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Cited by 70melanin and skin pigmentationZebrafish Biomedical Research ApplicationsDevelopmental Biology and Gene Regulation

In situ differentiation of iridophore crystallotypes underlies zebrafish stripe patterning

Open access
Gur, Dvir, Bain, Emily J., Johnson, Kory R., Aman, Andy J., Pasolli, H. Amalia, Flynn, Jessica D., Allen, Michael C., Deheyn, Dimitri D. +3 more·2020·Nature Communications
DOI 10.1038/s41467-020-20088-1PMC PMC7738553

Published version of a preprint

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Abstract

Skin color patterns are ubiquitous in nature, impact social behavior, predator avoidance, and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on light-reflecting cells called iridophores. It was suggested that the zebrafish’s color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. However, here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment. RNA-sequencing analysis further reveals that stripe and interstripe iridophores have different transcriptomic states, while cryogenic-scanning-electron-microscopy and micro-X-ray diffraction identify different crystal-arrays architectures, indicating that stripe and interstripe iridophores are different cell types. Based on these results, we present an alternative model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized, physiologically responsive, organelles arise in stripe and interstripe by in-situ differentiation.

AI snapshot

Written from the paper’s full text.

TL;DR

Zebrafish stripe and interstripe iridophores are not interconvertible migratory states but distinct cell types that differentiate in place from precursors according to local microenvironment. This “differentiation in situ” model is supported by time-lapse imaging, photoconversion fate mapping, crystallographic/optical differences, and distinct transcriptomic states.

Key findings

  • In >300 h of time-lapse recordings (14,475 total cells, including 1,637 at interstripe edges), no dense interstripe iridophores delaminated or assumed loose morphology; 981 divisions, including 160 at interstripe edges, produced daughters that remained in the interstripe. Photoconversion of primary interstripe iridophores at 7.5 SSL followed for 7 days to 8.6 SSL showed white nuclei in interstripe but only green nuclei in newly populated stripe, indicating no migration between zones (n = 8 fish).
  • Stripe and interstripe iridophores have distinct crystal architectures: stripe loose iridophores had ordered arrays with 20–30 layers, crystal thickness 27 ± 7 nm (n = 82), and cytoplasm spacing 131 ± 24 nm (n = 91); interstripe dense iridophores had disordered arrays, 30–40 crystals per cell, thickness 25 ± 8 nm (n = 130), and spacing 186 ± 81 nm (n = 145). Isolated stripe crystals were smaller (3.9 ± 0.4 vs. 5.3 ± 0.9 µm) and had smaller aspect ratios (1.9 ± 0.2 vs. 2.5 ± 0.3; n = 60 stripe, n = 57 interstripe; p < 0.0001, two-tailed Mann–Whitney), though both were β-guanine.
  • Optical properties are intrinsic: simulations predicted a blue peak at 450 nm approaching unity reflection for ordered stripe iridophores and broad reflection for disordered interstripe iridophores; color differences persisted in fish lacking melanin and carotenoids (tyrosinase and scarb1 mutants). Norepinephrine caused a ~120 nm shift in peak reflectance in stripe iridophores but no gross change in interstripe iridophores (n = 4 fish for optical response).
  • scRNA-seq recovered five clusters; cluster c5 was 85% stripe-derived and cluster c3 was 84% interstripe-derived, with 192 genes upregulated in c5 and 158 in c3. mitfaw2 mutants mostly lacked ordered-crystal diffractions, albino mutants retained wild-type-like alternating patterns, and ordered crystallotype diffractions appeared by ~6.9 SSL after disordered crystallotype at 6.0–6.5 SSL.

Methods

  • Zebrafish lines included Tg(pnp4a:palm-mcherry), Tg(pnp4a-nucEos), temperature-sensitive mitfavc7, mitfaw2, albino, and scarb1 mutants; experiments used time-lapse imaging, photoconversion, repeated daily imaging, and temperature shifts.
  • Structural and optical analyses used malachite green staining, cryo-SEM, TEM/electron diffraction, Raman microspectroscopy, synchrotron micro-X-ray diffraction, hyperspectral imaging, and Monte Carlo transfer-matrix reflectivity simulations.
  • scRNA-seq was performed on microdissected stripe and interstripe skin, enzymatically dissociated and FACS-purified for pnp4a-driven mCherry/Eos expression; 768 cells and 32,618 features were processed with Seurat/UMAP clustering and differential expression analysis.

