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Cited by 126melanin and skin pigmentationZebrafish Biomedical Research ApplicationsDevelopmental Biology and Gene Regulation

Gene Expression Analysis of Zebrafish Melanocytes, Iridophores, and Retinal Pigmented Epithelium Reveals Indicators of Biological Function and Developmental Origin

Open access
Higdon, Charles W., Mitra, Robi D., Johnson, Stephen L.·2013·PLoS ONE
DOI 10.1371/journal.pone.0067801PMC PMC3706446
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Abstract

In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

AI snapshot

Written from the paper’s full text.

TL;DR

The authors developed a density-gradient/FACS method to purify zebrafish melanocytes, iridophores, and retinal pigmented epithelium (RPE) and generated the first transcriptome-wide mRNA sequencing data for these three pigment cell types, including the first iridophore transcriptome. Comparative expression analysis identified shared and cell-type-specific gene sets, notably a guanine-synthesis cycle model in iridophores.

Key findings

  • RNA-seq detected 8,472 genes expressed in melanocytes, 8,096 in iridophores, and 9,053 in RPE at >1 RPKM; melanocyte–RPE expression was most correlated (r = 0.90), while melanocyte–iridophore and RPE–iridophore correlations were modest (r = 0.52 and r = 0.49), and each pigment cell type correlated with whole embryos at <0.02.
  • The authors found 214 genes co-enriched in melanocytes and RPE, 62 co-enriched in melanocytes and iridophores, and only one gene, alcama, co-enriched in RPE and iridophores; 28 genes were shared among all three pigment cell types at ≥4 RPKM, within 2-fold of each other, and ≥100-fold above whole embryos.
  • Iridophores showed extensive enrichment of guanine-production machinery: 5 glucose transporters, 7/11 glycolysis steps, and 9/9 de novo purine synthesis enzymes were enriched; impdh1b was 71-fold higher than in melanocytes, prtfdc1 was 198-fold higher, and the adenine-specific adssl was not significantly different from melanocytes (0.7-fold).
  • The iridophore-enriched list included 346 genes, with 30 genes expressed at least 30-fold above melanocytes and RPE and 100-fold above whole embryos; the authors propose a guanine cycle model in which PRPP is recycled during guanine pigment production.

Methods

  • Zebrafish homozygous for temperature-sensitive mitfavc7 were dissociated, pigment cells enriched by Percoll density gradient centrifugation, and sorted by FACS using iridophore autofluorescence; mRNA was extracted and Illumina cDNA libraries were sequenced for 11 melanocyte, 5 iridophore, and 5 RPE samples, plus whole 3 dpf embryos.
  • Sequence tags were aligned with Novoalign to a custom non-redundant database of 25,102 zebrafish genes; expression was normalized as RPKM, statistical tests were performed in R, and selected expression changes were validated by qRT-PCR.
  • Enrichment analyses used a 1 RPKM baseline and thresholds including ≥2-fold over the third cell type with p<0.05, ≥8-fold over whole embryos for co-enrichment/cell-specific genes, and stricter cutoffs for selected shared or iridophore-specific gene lists.

Limitations

  • Stated: mitfa may be partially compromised by aberrant splicing at permissive temperatures, though melanocytes appeared indistinguishable from wild-type; RPE preparations lacked separate markers for eye-associated neural crest-derived melanocytes or choroidal melanocytes, and one RPE library was prepared later than 5 dpf.
  • Stated: the authors did not have statistical power to confidently track developmental changes in gene expression within specific cell types, and xanthophores were not isolated by the procedure.
  • Evident: the method relies on density and autofluorescence rather than lineage markers, so purity depends on those physical properties; the reduced-representation cDNA strategy could miss short cDNAs lacking restriction sites, although the authors included enzyme sites in amplification primers to mitigate this.

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

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0067801

Cites

114 of 114 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

53 of 93 cited sources checked · 28 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Melanocytes, iridophores, and RPE develop normally at 25°C in mitfavc7.

    “Melanocytes, iridophores, and RPE develop normally at 25°C in mitfavc7.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] SL Johnson, 2011, mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell, Developmental Biology source

      The Results section (‘Identification of temperature-sensitive alleles of mitfa’; page unavailable) says mitfavc7 develops normal or nearly normal melanocyte patterns at 25°C, not unequivocally normal ones. The paper also says RPE has no requirement for mitfa, but it does not establish that iridophores develop normally in mitfavc7 at 25°C; its discussion of excess iridophores concerns temperature-sensitive alleles at restrictive temperatures. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    Purine synthesis is important for iridophore pigmentation.

    “Zebrafish bearing mutations in the de novo purine synthesis enzymes gart and paics have iridophore defects, indicating purine synthesis is important for iridophore pigmentation [30].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] A Ng, 2009, Zebrafish mutations in gart and paics identify crucial roles for de novo purine synthesis in vertebrate pigmentation and ocular development, Development source
  • Premisecitation

    mitfa is not required for RPE development in zebrafish.

    “This mutant facilitated the collection of RPE, as mitfa is not required for RPE development in zebrafish [41].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] SL Johnson, 2011, mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell, Developmental Biology source
  • Findingown data

    ltk is highly enriched in iridophores compared to melanocytes.

    “For instance, ltk, known to be a marker of iridophores, is highly enriched in iridophores compared to melanocytes (red square with x in Figure S4) [27].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [27] SS Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics source
  • Premisecitation

    TFEC is known to be expressed in iridophores.

