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Cited by 15melanin and skin pigmentationDevelopmental Biology and Gene RegulationZebrafish Biomedical Research Applications

Agouti and BMP signaling drive a naturally occurring fate conversion of melanophores to leucophores in zebrafish

Open access
Huang, Delai, Kapadia, Emaan H., Liang, Yipeng, Shriver, Leah P., Dai, Shengkun, Patti, Gary J., Humbel, Bruno M., Laudet, Vincent +1 more·2025·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2424180122PMC PMC11874323

Published version of a preprint

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Abstract

The often-distinctive pigment patterns of vertebrates are varied in form and function and depend on several types of pigment cells derived from embryonic neural crest or latent stem cells of neural crest origin. These cells and the patterns they produce have been useful for uncovering features of differentiation and morphogenesis that underlie adult phenotypes, and they offer opportunities to discover how patterns and the cell types themselves have diversified. In zebrafish, a body pattern of stripes arises by self-organizing interactions among three types of pigment cells. Yet these fish also exhibit white ornamentation on their fins that depends on the transdifferentiation of black melanophores to white cells, “melanoleucophores.” To identify mechanisms underlying this conversion we used ultrastructural, transcriptomic, mutational, and other approaches. We show that melanophore–melanoleucophore transition depends on regional BMP signals transduced through noncanonical receptors (Rgmb-Neo1a-Lrig2) as well as BMP-dependent signaling by Agouti genes, asip1 and asip2b . These signals lead to expression of transcription factor genes including foxd3 and runx3 that are necessary to induce loss of melanin, curtail new melanin production, and deploy a pathway for accumulating guanine crystals that, together, confer a white phenotype. These analyses uncover an important role for positional information in specifying ornamentation in zebrafish and show how tissue environmental cues and an altered gene regulatory program have allowed terminal addition of a distinct phenotype to a preexisting cell type.

AI snapshot

Written from the paper’s full text.

TL;DR

In zebrafish, white fin ornament cells called melanoleucophores (ML) arise from black melanophores through localized BMP and Agouti signaling, which drives melanosome degradation and guanine crystal accumulation. The study identifies Rgmb–Neo1a–Lrig2, Foxd3, and Runx3 as key mediators and shows ML adopt a transcriptomic state distinct from both melanophores and iridophores.

Key findings

  • ML arise from melanophores by degrading melanosomes and forming β-guanine-containing leucosomes. *pnp4a* mutants had reduced iridophore iridescence and lower ML leucosome/guanine contents, whereas *oca2* and *pmela* mutants had normal leucosome/guanine contents despite reduced or absent melanin, indicating distinct organellogenesis pathways.
  • scRNA-Seq resolved melanophore, transitional, and ML states; ML expressed fewer genes than melanophores or transitional cells and had reduced melanogenesis but increased purine synthesis signatures. Transcription factor genes constituted 4.4% of loci (120 of 2,612) expressed more highly in melanophores versus only 0.9% (5 of 534) in ML (χ² = 19.52, d.f. = 1, P < 0.0001), and ML lacked iridophore markers *tfec* and *ltk*.
  • *asip1* was expressed 4.5-fold higher at the fin edge than base (P = 2.4E-8, q = 0.0004); *asip1*/*asip2b* double mutants had significantly fewer ML, Asip2b overexpression increased ML, and Asip1 overexpression reduced ML and melanophores.
  • BMP signaling was required for ML development: pSmad immunoreactivity correlated with proximity to the fin edge (R² = 0.39), BMP inhibition reduced ML and increased melanophores in zebrafish and *D. albolineatus*, and *rgmb*, *neo1a*, and *lrig2* mutants had severe ML deficits with excess melanophores. *runx3* mutants lacked nearly all ML (WT: 144 ± 7 ML; *runx3*: 5 ± 8 ML; F₁,₁₄ = 163.6, P < 0.0001), and Foxd3/Runx3 were required and sufficient for ML development.

Methods

  • Zebrafish and other *Danio* species were reared and used for CRISPR/Cas9 mutagenesis, transgenesis (including heat-shock-inducible Asip1, Asip2b, and Foxd3), BMP inhibitor treatment (LDN-193189), imaging, FIB–SEM/transmission EM, and metabolomics.
  • Single-cell RNA-Seq was performed on FACS-isolated *tyrp1b*:PALM-mCherry+ melanophore–ML lineage cells and mCherry– fin environment cells from juvenile fish, using 10X Genomics, CellRanger, and Monocle3; spatial expression was assessed by in situ hybridization and pSmad immunostaining.
  • Statistical analyses included ANOVAs with post hoc Tukey–Kramer comparisons, controlling for standard length where noted.

