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Cited by 170Developmental Biology and Gene Regulationmelanin and skin pigmentationZebrafish Biomedical Research Applications

Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

Open access
Saunders, Lauren M, Mishra, Abhishek K, Aman, Andrew J, Lewis, Victor M, Toomey, Matthew B, Packer, Jonathan S, Qiu, Xiaojie, McFaline-Figueroa, Jose L +3 more·2019·eLife
DOI 10.7554/elife.45181PMC PMC6588384

Published version of a preprint

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Abstract

Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form.

AI snapshot

Written from the paper’s full text.

TL;DR

Thyroid hormone (TH) promotes maturation of both melanophore and xanthophore lineages in zebrafish, but through distinct mechanisms rather than by biasing fate specification or having discordant lineage-specific effects. Single-cell transcriptomics and functional assays show TH drives terminal differentiation/proliferative arrest in melanophores and carotenoid-dependent repigmentation in xanthophores, with unliganded TH receptors acting primarily as repressors.

Key findings

  • Single-cell RNA sequencing of 16,150 neural crest-derived cells from euthyroid and hypothyroid zebrafish identified melanophores, xanthophores, iridophores, pigment progenitors, neurons, Schwann cells, other glia, and chromaffin cells; hypothyroid and euthyroid trajectories were topologically equivalent, and progenitors were not depleted in hypothyroid fish.
  • Hypothyroid pigment cells were biased toward early pseudotime across all pigment lineages (Wilcoxon signed-rank tests: melanophores Z = −6.54, p<0.0001; xanthophores Z = −4.54, p<0.0001; iridophores Z = −13.55, p<0.0001), arguing against TH controlling fate specification or lineage-specific amplification/restraint.
  • TH promoted melanophore maturation: euthyroid melanophores had more melanin (F1,2710=271.2, p<0.0001; ~1.7× total melanin content by area estimate), greater lysosomal content, larger forward scatter, and more binucleation (~70% vs ~25% in stage-matched hypothyroid fish; χ2=230.3, d.f.=1, p<0.0001).
  • TH promoted xanthophore carotenoid repigmentation: xanthophores had fewer TH-dependent genes than melanophores (3.6% vs 9%; 160 vs 519 DEGs, only 58 overlapping), carotenoid pathway scores were higher in euthyroid xanthophores (p=1.5E-15), scarb1 was expressed more highly in euthyroid xanthophores (q=1.1E-10), and scarb1 mutants lacked mature yellow xanthophores; TR mutations rescued hypothyroid melanophore and xanthophore phenotypes.

Methods

  • Zebrafish were made hypothyroid by nitroreductase-mediated thyroid ablation in Tg(tg:nVenus-v2a-nfnB) fish; euthyroid and hypothyroid siblings were compared.
  • Neural crest-derived cells were lineage-labeled with Tg(sox10:Cre; ubi:switch), dissociated from trunks/skins, FACS-purified, and processed for 10X Chromium single-cell RNA sequencing across 5 dpf, 7.2 SSL, 8.6 SSL, 9.6 SSL, and 11 SSL; the gene-barcode matrix contained ~25,000 barcoded cells, with 16,150 cells assigned in Figure 2A.
  • Analyses included UMAP/louvain clustering, Monocle pseudotime and BEAM, differential expression across pseudotime, pathway signature scores, plus imaging, cell counts, HPLC for carotenoids, FACS for lysosomal content, TEM, and CRISPR/Cas9 mutants for scarb1 and TH receptors.

Limitations

  • Stated: the analyses cannot indicate whether TH acts directly on pigment cells through autonomous TR activity; TH could act on stromal or other cell types, and iridophores/stromal cells also regulate melanophore and xanthophore numbers.
  • Stated: roles for TRs are likely complex and context-dependent; loss of TRs in euthyroid fish did not cause precocious xanthophore pigmentation, suggesting other late-stage factors are required.
  • Stated/evident: SA-β-gal staining could not be scored reliably across TH conditions; FSC-A can be influenced by cell-size-independent factors; pseudotime is inferred rather than true developmental time, and the reconstructed trajectory topology differed from known lineage relationships.

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

Integrity signals

Registry

Crossref · 10.7554/elife.45181

Cites

137 of 137 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

27 of 40 cited sources checked · 9 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Adult melanophores arise from a transit-amplifying progenitor.