    “Known to be expressed in iridophores”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [77] JA Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec, Developmental Dynamics source

      In Results and Discussion, under “tfec” (page not provided), the authors observe tfec-expressing cells in locations associated with iridophores. They call the pattern “strongly suggestive” of iridophore pigment cells, but do not establish expression in identified iridophores as a known fact. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [78] MC Chung, 2001, TFEC can function as a transcriptional activator of the nonmuscle myosin II heavy chain-A gene in transfected cells, Biochemistry source
    • cites [79] KC Mansky, 2002, The microphthalmia transcription factor and the related helix-loop-helix zipper factors TFE-3 and TFE-C collaborate to activate the tartrate-resistant acid phosphatase, Journal of Leukocyte Biology source
  • Classified as probably not a substantial claim. 40
  • May combine two claims. 22
  • The quote only partly states this claim. 3

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

    Premisecitation

    Previous descriptions of gene expression in the RPE of zebrafish, chicken, and human have elucidated many of the genes playing roles in RPE biology.

    “Previous descriptions of gene expression in the RPE of zebrafish, chicken, and human have elucidated many of the genes playing roles in RPE biology [36]–[38].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [36] YF Leung, 2007, Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo, Investigative Ophthalmology & Visual Science source

      The Introduction says the study profiled the developing zebrafish retina, not its retinal pigment epithelium (RPE). The Materials and Methods describe removing the RPE before collecting the retinal tissue. For the zebrafish portion of this multi-citation sentence, the paper therefore describes gene expression in a different tissue. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [37] LJ Rizzolo, 2007, Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier, Mol Vis
    • cites [38] G Wistow, 2002, Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants, Mol Vis

  • Classified as probably not a substantial claim.

    Premisecitation

    Zebrafish bearing mutations in the de novo purine synthesis enzymes gart and paics have iridophore defects.

    “Zebrafish bearing mutations in the de novo purine synthesis enzymes gart and paics have iridophore defects, indicating purine synthesis is important for iridophore pigmentation [30].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] A Ng, 2009, Zebrafish mutations in gart and paics identify crucial roles for de novo purine synthesis in vertebrate pigmentation and ocular development, Development source

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

    Premisecitation

    Defects in melanin production are often associated with reduced visual function.

    “Many of these genes are responsible for producing melanin, and defects in melanin production are often associated with reduced visual function [39].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [39] K Grønskov, 2007, Oculocutaneous albinism, Orphanet Journal of Rare Diseases source

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

    Premisecitation

    Many RPE genes are responsible for producing melanin.

    “Many of these genes are responsible for producing melanin, and defects in melanin production are often associated with reduced visual function [39].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [39] K Grønskov, 2007, Oculocutaneous albinism, Orphanet Journal of Rare Diseases source

      The Etiology section identifies multiple genes involved in melanin biosynthesis, including TYR and TYRP1, and the Definition section links reduced ocular melanin to reduced visual acuity. The paper discusses these genes in melanocytes; it does not establish that they are specifically RPE genes. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    The RPE develops from the eye primordium and is continuous with the layer of cells that forms the iris.

    “The RPE develops from the eye primordium, and is continuous with the layer of cells that forms the iris [31].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [31] K Bharti, 2006, The other pigment cell: specification and development of the pigmented epithelium of the vertebrate eye, Pigment Cell Res source

  • May combine two claims.

    Study design and conductcitation

    Following development at 25°C, melanocyte numbers, morphology, and pigmentation in mitfavc7 are indistinguishable from wild-type zebrafish.

    “We note the possibility that mitfa may be partially compromised by aberrant splicing products in this mutant at permissive temperatures, but following development at 25°C, melanocyte numbers, morphology, and pigmentation are indistinguishable from wild-type zebrafish [41].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [41] SL Johnson, 2011, mitfa is required at multiple stages of melanocyte differentiation but not to establish the melanocyte stem cell, Developmental Biology source

      The Results section, “Identification of temperature-sensitive alleles of mitfa” (page unavailable), says mitfavc7 developed a normal or nearly normal melanocyte pattern at 25°C. It also says the allele appeared somewhat compromised even at 23°C. The source therefore supports a near-normal pattern at 25°C, but not the stronger claim that melanocyte numbers, morphology, and pigmentation were all indistinguishable from wild type. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    Several neural crest-derived pigment cells are found in zebrafish and other poikilotherms, including reflective iridophores.

    “In contrast, several neural crest-derived pigment cells are found in zebrafish and other poikilotherms, including reflective iridophores [5].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] DM Parichy, 2000, Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio, Developmental Biology source

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

    Premisecitation

    Several requirements for iridophore development from the neural crest are known.

    “Several requirements for iridophore development from the neural crest are known [5], [26], [27].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [5] DM Parichy, 2000, Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio, Developmental Biology source
    • cites [26] K Curran, 2009, Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf, Developmental Biology source
    • cites [27] SS Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics source

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

    Premisecitation

    mitfa has the reported ability to promote pigmented fate in zebrafish retinas.

    “consistent with the reported ability of mitfa to promote pigmented fate in zebrafish retinas [59].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [59] BM Lane, 2012, Otx but not mitf transcription factors are required for zebrafish retinal pigment epithelium development, PLoS ONE source

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

    Premisecitation

    Formation of COPI transport vesicles is mediated by the interaction of GAPDH with RAB2.