Limitations

  • Stated: the authors “cannot exclude the possibility that some preexisting iridophores were incorporated into remodeled pattern elements” in temperature-shift experiments; they also state that whether iridoblasts differentiate within the skin plane or arrive from peripheral nervous system niches “remains to be determined.”
  • Stated: sample size was based on previous experience, with no statistical method used to predetermine sample size; Raman spectroscopy failed, within measurement accuracy, to reveal other crystal components.
  • Evident from the text: photoconversion tracked differentiated pnp4a-expressing iridophores, not earlier progenitors, so the study cannot directly resolve the behavior of uncommitted precursors; scRNA-seq clusters c2 and c4 had lower pigment-cell marker expression and were suggested to potentially represent not fully differentiated cells.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-020-20088-1

Cites

60 of 60 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

25 of 49 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Mutants for mitfa, which lack melanophores, have the dense morphology iridophores over a broader area than wild-type fish.

    “Prior work has hinted at this possibility as mutants for melanophore-inducing transcription factor (mitfa), which lack melanophores, have the dense morphology iridophores (characteristic of interstripe zones) over a broader area than wild-type fish14,27.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [14] Frohnhofer, H. G., Krauss, J., Maischein, H. M. & Nusslein-Volhard, C. Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997–3007 (2013). source
    • cites [27] Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. & Raible, D. W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999). source
  • Findingcitation

    Proliferation of loose iridophores within stripes was greater than that of dense iridophores within interstripes.

    “We found that proliferation of loose iridophores within stripes was greater than dense iridophores within interstripes, as noted previously18 (Supplementary Fig. 2a).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Spiewak, J. E. et al. Evolution of endothelin signaling and diversification of adult pigment pattern in Danio fishes. PLoS Genet. 14, e1007538 (2018). source
  • Findingown data

    The few of these cells that transitioned from a nascent dense morphology to a loose morphology occurred already within prospective stripe regions.

    “Contrary to the expectations of the morphogenetic respecification model12,20, the few of these cells that transitioned from a nascent dense morphology to a loose morphology occurred already within prospective stripe regions.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [12] Singh, A. P., Schach, U. & Nusslein-Volhard, C. Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish. Nat. Cell Biol. 16, 607–614 (2014). source
    • cites [20] Volkening, A. & Sandstede, B. Iridophores as a source of robustness in zebrafish stripes and variability in Danio patterns. Nat. Commun. 9, 3231 (2018). source

      The Results section (‘Deriving proposed iridophore form change mechanisms’; page unavailable) describes dense iridophores becoming loose and spreading outward during stripe development, supporting the model expectation invoked by the citation. It does not report that only a few cells made this transition or that they were already within prospective stripe regions when they did so. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingcitation

    Iridophores within interstripes tended to divide along an anterior-posterior plane.

    “Moreover, iridophores within interstripes tended to divide along an anterior–posterior plane, consistent with the known faster growth along this axis than dorsoventrally24 (Supplementary Figs. 1a and 2b).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [24] Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N. & Engeszer, R. E. Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish. Dev. Dyn. 238, 2975–3015 (2009). source

      Citation 24 is attached to the statement that anterior–posterior growth is faster than dorsoventral growth, rather than to the authors’ observation of iridophore division. The paper’s “Measurements of Larval Size” section compares length and height measures but does not state which axis grows faster. Its pigment-pattern section describes where iridophores appear, not their division orientation. No page numbers are available in our copy. (LLM, conf=0.70)

      Experimental — accuracy not yet measured
  • Premisecitation

    An evolutionary truncation in iridophore development leads to an attenuated stripe pattern in the zebrafish relative D. nigrofasciatus.

    “Likewise, an evolutionary truncation in iridophore development leads to an attenuated stripe pattern in the zebrafish relative D. nigrofasciatus18.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [18] Spiewak, J. E. et al. Evolution of endothelin signaling and diversification of adult pigment pattern in Danio fishes. PLoS Genet. 14, e1007538 (2018). source

      The Results report curtailed iridophore expansion and fewer reiterated stripe elements in D. nigrofasciatus than in D. rerio. The Discussion proposes that early cessation of iridophore expansion led to the attenuated pattern, but frames that causal explanation as a suggestion; the citing sentence states it as fact. No page number is available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • May combine two claims. 39
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Premisecitation

    Genetically or experimentally induced deficiencies in iridophores cause pattern defects, including alterations in primary stripe positioning and boundary formation, and also lead to reductions or losses of secondary interstripes and stripes.