    “In contrast to adult melanophores that arise from a transit-amplifying progenitor”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites McMenamin et al., 2014 source

      The passage labeled Abstract (page unavailable) says adult body melanophores arise from extra-hypodermal, peripheral nerve-associated precursors and describes presumptive melanophore precursors differentiating. It does not identify those precursors as transit-amplifying, so the specific progenitor type is unsupported. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites Patterson et al., 2014 source

      The Discussion (page unavailable in our copy) says adult melanophores arise from motile precursors that migrate to the hypodermis and begin differentiating. It does not describe those precursors as transit-amplifying, so the specific progenitor type is unsupported. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The findings are broadly consistent with a role for thyroid hormone in balancing proliferation and differentiation.

    “Our findings are broadly consistent with a role for TH in balancing proliferation and differentiation (Brent, 2012)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Brent, 2012 source
  • Premisecitation

    Thyroid hormone promotes a cessation of proliferative activity in vivo.

    “TH similarly promotes the melanization of melanoblasts ex vivo and a cessation of proliferative activity in vivo (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The passage discussing Fig. 4A reports that melanophores rarely divided in euthyroid larvae but divided frequently in hypothyroid larvae. It concludes that TH represses melanophore proliferation in vivo; it does not show a complete cessation of proliferation. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Addition of exogenous thyroid hormone to hypothyroid cells ex vivo can promote differentiation of unpigmented melanoblasts to melanophores.

    “Indeed, prior analyses showed that addition of exogenous TH to hypothyroid cells ex vivo can promote differentiation of unpigmented melanoblasts to melanophores (McMenamin et al., 2014), contrary to the idea that TH specifically blocks melanophore development.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      In the passage labeled Abstract (page unavailable), the paper reports that melanophore precursors in hypothyroid larvae were less likely to differentiate and concludes that TH promotes their differentiation. It does not report adding TH to hypothyroid cells ex vivo or observing unpigmented melanoblasts become melanophores in that setting. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Thyroid hormone promotes the melanization of melanoblasts ex vivo.

    “TH similarly promotes the melanization of melanoblasts ex vivo and a cessation of proliferative activity in vivo (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The passage on melanophore lineage development (section labeled Abstract; page unavailable) says thyroid hormone promotes melanophore precursor differentiation and represses melanophore proliferation in larvae. It does not report that thyroid hormone promotes melanoblast melanization ex vivo; the experiments described were in fish. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 22
  • May combine two claims. 45

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

    Study design and conductcitation

    Forward scatter measurements can be influenced by cell-size-independent factors.

    “although FSC-A can be influenced by cell-size-independent factors as well (Tzur et al., 2011).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Tzur et al., 2011 source

  • May combine two claims.

    Premisecitation

    Fish lacking thyroid hormone have gross defects in pigment cell numbers and pattern, with approximately twice the normal complement of melanophores and a simultaneous absence of visible xanthophores.

    “Fish lacking TH have gross defects in pigment cell numbers and pattern with ~two fold the normal complement of melanophores and the simultaneous absence of visible xanthophores (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The paper’s thyroid-ablation passage (section labeled Abstract; page unavailable) reports excess melanophores and a lack of adult body xanthophores in juvenile fish. It also says some xanthophores developed later, and the passages do not establish the claimed approximately twofold melanophore count. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Fish lacking thyroid hormone have about twice the normal number of melanophores and lack visible xanthophores.

    “fish lacking TH have about twice the normal number of melanophores and lack visible xanthophores (Figure 1A) (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The passage labeled Abstract (no page supplied), describing Fig. 1, reports that fish thyroid-ablated early in development lacked adult body xanthophores and had excess melanophores. It does not quantify the excess as about twice the normal number. It also notes that some xanthophores developed later, so their absence is not permanent. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Melanophores of euthyroid fish fail to divide whereas those of hypothyroid fish continue to divide.

    “Prior analyses indicated that melanophores of euthyroid fish fail to divide whereas those of hypothyroid fish continue to do so (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The indexed passage labeled Abstract reports that melanophores in euthyroid larvae rarely divided, while those in hypothyroid larvae divided frequently. It supports the comparison, but “fail to divide” overstates the finding that division was rare, not absent. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Multipotent progenitors that give rise to adult melanophores, some xanthophores, and iridophores are established in the embryo and reside within peripheral nerves as development progresses.