Limitations

  • The paper states that the relative importance of positional information versus self-organizing processes across pattern elements and species remains an open question, and that direct targets of Foxd3, Runx3, and Mitfa in repressing melanization or promoting leucistic fate are not yet identified.
  • Because *foxd3* homozygotes are lethal by ~7 days postfertilization, the *foxd3* requirement was tested using F0 mosaic CRISPR/Cas9 mutants rather than germline homozygous mutants.
  • The text indicates unresolved direct versus indirect effects: BMP may act indirectly by promoting dermal development and Agouti gene expression, while direct effects on melanophores–ML are inferred; the reduced Asip2b effect in *rgmb* mutants (about half that in WT siblings) suggests BMP roles beyond stimulating Agouti expression.

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

Integrity signals

Registry

Crossref · 10.1073/pnas.2424180122

Cites

109 of 109 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

37 of 50 cited sources checked · 20 supported · 0 flagged · accuracy not yet measured

  • Premiseown data

    In zebrafish, white highlights on fins arise through the transformation of black pigment cells into white ones.

    “yet the white highlights on their fins arise differently—through the transformation of black pigment cells into white ones”
    • Also stated in: § Abstract · plain
      “Remarkably, these cells arise from melanophores that lose their melanin over just a few days while acquiring crystalline deposits of guanine.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] V. M. Lewis, Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores. Proc. Natl. Acad. Sci. U.S.A. 116, 11806– 11811 (2019). 31138706 PMC6575160 source
  • Findingown data

    Trajectory analyses bridged states in pseudotime and represented an inferred pathway of differentiation concordant with previous fate mapping in vivo.

    “with trajectory analyses bridging states in pseudotime and representing an inferred pathway of differentiation concordant with previous fate mapping in vivo (Fig. 1I and SI Appendix, Fig. S4 A–F and Datasets S2–S4) (9).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] V. M. Lewis, Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores. Proc. Natl. Acad. Sci. U.S.A. 116, 11806– 11811 (2019). 31138706 PMC6575160 source
  • Findingown data

    rgmb was expressed relatively prominently in melanophores and transitional cells.

    “we found that rgmb, encoding Repulsive Guidance Molecule b (RGMb) (43, 44), was expressed relatively prominently in melanophores and transitional cells”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [43] J. Zhang, Repulsive guidance molecules b (RGMb): Molecular mechanism, function and role in diseases. Expert Rev. Mol. Med. 26, e24 (2024). 39375839 PMC11488336 source
    • cites [44] C. Siebold, T. Yamashita, P. P. Monnier, B. K. Mueller, R. J. Pasterkamp, RGMs: Structural insights, molecular regulation, and downstream signaling. Trends Cell Biol. 27, 365– 378 (2017). 28007423 PMC5404723 source
  • Premisecitation

    Iridophores contain crystals of β-guanine.

    “Since iridophores also contain crystals of β-guanine (14, 15)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [14] R. Deis, Genetic control over biogenic crystal morphogenesis in zebrafish. Nat. Chem. Biol., 10.1038/s41589-024-01722-1 (2024). PMC11867974 39215102 source
    • cites [15] D. Gur, In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning. Nat. Commun. 11, 6391 (2020). 33319779 PMC7738553 source
  • Premisecitation

    mitfa and kita have essential functions in ML independent of their earlier requirements in melanophores.

    “both having essential functions in ML independent of their earlier requirements in melanophores (9)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [9] V. M. Lewis, Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores. Proc. Natl. Acad. Sci. U.S.A. 116, 11806– 11811 (2019). 31138706 PMC6575160 source
  • May combine two claims. 36
  • Classified as probably not a substantial claim. 23
  • The quote only partly states this claim. 2

  • May combine two claims.

    Findingown data

    Neither transitional cells nor ML expressed genes for Tfec or Ltk.

    “neither transitional cells nor ML expressed genes for Tfec, a transcription factor essential for iridophore specification (21) or Ltk, a receptor tyrosine kinase essential for iridophore differentiation and morphogenesis (22)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [21] K. Petratou, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest. PLoS Genet. 14, e1007402 (2018). 30286071 PMC6191144 source

      The Results section on the Tfec–Ltk feedback loop and the Discussion support Tfec’s role in iridophore specification. They do not report whether the citing document’s “transitional cells” or “ML” express tfec or ltk, so the claimed absence of expression in those groups is not established by this paper. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [22] S. S. Lopes, Leukocyte tyrosine kinase functions in pigment cell development. PLoS Genet. 4, e1000026 (2008). 18369445 PMC2265441 source

      Citation (22) is attached to the description of Ltk, not to the citing authors’ observation that transitional cells and ML lack Ltk expression. The Discussion (page unavailable) identifies Ltk as a receptor tyrosine kinase crucial for iridophore specification, but says the early mutant phenotype prevents direct examination of its role in differentiating cells. Our copy does not establish that Ltk is essential for iridophore differentiation or morphogenesis specifically. (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

    Asip1 and the orthologous protein of mammals, Asip, are Melanocortin-1 receptor (Mc1r) antagonists and their ventral expression inhibits differentiation of melanophores and melanocytes, respectively, resulting in light bellies.