    “The iridophore’s importance in skin patterning has been demonstrated in experiments showing that genetically or experimentally induced deficiencies in iridophores cause pattern defects, including alterations in primary stripe positioning and boundary formation, and also lead to reductions or losses of secondary interstripes and stripes13–17.”
    Quote in the paperSubstantial claim5 of 5 sources checked
    • cites [13] Krauss, J. et al. Endothelin signalling in iridophore development and stripe pattern formation of zebrafish. Biol. Open 3, 503–509 (2014). 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
    • cites [14] Frohnhofer, H. G., Krauss, J., Maischein, H. M. & Nusslein-Volhard, C. Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997–3007 (2013). 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
    • cites [15] Patterson, L. B. & Parichy, D. M. Interactions with iridophores and the tissue environment required for patterning melanophores and xanthophores during zebrafish adult pigment stripe formation. PLoS Genet. 9, e1003561 (2013). 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
    • cites [16] Parichy, D. M. et al. Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio. Dev. Biol. 227, 294–306 (2000). 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 [17] Patterson, L. B., Bain, E. J. & Parichy, D. M. Pigment cell interactions and differential xanthophore recruitment underlying zebrafish stripe reiteration and Danio pattern evolution. Nat. Commun. 5, 5299 (2014). source

  • May combine two claims.

    Premisecitation

    High-angular distribution diffractions having a full-ring signal are indicative of crystal orientations that vary (disordered), whereas low-angular distribution diffractions having a punctuated-ring signal are indicative of crystals that are consistently oriented (ordered).

    “high-angular distribution diffractions having a full-ring signal are indicative of crystal orientations that vary (i.e., disordered), whereas low-angular distribution diffractions having a punctuated-ring signal are indicative of crystals that are consistently oriented (i.e., ordered)10”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [10] Gur, D. et al. The dual functional reflecting Iris of the zebrafish. Adv. Sci. 5, 1800338 (2018). 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

  • May combine two claims.

    Findingown data

    Crystal plates in iridophores of the stripe zone were well oriented (ordered), whereas those in the interstripe zone were nonaligned (disordered).

    “crystal plates in iridophores of the stripe zone were well oriented (i.e., ordered; Fig. 4a, panels 1 and 3, and Supplementary Fig. 10), whereas those in the interstripe zone were nonaligned (i.e., disordered; Fig. 4a, panels 2 and 4, and Supplementary Fig. 10)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [10] Gur, D. et al. The dual functional reflecting Iris of the zebrafish. Adv. Sci. 5, 1800338 (2018). source

      The passage on cryo-SEM imaging (indexed under Abstract; page unavailable) describes ordered crystals in an outer iris iridophore layer and randomly arranged crystals with no preferred orientation in an adjacent iris layer. It does not place those layers in stripe and interstripe zones, so the claimed zone-specific comparison is unsupported. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [35] Gur, D. et al. Guanine-based photonic crystals in fish scales form from an amorphous precursor. Angew. Chem. Int. Ed. 52, 388–391 (2013). source

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

    Premisecitation

    tjp1a is highly expressed only in dense iridophores (found in the interstripe), and knocking out tjp1a results in disruption of the stripe pattern due to dense (interstripe) iridophores invading the stripe regions.

    “it was recently shown using immuno-labeling that Tight Junction Protein 1a (tjp1a) is highly expressed only in dense iridophores19 (found in the interstripe), and that knocking out tjp1a results in the disruption of the stripe pattern due to dense (interstripe) iridophores invading the stripe regions19”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Fadeev, A., Krauss, J., Frohnhofer, H. G., Irion, U. & Nusslein-Volhard C. Tight Junction Protein 1a regulates pigment cell organisation during zebrafish colour patterning. Elife 4, e06545 (2015). source

      The Results section reports antibody staining in dense iridophores of the light stripe, but not in loose iridophores or other pigment cells. The Discussion reports that Tjp1a-deficient mutants develop dark-stripe interruptions as dense iridophores invade those regions. The unqualified claim that Tjp1a is expressed only in dense iridophores goes beyond the paper: the Results also detect it in epithelial cells and blood vessels. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    The authors' data, together with those of others, suggest that some of these signals depend on melanophores, promoting differentiation of iridoblasts toward a state having ordered reflecting crystal platelets that are physiologically responsive (blue ⟷ yellow) in stripes, and away from an alternative state of disordered-crystal platelets lacking physiological responsiveness (silvery-yellow) in the

    “Our data, together with those of others, suggest that some of these signals depend on melanophores14,15,25,47, promoting differentiation of iridoblasts toward a state having ordered reflecting crystal platelets that are physiologically responsive (blue ⟷ yellow) in stripes, and away from an alternative state of disordered-crystal platelets lacking physiological responsiveness (silvery-yellow) in”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [14] Frohnhofer, H. G., Krauss, J., Maischein, H. M. & Nusslein-Volhard, C. Iridophores and their interactions with other chromatophores are required for stripe formation in zebrafish. Development 140, 2997–3007 (2013). source

