The MITF paralog tfec is required in neural crest development for fate specification of the iridophore lineage from a multipotent pigment cell progenitor

May combine two claims.

In shady/ltk mutants, fate specification and proliferation of iridophores, but not other pigment cells, are impeded.

“In shady/ltk mutants fate specification and proliferation of iridophores, but not other pigment cells, are impeded [11, 36].”

• cites [11] S. S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics sourcePartly supported

  The Results sections on iridophore markers and the shd allelic series support impaired iridophore fate specification, with melanophores and xanthophores generally comparable to wild-type siblings. But the Results section on iridophore markers says a primary role in iridoblast proliferation was unlikely and reports no significant effect on neural crest cell proliferation. Thus, the attributed proliferation defect is not supported. (LLM, conf=0.91)

  Experimental — accuracy not yet measured
• cites [36] F. S. L. M. Rodrigues, 2012, A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors, ACS Chemical Biology sourcePartly supported

  The Introduction (page unavailable) says ltk mutants lack iridophores because iridophore fate specification fails. It does not say that proliferation is impeded. The Results and Discussion report no effects on other pigment cells in inhibitor-treated embryos, but do not establish that finding for ltk mutants. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Tfap2a acts as a key co-factor supporting Mitfa.

“However, in this role, Mitfa is supported by Tfap2, especially Tfap2a which acts as a key co-factor [27].”

• cites [27] H. E. Seberg, 2017, TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF, PLOS Genetics sourcePartly supported

  The Results report a synergistic genetic interaction between tfap2a and mitfa, and the Discussion reports widespread TFAP2A–MITF co-occupancy at regulatory elements. These findings support a role for Tfap2a alongside Mitfa in melanocyte development, but the Discussion says it is unknown whether they bind cooperatively or physically interact. Calling Tfap2a a ‘key co-factor’ states that relationship more definitively than the paper does. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

May combine two claims.

By 24 hpf, tfec labels migrating progenitors which co-express mitfa and ltk markers, considered fate-specified iridoblasts but likely retain at least melanocyte potential.

“By 24 hpf, tfec labels migrating progenitors which co-express mitfa and ltk markers, and which we consider fate-specified iridoblasts, but which likely retain at least melanocyte potential too (Fig 1; Ib(sp); [14]).”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourceSupported

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

Sox10, in conjunction with Wnt signalling, is required to activate and maintain mitfa transcription.

“In this lineage, Sox10, in conjunction with Wnt signalling, is required to activate and maintain mitfa transcription [13, 20, 24–26].”

• cites [13] E. R. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development, PLoS Genetics sourcePartly supported

  The Results section, “Overexpression of Mitfa, but not Sox10,” reports that Sox10 induces mitfa transcription, supporting the activation part. But the Discussion attributes maintenance of mitfa expression to Mitfa-dependent feedback, and the modelling Results describe that feedback as Sox10-independent. The paper mentions Wnt-related factors as possible inputs to sox10 regulation, not as an established joint requirement with Sox10 for activating and maintaining mitfa. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [20] K. A. Dutton, 2001, Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates, Development sourceNot checked — no full text of the source
• cites [24] R. I. Dorsky, 2000, Direct regulation of nacre, a zebrafish MITF homolog required for pigment cell formation, by the Wnt pathway, Development sourcePartly supported

  The Results (page not provided) support a role for Wnt signalling in activating nacre, the zebrafish MITF homolog: Tcf/Lef-site experiments support activation of nacre transcription in melanophore precursors. The cited paper does not establish that Sox10 acts in conjunction with Wnt or that the two are required to maintain transcription. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [25] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development, Development sourceNot checked — no full text of the source
• cites [26] L. Vibert, 2017, An ongoing role for Wnt signaling in differentiating melanocytes in vivo, Pigment Cell & Melanoma Research sourcePartly supported

  The Introduction says Sox10 and Wnt signaling act together to drive mitfa expression during melanocyte specification. The Results distinguish maintenance from that early activation: later Wnt input acts through a Sox10-independent, Mitfa-dependent positive-feedback loop. The source therefore does not support a continuing requirement for Sox10 in maintaining mitfa transcription. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

May combine two claims.

tfec is first expressed extensively throughout the premigratory NC progenitors of the trunk and tail at 18 hpf, and then exclusively in the developing iridophore lineage.