    “Asip1 and the orthologous protein of mammals, Asip, are Melanocortin-1 receptor (Mc1r) antagonists and their ventral expression inhibits differentiation of melanophores and melanocytes, respectively, resulting in light bellies (24–27).”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [24] M. M. Ollmann, M. L. Lamoreux, B. D. Wilson, G. S. Barsh, Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo. Genes Dev. 12, 316– 330 (1998). 9450927 PMC316484 source

      The Discussion (page unavailable) states that Agouti antagonizes Mc1r, and the paper describes Agouti shifting pigment production in mammalian melanocytes toward yellow. It does not establish that Agouti inhibits melanocyte differentiation or that ventral expression produces light bellies. The Concluding remarks (page unavailable) treat an Agouti homolog involved in amphibian dorsal–ventral pigmentation as speculative; the paper does not establish the claim about Asip1 in fish. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [25] M. Manceau, V. S. Domingues, R. Mallarino, H. E. Hoekstra, The developmental role of Agouti in color pattern evolution. Science 331, 1062– 1065 (2011). 21350176 source
    • cites [26] L. Cal, Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation. Sci. Rep. 9, 3449 (2019). 30837630 PMC6401153 source

      The Discussion supports the zebrafish portion: it reports that Asip1 binds Mc1r as an antagonist and represses ventral melanophore differentiation. But the Introduction and Discussion describe mammalian ASIP as changing the type of melanin produced by melanocytes, not inhibiting melanocyte differentiation. The claimed mechanism for mammalian light bellies is therefore not supported. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites [27] L. Cal, Loss-of-function mutations in the melanocortin 1 receptor cause disruption of dorso-ventral countershading in teleost fish. Pigment Cell Melanoma Res. 32, 817– 828 (2019). 31251842 source

      The available passages are bibliographic metadata and keywords under the Abstract label, not substantive text about Asip1 or Asip. Searches for MC1R antagonism, ventral expression, and melanophore or melanocyte differentiation found no passage establishing the claimed mechanism. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The self-organizing properties of pigment cell interactions are revealed by perturbations, with cellular responses that regenerate, reposition, or even reorient the pattern.

    “Yet the self-organizing properties of pigment cell interactions are revealed by perturbations, with cellular responses that regenerate, reposition, or even reorient the pattern (7, 8).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [7] A. Nakamasu, G. Takahashi, A. Kanbe, S. Kondo, Interactions between zebrafish pigment cells responsible for the generation of turing patterns. Proc. Natl. Acad. Sci. U.S.A. 106, 8429– 8434 (2009). 19433782 PMC2689028 source

      The Results describe laser-ablation experiments in which pigment cells regenerate and melanophores develop in a region previously occupied by xanthophores. The paper’s introductory section also says regenerated patterns sometimes change conformation, supporting self-organization after perturbation. It does not specifically establish that a pattern reorients. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [8] D. M. Parichy, J. M. Turner, Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development. Development 130, 817– 833 (2003). 12538511 source

  • May combine two claims.

    Premisecitation

    pnp4a encodes a purine nucleoside phosphorylase of the purine salvage pathway expressed in both iridophores and ML.

    “pnp4a, which encodes a purine nucleoside phosphorylase of the purine salvage pathway expressed in both cell types (9).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] V. M. Lewis, Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores. Proc. Natl. Acad. Sci. U.S.A. 116, 11806– 11811 (2019). 31138706 PMC6575160 source

      The Results section, “Dual Classes and Origins of Leucophores in Danio” (page unavailable), identifies pnp4a as purine nucleoside phosphorylase 4a, reports its strong expression in iridophores, and says melanoleucophores expressed a pnp4a reporter. The paper does not identify pnp4a as part of the purine salvage pathway; its discussion of purine-synthesis genes concerns de novo synthesis. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Ectothermic vertebrates have multiple types of pigment cells that retain their pigmentary materials intracellularly: black melanophores, with melanin; yellow or orange xanthophores and red erythrophores, with pteridines, carotenoids, or both; and iridescent iridophores, with crystalline deposits of guanine in stacks of reflecting platelets.