      The Results (‘Mutants lacking xanthophores and melanophores display abnormal iridophore distribution’; page unavailable) show altered iridophore distribution when melanophores are absent, supporting a role for melanophores in their patterning. The paper also describes blue iridophores in stripes, but it does not establish that melanophore-dependent signals direct iridoblast differentiation toward ordered, physiologically responsive crystal platelets rather than disordered, unresponsive ones. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [15] Patterson, L. B. & Parichy, D. M. Interactions with iridophores and the tissue environment required for patterning melanophores and xanthophores during zebrafish adult pigment stripe formation. PLoS Genet. 9, e1003561 (2013). source

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

      Experimental — accuracy not yet measured
    • cites [25] McMenamin, S. K. et al. Thyroid hormone-dependent adult pigment cell lineage and pattern in zebrafish. Science 345, 1358–1361 (2014). 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 [47] Mahalwar, P., Singh, A. P., Fadeev, A., Nusslein-Volhard, C. & Irion, U. Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish. Biol. Open 5, 1680–1690 (2016). source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 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.

    Interpretationcitation

    The resulting iridophore subtypes likely allow the zebrafish to alter its skin patterning to make it more or less distinctive, a trait crucial for the fish to be able to join shoals or obscure itself.

    “The resulting iridophore subtypes likely allow the zebrafish to alter its skin patterning to make it more or less distinctive, a trait crucial for the fish to be able to join shoals or obscure itself48,49”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [48] Rosenthal, G. G. & Ryan, M. J. Assortative preferences for stripes in danios. Anim. Behav. 70, 1063–1066 (2005). source
    • cites [49] Engeszer, R. E., Wang, G., Ryan, M. J. & Parichy, D. M. Sex-specific perceptual spaces for a vertebrate basal social aggregative behavior. Proc. Natl Acad. Sci. USA 105, 929–933 (2008). source

      The paper reports that adult pigment-pattern variation affects shoaling preferences, particularly males’ responses to stripe patterning (p. 1; Results, p. 2). It describes iridophores as components of zebrafish stripes and interstripes (Results, p. 1), but does not show that iridophore subtypes let fish alter their patterns or obscure themselves. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Dense iridophores moved little, whereas loose iridophores could migrate up to several cell diameters and these movements tended to be biased away from the first interstripe.

    “Dense iridophores also moved little, whereas loose iridophores could migrate up to several cell diameters and these movements tended to be biased away from the first interstripe (Supplementary Fig. 2c, d and Supplementary Movie S5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Malachite green labeling revealed remarkably stacked arrays of crystals in loose iridophores from the stripe, but markedly disordered arrays of crystals in dense iridophores from the interstripe.

    “Notably, malachite green labeling of guanine crystals within iridophores in these two zones revealed remarkably stacked arrays of crystals in loose iridophores from the stripe, but markedly disordered arrays of crystals in dense iridophores from the interstripe (Fig. 3a, b, upper panels).”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Cryo-SEM preserves near-physiological crystal organization and cytoplasmic spacing.

  • May combine two claims.

    Findingown data

    Reciprocal temperature shifts to ablate melanophores led to a similar loss of preexisting loose iridophores and the differentiation of new dense iridophores.

    “Reciprocal temperature shifts to ablate melanophores led to a similar loss of preexisting loose iridophores and the differentiation of new dense iridophores (Supplementary Fig. 6b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Crystal arrays of loose iridophores from stripes were remarkably ordered, with 20-30 layers of parallel crystals having an average thickness of 27 ± 7 nm (n = 82), neatly separated by thin layers of cytoplasm of average thickness 131 ± 24 nm (n = 91).

    “Crystal arrays of loose iridophores from stripes were remarkably ordered, with 20–30 layers of parallel crystals having an average thickness of 27 ± 7 nm (n = 82), neatly separated by thin layers of cytoplasm of average thickness 131 ± 24 nm (n = 91; Fig. 3a, lower panel).”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Cryo-SEM preserves near-physiological crystal organization and cytoplasmic spacing.

  • May combine two claims.

    Findingown data

    Extended analyses revealed extensive differentiation of iridophores without indications of EMT.

    “These analyses also revealed extensive differentiation of iridophores without indications of EMT (Supplementary Figs. 4 and 5a, b, and Supplementary Movie S6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Incident illumination images showed blue, loosely distributed iridophores on top of black melanophores in the stripe zone, whereas the interstripe zone showed dense silvery iridophores covered by yellow xanthophores.

    “Incident illumination images of the stripe zone showed blue, loosely distributed iridophores on top of black melanophores, whereas images of the interstripe zone showed dense silvery iridophores covered by yellow xanthophores (Fig. 3a, b, incident illumination).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    After photoconversion of the interstripe, after 7 days only iridophores in the interstripe zone had white nuclei, whereas newly formed iridophores with green nuclei were mostly in the stripe zone.