“we concluded that tfec is first expressed extensively throughout the premigratory NC progenitors of the trunk and tail at 18 hpf, and then exclusively in the developing iridophore lineage.”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourcePartly supported

  The Results section, “Identification of the iridophore lineage throughout zebrafish embryogenesis” (page not provided), reports tfec expression in premigratory neural crest cells at 18 hpf and later in iridoblasts and differentiated iridophores. But “then exclusively” overstates the pattern: at 24 hpf tfec remains detectable in multipotent premigratory cells, and the authors interpret some tfec-positive migrating cells as at least bipotent. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

May combine two claims.

ltk expression is lacking in tfec mutants in premigratory late Cbls, migrating Ib(sp), the medial migration pathway, and eye iridoblasts.

“ltk expression was completely lacking on the medial migration pathway in homozygous mutants at 24 hpf (Fig 5S and 5T; [14]), consistent with an early defect in iridophore specification.”

• Also stated in: § Loss of tfec function affects the development of all embryonic pigment cells · plain

  “(S, T) tfec mutants lack ltk expression in premigratory late Cbls (arrowhead), in migrating Ib(sp) (arrow) and in iridoblasts of the eye (insets).”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourceSupported

May combine two claims.

At approximately 22 hpf, ltk and mitfa are upregulated in the tfec+; foxd3- premigratory cells of the trunk, indicating they correspond to partially restricted progenitors capable of generating pigment cells.

“At approximately 22 hpf, ltk and mitfa are upregulated in the tfec+; foxd3- premigratory cells of the trunk [11, 14, 21], indicating that they correspond to partially restricted progenitors, capable of generating pigment cells”

• cites [11] S. S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics sourcePartly supported

  The Results report ltk expression in premigratory trunk neural crest cells and place it after expression of foxd3; the Discussion describes foxd3-negative, ltk-expressing cells in sox10 mutants as possible partially restricted progenitors. The Discussion also places the onset of mitfa expression in the posterior trunk at approximately 21 hpf. The paper does not establish that ltk and mitfa are upregulated together at approximately 22 hpf in tfec-positive, foxd3-negative cells, or demonstrate the developmental potential of that specific cell population. No page numbers were supplied for these sections. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourcePartly supported

  The Results (Fig. 1 legend; page unavailable) describe tfec-positive premigratory neural crest cells that have downregulated foxd3 at 18 hpf but have not yet detectably activated ltk or mitfa. The Discussion (page unavailable) proposes that these cells then turn on both genes and enter a pigment-cell progenitor phase. The paper supports the developmental sequence, but does not establish that the upregulation occurs at approximately 22 hpf; its reported observations are at 18 and 24 hpf. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [21] J. A. Lister, 1999, nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate, Development sourceNot checked — no full text of the source

May combine two claims.

Ltk signalling is crucial to fate specification, proliferation and differentiation of iridophores.

“a signalling pathway crucial to fate specification, proliferation and differentiation of iridophores has been highlighted by mutations in the gene encoding the Leukocyte Tyrosine Kinase (Ltk; [11, 36]) receptor tyrosine kinase”

• cites [11] S. S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics sourcePartly supported

  The Discussion states that Ltk is crucial for iridophore fate specification in zebrafish. It proposes proliferation as a likely late function but says the early mutant phenotype prevents direct examination of Ltk’s role in differentiating cells. The Results report no significant effect on neural crest cell proliferation, so the claim overstates the evidence for proliferation and differentiation. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [36] F. S. L. M. Rodrigues, 2012, A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors, ACS Chemical Biology sourcePartly supported

  The Introduction says Ltk drives iridophore fate specification, and the Results and Discussion report that activated Ltk rescues iridophore development and produces additional iridophores. Our copy does not establish separate roles for Ltk signalling in iridophore proliferation and differentiation; those parts of the claim go beyond the reported findings. The indexed passages provide section labels but no page numbers. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Mitfa is necessary and sufficient to upregulate numerous melanocyte differentiation genes, including those controlling melanin synthesis.

“Like its mammalian counterpart, MITF, Mitfa has been proven necessary and sufficient to upregulate numerous melanocyte differentiation genes, including those controlling melanin synthesis (e.g. dct, silva and tyrosinase).”