    “In addition, the formation of COPI transport vesicles is known to be mediated by the interaction of GAPDH with RAB2 [75].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [75] EJ Tisdale, 2004, Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity, Journal of Biological Chemistry source

      The Results show that activated Rab2 interacts directly with GAPDH, and the Abstract links Rab2 to vesicle formation from vesicular tubular clusters. But the Abstract says GAPDH’s precise function there is unknown; the cited paper does not establish that the interaction mediates formation of COPI vesicles. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation

    ltk, ednrb1, and pnp4a are known to be important for iridophore development.

    “Included in this enrichment list are several factors known to be important for iridophore development, including ltk, ednrb1, and pnp4a [5], [26], [27].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [5] DM Parichy, 2000, Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio, Developmental Biology source
    • cites [26] K Curran, 2009, Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf, Developmental Biology source

      The Discussion says Foxd3 appears to promote iridophore specification, but it does not address whether ltk, ednrb1, or pnp4a are important for iridophore development. Searches of our copy for those gene names and their expanded terms found no passage establishing the attributed claim. The indexed passages provide no page numbers. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [27] SS Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics source

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

    Premisecitation

    Roles for GPNMB have been described for melanocytes, melanoma, and the pigmented iris in mammals, and it has been suggested to act both as a plasma membrane protein and a component of the melanosome.

    “Roles for GPNMB have been described for melanocytes, melanoma, and the pigmented iris in mammals, and it has been suggested to act both as a plasma membrane protein and a component of the melanosome [70].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [70] M Tomihari, 2009, Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion, Experimental Dermatology source

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

    Premisecitation

    In humans, Rab27 mutations cause hypopigmentation associated with the immunodeficiency disorder Griscelli syndrome type II.

    “In humans, Rab27 mutations cause hypopigmentation associated with the immunodeficiency disorder Griscelli syndrome type II [74].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [74] P Bahadoran, 2001, Rab27a: A key to melanosome transport in human melanocytes, The Journal of Cell Biology source

      The Abstract describes Griscelli syndrome as involving immune deficiency and partial albinism and says a Rab27a mutation can cause it. The Discussion also links absent Rab27a to pigmentary dilution in human patients. Our copy does not identify the condition specifically as Griscelli syndrome type II. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    rxrga was previously reported to be expressed in neural crest tissues.

    “previously reported to be expressed in neural crest tissues [64]–[67].”
    Quote in the paperNot a substantial claim1 of 4 sources checked
    • cites [64] Thisse C, Thisse B (2005) High Throughput Expression Analysis of ZF-Models Consortium Clones. ZFIN Direct Data Submission (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://zfin.org" xlink:type="simple">http://zfin.org</ext-link>).

      Not checked in the free run.

    • cites [65] A Tallafuss, 2006, Characterization of retinoid-X receptor genes rxra, rxrba, rxrbb and rxrg during zebrafish development, Gene Expression Patterns source
    • cites [66] JS Waxman, 2007, Comparison of the expression patterns of newly identified zebrafish retinoic acid and retinoid X receptors, Developmental Dynamics source
    • cites [67] C He, 2009, Tissue-specific and embryonic expression of the retinoid X receptors in Sebastiscus marmoratus. Comparative Biochemistry and Physiology, Part B, Biochemistry & Molecular Biology source

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

    Premisecitation

    Phosphorylation of GAPDH occurs through the Src/PI3K/AKT pathway, often downstream of receptor tyrosine kinase activation.

    “Phosphorylation of GAPDH occurs through the Src/PI3K/AKT pathway, often downstream of receptor tyrosine kinase activation [76].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [76] Q Huang, 2011, AKT2 suppresses GAPDH mediated-apoptosis in ovarian cancer cells via phosphorylating GAPDH at threonine 237 and decreasing its nuclear translocation, Journal of Biological Chemistry source

      The Results (page not provided) show that Akt2 phosphorylates GAPDH and that wortmannin or Akt2 siRNA inhibits the observed phosphorylation; the paper also places Akt downstream of PI3K. The cited passages do not establish a Src/PI3K/Akt route to GAPDH phosphorylation or show that it often follows receptor tyrosine kinase activation. (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

    pcdh10a is expressed by migrating zebrafish neural crest cells and acts as a tumor suppressor in several cancers.

    “which is expressed by migrating zebrafish neural crest cells, and acts as a tumor suppressor in several cancers [61]-[63].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [61] Zhong X, Zhu Y, Mao J, Zhang J, Zheng S (2012) Frequent epigenetic silencing of PCDH10 by methylation in human colorectal cancer. Journal of Cancer Research and Clinical Oncology doi 10.1007/s00432–012–1353–5. source

      The Discussion (p. 5) identifies human PCDH10 as a functional tumor-suppression gene in colorectal cancer and discusses findings in other tumors. The paper does not establish that zebrafish pcdh10a is expressed in migrating neural crest cells, or that the zebrafish gene acts as a tumor suppressor. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [62] KC Bertrand, 2011, PCDH10 is a candidate tumour suppressor gene in medulloblastoma, Child’s Nervous Syst source
    • cites [63] Z Li, 2011, Epigenetic inactivation of PCDH10 in human prostate cancer cell lines, Cell Biology International source

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

    Premisecitation

    TFEC forms hetero and homodimers with other bHLH members and can function as a transcriptional activator or repressor.