    “Ectothermic vertebrates have multiple types of pigment cells that retain their pigmentary materials intracellularly: black melanophores, with melanin; yellow or orange xanthophores and red erythrophores, with pteridines, carotenoids, or both; and iridescent iridophores, with crystalline deposits of guanine in stacks of reflecting platelets (3).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [3] D. M. Parichy, Evolution of pigment cells and patterns: Recent insights from teleost fishes. Curr. Opin. Genet. Dev. 69, 88– 96 (2021). 33743392 PMC8364858 source

      The ‘Chromatophore origins and arrangements’ section says that teleosts and other ectotherms have several chromatophore classes that typically retain pigments intracellularly; it identifies black melanophores, xanthophores with pteridines and carotenoids, and iridescent iridophores with reflective guanine crystals. ‘Diversification of chromatophore type’ identifies red erythrophores, but does not specify their pigment materials. It describes stacked guanine platelets in a matte-white clownfish iridophore subtype, rather than establishing that arrangement for iridescent iridophores generally. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The presence of leucophores impacts the propensity of fish to shoal with one another in the laboratory.

    “their presence impacts the propensity of fish to shoal with one another in the laboratory (9)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [9] V. M. Lewis, Fate plasticity and reprogramming in genetically distinct populations of Danio leucophores. Proc. Natl. Acad. Sci. U.S.A. 116, 11806– 11811 (2019). 31138706 PMC6575160 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Teleosts have several Agouti-related genes due to the teleost specific genome duplication.

    “Due to the teleost specific genome duplication, teleosts have several Agouti-related genes (29–31)”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [29] Y. Liang, M. Grauvogl, A. Meyer, C. F. Kratochwil, Functional conservation and divergence of color-pattern-related agouti family genes in teleost fishes. J. Exp. Zool. B Mol. Dev. Evol. 336, 443– 450 (2021). 33755299 source
    • cites [30] L. Cal, P. Suarez-Bregua, J. M. Cerda-Reverter, I. Braasch, J. Rotllant, Fish pigmentation and the melanocortin system. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 211, 26– 33 (2017). 28599948 source

      The Introduction identifies four teleost agouti-family genes and describes a model in which the teleost genome duplication produced the asip2a/asip2b pair. It also says other family members arose before that duplication and that the family's evolutionary history is contested. Thus, the paper supports a contribution from the teleost duplication, but not the unqualified claim that it explains the presence of several Agouti-related genes. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [31] I. Braasch, J. H. Postlethwait, The teleost agouti-related protein 2 gene is an ohnolog gone missing from the tetrapod genome. Proc. Natl. Acad. Sci. U.S.A. 108, E47– 48 (2011). 21406593 PMC3069192 source

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

    Premisecitation

    Mammals and birds have a single skin pigment cell, the melanocyte, which can transfer melanin-containing melanosomes to keratinocytes for deposition in hair or feathers.

    “Mammals and birds have a single skin pigment cell, the melanocyte, which can transfer melanin-containing melanosomes to keratinocytes for deposition in hair or feathers (2).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [2] A. Brombin, E. E. Patton, Melanocyte lineage dynamics in development, growth and disease. Development 151, dev201266 (2024). 39092608 PMC11317096 source

      The Introduction (page not available in our copy) says melanocytes produce melanin in melanosomes, that mammalian melanosomes are distributed to skin keratinocytes, and that melanocytes transfer pigment to growing hair. It does not establish that melanocytes are the single skin pigment-cell type in mammals and birds, or describe transfer to keratinocytes for feather pigmentation. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Analyses controlled for variation in standard length, a proxy for stage of development.

    “after controlling for variation in standard length (SL), a proxy for stage of development (41)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] D. M. Parichy, M. R. Elizondo, M. G. Mills, T. N. Gordon, R. E. Engeszer, Normal table of postembryonic zebrafish development: Staging by externally visible anatomy of the living fish. Dev. Dyn. 238, 2975– 3015 (2009). 19891001 PMC3030279 source

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

    Premisecitation

    RGM proteins have high affinity for BMPs and can enhance BMP signaling in ways still incompletely understood but involving formation and internalization of a cell surface complex in which RGMb forms a bridge between BMP ligands and Neogenin (Neo).

    “RGM proteins have high affinity for BMPs and can enhance BMP signaling in ways still incompletely understood but involving formation and internalization of a cell surface complex in which RGMb forms a bridge between BMP ligands and Neogenin (Neo) (45, 46).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [45] C. H. Bell, Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub. Science 341, 77– 80 (2013). 23744777 PMC4730555 source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [46] E. G. Healey, Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat. Struct. Mol. Biol. 22, 458– 465 (2015). 25938661 PMC4456160 source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

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

    Premisecitation

    Foxd3 maintains pluripotency of neural crest cells and later contributes to specifying neuronal, glial, and iridophore fates over melanocyte or melanophore fates.