    “Multipotent progenitors that give rise to adult melanophores, some xanthophores, and iridophores are established in the embryo and reside within peripheral nerves as development progresses (Budi et al., 2011; Budi et al., 2008; Camargo-Sosa et al., 2019; Dooley et al., 2013a; Singh et al., 2016).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Budi et al., 2011 source

      The Discussion (page unavailable) describes embryonically established precursors to adult melanophores and iridophores that subsequently associate with peripheral nerves and ganglia. It does not include xanthophores among their descendants; the Figure 12 discussion instead says metamorphic xanthophores presumably arise from a different precursor population. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Budi et al., 2008 source
    • cites Camargo-Sosa et al., 2019 source

      The Introduction says adult pigment stem cells are set aside from neural crest cells during embryogenesis, with at least some occupying a niche within dorsal root ganglia. The Discussion says these cells produce adult melanophores and iridophores, with a small contribution to xanthophores. The claim overstates their location: the paper places at least some in ganglia and links others to the peripheral nervous system, but does not establish that the progenitors generally reside within peripheral nerves. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In zebrafish, thyroid hormone has opposite effects on neural crest-derived pigment cells of the adult stripe pattern: it limits melanophore population expansion yet increases yellow/orange xanthophore numbers.

    “In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers.”
    • Also stated in: § Introduction · hedged
      “Remarkably, TH has seemingly opposite effects on two classes of NC-derived pigment cells, curtailing the population of black melanophores yet promoting development of yellow/orange xanthophores; fish lacking TH have about twice the normal number of melanophores and lack visible xanthophores (Figure 1A) (McMenamin et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

  • May combine two claims.

    Premisecitation

    Melanoblasts derived from peripheral-nerve associated progenitors are proliferative during adult pigment pattern formation, but this activity largely ceases as the cells differentiate.

    “Melanoblasts derived from peripheral-nerve associated progenitors are proliferative during adult pigment pattern formation yet this activity largely ceases as the cells differentiate (Budi et al., 2011; McMenamin et al., 2014).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Budi et al., 2011 source
    • cites McMenamin et al., 2014 source

      The passage labeled Abstract says adult melanophores arise from peripheral nerve-associated precursors. It also reports that melanophores rarely divided in euthyroid larvae, but it does not establish that their precursors proliferate or that proliferation ceases as they differentiate; melanophores divided frequently in hypothyroid larvae. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Findingown data

    mitfa was expressed in melanophores and progenitors, but also in xanthophores, consistent with prior reports.

    “for example mitfa, encoding a transcription factor required for melanophore fate specification (Lister et al., 1999) was expressed in melanophores and progenitors, but also xanthophores (Figure 3C), consistent with prior reports (Eom et al., 2012; Parichy et al., 2000b).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Eom et al., 2012 source

      In the Results section on igsf11 expression (Figure 5C,E), mitfa:GFP marks pigment-cell precursors, and the paper also uses a mitfa-driven transgene in melanophores (Figure 4). It does not establish mitfa expression in xanthophores: Figure 5F measures igsf11, not mitfa, in xanthophore-enriched cells. Page numbers were not provided in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites Parichy et al., 2000b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Study design and conductcitation

    Ablated thyroid glands fail to regenerate, and absence of regeneration in this study was confirmed by continued absence of nVenus expression.

    “Ablated thyroid glands fail to regenerate (McMenamin et al., 2014) and absence of regeneration in this study was confirmed by continued absence of nVenus expression.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites McMenamin et al., 2014 source

      The paper’s section labeled Abstract (page unavailable) reports that fish thyroid-ablated at 4 days post-fertilization still lacked tg mRNA and T4 as juveniles, supporting persistent loss of thyroid function. It does not establish that the glands themselves cannot regenerate. The nVenus observation is described as a finding of the citing study, not attributed to McMenamin et al. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The yellow-orange color of xanthophores can depend on pteridine pigments, carotenoid pigments, or both.

    “The yellow-orange color of xanthophores can depend on pteridine pigments, carotenoid pigments, or both”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Bagnara and Matsumoto, 2006 source
    • cites Granneman et al., 2017 source
    • cites Lister, 2019 source

  • May combine two claims.