    “TFEC forms hetero and homodimers with other bHLH members and can function as a transcriptional activator or repressor [77]–[79].”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [77] JA Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec, Developmental Dynamics source

      The Introduction (page not provided) says MiT proteins, including TFEC, can form homodimers and heterodimers with one another, but explicitly says they do not dimerize with other bHLH or bHLH-ZIP proteins outside that family. The paper says TFEC regulates gene expression and lists a study of its inhibition of TFE3-dependent activation, but it does not establish the claimed ability to function as both a transcriptional activator and a repressor. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [78] MC Chung, 2001, TFEC can function as a transcriptional activator of the nonmuscle myosin II heavy chain-A gene in transfected cells, Biochemistry source
    • cites [79] KC Mansky, 2002, The microphthalmia transcription factor and the related helix-loop-helix zipper factors TFE-3 and TFE-C collaborate to activate the tartrate-resistant acid phosphatase, Journal of Leukocyte Biology source

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

    Premisecitation

    Melanocyte and RPE gene expression have been previously characterized.

    “Because melanocyte and RPE gene expression have been previously characterized, we do not discuss them in detail here, but present the data as the supplemental tables listed below [36]–[38], [68], [69].”
    Quote in the paperNot a substantial claim3 of 5 sources checked
    • cites [36] YF Leung, 2007, Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo, Investigative Ophthalmology & Visual Science source

      The Materials and Methods describe removing the RPE before profiling retinal RNA, and the Results compare retinal with whole-embryo gene expression. Searches for RPE and melanocyte expression found no passage in our copy characterizing gene expression in either cell type. No page numbers were available for these passages. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [37] LJ Rizzolo, 2007, Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier, Mol Vis
    • cites [38] G Wistow, 2002, Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants, Mol Vis
    • cites [68] R Flockhart, 2012, BRAFV600E remodels the melanocyte transcriptome and induces BANCR to regulate melanoma cell migration, Genome Research source

      The Results (page unavailable) characterize gene expression in normal human melanocytes, including BRAF-regulated transcripts. The cited paper does not address RPE gene expression, so it supports only the melanocyte part of the statement. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [69] J An, 2011, A Comparative Transcriptomic Analysis of Uveal Melanoma and Normal Uveal Melanocyte, PLoS ONE source

      The Results section (‘Gene profiling and the category of highly-expressed genes in two libraries’; page unavailable) reports gene-expression profiling of normal uveal melanocytes. The cited paper does not characterize RPE gene expression, so it supports only the melanocyte part of the claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Endothelin receptor (ednrb1) signaling is required for iridophore development in zebrafish.

    “Relatedly, endothelin receptor (ednrb1) signaling is required for iridophore development in zebrafish [5].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [5] DM Parichy, 2000, Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio, Developmental Biology source

      The Abstract states that zebrafish ednrb1 is required for a subset of adult iridophores. The section ‘ednrb1 Is Expressed during Pigment Pattern Metamorphosis’ reports a severe iridophore deficit in mutant adults but no gross pigment-pattern defect in mutant embryos. The claim omits the adult-stage and subset qualifications, making the requirement sound general. (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

    Several mouse coat color mutants are ribosomal proteins, although they are not the ribosomal components enriched here.

    “Pigment cell enrichment of ribosomal components is not surprising, considering several mouse coat color mutants are ribosomal proteins, although they are not the ribosomal components enriched here [50]–[53].”
    Quote in the paperNot a substantial claim2 of 4 sources checked
    • cites [50] ER Oliver, 2004, Ribosomal protein L24 defect in belly spot and tail (Bst), a mouse Minute, Development (Cambridge, England) source
    • cites [51] N Draptchinskaia, 1999, The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia, Nature Genetics source
    • cites [52] WC Skarnes, 2011, A conditional knockout resource for the genome-wide study of mouse gene function, Nature source

      The Abstract and Discussion describe a resource for generating mouse knockout alleles, not coat-color phenotypes or ribosomal proteins. Searches of our copy for coat-color mutants and ribosomal pigmentation found no passage addressing the attributed relationship; the claim about which components were enriched also cannot be checked against this source. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites [53] MH Hrabe de Angelis, 2000, Genome-wide, large-scale production of mutant mice by ENU mutagenesis, Nature Genetics source

      The indexed copy's bibliographic entry (section ‘1999 (7)’, no page) identifies the paper but provides no findings about coat color mutants or ribosomal proteins. Searches for those terms and for pigmentation returned only catalog material, so the claim cannot be checked against the available text. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

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

    Premisecitation

    alcama has been found to mediate endothelin 1 signaling in cartilage development.

    “It is intriguing that alcama has been found to mediate endothelin 1 signaling in cartilage development [58].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [58] P Choudhry, 2011, Alcama mediates Edn1 signaling during zebrafish cartilage morphogenesis, Developmental Biology source

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

    Premisecitation

    Ets-1 is an oncogene shown to regulate glycolysis and associated feeder pathways.

    “Another oncogene shown to regulate glycolysis and associated feeder pathways is Ets-1 [83].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [83] ML Verschoor, 2010, Ets-1 regulates energy metabolism in cancer cells, PLoS ONE source

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

    Premisecitation

    MYC upregulates glycolysis, DNA-synthesis, and nucleotide metabolism.