    “Foxd3 maintains pluripotency of neural crest cells and later contributes to specifying neuronal, glial, and iridophore fates over melanocyte or melanophore fates (51, 52, 60) and is regulated by BMP signals in other contexts (61, 62).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [51] A. J. Thomas, C. A. Erickson, FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. Development 136, 1849– 1858 (2009). 19403660 PMC2680109 source
    • cites [52] K. Curran, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest. Dev. Biol. 344, 107– 118 (2010). 20460180 PMC2909359 source

      The Introduction (page unavailable) reports evidence that Foxd3 promotes iridophore development and blocks melanophore development. The Results section, “Foxd3 is necessary for pnp4a expression” (page unavailable), notes Foxd3 expression in glia and neurons, but does not establish that it specifies those fates. The paper describes neural crest cells as multipotent; it does not show that Foxd3 maintains their pluripotency. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [60] E. Nitzan, E. R. Pfaltzgraff, P. A. Labosky, C. Kalcheim, Neural crest and Schwann cell progenitor-derived melanocytes are two spatially segregated populations similarly regulated by Foxd3. Proc. Natl. Acad. Sci. U.S.A. 110, 12709– 12714 (2013). 23858437 PMC3732929 source

      In “Foxd3 Acts as a Fate Switch Between SCPs and Hypaxial Melanocytes” (page unavailable), the paper says Foxd3 maintains early multipotent neural crest progenitors in an undifferentiated state, supports glial development, and opposes melanocyte differentiation. It does not establish pluripotency or address iridophore fate. Contrary to the neuronal-fate portion, it says Foxd3 must be down-regulated for sensory neurons to differentiate. (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

    Both Foxd3 and Runx3 gene products can function as pioneer factors and both can activate or repress transcription depending on context.

    “Intriguingly both gene products can function as pioneer factors and both can activate or repress transcription depending on context (50, 59).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [50] M. Lukoseviciute, From pioneer to repressor: Bimodal foxd3 activity dynamically remodels neural crest regulatory landscape in vivo. Dev. Cell 47, 608– 628.e606 (2018). 30513303 PMC6286384 source
    • cites [59] J. W. Lee, RUNX3 regulates cell cycle-dependent chromatin dynamics by functioning as a pioneer factor of the restriction-point. Nat. Commun. 10, 1897 (2019). 31015486 PMC6479060 source

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

    Premisecitation

    BMP also promotes postnatal differentiation of WNT-activated hair follicle stem cells as melanocytes, with progenitors accumulating in excess if BMP signaling is blocked.

    “Yet BMP also promotes postnatal differentiation of WNT-activated hair follicle stem cells as melanocytes, with progenitors accumulating in excess if BMP signaling is blocked (37).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] N. R. Infarinato, BMP signaling: At the gate between activated melanocyte stem cells and differentiation. Genes Dev. 34, 1713– 1734 (2020). 33184221 PMC7706702 source

      The Results report that BMP signaling promotes maturation of melanocyte stem-cell progeny and that blocking it increases the number of less-differentiated cells in mouse hair bulbs. However, the paper distinguishes melanocyte stem cells from epithelial hair follicle stem cells; it does not say that the latter differentiate into melanocytes. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Foxd3 is regulated by BMP signals in other contexts.

    “Foxd3 maintains pluripotency of neural crest cells and later contributes to specifying neuronal, glial, and iridophore fates over melanocyte or melanophore fates (51, 52, 60) and is regulated by BMP signals in other contexts (61, 62).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [61] Q. Shen, L. B. Toulabi, H. Shi, E. E. Nicklow, J. Liu, The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm. Dev. Biol. 433, 75– 83 (2018). 29155044 PMC5722696 source
    • cites [62] E. Nitzan, Dynamics of BMP and Hes1/Hairy1 signaling in the dorsal neural tube underlies the transition from neural crest to definitive roof plate. BMC Biol. 14, 23 (2016). 27012662 PMC4806459 source

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

    Premisecitation

    BMP signaling in zebrafish biases the fates of progenitor cells away from melanophores and toward iridophores, at least in part by repressing mitfa and other genes required for melanogenesis.

    “BMP signaling in zebrafish biases the fates of progenitor cells away from melanophores and toward iridophores, at least in part by repressing mitfa and other genes required for melanogenesis (38).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [38] A. K. Gramann, A. M. Venkatesan, M. Guerin, C. J. Ceol, Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling. eLife 8, e50047 (2019). 31868592 PMC6968919 source

      The Results section on lineage tracing reports that BMP signaling promotes iridophore rather than melanocyte fate in mitfa-expressing progenitors; the section on mitfa expression reports that BMP signaling suppresses mitfa. The Results also show higher expression of the melanocyte markers mc1r, tyr, and tyrp1b when BMP signaling is inhibited, but describe this as consistent with increased melanocyte numbers, not as evidence that BMP represses those genes to determine progenitor fate. The indexed passages have no page numbers. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Premature termination alleles of rgmb did not have defects in fin growth but did develop fewer ML than WT siblings.