    “After 7 days, only iridophores in the interstripe zone had white nuclei, whereas newly formed iridophores, having green nuclei (indicative of their acquiring pnp4a expression), could be seen mostly in the stripe zone (Fig. 2b, after 7 day).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Iridophores differentiate and proliferate in-place.

    “here we find that iridophores do not migrate between stripes and interstripes but instead differentiate and proliferate in-place, based on their micro-environment.”
    • Also stated in: § Role of melanophores in iridophore pattern remodeling · plain
      “new iridophores differentiated into a loose arrangement in regions where melanophore differentiation had occurred”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Crystal arrays of dense iridophores from interstripes were disordered, varying in both orientations and spacings between crystals, with 30-40 crystals per cell, and a similar average crystal thickness of 25 ± 8 nm (n = 130) and an average cytoplasm spacing of 186 ± 81 (n = 145).

    “By contrast, crystal arrays of dense iridophores from interstripes were disordered, varying in both orientations and spacings between crystals (Fig. 3b, lower panel), with 30–40 crystals per cell, and a similar average crystal thickness of 25 ± 8 nm (n = 130) and an average cytoplasm spacing of 186 ± 81 (n = 145).”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Cryo-SEM preserves near-physiological crystal organization and cytoplasmic spacing.

  • May combine two claims.

    Findingown data

    Upon NE treatment, stripe-derived iridophores exhibited a ~120 nm shift in peak reflectance, whereas interstripe-derived iridophores did not exhibit gross changes in reflected light spectra.

    “Upon NE treatment, stripe-derived iridophores exhibited a ~120 nm shift in peak reflectance, whereas interstripe-derived iridophores did not exhibit gross changes in reflected light spectra, measured by hyperspectral imaging (Fig. 5e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Scanning the entire mitfaw2 fish showed some diffraction patterns corresponding to ordered iridophores, but these were located toward the posterior and were a minor component of the diffractions.

    “Scanning the entire fish showed some diffraction patterns corresponding to ordered iridophores, but these were located toward the posterior and were a minor component of the diffractions (Supplementary Fig. 12)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    tjp1a is highly expressed in cluster c3 (mostly interstripe iridophores) and expressed at lower levels in cluster c5 (mostly stripe iridophores).

    “we found that tjp1a is highly expressed in cluster c3 (mostly interstripe iridophores) and expressed at lower levels in cluster c5 (mostly stripe iridophores)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    rdh5 and rdh10a were upregulated in cluster c3 and cluster c5, respectively.

    “Other interesting genes that were differentially expressed at high levels were retinol dehydrogenase 5 (rdh5) and retinol dehydrogenase 10a (rdh10a), upregulated in cluster c3 and cluster c5, respectively”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Reflection from ordered-crystal iridophores was highly dependent on the angle of incident light, whereas reflection from disordered-crystal iridophores was not.

    “while the reflection from the ordered-crystal iridophores was highly dependent on the angle of incident light, the reflection from disordered-crystal iridophores was not (Supplementary Fig. 9)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Gene-set enrichment analysis highlighted many differentially regulated pathways, including responses to purine-containing compounds and cAMP pathways, which were both upregulated in cluster c5.

    “The gene enrichment analysis highlighted many differentially regulated pathways, including responses to purine-containing compounds, and cAMP pathways (Supplementary Table 1), which were both upregulated in cluster c5 (mostly stripe iridophores)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Cluster c3 was mostly interstripe specific, with 84% of cells originating from interstripes.

    “whereas cluster c3 was mostly interstripe specific, as 84% of cells in cluster c3 originated from interstripes (Fig. 5c)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Such behaviors occurred within prospective stripes, rather than at boundaries between interstripes and stripes, and involved cells that had not yet fully differentiated.

    “Such behaviors occurred within prospective stripes, rather than at boundaries between interstripes and stripes, as previously postulated5,12, and involved cells that had not yet fully differentiated”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Irion, U., Singh, A. P. & Nusslein-Volhard, C. The developmental genetics of vertebrate color pattern formation: lessons from zebrafish. Curr. Top. Dev. Biol. 117, 141–169 (2016). source
    • cites [12] Singh, A. P., Schach, U. & Nusslein-Volhard, C. Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish. Nat. Cell Biol. 16, 607–614 (2014). source

  • May combine two claims.

    Findingown data

    Simulations for ordered-crystal iridophores from stripes predicted a peak in the blue region at 450 nm approaching unity reflection, whereas simulations for disordered-crystal iridophores from interstripes predicted a broad wavelength reflection.