• cites [13] E. R. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development, PLoS Genetics sourceSupported
• cites [21] J. A. Lister, 1999, nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate, Development sourceNot checked — no full text of the source
• cites [25] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development, Development sourceNot checked — no full text of the source

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

Mitfa is the master regulator of melanocyte fate.

“Mitfa is thus dubbed the ‘master regulator’ of melanocyte fate choice [13, 21, 25].”

• cites [13] E. R. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development, PLoS Genetics sourceSupported
• cites [21] J. A. Lister, 1999, nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate, Development sourceNot checked — no full text of the source
• cites [25] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development, Development sourceNot checked — no full text of the source

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

Our tfec mutants fail to inflate the swim bladder and die after approximately 12 days, apparently from lack of ability to feed.

“As with the CRISPR-generated exon 3 allele reported by Mahony and colleagues, our mutants fail to inflate the swim bladder (which, along with the caudal hematopoietic tissue, is another site of tfec expression; [39]) and die after approximately 12 days, apparently from lack of ability to feed.”

• cites [39] J. A. Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec, Developmental Dynamics sourceSupported

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

In sox10 mutants, NC derivatives fail to become specified and to enter the migration pathways.

“Specifically, as was previously reported for a single time point [14], in sox10 mutants, where NC derivatives fail to become specified and to enter the migration pathways [11, 20], tfec expression is maintained in trapped late Cbls, extending to the hindbrain/trunk boundary (Fig 7A and 7B).”

• cites [11] S. S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics sourcePartly supported

  The Results section (page unavailable) describes a general failure of fate specification in non-ectomesenchymal neural-crest derivatives and reports that a large proportion of neural-crest cells in sox10 mutants fail to migrate. The claim omits those limits: the paper does not say that all neural-crest derivatives fail to specify or enter migration pathways, and it notes occasional differentiated escapees. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [20] K. A. Dutton, 2001, Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates, Development sourceNot checked — no full text of the source

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

Previous work established tfec as a marker during NC development and iridophore fate choice.

“Our previous work established tfec as a marker during NC development and iridophore fate choice [14, 39].”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourceSupported
• cites [39] J. A. Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec, Developmental Dynamics sourcePartly supported

  The Results and Discussion (‘tfec’; page unavailable) documents tfec expression in cranial and trunk neural crest and in cell clusters the authors describe as strongly suggestive of neural crest-derived iridophores. This supports its use as a developmental marker, but the paper does not establish that tfec marks the point of iridophore fate choice. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Previous work identified the GRN governing maintenance of tfec in the iridophore lineage.

“Finally, our previous work identified the GRN governing maintenance of tfec in the iridophore lineage [14].”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourceSupported

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

Mitfa is able to efficiently rescue melanocyte development in the absence of sox10 through triggering both an Mitfa autoregulatory feedback loop and expression of melanocyte differentiation genes.

“For example, Mitfa is able to efficiently rescue melanocyte development in the absence of sox10 through triggering both an Mitfa autoregulatory feedback loop and expression of melanocyte differentiation genes [13, 25].”

• cites [13] E. R. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development, PLoS Genetics sourcePartly supported

  The Results (pages unavailable) show that Mitfa overexpression induces melanocyte differentiation genes and endogenous mitfa expression; the Discussion interprets the latter as a positive feedback loop that may be indirect. But the paper does not show that Mitfa efficiently rescues melanocyte development in sox10 mutants. Its Results instead describe weak differentiation-gene expression in sox10;mitfa double mutants as Mitfa-independent. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [25] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development, Development sourceNot checked — no full text of the source

May combine two claims.

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

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

• cites [45] L. M. Saunders, 2019, Thyroid hormone regulates distinct paths to maturation in pigment cell lineages, eLife sourcePartly supported

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

  Experimental — accuracy not yet measured

May combine two claims.

Ib(sp) retain melanocyte potential and can be considered both specified melanoblasts and specified iridoblasts.

“In addition, these data support our previous suggestion [14] that these Ib(sp) retain melanocyte potential (i.e. they can be considered both specified melanoblasts as well as specified iridoblasts), and show that melanocyte fate specification is delayed in the tfec mutant.”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourceSupported

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

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].”