    “Premature termination alleles of rgmb did not have defects in fin growth but did develop fewer ML than WT siblings”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Anti-pSmad immunoreactivity was indistinguishable between WT and asip1 or asip2b mutants, as well as between WT and fish overexpressing Asip1 and Asip2b.

    “Anti-pSmad immunoreactivity was indistinguishable between WT and asip1 or asip2b mutants, as well as between WT and fish overexpressing Asip1 and Asip2b”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    pnp4a homozygotes had reduced iridescence of iridophores and lower leucosome/guanine contents of ML, indicating overlapping requirements between cell types.

    “homozygotes had reduced iridescence of iridophores and lower leucosome/guanine contents of ML, indicating overlapping requirements between cell types”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transgenic fish expressing heat-shock inducible Asip1 developed fewer ML, but also fewer melanophores.

    “Transgenic fish expressing heat-shock (hsp70l) inducible Asip1 developed fewer ML, but also fewer melanophores”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A Wnt signaling activity reporter was not detectably expressed by melanophores-ML and inhibition of Wnt signaling did not affect ML number independently of fin outgrowth.

    “a Wnt signaling activity reporter was not detectably expressed by these cells and inhibition of Wnt signaling did not affect ML number independently of fin outgrowth”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutants for premature termination alleles of rgmb, neo1a, and lrig2 had more melanophores and were severely deficient for ML.

    “Mutants for premature termination alleles of rgmb, neo1a, and lrig2 had more melanophores and were severely deficient for ML.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    asip1 mutants had mild perturbations to fin melanophore pattern, but ML complements equivalent to wild-type.

    “asip1 mutants had mild perturbations to fin melanophore pattern, but ML complements equivalent to wild-type(WT)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fish transgenic for heat-shock inducible Asip2b developed excess ML, with a corresponding, albeit nonsignificant reduction in melanophores.

    “fish transgenic for heat-shock inducible Asip2b developed excess ML, with a corresponding, albeit nonsignificant reduction in melanophores”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BMP signaling activity assayed by anti-pSmad immunoreactivity was apparent in melanophores, transitional cells, and ML, and its strength was correlated with proximity to the fin edge.

    “BMP signaling activity assayed by anti-pSmad immunoreactivity was apparent in melanophores, transitional cells, and ML, and its strength was correlated with proximity to the fin edge”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    ML of adult fish contained larger, jagged leucosomes bearing crystalline material but also small numbers of irregular melanosomes similar to those of transitional cells.

    “ML of adult fish contained larger, jagged leucosomes bearing crystalline material (Fig. 1 F and H) but also small numbers of irregular melanosomes similar to those of transitional cells”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    White ornaments and the melanoleucophores that comprise them are unperturbed in mutants that lack xanthophores or iridophores, indicating pattern-forming mechanisms other than self-organizing pigment cell interactions.

    “In contrast to stripe pattern, white ornaments and the “melanoleucophores”(ML) that comprise them are unperturbed in mutants that lack xanthophores or iridophores, indicating pattern-forming mechanisms other than self-organizing pigment cell interactions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By scRNA-Seq of whole fin, 592 genes were expressed at higher levels (q < 0.05) at the fin edge, among which asip1 had the largest difference in transcript abundance.

    “By scRNA-Seq of whole fin, 592 genes were expressed at higher levels (q < 0.05) at the fin edge, among which agouti signaling protein 1 (asip1) had the largest difference in transcript abundance”
    Quote in the paperSubstantial claim

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

    Findingown data

    In zebrafish, populations of white pigment cells-leucophores-are situated at the dorsal fin edge and caudal fin tips.

    “An example in zebrafish itself is ornamentation of the dorsal fin edge and caudal fin tips, where populations of white pigment cells—leucophores—are situated (Fig. 1 A and B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Fish mosaically expressing both Asip1 and Asip2b transgenes developed extra ML, yet very few melanophores.

    “Fish mosaically expressing both transgenes developed extra ML, yet very few melanophores.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    BMP inhibition in D. albolineatus led to fewer ML and more melanophores.

    “an evolutionarily conserved function for BMP signaling in ML development was indicated by a corresponding effect of BMP inhibition in D. albolineatus”
    Quote in the paperSubstantial claim

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

    Findingnone given

    Leucophores are highly reflective and prominent when fins are flared during behavioral interactions.

    “These highly reflective cells are prominent when fins are flared during behavioral interactions”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Pathway enrichment analyses confirmed a strong signature of MAPK signaling in ML, consistent with Kita activity, as well as signatures for autophagy, proteolysis, and other pathways suggestive of cellular remodeling.