    “simulations for ordered-crystal iridophores from stripes predicted a peak in the blue region at 450 nm approaching unity reflection, whereas simulations for disordered-crystal iridophores from interstripes predicted a broad wavelength reflection”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premiseown data

    In light interstripes, iridophores have a cuboidal shape and form an epithelial-like mat, presenting a dense morphological arrangement.

    “In the light interstripes, iridophores have a cuboidal shape and form an epithelial-like mat, presenting a “dense” morphological arrangement (Fig. 1b).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    This process would depend on factors in the iridophore environment that impact the specification and subcellular organization of specialized organelles within iridophore precursors.

    “This process would depend on factors in the iridophore environment that impact the specification and subcellular organization of specialized organelles within iridophore precursors.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Raman spectroscopy failed to reveal other components in crystals, suggesting differences in crystal morphology are not related to their chemistry.

    “failed, within the accuracy afforded by these measurements, to reveal other components, suggesting that differences in crystal morphology are not related to their chemistry (Supplementary Fig. 8)”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In dark stripes, iridophores are sparse in number and stellate in shape, sometimes referred to as having a loose morphology.

    “In the dark stripes, by contrast, iridophores are sparse in number and stellate in shape, and are sometimes referred to as having a “loose” morphology12 (Fig. 1b).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Singh, A. P., Schach, U. & Nusslein-Volhard, C. Proliferation, dispersal and patterned aggregation of iridophores in the skin prefigure striped colouration of zebrafish. Nat. Cell Biol. 16, 607–614 (2014). source

  • May combine two claims.

    Interpretationown data

    In this model, iridophore precursors in developing stripes and interstripes differentiate based on their microenvironment into distinct iridophore crystallotypes, with different subcellular organization and physiological responsiveness.

    “In this model, iridophore precursors in developing stripes and interstripes differentiate based on their microenvironment into distinct iridophore crystallotypes, with different subcellular organization and physiological responsiveness”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The beam center in the detector and the sample-detector distance were calibrated using a powder X-ray diffraction pattern of synthetic guanine powder standard.

    “The beam center in the detector and the sample–detector distance were calibrated, using a powder X-ray diffraction pattern of synthetic guanine powder standard (Sigma-Aldrich).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    In this model, latent progenitors associated with the peripheral nervous system that have transited to the skin during the larva-to-adult transformation expand clonally as early iridoblasts-not specified to subtype-and subsequently differentiate according to cues in the microenvironment they encounter.

    “In this model, latent progenitors associated with the peripheral nervous system that have transited to the skin during the larva-to-adult transformation3 expand clonally as early iridoblasts—not specified to subtype—and subsequently differentiate according to cues in the microenvironment they encounter”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Patterson, L. B. & Parichy, D. M. Zebrafish pigment pattern formation: insights into the development and evolution of adult form. Annu. Rev. Genet. 53, 505–530 (2019). source

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

    Study design and conductnone given

    The data were normalized with respect to the primary beam monitor and corrected for background caused by pinhole and air scattering.

    “The data were normalized with respect to the primary beam monitor (ionization chamber) and corrected for background caused by pinhole and air scattering.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    In this case, clonal spread over the flank from interstripe-to-stripe, and stripe-to-interstripe, is apparent as progeny of individual progenitors migrate to the skin or expand their numbers at the hypodermis.

    “In this case, clonal spread over the flank from interstripe-to-stripe, and stripe-to-interstripe, is indeed apparent as progeny of individual progenitors migrate to the skin or expand their numbers at the hypodermis”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Reflectance spectra were normalized using a standard silver mirror.

    “Reflectance spectra were normalized using a standard silver mirror (Thorlabs Inc.).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All datasets were tested for Gaussian distribution using the Kolmogorov-Smirnov test.

    “All datasets were tested for Gaussian distribution using the Kolmogorov–Smirnov test.”
    Quote in the paperNot a substantial claim

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

    Interpretationcitation

    rdh5 and rdh10a code for proteins that catalyze key oxidation-reduction reactions in the visual cycle, and could be related to the presence of visual pigments in these cells and their reported ability to sense light.

    “These genes code for proteins that catalyze key oxidation–reduction reactions in the visual cycle, and could be related to the presence of visual pigments in these cells and their reported ability to sense light38”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Lythgoe, J. N., Shand, J. & Foster, R. G. Visual pigment in fish iridocytes. Nature 308, 83–84 (1984). source

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

    Study design and conductnone given

    Unless otherwise noted, each experiment was repeated three or more times.

    “Unless otherwise noted, each experiment was repeated three or more times.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The population of zebrafish skin iridophores was designated based on their FSC and SSC characteristics and back-gating on fluorescence.

    “The population of zebrafish skin iridophores was designated based on their FSC and SSC characteristics and back-gating on fluorescence.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    The authors' analyses employed pnp4a transgenes, expressed as iridophores begin to differentiate, and their photoconversion approach, tracking individual pnp4a expressing cells, allowed them to map the fate of differentiated iridophores during development.

