalling analyses suggest TGFβ superfamily, PDGF, FGF, and mechanical signalling may play a role in the activation of this cell type.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Cells were filtered based on unique features (>2000 genes) and mitochondrial RNA content (<20%).

    “cells were subsequently filtered based on unique features (>2000 genes) and mitochondrial RNA content (<20%).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    At least two distinct processes function to promote AV EndMT: a SOX9-independent process involving adoption of EMP possibly due in part to fluid shear stress, and a SOX9-dependent process involving complete transdifferentiation to AV mesenchyme.

    “This suggests that at least two distinct processes function to promote AV EndMT. The first is SOX9-independent, involving the adoption of EMP possibly in part due to fluid shear stress, while the second is SOX9-dependent and involves the complete transdifferentiation to AV mesenchyme.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    EndMT and EpiMT are both initiated within an epithelium and exist transiently in a stratified state, where induction of EMP is associated with high cell lineage plasticity.

    “EndMT and EpiMT are both initiated within an epithelium and exist transiently in a stratified state, where the induction of EMP is associated with high cell lineage plasticity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    Differences exist between EndMT and EpiMT, including presence of hemodynamic forces in promoting EndMT, differences in cell signalling microenvironments, and process-specific expression of transcription factors including Nfatc1 and Hic1 in EndMT and EpiMT, respectively.

    “However, differences also exist between these processes. This includes the presence of hemodynamic forces in promoting EndMT, differences in the cell signalling microenvironments, and process-specific expression of transcription factors, including Nfatc1 and Hic1 in EndMT and EpiMT, respectively.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    NicheNet predicts which ligands produced by a sender cell are the most active in affecting gene expression in a receiver cell, through the correlation of ligand activity with genes previously characterized as targets of their downstream pathways.

    “NicheNet predicts which ligands produced by a sender cell are the most active in affecting gene expression in a receiver cell, through the correlation of ligand activity with genes previously characterized as targets of their downstream pathways.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [66] Browaeys, R., Saelens, W. & Saeys, Y. NicheNet: modeling intercellular communication by linking ligands to target genes. Nat. Methods 17, 159–162 (2020). source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Classification of cells to gene profile categories was done through determining conservative minimum thresholds in each gene profile for meeting endothelial, epicardial, or mesenchymal identity.

    “Classification of cells to gene profile categories (e.g. exhibiting EMP), was done through determining conservative minimum thresholds in each gene profile for meeting endothelial, epicardial, or mesenchymal identity (Supplementary Figs. 3a and 6a).”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Researchers were not blinded during data acquisition and analysis.

    “Researchers were not blinded during data acquisition and analysis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    No data was excluded from analysis.

    “The experiments were not randomized and no data was excluded from analysis.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    No statistical method was used to predetermine sample size.

    “No statistical method was used to predetermine sample size.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

    Study design and conductnone given

    Globally enriched genes were identified based on log fold-change of 0.25 with expression threshold >25% using two-sided Wilcoxon rank-sum test, and only positively enriched genes with FDR 0.05 or lower were included.

    “Globally enriched genes within a cluster were identified based on a log fold-change of 0.25 with an expression threshold of >25% within a population using two-sided Wilcoxon rank-sum test. Only positively enriched genes with a false-discovery rate (FDR) of 0.05 or lower are included.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All mice were maintained in accordance with the University of British Columbia's Animal Care Committee's standards under protocol numbers A20-0281, A20-0282, and A19-0221 in pathogen-free conditions.

    “All mice were maintained in accordance with the University of British Columbia’s Animal Care Committee’s standards under protocol numbers A20-0281, A20-0282 (principal investigator, P.A.H.), and A19-0221 (principal investigator T.M.U.) in pathogen-free conditions.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The experiments were not randomized.

    “The experiments were not randomized and no data was excluded from analysis.”
    Quote in the paperNot a substantial claim
60 of 60

  • The paper relies on this without support.

    Many aspects of AV EMTs are incompletely characterized, mainly due to limitations related to the size of the embryonic valves.

    “While our understanding of atrioventricular development has improved greatly with the advent of genomics research, the details underlying many aspects of AV EMTs are incompletely characterized, mainly due to limitations related to the size of the embryonic valves.”

  • The paper relies on this without support.

    Lineage divergence of epicardial and mesenchymal populations during AV EpiMT remains uncharacterized.

    “lineage divergence of epicardial and mesenchymal populations during AV EpiMT remains uncharacterized.”

  • The paper relies on this without support.

    Little is known about Sox9’s molecular mechanisms during valve development beyond terminal phenotypes.

    “surprisingly little is known about Sox9’s molecular mechanisms during valve development beyond what can be seen in the terminal phenotypes.”

  • The paper relies on this without support.

    The early endocardium requires maintenance of tight junctions to sustain endothelial barrier and cardiac functions.

    “The early endocardium requires the maintenance of tight junctions to sustain endothelial barrier and cardiac functions.”
4 of 4

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.

  • Conflicting gene-count quality-control cutoffs

    Major

    One methods section says cells were filtered to retain those with more than 2000 genes; another says only cells with fewer than 1000 expressed genes were excluded as low quality. The stated rules disagree about cells with 1000-2000 genes.

  • Fig. 2e is assigned different trajectory termini

    Minor

    The results describe Fig. 2e as differentiation potential toward the AV mesenchymal terminus, but the figure legend describes that panel as progressing toward a VEC terminus.

  • Conflicting Cell Ranger versions

    Minor

    The read-alignment method specifies Cell Ranger version 6.0.0, while the statistics section says version 5.0 was used for alignment and UMI counting.

  • EpiMT heatmap is labelled Fig. 7b and Fig. 7c

    Minor

    The Fig. 7 legend assigns the EpiMT ligand-receptor heatmap to panel b, whereas the results cite panel c for the EpiMT signalling findings.

4 of 4

Cites

Flagged first, then by citations

1 of the 95 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Lotto, J., Cullum, R., Drissler, S., Arostegui, M., Garside, V. C., Fuglerud, B. M., Clement-Ranney, M., Thakur, A., Underhill, T. M., & Hoodless, P. A.
Affiliations
  • BC Cancer Agency (CA)
  • University of British Columbia (CA)
Corresponding author
Hoodless, Pamela A.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2023-09-09
Volume
14
Issue
1
Pages
5567-5567
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-023-41279-6
PMC
PMC10492828
OpenAlex
W4386568498
Semantic Scholar
c05cfc09413a1acc253bbe470242e1585d3713cf
Cited by
13
References
95
Citations by year
2026: 5 · 2025: 2 · 2024: 4 · 2023: 2
FWCI
0.97
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Gouvernement du Canada | Canadian Institutes of Health Research (PJT-159512, PJT-149026)
  • Canadian Foundation for Innovation British Columbia Knowledge Development Fund
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Congenital heart defects research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
epithelial-mesenchymal transition, epithelial-mesenchymal plasticity, endocardium, epicardium, SOX9, single-cell transcriptomics, heart development, cell fate determination, cell-cell interactions
MeSH terms
Animals, Biology, Cell Communication, Cell Differentiation, Endocardium, Heart Valves, 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.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review