    Findingown data

    Measurements of forward scatter suggested that melanophores from juvenile euthyroid fish were larger than those from hypothyroid fish, although forward scatter can be influenced by cell-size-independent factors.

    “Measurements of forward scatter (FSC-A) also suggested that melanophores from juvenile euthyroid fish were larger than melanophores from hypothyroid fish (Figure 5—figure supplement 1D), although FSC-A can be influenced by cell-size-independent factors as well (Tzur et al., 2011).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In stage-matched comparisons, approximately 70% of melanophores from euthyroid fish were binucleated, whereas only approximately 25% of melanophores from hypothyroid fish were binucleated.

    “In stage-matched comparisons for TH status, ~70% of melanophores from euthyroid fish were binucleated, whereas only ~25% of melanophores from hypothyroid fish were in this state (Figure 5D and E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pteridine pathway genes tended to be expressed similarly across stages regardless of thyroid hormone status and were even moderately upregulated in hypothyroid xanthophores.

    “By contrast, pteridine pathway genes tended to be expressed similarly across stages regardless of TH status and were even moderately upregulated in hypothyroid xanthophores (Figure 6C, Figure 6—figure supplement 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Melanophore subcluster 2 cells exhibited low transcriptional activity and expressed fewer genes than melanophore 1 cells, suggesting a more advanced state of differentiation.

    “For example, cells of subcluster melanophore 2 exhibited low levels of transcriptional activity and expressed fewer genes, suggesting a more advanced state of differentiation, as compared to cells of melanophore 1 (Figure 3—figure supplement 1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Neural crest-derived cell types from embryonic-early larval stages and middle larval-juvenile stages did not form markedly different transcriptomic programs; combining profiles failed to reveal non-contiguous, life-stage-specific clusters.

    “These data do not indicate markedly different transcriptomic programs of NC-derived cell types across life stages, despite the existence of some stage-specific requirements for particular genes and pathways.”
    • Also stated in: § Post-embryonic NC-derived subpopulations revealed by single-cell RNA sequencing · plain
      “Combining profiles for cells at different stages failed to reveal non-contiguous, life-stage-specific clusters, although some EL cells occupied subsets of transcriptomic space relative to their broader cell type (e.g. melanophores) (Figure 2—figure supplement 5B–D).”
    Quote in the paperSubstantial claim

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

    Findingown data

    Some stripe melanophores plated ex vivo exhibited senescence-associated β-galactosidase activity, but staining could not be scored reliably for comparisons across thyroid hormone conditions.

    “When plated ex vivo, some stripe melanophores exhibited senescence-associated β-galactosidase (SA-β-gal) activity (Figure 5—figure supplement 1A), although we were unable to score such staining reliably, precluding comparisons across TH conditions.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In hypothyroid fish, loss of thrab alone or with thraa partially restored carotenoid deposition in interstripe xanthophores; mutation of all three receptors fully rescued the number of carotenoid-containing xanthophores.

    “Loss of thrab, on its own or in conjunction with loss of thraa, partially restored the deposition of carotenoids in interstripe xanthophoes; mutation of all three receptors fully rescued the number of carotenoid-containing xanthophores (Figure 7B and C; Figure 7—figure supplement 1E and F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In euthyroid fish, approximately 20% of melanophores were binucleate near the onset of adult melanophore differentiation but more than 50% were binucleate by juvenile stages, confirming an overall increase in binucleation with somatic stage and melanophore age.

    “In euthyroid fish, ~20% of melanophores were binucleate near the onset of adult melanophore differentiation but >50% were binucleate by juvenile stages, confirming an overall increase in binucleation with somatic stage and melanophore age (Figure 5—figure supplement 1E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Carotenoids were undetectable in euthyroid 5 dpf larvae, and carotenoid-related genes were expressed at lower levels in embryonic-early larval xanthophores than in adult xanthophores.

    “Carotenoids were undetectable in euthyroid 5 dpf larvae, and carotenoid-related genes were expressed at lower levels in EL xanthophores than adult xanthophores (Figure 6—figure supplement 4B and C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transcripts per cell declined in melanophores but not iridophores, consistent with reduced RNA abundance as melanophores, but not iridophores, exit the cell cycle with maturation.