    “By contrast our analyses employed pnp4a transgenes, expressed as iridophores begin to differentiate and our photoconversion approach, tracking individual pnp4a expressing cells, allowed us to map the fate of differentiated iridophores during development”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The union set of cells identified as low quality were removed and counts for surviving cells, minus features representing mitochondria genes or spike-in controls, were used to create a new cell data object.

    “The union set of cells identified as low quality were then removed and counts for surviving cells, minus features representing mitochondria genes or spike-in controls, used to create a new cell data object via the “CreateSeuratObject” function supported in the “Seurat” library (v3.1.4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    These results supported the idea that precursor cells differentiate into ordered-crystallotype iridophores only after the differentiation of disordered-crystallotype iridophores and in the presence of melanophores.

    “These results supported the idea that precursor cells differentiate into ordered-crystallotype iridophores only after the differentiation of disordered-crystallotype iridophores and in the presence of melanophores”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Spectral calibration was performed using a MIDL Hg+/Ar+ wavelength calibration lamp with accuracy better than 2 nm.

    “Spectral calibration was performed using a MIDL® Hg+/Ar+ wavelength calibration lamp (Lightform Inc.) with accuracy better than 2 nm.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Development and remodeling of interstripes and stripes involved de novo differentiation, subsequent proliferation, and in some cases migration of iridophores with morphologies appropriate to their location within the pattern.

    “Instead, development and remodeling of interstripes and stripes involved de novo differentiation, subsequent proliferation, and in some cases migration of iridophores with morphologies appropriate to their location within the pattern”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    If individual cells appear to switch between dense and loose morphologies, these may represent iridophore precursors transiently associated with one pattern element or another, but not yet committed to crystallotype.

    “If individual cells appear to switch between dense and loose morphologies, these may represent iridophore precursors transiently associated with one pattern element or another, but not yet committed to crystallotype”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Calculation of quality control metrics and screening for low quality cells was accomplished using a combination of functions supported in the scater library.

    “Calculation of quality control metrics and screening for low quality cells was accomplished using a combination of functions supported in the “scater” library (v1.14.6).”
    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

    Daily calibration of the imaging spectrometer was done using neat cyclohexane, and bandpass and accuracy were found to be <12 and ±1 cm-1, respectively.

    “Daily calibration of imaging spectrometer was done using neat cyclohexane (20 μl in a sealed capillary tube). Bandpass and accuracy were found to be <12 and ± 1 cm–1, respectively.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Color pattern can be influenced physiologically, as some types of pigment cells disperse or contract pigment granules in response to endocrine and neuroendocrine factors, including norephinephrine (NE).

    “Color pattern can be influenced physiologically41,42, as some types of pigment cells disperse or contract pigment granules in response to endocrine and neuroendocrine factors, including norephinephrine (NE)43–45”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [41] Gur, D. et al. Light-induced color change in the Sapphirinid copepods: tunable photonic crystals. Adv. Funct. Mat. 26, 1393–1399 (2016). source
    • cites [42] Gur, D. et al. The mechanism of color change in the Neon Tetra fish: a light-induced tunable photonic crystal array. Angew. Chem. Int. Ed. 54, 12426–12430 (2015). source
    • cites [43] Oshima, N. & Kasai, A. Iridophores involved in generation of skin color in the zebrafish Brachydanio rerio. Forma 17, 91–101 (2002).
    • cites [44] Fujii, R. The regulation of motile activity in fish chromatophores. Pigment Cell Res. 13, 300–319 (2000). source
    • cites [45] Fujii, R. Cytophysiology of fish chromatophores. Int. Rev. Cytol. 143, 191–255 (1993). source

  • May combine two claims.

    Premisecitation

    In mitfaw2 mutants, melanophores are missing owing to a defect in their specification; in albino mutants, melanophores are present but lack melanin.

    “In mitfaw2 mutants, melanophores are missing owing to a defect in their specification; in albino mutants, melanophores are present but lack melanin27,36”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [27] Lister, J. A., Robertson, C. P., Lepage, T., Johnson, S. L. & Raible, D. W. nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. Development 126, 3757–3767 (1999). source
    • cites [36] Dooley, C. M. et al. Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease. Pigment Cell Melanoma Res. 26, 205–217 (2013). source

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

    Study design and conductnone given

    No statistical method was used to predetermine sample size.

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

  • May combine two claims.

    Interpretationown data

    These results support prior work showing that iridophores of different shapes respond differently to NE, and further demonstrate that only stripe iridophores change their color upon NE treatment.