    “It is established that MYC upregulates glycolysis, DNA-synthesis, and nucleotide metabolism [80]–[82].”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [80] SJ Yeung, 2008, Roles of p53, MYC and HIF-1 in regulating glycolysis–the seventh hallmark of cancer, Cellular and Molecular Life Sciences source

      The section “Coordinated Regulation of Proteins that Control the Glycolytic Flux” (pp. 7–8) says increased c-MYC activity helps upregulate glycolytic-enzyme genes and glycolytic capacity. Our copy does not say that MYC upregulates DNA synthesis or nucleotide metabolism; searches for those terms found no supporting passage. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [81] M Albajar, 2011, MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib, Molecular Cancer Research source

      The Introduction (page not provided) describes MYC-induced DNA synthesis and mentions enhanced energetic metabolism. The section “Ectopic MYC increased DNA synthesis in K562 cells treated with imatinib” (page not provided) reports increased DNA synthesis after MYC induction. Neither passage establishes the more specific claims that MYC upregulates glycolysis or nucleotide metabolism. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [82] Y Liu, 2008, Global regulation of nucleotide biosynthetic genes by c-Myc, PLoS ONE source

      The Discussion states that MYC upregulates glycolytic genes, and the Results show that MYC induction upregulates genes for nucleotide biosynthesis. The Discussion links these effects to providing energy and nucleotides for DNA synthesis, but our copy does not establish that MYC upregulates DNA synthesis itself. Page numbers are unavailable in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Members of the Cart1/Alx3/Alx4 family of homeodomain proteins are known to regulate formation of skeletal elements in organisms ranging from sea urchin to mammals.

    “Members of the Cart1/Alx3/Alx4 family of homeodomain proteins are known to regulate formation of skeletal elements in organisms ranging from sea urchin to mammals [86], [87].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [86] CA Ettensohn, 2003, Alx1, a member of the Cart1/Alx3/Alx4 subfamily of Paired-class homeodomain proteins, is an essential component of the gene network controlling skeletogenic fate specification in the sea urchin embryo, Development source
    • cites [87] A Beverdam, 2001, Severe nasal clefting and abnormal embryonic apoptosis in Alx3/Alx4 double mutant mice, Development source

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

    Premisecitation

    In humans, mutation and haploinsufficiency of ALX4 are associated with skull ossification defects.

    “In humans, mutation and haploinsufficiency of ALX4 are associated with skull ossification defects [88], [89].”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [88] LA Mavrogiannis, 2001, Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects, Nature Genetics source
    • cites [89] W Wuyts, 2000, The ALX4 homeobox gene is mutated in patients with ossification defects of the skull (foramina parietalia permagna, OMIM 168500), Journal of Medical Genetics source

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

    Premisecitation

    gbx2 is known to have several roles in the embryo and is an early specifier of posterior neural crest in Xenopus.

    “Known to have several roles in the embryo and an early specifier of posterior neural crest in Xenopus”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [85] B Li, 2009, The posteriorizing gene Gbx2 is a direct target of Wnt signalling and the earliest factor in neural crest induction, Development source

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

    Findingown data

    The non-redundant zebrafish cDNA database includes 7% (10/126) records of dubious utility.

    “In summary, our efforts have generated a non-redundant zebrafish cDNA database that identifies most (∼99%) of annotated genes, and includes 7% (10/126) records of dubious utility, but would tend not to confound RNA-seq analysis such as reported here.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    The 15 genes expressed at least 5-fold greater than RPE and 10-fold greater than whole embryos in melanocytes and iridophores include well-known regulators and markers of neural crest and pigment cell development, including sox10 and crestin.

    “Included in this group are several well-known regulators and markers of neural crest and pigment cell development, including the transcription factors sox10 and the expressed repetitive element crestin [60].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [60] AL Rubinstein, 2000, Genes dependent on zebrafish cyclops function identified by AFLP differential gene expression screen, Genesis source

  • May combine two claims.

    Findingown data

    Melanocytes, RPE, and iridophores are distinct from whole embryos, with average correlation values less than 0.02.

    “By this general assessment, melanocytes, RPE, and iridophores are distinct from whole embryos, with average correlation values less than 0.02.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When comparing iridophores to melanocytes and RPE, 5 facilitated glucose transporters, 7/11 steps of glycolysis, and 9/9 enzymes for de novo purine synthesis are enriched.

    “When comparing iridophores to melanocytes and RPE, we find 5 facilitated glucose transporters, 7/11 steps of glycolysis, and 9/9 enzymes for de novo purine synthesis to be enriched.”
    Quote in the paperSubstantial claim

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

    Findingown data

    mycl1a is another bHLH enriched in iridophores compared to melanocytes and RPE, but it did not meet the 8-fold requirement over whole embryos.

    “Interestingly, there is one other bHLH in enriched in iridophores when compared to melanocytes and RPE, but it did not meet the 8-fold requirement over whole embryos.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.

    “This study was carried out in accordance with the Washington University Animal Use Committee guidelines under approved protocol #20110236.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The RPE forms part of the blood-retina barrier, providing the eye with an immune-privileged status.

    “The RPE forms part of the blood-retina barrier, providing the eye with an immune-privileged status [33].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] EA Runkle, 2011, The blood-retinal barrier: structure and functional significance, Methods Mol Biol source

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

    Study design and conductnone given

    All sequencing data used in this study are publicly available at NCBI's GEO database under accession GSE46387.

    “All sequencing data used in this study are publicly available at NCBI’s GEO database under accession GSE46387 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE46387).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Defects in the RPE contribute to diseases such as macular degeneration and retinitis pigmentosa, which result in vision problems.