    “For example, transcripts per cell declined in melanophores but not iridophores, consistent with an expectation of reduced RNA abundance as melanophores—but not iridophores—exit the cell cycle with maturation (Figure 3—figure supplement 5B) (Budi et al., 2011; Darzynkiewicz et al., 1980; McMenamin et al., 2014; Spiewak et al., 2018).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The collected cell types include immature and mature glia, differentiating pigment cells, and presumptive progenitors of different types, and identify new candidate genes for promoting and recognizing distinct states of differentiation and morphogenetic activities.

    “Our collected cell-types, which include immature and mature glia, differentiating pigment cells, and presumptive progenitors of different types identify new candidate genes for promoting—and recognizing—distinct states of differentiation and morphogenetic activities”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    Xanthophore 1 cells had fewer transcripts and expressed fewer genes than xanthophore 2 cells, suggesting they may represent undifferentiated, cryptic xanthophores and actively differentiating populations, respectively.

    “Likewise cells of xanthophore 1 had fewer transcripts and expressed fewer genes than cells of xanthophore 2, suggesting they may represent undifferentiated, cryptic xanthophores and actively differentiating populations, respectively (McMenamin et al., 2014).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    An additional cluster expressed markers suggestive of proliferative, non-pigmentary progenitors, and one large cluster ('unknown') was not readily assignable to previously described neural crest-derived populations.

    “An additional cluster expressed markers suggestive of proliferative, non-pigmentary progenitors, and one large cluster (‘unknown’) was not readily assignable to NC-derived populations described previously.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The survey captured numerous neural crest-derived cell types, including abundant pigment cells and progenitors, and revealed substantial variation in gene expression programs among them.

    “Overall, our survey captured numerous NC-derived cell types, including abundant pigment cells and progenitors, and revealed substantial variation in gene expression programs among them.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Genes encoding each of the three zebrafish thyroid hormone receptors (thraa, thrab, thrb) were expressed by melanophores and xanthophores, yet presumptive null alleles for each had pigment cell complements and patterns resembling wild type.

    “Genes encoding each of the three zebrafish TRs (thraa, thrab, thrb) were expressed by melanophores and xanthophores, yet presumptive null alleles for each unexpectedly had pigment cell complements and patterns that resembled the wild type (Figure 7A; Figure 7—figure supplement 1A–D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Euthyroid melanophores tended to be highly melanized and stellate, whereas hypothyroid melanophores were variably melanized and often dendritic.

    “Euthyroid melanophores tended to be highly melanized and stellate, whereas hypothyroid melanophores were variably melanized and often dendritic.”
    • Also stated in: § TH promotes a melanophore maturation program · hedged
      “Melanophores of juvenile euthyroid fish tended to be uniformly well-melanized and stellate, whereas melanophores of juvenile hypothyroid fish were variably melanized and dendritic (Figure 5B), reminiscent of earlier stages of melanophore development in wild-type (Eom et al., 2012; Parichy and Turner, 2003).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The pseudotemporal topology differed from known lineage relationships but was consistent with similarity of embryonic-early larval and mid-larval/juvenile gene expression programs.

    “This topology differed from known lineage relationships (Figure 3A) but was consistent with similarity of EL and mid-larval/juvenile gene expression programs (Figure 2—figure supplement 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Additional surveys revealed new markers of xanthophore and iridophore lineages and cell-type-specific expression of some previously identified markers.

    “Additional surveys of these data revealed new markers of xanthophore and iridophore lineages (Figure 3—figure supplements 2 and 3), and cell-type-specific expression of some previously identified markers [e.g. tyrp1b, aox5, tfec (Lister et al., 2011; McMenamin et al., 2014) (Figure 3C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    scarb1 is required in zebrafish for carotenoid deposition, although not for xanthophore cell persistence.

    “Among carotenoid genes, scavenger receptor B1 (scarb1) encodes a high-density lipoprotein receptor essential for carotenoid accumulation in birds and invertebrates (Kiefer et al., 2002; Toomey et al., 2017) and we found it to be required in zebrafish for carotenoid deposition, although not cell persistence (Figure 6—figure supplement 3A and B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Exogenous thyroid hormone was sufficient to rescue both scarb1 expression and carotenoid deposition.