    “Pathway enrichment analyses confirmed a strong signature of MAPK signaling in ML, consistent with Kita activity, as well as signatures for autophagy, proteolysis, and other pathways suggestive of cellular remodeling”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    neo1a was expressed in melanophores-ML and mutants homozygous for premature termination alleles had fewer ML, and also more melanophores, than WT.

    “Similar to rgmb, neo1a was expressed in melanophores–ML and mutants homozygous for premature termination alleles had fewer ML, and also more melanophores, than WT”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    lrig2 was expressed in melanophores and transitional cells and fish homozygous for premature termination alleles had ML deficits indistinguishable from rgmb or neo1a mutants, along with an excess of melanophores.

    “lrig2 was expressed in melanophores and transitional cells and fish homozygous for premature termination alleles had ML deficits indistinguishable from rgmb or neo1a mutants, along with an excess of melanophores”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The crystals in ML are similar chemically to those of iridophores, yet lack the disc-like structure or spatial organization of reflecting platelets and thereby confer a white rather than iridescent appearance.

    “These crystals are similar chemically to those of iridophores, yet lack the disc-like structure or spatial organization of reflecting platelets and thereby confer a white rather than iridescent appearance.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Several Wnt ligand genes were expressed at higher levels at the fin edge than base and melanophores-ML expressed Wnt receptor genes.

    “Several Wnt ligand genes were expressed at higher levels at the fin edge than base and melanophores–ML expressed Wnt receptor genes”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    runx3 mutants were viable and had normal fins.

    “runx3 mutants were viable, had normal fins, and lacked nearly all ML (means ± SE ML, WT: 144 ± 7; runx3: 5 ± 8 ML; F1,14 = 163.6, P < 0.0001).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Melanin and melanosomes are stable under a variety of conditions and persist when melanogenesis is inhibited.

    “Melanin and melanosomes are stable under a variety of conditions and persist when melanogenesis is inhibited (10, 11).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] J. Borovansky, M. Elleder, Melanosome degradation: Fact or fiction. Pigment Cell Res. 16, 280– 286 (2003). 12753402 source
    • cites [11] J. F. Rawls, S. L. Johnson, Zebrafish kit mutation reveals primary and secondary regulation of melanocyte development during fin stripe regeneration. Development 127, 3715– 3724 (2000). 10934016 source

  • May combine two claims.

    Interpretationown data

    BMP signals promote the development of dermal cells and their expression of Agouti genes.

    “Together these observations suggested that BMP signals promote the development of dermal cells and their expression of Agouti genes.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Fin lengths for a given standard length did not differ among genotypes.

    “Fin lengths for a given SL did not differ among genotypes (SI Appendix, Fig. S8G).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Even fish doubly mutant for asip1 and asip2b retained some ML, indicating essential roles for additional factors.

    “Even fish doubly mutant for asip1 and asip2b retained some ML indicating essential roles for additional factors as well.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Cells were sequenced to similar depth.

    “even when sequenced to similar depth”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Local signals in tissues can drive the development of ornamental features and provide insights into how new cell types evolve.

    “This work shows how local signals in tissues can drive the development of ornamental features and provides insights into how new cell types evolve.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. The quote only partly states this claim.

    Study design and conductnone given

    BMP inhibitor was used at a dose permissive for fin outgrowth.

    “at a dose permissive for fin outgrowth”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In zebrafish, interactions among chromatophores generate and maintain a pattern of dark and light horizontal stripes.

    “In the teleost most studied for its pigmentation, zebrafish Danio rerio, interactions among chromatophores generate and maintain a pattern of dark and light horizontal stripes (4)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] L. B. Patterson, D. M. Parichy, Zebrafish pigment pattern formation: Insights into the development and evolution of adult form. Annu. Rev. Genet. 53, 505– 530 (2019). 31509458 source

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

    Study design and conductown data

    ML counts were square-root transformed to control for increased variance in residuals with larger counts.

    “numbers of ML square-root transformed to control for increased variance in residuals with larger counts”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Zebrafish stripe pattern formation has features resembling a Turing process.

    “with features of pattern formation resembling a Turing process (5)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] S. Kondo, T. Miura, Reaction-diffusion model as a framework for understanding biological pattern formation. Science 329, 1616– 1620 (2010). 20929839 source

  • May combine two claims.

    Interpretationnone given

    Melanin and melanosomes are degraded while new melanogenesis is curtailed by downregulation of Oca2 and other factors.

    “iv) melanin and melanosomes are degraded by while new melanogenesis is curtailed by downregulation of Oca2 and other factors;”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    WNT and BMP signals promote fin outgrowth and BMP contributes to a landscape of positional information available to pigment cells.