    “Defects in the RPE contribute to diseases such as macular degeneration and retinitis pigmentosa, which result in vision problems [34], [35].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [34] S Dridi, 2012, ERK1/2 activation is a therapeutic target in age-related macular degeneration, Proc Natl Acad Sci USA source
    • cites [35] DA Thompson, 2003, Genetic defects in vitamin A metabolism of the retinal pigment epithelium, Developments in Ophthalmology source

  • May combine two claims.

    Premisereasoning

    When held at 32°C, neural crest-derived melanocytes do not develop, but RPE and iridophores develop normally.

    “When held at 32°C, the neural crest-derived melanocytes do not develop, but RPE and iridophores develop normally.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    RPE65 is known to be present in keratinocytes, melanocytes, and melanoma, in addition to the RPE.

    “This is not entirely surprising, as RPE65 is known to be present in keratinocytes, melanocytes, and melanoma, in addition to the RPE [46], [47].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [46] G Hinterhuber, 2004, RPE65 of retinal pigment epithelium, a putative receptor molecule for plasma retinol-binding protein, is expressed in human keratinocytes. J Invest Dermatol, Feb source
    • cites [47] PM Amann, 2012, Vitamin A metabolism in benign and malignant melanocytic skin cells: importance of lecithin/retinol acyltransferase and RPE65, J Cell Physiol. Feb source

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

    Study design and conductown data

    The non-redundant zebrafish cDNA database identifies most (∼99%) of annotated genes.

    “In summary, our efforts have generated a non-redundant zebrafish cDNA database that identifies most (∼99%) of annotated genes, and includes 7% (10/126) records of dubious utility, but would tend not to confound RNA-seq analysis such as reported here.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Study design and conductreasoning

    The purification procedure relies on the inherent densities of melanin-filled melanocytes and RPE and guanine-filled iridophores.

    “This purification procedure relies on the inherent densities of melanin and guanine-filled cells; hence it can be used without other complicated lineage markers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    The other pigment cells in zebrafish, pteridine-containing xanthophores, do not pass through the density gradient and are not isolated by this method.

    “The other pigment cells in zebrafish, the pteridine-containing xanthophores, do not pass through the density gradient and are not isolated by this method.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The RPE provides trophic support and recycles wastes from the photoreceptors of the retina.

    “It provides trophic support and recycles wastes from the photoreceptors of the retina [32].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] O Strauss, 2005, The retinal pigment epithelium in visual function, Physiological Reviews source

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

    Premisecitation

    Rab GTPases regulate many aspects of membranous vesicle formation and traffic.

    “Rab GTPases regulate many aspects of membranous vesicle formation and traffic [73].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [73] BL Grosshans, 2006, Rabs and their effectors: achieving specificity in membrane traffic, Proc Natl Acad Sci USA source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    To independently validate the enrichment sets, separate biological samples were generated for each cell type and expression of selected genes was compared via qRT-PCR.

    “To independently validate these enrichment sets, we generated separate biological samples for each cell type and compared expression of selected genes from the enrichment sets via qRT-PCR.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    ARFs are a large family of ras-related GTPases that regulate membrane trafficking and organelle structure.

    “ARFs are a large family of ras-related GTPases that regulate membrane trafficking and organelle structure (For review see [72]).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [72] JG Donaldson, 2005, Localization and function of Arf family GTPases, Biochemical Society Trans source

  • May combine two claims.

    Study design and conductown data

    RNA-seq and qRT-PCR relative expression levels showed good correlation (r2 = 0.75), indicating RNA-seq data is a reliable indicator of gene expression in these pigment cells.

    “We observed good correlation of RNA-seq and qRT-PCR relative expression levels, indicating our RNA-seq data is a reliable indicator of the gene expression in these pigment cells (r2 = 0.75, Figure S4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    slc45a2 expression is not shared by all pigment cells, as in situ analysis reveals no enriched expression in xanthophores.

    “However, in situ analysis reveals no enriched expression in xanthophores, suggesting that slc45a2 expression is not shared by all pigment cells [55].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [55] Dooley CM, Schwarz H, Mueller K, Mongera A, Konantz M, <etal>et al</etal>.. (2012) Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease. Pigment Cell &amp; Melanoma Research doi: 10.1111/pcmr.12053. source

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

    Interpretationreasoning

    slc23l may act as a guanine transporter.

    “The third highest expressed gene on this list, slc23l, may act as a guanine transporter.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: In mammals, the SLC23 gene family has roles in transporting nucleobases, such as guanine, as well as vitamin C.

  • May combine two claims.

    Interpretationown data

    The lists of co-expressed genes are likely enriched for common metabolic functions (melanocytes and RPE) or developmental origins (melanocytes and iridophores).

    “Therefore, we suggest these lists of co-expressed genes are likely enriched for common metabolic functions, in the case of melanocytes and RPE, or developmental origins, in the case of melanocytes and iridophores.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Genes co-enriched among pigment cell types indicate shared pigment cell functions.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    To avoid artificial skewing by outlying data points, average correlation was calculated from 24,102 overlapping windows of 1000 genes sorted by increasing whole embryo expression.

    “Because correlation values can be artificially skewed by outlying data points [49], we calculated the average correlation from 24,102 overlapping windows of 1000 genes, sorted by increasing whole embryo expression (Figure S6).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] A Fujita, 2009, Comparing Pearson, Spearman and Hoeffding’s D measure for gene expression association analysis, Journal of Bioinformatics and Computational Biology source

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

    Interpretationreasoning

    The low correlation value (0.02) between pigment cells and whole embryos reflects the baseline correlation component from expression of housekeeping genes shared by all cells.