    “exogenous TH was sufficient to rescue both expression and carotenoid deposition (Figure 6F; Figure 6—figure supplement 3C).”
    Quote in the paperSubstantial claim

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

    Findingown data

    In the three-branch BEAM analysis, all selected genes except pax3a had q < 6.0E-11.

    “q < 6.0E-11 for all genes, except for pax3a”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Thyroid hormone directly or indirectly regulates carotenoid-associated genes and carotenoid deposition, allowing cryptic xanthophores to reacquire visible pigmentation.

    “We showed that TH directly or indirectly regulates carotenoid-associated genes and carotenoid deposition, allowing cryptic xanthophores to reacquire visible pigmentation.”
    Quote in the paperSubstantial claim

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

    Findingown data

    In the three-branch BEAM analysis, pax3a had q = 0.03.

    “except for pax3a (starred, q = 0.03)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    In melanophores, thyroid hormone drives terminal differentiation, limiting final cell numbers.

    “In melanophores, TH drives terminal differentiation, limiting final cell numbers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Thyroid hormone promotes maturation of both melanophore and xanthophore lineages, but through distinct mechanisms.

    “We show that TH promotes maturation of both cell types but in distinct ways.”
    • Also stated in: § Introduction · plain
      “Our analyses showed that TH drives maturation of cells committed to melanophore and xanthophore fates through different mechanisms, promoting terminal differentiation and proliferative arrest in melanophores, and carotenoid-dependent repigmentation in xanthophores.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Thyroid hormone has an essential role in carotenoid pigmentation, and thyroid hormone modulation of a suite of carotenoid pathway genes is required for cryptic xanthophores to re-pigment during adult pattern formation.

    “Together these findings demonstrate an essential role for TH in carotenoid pigmentation and suggest that TH modulation of a suite of carotenoid pathway genes is required for cryptic xanthophores to re-pigment during adult pattern formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Repression by unliganded thyroid hormone receptors contributes to pigment-associated phenotypes in hypothyroid fish, implying a function for thyroid hormone receptors in repressing the repigmentation of xanthophores and terminal differentiation of melanophores until late stages in adult pigment pattern development.

    “These findings suggest that repression by unliganded TRs contributes to pigment-associated phenotypes in hypothyroid fish, implying a function for TRs in repressing the repigmentation of xanthophores and terminal differentiation of melanophores until late stages in adult pigment pattern development.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Thyroid hormone drives melanophores into a terminally differentiated state characterized by increased melanization, larger size, increased lysosomal content, binucleation, and proliferative cessation.

    “Collectively, our observations and those of McMenamin et al. (2014) suggest a model in which TH drives melanophores into a terminally differentiated state of increased melanization, larger size and lysosomal content, binucleation, and proliferative cessation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Thyroid hormone drives maturation of both xanthophores and melanophores yet has markedly different roles in each lineage.

    “TH therefore drives maturation of both xanthophores and melanophores yet has markedly different roles in each lineage.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Human melanoma is associated with hypothyroidism and recurrent thyroid hormone pathway mutations.

    “human melanoma is associated with hypothyroidism and recurrent TH pathway mutations (Ellerhorst et al., 2003; Shah et al., 2006; Sisley et al., 1993)”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Ellerhorst et al., 2003 source
    • cites Shah et al., 2006 source
    • cites Sisley et al., 1993 source

  • May combine two claims.

    Interpretationreasoning

    Thyroid hormone induces new, carotenoid-based pigmentation, allowing transiently cryptic xanthophores to reacquire coloration during adult pattern development.

    “Together, these observations imply that TH induces new, carotenoid-based pigmentation, allowing transiently cryptic xanthophores to reacquire coloration during adult pattern development.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    In euthyroid fish homozygous for thrab mutation, there was no evidence for early pigmentation of xanthophores, suggesting essential roles for other factors present only at later stages.

    “Yet, we found no evidence for early pigmentation of xanthophores in euthyroid fish homozygous for thrab mutation (Figure 7—figure supplement 1G), suggesting essential roles for other factors present only at later stages (Patterson and Parichy, 2013).”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Senescence-associated β-galactosidase staining could not be scored reliably, precluding comparisons across thyroid hormone conditions.