    “These results support prior work showing that iridophores of different shapes respond differently to NE43, and further demonstrate that only stripe iridophores change their color upon NE treatment”
    • Also stated in: § Discussion · plain
      “Physiological responses also differed: disordered-crystal platelets of interstripe iridophores were refractory to NE, whereas, ordered-crystal platelets of stripe iridophores changed their cellular organization upon NE treatment”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [43] Oshima, N. & Kasai, A. Iridophores involved in generation of skin color in the zebrafish Brachydanio rerio. Forma 17, 91–101 (2002).

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).

    Interpretationnone given

    Additional signals from xanthophores, iridophores, and other cell types likely contribute as well.

    “Additional signals from xanthophores, iridophores, and other cell types likely contribute as well”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Features observed to not have a count greater than zero for at least one cell were removed.

    “Features observed to not have a count greater than zero for at least one cell were removed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    Sample size was based on previous experience.

    “Sample size was based on previous experience.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All Raman spectra were corrected by applying a baseline polynomial fit.

    “All Raman spectra were corrected by applying a baseline polynomial fit (Lab Spec 6 software).”
    Quote in the paperNot a substantial claim

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

    Interpretationown data

    The purine and cAMP pathways might be involved in iridophore environmental sensing and reaction to specific stimuli.

    “and might be involved in iridophore environmental sensing and reaction to specific stimuli (see below)”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Control zebrafish skin samples were used to gate out highly autofluorescent cells among the iridophore population.

    “Control zebrafish skin samples were used to gate out the highly auto fluorescent cells among the members of this population.”
    Quote in the paperNot a substantial claim
56 of 56

  • The authors state this as an assumption.

    Cryo-SEM preserves near-physiological crystal organization and cytoplasmic spacing.

    “To assess the ultrastructural organization of iridophores under near-physiological conditions, in which crystal organization and cytoplasmic spacing are likely to be retained, we used cryogenic scanning electron microscopy (cryo-SEM).”

  • The authors state this as an assumption.

    The weak dependence of the refractive index on wavelength can be neglected.

    “We neglected the weak dependence of the refractive index on wavelength”

  • The authors state this as an assumption.

    The interfaces between layers are parallel.

    “and assumed that the interfaces were parallel.”
3 of 3

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.

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

  • Stripe and interstripe iridophore morphologies reversed

    Minor

    The stated comparison places dense/cuboidal iridophores in stripes and loose/stellate iridophores in interstripes, reversing the locations assigned to those morphologies earlier in the paper.

  • Stripe cryo-SEM arrays described as both disordered and ordered

    Minor

    The Fig. 3a caption calls the stripe-iridophore crystal arrays highly disordered, whereas the results describe those arrays as remarkably ordered.

2 of 2

Cites

Flagged first, then by citations

Research works printed but not linked: 2

  1. Saunders, L. M. et al. Thyroid hormone regulates distinct paths to maturation in pigment cell lineages. eLife 8, e45181 (2019). · DOI · Paper page
  2. Eom, D. S., Bain, E. J., Patterson, L. B., Grout, M. E. & Parichy D. M. Long-distance communication by specialized cellular projections during pigment pattern development and evolution. Elife 4, e12401 (2015). · DOI

Cited by

Sorted by citations

Related

Authors
Gur, D., Bain, E. J., Johnson, K. R., Aman, A. J., Pasolli, H. A., Flynn, J. D., Allen, M. C., Deheyn, D. D., Lee, J. C., Lippincott-Schwartz, J., & Parichy, D. M.
Affiliations
  • National Institutes of Health (US)
  • University of Virginia (US)
  • Howard Hughes Medical Institute (US)
  • National Heart, Lung, and Blood Institute (US)
  • Scripps Institution of Oceanography (US)
Corresponding author
Parichy, David M.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-12-15
Volume
11
Issue
1
Pages
6391-6391
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-20088-1
PMC
PMC7738553
OpenAlex
W3112530233
Cited by
69
References
60
Citations by year
2026: 8 · 2025: 12 · 2024: 17 · 2023: 13 · 2022: 4 · 2021: 15
FWCI
4.12
Type
journal article
Language
en
Update notices
correction (2022-07-26)
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • HZB Repository (Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB)) (submittedVersion, other-oa)
Funders
Howard Hughes Medical Institute
Topics
  • 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
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
Keywords
iridophores, zebrafish, in situ differentiation, crystal arrays, transcriptomics, cell proliferation
MeSH terms
Animals, Cell Differentiation, Chromatophores, Skin, Skin Pigmentation, X-Ray Diffraction, Zebrafish, Mutagenesis, Zebrafish Proteins, Cell Proliferation, Microphthalmia-Associated Transcription Factor, Transcriptome

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.

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