    “i) WNT and BMP signals promote fin outgrowth and the latter contributes to a landscape of positional information available to pigment cells;”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    BMP signaling promotes dermal development and expression of Agouti genes asip1 and asip2b by subsets of dermal cells.

    “ii) BMP signaling promotes dermal development and expression of Agouti genes asip1 and asip2b by subsets of these dermal cells;”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Runx3 has roles in cancers including melanoma but has not previously been ascribed roles in normal pigment cells.

    “Runx3 has roles in cancers including melanoma but has not previously been ascribed roles in normal pigment cells (63–66).”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [63] R. Mevel, J. E. Draper, A. L. M. Lie, V. Kouskoff, G. Lacaud, RUNX transcription factors: Orchestrators of development. Development 146, dev148296 (2019). 31488508 source
    • cites [64] Y. Ito, S. C. Bae, L. S. Chuang, The RUNX family: Developmental regulators in cancer. Nat. Rev. Cancer 15, 81– 95 (2015). 25592647 source
    • cites [65] X. Zhang, L. Wang, X. Zeng, T. Fujita, W. Liu, Runx3 inhibits melanoma cell migration through regulation of cell shape change. Cell Biol. Int. 41, 1048– 1055 (2017). 28699302 source
    • cites [66] Y. Ito, K. Miyazono, RUNX transcription factors as key targets of TGF-beta superfamily signaling. Curr. Opin. Genet. Dev. 13, 43– 47 (2003). 12573434 source

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

    Interpretationreasoning

    Consequences of BMP signaling for cell fate transitions depend on cellular context including collaborating pathways such as WNT versus Agouti.

    “Comparisons across systems thereby suggest that consequences of BMP signaling for cell fate transitions depend on cellular context including collaborating pathways (e.g., WNT vs. Agouti).”
    Quote in the paperNot a substantial claim
52 of 52

  • The paper relies on this without support.

    The effects of transgenic overexpression of Agouti genes reflect their endogenous functions.

    “To assess functional consequences of these interactions for ML development, we overexpressed Agouti in WT and rgmb mutants.”

  • The paper relies on this without support.

    CRISPR/Cas9-induced somatic mutations in foxd3 cause loss of foxd3 function, and mutagenesis efficacy assayed by Sanger sequencing of whole fins reflects functional loss.

    “Increasing efficacies of foxd3 mutagenesis, as assayed by Sanger sequencing of whole fins, were associated with fewer ML”

  • The paper relies on this without support.

    Premature termination alleles of runx3 are loss-of-function alleles.

    “premature termination alleles of one candidate, transcription factor gene runx3 (SI Appendix, Fig. S12 D and F and Dataset S16), led to severe ML deficiencies when homozygous”

  • The paper relies on this without support.

    pSmad immunoreactivity reflects BMP signaling activity.

    ““BMP” refers to pathway signaling activity overall, as inferred by pSmad immunostaining (SI Appendix, Fig. S3B)”
4 of 4

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?

Self-check

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

  • Melanophore transcription-factor percentage is miscalculated

    Minor

    120 of 2,612 loci is approximately 4.6%, not the reported 4.4%.

    Reported 4.4 · recomputed 4.594181

1 of 1

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Authors
Huang, D., Kapadia, E. H., Liang, Y., Shriver, L. P., Dai, S., Patti, G. J., Humbel, B. M., Laudet, V., & Parichy, D. M.
Affiliations
  • University of Virginia (US)
  • Washington University in St. Louis (US)
  • Okinawa Institute of Science and Technology Graduate University (JP)
Corresponding author
Parichy, David M.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2025-02-18
Volume
122
Issue
8
Pages
e2424180122-e2424180122
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2424180122
PMC
PMC11874323
OpenAlex
W4407695406
Semantic Scholar
290f9b2f291a04a675a5d6d58574f39b5a29b0cd
Cited by
15
References
109
Citations by year
2026: 7 · 2025: 8
FWCI
7.43
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by-nc-nd)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion)
  • Okinawa Institute of Science and Technology Graduate University (Okinawa Institute of Science and Technology Graduate University) (submittedVersion, other-oa)
Funders
HHS | NIH | National Institute of General Medical Sciences (R35 GM122471)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › melanin and skin pigmentation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Zebrafish Biomedical Research Applications
Keywords
BMP signaling, FOXD3, RUNX3, pigment cell differentiation, positional information
MeSH terms
Animals, Cell Differentiation, Melanophores, Pigmentation, Zebrafish, Signal Transduction, Gene Expression Regulation, Developmental, Bone Morphogenetic Proteins, Zebrafish Proteins, Agouti Signaling Protein

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.

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