    “although we were unable to score such staining reliably, precluding comparisons across TH conditions.”
    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

    Single transgene controls and wild-type cells were used to adjust voltage and gating.

    “Single transgene controls and wild-type cells were used to adjust voltage and gating.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Guides were tested for mutagenicity by Sanger sequencing.

    “Guides were tested for mutagenicity by Sanger sequencing”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Cryptic xanthophores appear poised to re-differentiate, which likely explains the smaller proportion of thyroid-hormone-dependent genes in these cells compared to melanophores.

    “That cryptic xanthophores appear poised to re-differentiate likely explains the smaller proportion of genes that were TH-dependent in these cells as compared to melanophores.”
    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

    Images were corrected for color balance and adjusted for display levels as necessary, with conditions within analyses treated identically.

    “Images were corrected for color balance and adjusted for display levels as necessary with conditions within analyses treated identically.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For all thyroid ablations, treated individuals were assessed for loss of nuclear-localizing Venus (nVenus) the following day.

    “For all thyroid ablations, treated individuals were assessed for loss of nuclear-localizing Venus (nVenus) the following day.”
    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

    Final cellular barcodes and UMIs were determined using Cell Ranger 2.0.2, and cells were filtered to include only high-quality cells.

    “Final cellular barcodes and UMIs were determined using Cell Ranger 2.0.2 (10X Genomics) and cells were filtered to include only high-quality cells.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The experiment was replicated three times.

    “We replicated the experiment three times.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    To correct for bias in aligning reads to the transcriptome, all 3' UTR annotations were extended by 500 bp.

    “To correct for this bias in aligning reads to the transcriptome, we extended all 3' UTR annotations by 500 bp.”
    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 procedures involving live animals followed federal, state and local guidelines for humane treatment and protocols approved by Institutional Animal Care and Use Committees.

    “All procedures involving live animals followed federal, state and local guidelines for humane treatment and protocols approved by Institutional Animal Care and Use Committees of University of Virginia and University of Washington.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Individual genotypes of fish assessed were confirmed using PCR or Sanger sequencing.

    “Individual genotypes of fish assessed were confirmed using PCR or Sanger sequencing.”
    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 conductown data

    For parametric analyses, residuals were assessed for normality and homoscedasticity to meet model assumptions, and no transformations were found to be warranted.

    “For parametric analyses, residuals were assessed for normality and homoscedasticity to meet model assumptions and no transformations were found to be warranted.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Clusters were assigned to cell types based on the detection of published marker genes.

    “We assigned clusters to cell types based on the detection of published marker genes.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Cells with more than 15,000 UMIs were discarded as possible doublets.

    “Cells with more than 15,000 UMIs were discarded as possible doublets.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Batch correction methods were not used between the two groups or across samples because no sample-specific separation or clustering was observed in UMAP space.

    “Batch correction methods were not used between the two groups or across samples because we did not observe sample-specific separation or clustering in UMAP space.”
    Quote in the paperSubstantial claim
57 of 57

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • tfec expression is maintained at detectable levels only in mature iridophores, but neither in melanocytes nor in xanthophores.

    “The first aim of the present study was to determine whether iridophores are indeed the only embryonic differentiated pigment cell expressing tfec; to this end, we conducted analyses showing that tfec expression is maintained at detectable levels only in mature iridophores, but neither in melanocytes nor in xanthophores, in agreement with recently published single cell transcriptomics analyses [45]”

    From The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor (2021) · cites this paper as [45]

    The Results section, “Pigment cell sub-classes and gene expression dynamics across differentiation” (page unavailable), identifies tfec as a cell-type-specific pigment-cell marker, consistent with the claimed distinction among pigment-cell types. It does not establish that expression is maintained only in mature iridophores specifically among embryonic differentiated pigment cells; the citing authors describe that embryonic finding as their own analysis. (LLM, conf=0.83)

    Experimental — accuracy not yet measured

  • Clusters c1, c3, and c5 contained cells expressing high levels of known iridophore markers gpnmb and pnp4a, as well as recently identified iridophore markers alx4a and alx4b.