    “Presumably, this low value (0.02) reflects the baseline correlation component from expression of housekeeping genes shared by all cells”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Synthesis of guanine from GMP likely results in the recycling of PRPP, the rate-limiting substrate in purine synthesis.

    “Upon inspection of the known pathway of guanine production, we observed synthesis of guanine from GMP likely results in the recycling of 5-Phosphoribosyl 1-Pyrophosphate (PRPP), the rate-limiting substrate in purine synthesis (KEGG Pathway: dre00230).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites KEGG Pathway: dre00230

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

  • May combine two claims.

    Study design and conductnone given

    The authors developed a method for rapidly and reliably purifying melanocytes, iridophores, and retinal pigmented epithelium from zebrafish embryos, followed by global gene expression analysis by mRNA sequencing.

    “To contribute to the understanding of pigment cell biology, we have developed a method for rapidly and reliably purifying melanocytes, iridophores, and retinal pigmented epithelium from zebrafish embryos, followed by global gene expression analysis by mRNA sequencing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The comparison uniquely allowed identification of co-expressed genes indicative of shared function or developmental origin, as well as genes specifically enriched in single cell types.

    “which uniquely allowed us to identify co-expressed genes indicative of shared function or developmental origin, as well as those that are specifically enriched in single cell types.”
    Quote in the paperSubstantial claim
53 of 53

  • The paper relies on this without support.

    This is the first analysis of the iridophore transcriptome.

    “This is also the first analysis of the iridophore transcriptome.”
    “Furthermore, this is the first characterization of the iridophore transcriptome.”

  • The paper relies on this without support.

    The purification procedure and expression analysis provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types.

    “The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.”

  • The paper relies on this without support.

    It is unknown whether markers of neural crest identity persist in iridophores following development, and whether these markers are shared by other neural crest-derived pigment cells, such as melanocytes.

    “However, it is unknown if markers of neural crest identity persist in iridophores following development, and whether these markers are shared by other neural crest-derived pigment cells, such as melanocytes.”

  • The paper relies on this without support.

    It is unknown whether RPE and melanocytes use different pathways of melanin production, or if they are essentially identical.

    “However, it is unknown whether RPE and melanocytes use different pathways of melanin production, or if they are essentially identical.”

  • The paper relies on this without support.

    The percentage of melanocytes, iridophores, and RPE compared to all other cells in the fish is less than 1%.

    “However, the percentage of melanocytes, iridophores, and RPE compared to all other cells in the fish is less than 1%, making cell-specific gene expression analysis from the whole organism difficult.”

  • The paper relies on this without support.

    Genes co-enriched among pigment cell types indicate shared pigment cell functions.

    “We reasoned that genes co-enriched among pigment cell types would indicate shared pigment cell functions.”

  • The paper relies on this without support.

    RPE and iridophores do not share a pigment-type production or developmental origin.

    “Given that the RPE and iridophores do not share a pigment-type production or developmental origin, we expected fewer genes to be co-enriched in these cells, but not expressed by neural-crest melanocytes.”

  • The paper relies on this without support.

    Melanocytes and RPE share the function of producing melanin and melanosomes.

    “Due to their shared function of producing melanin and melanosomes, we expected many genes to be shared between melanocytes and the RPE when compared to iridophores.”

  • The paper relies on this without support.

    mitfa is not required for RPE development in zebrafish.

    “Although not required for RPE development in zebrafish, mitfa is expressed in the RPE at a relatively high level, consistent with the reported ability of mitfa to promote pigmented fate in zebrafish retinas [59].”

  • The paper relies on this without support.

    Melanocytes and iridophores share a common developmental origin from the neural crest.

    “Because of the common developmental origin from the neural crest for melanocytes and iridophores, we speculated that neural crest-specific gene expression would be readily identifiable.”

  • The paper relies on this without support.

    The iridophore transcriptome has not previously been explored.

    “In contrast to melanocytes and RPE, the iridophore transcriptome has not previously been explored.”

  • The paper relies on this without support.

    Iridophores produce a guanine-based pigment, whereas melanocytes and RPE produce melanin.

    “Since iridophore produce a guanine-based pigment, rather than the melanin characteristic of melanocytes and RPE, we expected to find many genes to be specifically enriched in this cell type.”

  • The paper relies on this without support.

    In mammals, the SLC23 gene family has roles in transporting nucleobases, such as guanine, as well as vitamin C.

    “In mammals, the SLC23 gene family has roles in transporting nucleobases, such as guanine, as well as vitamin C.”

  • The paper relies on this without support.

    Iridophores are relatively less-studied than melanocytes and RPE, and aside from requirements for foxd3, ednrb1, and ltk, not much is known about transcriptional regulation of iridophore identity.

    “Iridophores are a relatively less-studied pigment cell than melanocytes and RPE, and aside from the requirements for foxd3, ednrb1, and ltk, not much is known regarding the transcriptional regulation of iridophore identity.”

  • The paper relies on this without support.

    mycl1a is a paralog of the classic oncogenic protein MYC.

    “This gene, mycl1a, is a paralog of the classic oncogenic protein MYC.”
15 of 15

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?

  • pnp4a encodes purine nucleoside phosphorylase 4a, an enzyme converting guanosine mono-phosphate to guanine.