• cites [40] C. W. Higdon, 2013, Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin, PLoS One sourcePartly supported

  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

May combine two claims.

In tfec mutants, the ability to express the lineage markers of melanocytes and xanthophores is delayed but otherwise unaffected, whereas ltk expression is completely missing, even in premigratory cells, contrasting the phenotype of sox10 mutants.

“We show here that in tfec mutants the ability to express the lineage markers of melanocytes and xanthophores is delayed but otherwise unaffected, whereas ltk expression is completely missing, even in premigratory cells, also contrasting the phenotype of sox10 mutants [11].”

• cites [11] S. S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development, PLoS Genetics sourcePartly supported

  Split into 2 atoms — 1 confirmed, 0 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).

Sox10 and Sox9b have previously been reported to act redundantly during zebrafish development.

“Sox10 and Sox9b have previously been reported to act redundantly during zebrafish development [13, 22].”

• cites [13] E. R. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development, PLoS Genetics sourcePartly supported

  In Results (‘Sox9b has the properties of Factor Z’; page unavailable), Sox9b knockdown reduces residual melanisation in sox10-mutant zebrafish embryos, showing that Sox9b can contribute when Sox10 is absent. The finding concerns early melanocyte development; the paper does not establish that the two act redundantly throughout zebrafish development. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [22] T. J. Carney, 2006, A direct role for Sox10 in specification of neural crest-derived sensory neurons, Development (Cambridge, England) sourceNot checked — no full text of the source

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

Redundant activities of Tfap2a and Tfap2c are required for NC induction and development of other non-neural ectoderm derivatives in zebrafish embryos.

“Redundant activities of Tfap2a and Tfap2c are required for NC induction and development of other non-neural ectoderm derivatives in zebrafish embryos [52, 53].”

• cites [52] W. Li, 2007, Redundant activities of Tfap2a and Tfap2c are required for neural crest induction and development of other non-neural ectoderm derivatives in zebrafish embryos, Developmental Biology sourceNot checked — no full text of the source
• cites [53] W. Der Wang, 2011, Tfap2a and Foxd3 regulate early steps in the development of the neural crest progenitor population, Developmental Biology sourcePartly supported

  The Discussion reports that Tfap2a and Foxd3 cooperate in neural crest induction in zebrafish embryos. It does not establish redundant activity of Tfap2a and Tfap2c or their requirement for development of other non-neural ectoderm derivatives; a search for Tfap2c found no supporting passage. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Specification and differentiation of the third pigment cell type of zebrafish, the xanthophore, depends upon the paired-box transcription factors Pax3 and Pax7a/b.

“Specification and differentiation of the third pigment cell type of zebrafish, the xanthophore, has been shown to depend upon the paired-box transcription factors Pax3 and Pax7a/b [59, 60].”

• cites [59] J. E. N. Minchin, 2008, Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest, Developmental Biology sourcePartly supported

  The Discussion identifies Pax3 as required for xanthophore specification and links Pax7 reduction to later differentiation defects. It says Pax7 is dispensable for specification, while Pax7b’s role is probable rather than demonstrated. Thus the paper supports distinct roles for Pax3 and Pax7, but not an established dependence on both Pax7a and Pax7b. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [60] H. Nord, 2016, Pax7 is required for establishment of the xanthophore lineage in zebrafish embryos, Molecular Biology of the Cell sourcePartly supported

  The Introduction reports evidence that Pax3 is required for xanthoblast specification, and the Discussion concludes that Pax7a or Pax7b is required to establish xanthophores. The Discussion explicitly leaves unresolved whether Pax7 acts through differentiation, xanthoblast survival, or a fate decision, so the claim that differentiation depends on Pax7a/b is stronger than this paper establishes. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

tfec was recently identified as a likely direct target of Tfap2a/2c, through analysis of gene expression changes in mutants combining different numbers of tfap2a and tfap2c mutant alleles.

“In this context, it is interesting that tfec was recently identified as a likely direct target of Tfap2a/2c, through analysis of gene expression changes in mutants combining different numbers of tfap2a and tfap2c mutant alleles [54].”