    “three of which (clusters c1, c3, and c5) contained cells expressing high levels of known markers of iridophores (i.e., gpnmb, pnp4a)37 (Supplementary Figs. 17 and 18a, b), as well as genes that were recently identified as iridophore markers (i.e., alx4a, alx4b)23 (Supplementary Figs. 17 and 18c, d)”

    From In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning (2020) · cites this paper as [23]

    The Results section (‘Pigment cell sub-classes and gene expression dynamics across differentiation’; page unavailable) reports newly identified iridophore-lineage markers, and the Materials and methods key resources table lists alx4a and alx4b as genes used in this study. The available passages do not explicitly identify those two genes as the new iridophore markers. Citation 23 is attached to that marker identification, not to the citing authors’ c1, c3, and c5 cluster observations or the markers cited to reference 37. (LLM, conf=0.78)

    Experimental — accuracy not yet measured

  • Differences in color between stripes and interstripes (blue versus silvery-yellow) persisted in fish lacking both melanin in melanophores and carotenoids in xanthophores.

    “Here, differences in color between stripes and interstripes (i.e., blue versus silvery-yellow) persisted even in the absence of other pigments (Fig. 3h)”

    From In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning (2020) · cites this paper as [23]

    The Results section on xanthophore re-pigmentation (page unavailable) reports carotenoid-deficient fish, and Materials and methods describes a melanin-free mutant. Searches for blue versus silvery-yellow stripes, iridophores, and fish lacking both pigments found no passage reporting that the color difference persisted under the claimed conditions. (LLM, conf=0.76)

    Experimental — accuracy not yet measured

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Consistentz = -6.54reported p < .0001 · recomputed p < .001

    “Z = −6.54, p < .0001”

  • Consistentz = -4.54reported p < .0001 · recomputed p < .001

    “Z = −4.54, p < .0001”

  • Consistentz = -13.55reported p < .0001 · recomputed p < .001

    “Z = −13.55, p < .0001”

3 of 3

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.

  • RT-PCR cell-sorting markers are assigned to the opposite cell types

    Minor

    The RT-PCR method assigns tyrp1b:palm-mCherry to xanthophores, whereas the melanophore maturation assay uses that transgene to isolate melanophores.

  • RT-PCR protocol gives two amplification cycle counts

    Minor

    The same RT-PCR protocol states both 40 and 38 amplification cycles.

2 of 2

Cites

Flagged first, then by citations

3 of the 140 references couldn’t be matched to a known paper.

Cited by

Sorted by citations

Related

Authors
Saunders, L. M., Mishra, A. K., Aman, A. J., Lewis, V. M., Toomey, M. B., Packer, J. S., Qiu, X., McFaline-Figueroa, J. L., Corbo, J. C., Trapnell, C., & Parichy, D. M.
Affiliations
  • University of Washington (US)
  • University of Virginia (US)
  • Washington University in St. Louis (US)
  • Department of Genome Sciences, University of Washington, Seattle, United States
Corresponding author
  • Saunders, Lauren M
  • Mishra, Abhishek K
  • Aman, Andrew J
  • Lewis, Victor M
  • Toomey, Matthew B
  • Packer, Jonathan S
  • Qiu, Xiaojie
  • Corbo, Joseph C
  • Trapnell, Cole
  • Parichy, David M
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2019-05-29
Volume
8
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.45181
PMC
PMC6588384
OpenAlex
W2911328426
Cited by
170
References
140
Citations by year
2026: 21 · 2025: 25 · 2024: 22 · 2023: 28 · 2022: 17 · 2021: 32 · 2020: 16 · 2019: 9
FWCI
6.20
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Institute of General Medical Sciences (R35 GM122471, T32 GM007067)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (DP2 HD088158)
  • National Eye Institute (EY024958, EY025196, EY026672)
  • W. M. Keck Foundation
  • Alfred P. Sloan Foundation
  • Paul G Allen Frontiers Group
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › melanin and skin pigmentation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Zebrafish Biomedical Research Applications
Keywords
thyroid hormones, melanophores, xanthophores, neural crest, stripe patterns, terminal differentiation, carotenoid accumulation, thyroid hormone receptor, developmental trajectories, zebrafish
MeSH terms
Animals, Cell Differentiation, Neural Crest, Pigments, Biological, Thyroid Hormones, Zebrafish, Gene Expression Regulation, Developmental, Gene Expression Profiling

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 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

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 ›

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