    “This gene encodes purine nucleoside phosphorylase 4a, an enzyme converting guanosine mono-phosphate to guanine [48].”

    From A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest (2018) · cites this paper as [48]

    In Results and Discussion, under “Iridophore Gene Enrichment” (page unavailable), the paper identifies prtfdc1—not pnp4a—as the enzyme responsible for the final step of guanine synthesis from GMP. It mentions pnp4a as a factor important for iridophore development, but does not assign it the claimed conversion. (LLM, conf=0.85)

    Checked by a reasoning model — may be wrong.

    Experimental — accuracy not yet measured

  • Higdon et al. detected low levels of tfec expression in purified melanocytes.

    “Although Higdon et al showed that tfec expression was prominent in the RNA-seq profiles of iridophores, they also detected low levels of expression in purified melanocytes [40].”

    From The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor (2021) · cites this paper as [40]

    The Results and Discussion section on iridophore gene enrichment says tfec is specifically enriched in iridophores. The passages from our copy do not report a tfec expression level in purified melanocytes, so they do not establish the claimed low-level detection there. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • The final lower Mitfa level reflects expression levels undetectable by in situ hybridisation in differentiated iridophores, although still measurable by microarray in pooled isolated iridophores.

    “We note that the Mitfa concentration is not required to attain zero, but simply to drop to a lower steady-state value; given the expectation that in situ hybridisation techniques have a ‘detection threshold’, we consider that this final lower value would reflect expression levels undetectable by our detection methods in differentiated iridophores, although they would still be measurable by micro”

    From A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest (2018) · cites this paper as [48]

    Table 3 in Results and Discussion (Melanocyte and RPE Co-Expression; page unavailable) reports mitfa expression of 16.22 RPKM in iridophores, much lower than the 1122.44 RPKM reported for melanocytes. The paper describes mRNA sequencing, not microarray measurement, and the cited passages do not establish that mitfa is undetectable by in situ hybridisation in differentiated iridophores. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • pnp4a is expressed at high levels in differentiated iridophores and at lower levels in melanocytes.

    “RNA-seq analysis of gene expression in purified iridophores and melanocytes has shown that it is expressed at high levels not only in differentiated iridophores, but also, albeit at lower levels, in melanocytes [48].”

    From A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest (2018) · cites this paper as [48]

    The Results and Discussion section on Iridophore Gene Enrichment lists pnp4a among genes enriched in purified iridophores, supporting higher expression there than in melanocytes. The passage does not establish detectable pnp4a expression in melanocytes or specify that the iridophores were differentiated; no page number is available in our copy. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Higdon et al. found tfec enriched in the iridophore lineage.

    “Higdon et al. performed transcriptomic analysis to compare FACS-purified iridophores, melanocytes, and retinal pigment epithelium (RPE), and found tfec to be one of several transcription factor genes with enriched expression specifically in the iridophore lineage [40].”

    From The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor (2021) · cites this paper as [40]

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.

  • Dubious-record percentage is miscalculated

    Minor

    Ten records out of 126 is about 7.94%, which rounds to 8%, not 7%.

    Reported 7 · recomputed 7.936508

  • Library preparation both eliminates and includes end-repair

    Minor

    The methods say enzymatic fragmentation eliminated the Illumina end-repair step, but the Figure 2 caption says end-repair was performed after that fragmentation.

2 of 2

Cited works that were retracted

Works this paper cites that were later retracted, from the retraction notices publishers deposit. Major when the notice came out before this paper was published.

  • Cites a retracted work: 10.1007/s11010-011-0880-8

    Info

    The cited work was retracted on 2024-04-24 (notice 10.1007/s11010-024-05015-y), not before this paper was published (2013-07-09).

    Cited work 10.1007/s11010-011-0880-8 · retracted 2024-04-24 · notice 10.1007/s11010-024-05015-y · this paper published 2013-07-09

1 of 1

Cites

1 retracted

Flagged first, then by citations

Cited by

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Related

Authors
Higdon, C. W., Mitra, R. D., & Johnson, S. L.
Affiliations
Washington University in St. Louis (US)
Corresponding author
  • Higdon, Charles W.
  • Johnson, Stephen L.
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2013-07-09
Volume
8
Issue
7
Pages
e67801
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0067801
PMC
PMC3706446
OpenAlex
W2144853273
Cited by
125
References
114
Citations by year
2026: 8 · 2025: 8 · 2024: 10 · 2023: 16 · 2022: 5 · 2021: 12 · 2020: 12 · 2019: 11 · 2018: 11 · 2017: 18 · 2016: 4 · 2015: 9 · 2014: 1
FWCI
2.71
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Commons@Becker (Washington University School of Medicine) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • W. M. Keck Foundation
  • Alvin J. Siteman Cancer Center
  • National Institutes of Health (RO1-GM56988)
  • National Institute of General Medical Sciences (R01GM056988)
  • National Institute of Neurological Disorders and Stroke (R01NS076993)
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
zebrafish, melanocytes, iridophores, retinal pigment epithelium, transcriptome, cell purification, neural crest, purine biosynthesis, phosphoribosylpyrophosphate, melanogenesis, developmental origins
MeSH terms
Animals, Cell Differentiation, Embryo, Nonmammalian, Guanine, Melanocytes, Organ Specificity, Zebrafish, Gene Expression Regulation, Developmental, Gene Expression Profiling, Biosynthetic Pathways, Retinal Pigment Epithelium

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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