• cites [54] C. M. Dooley, 2019, The gene regulatory basis of genetic compensation during neural crest induction, PLOS Genetics sourcePartly supported

  The Results section on neural crest rescue (Fig. 4; page unavailable) compares gene expression across mutants with different numbers of tfap2a and tfap2c alleles and identifies a set enriched for possible AP-2 targets. The passages in our copy do not identify tfec as a likely direct target; a search for “tfec” found no passage that does so. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

tfap2a and foxd3 mutant embryos were identified using previously described genotyping protocols.

“tfap2a and foxd3 mutant embryos were identified using previously described genotyping protocols [29, 79] following imaging and preparation of genomic DNA [71].”

• cites [29] R. A. Stewart, 2006, Zebrafish foxd3 is selectively required for neural crest specification, migration and survival, Developmental Biology sourceSupported
• cites [79] R. D. Knight, 2003, lockjaw encodes a zebrafish tfap2a required for early neural crest development, Development sourceNot checked — no full text of the source

May combine two claims.

Single mutations in tfap2a and foxd3 affect the melanocyte and iridophore lineages, respectively.

“Single mutations in tfap2a and foxd3 affect the melanocyte and iridophore lineages, respectively [28, 29, 55], likely in a manner dependent upon distinct regulatory interactions with Mitfa.”

• cites [28] M. Montero-Balaguer, 2006, The mother superior mutation ablates foxd3 activity in neural crest progenitor cells and depletes neural crest derivatives in zebrafish, Developmental dynamics an official publication of the American Association of Anatomists sourceSupported
• cites [29] R. A. Stewart, 2006, Zebrafish foxd3 is selectively required for neural crest specification, migration and survival, Developmental Biology sourceSupported
• cites [55] R. D. Knight, 2004, Skeletal and pigment cell defects in the lockjaw mutant reveal multiple roles for zebrafish tfap2a in neural crest development, Developmental Dynamics sourceSupported

Classified as probably not a substantial claim.

Singh et al. identified progenitor cells proposed to generate all 3 pigment cell types throughout embryogenesis and into adulthood.

“In a recent single cell lineage tracing study, Singh et al., identified progenitor cells proposed to generate all 3 pigment cell types throughout embryogenesis and into adulthood.”

• cites [12] A. P. Singh, 2016, Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis, Developmental Cell sourcePartly supported

  The Introduction (page not supplied) says Singh et al. induced clones from embryogenesis to early metamorphosis and analyzed them in 2- to 3-month-old fish. The Discussion (page not supplied) says the progenitors generate all three pigment cell types of the adult color pattern. The study supports their multipotency through early metamorphosis, but does not show that they continue generating all three types throughout adulthood. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

Marker analyses and lineage-tracing experiments support the existence of a bipotent melanocyte-iridophore (MI) precursor, the fate of which is influenced by foxd3/mitfa interaction.

“In the absence of foxd3, iridophore numbers are reduced in a manner that is at least partially mitfa-dependent, and marker analyses and lineage-tracing experiments support the existence of a bipotent melanocyte-iridophore (MI) precursor, the fate of which is influenced by this foxd3/mitfa interaction [12, 17].”

• cites [12] A. P. Singh, 2016, Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis, Developmental Cell sourcePartly supported

  In “Progenitors of Adult Pigment Cells Remain Multipotent from Embryogenesis to Early Metamorphosis” (page unavailable), lineage-traced MI clones support a progenitor capable of producing both melanophores and iridophores. The cited paper does not establish that a foxd3/mitfa interaction influences that progenitor’s fate; a search of our copy for foxd3 found no such evidence. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [17] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest, Developmental Biology sourceSupported

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

tfap2a and mitfa work in parallel to promote melanocyte differentiation.

“While tfap2a and mitfa work in parallel to promote melanocyte differentiation [27], foxd3 has been suggested to repress mitfa transcription [16, 56], at least in some contexts [14].”

• cites [27] H. E. Seberg, 2017, TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF, PLOS Genetics sourceSupported

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

Mitfa and Tfec have the potential to physically interact as a heterodimer.

“Notably, Mitfa and Tfec have the potential to physically interact as a heterodimer [57, 58], which adds an additional layer of complexity when attempting to elucidate the mechanism of cell fate choice.”

• cites [57] T. J. Hemesath, 1994, microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family, Genes & development sourcePartly supported

  The ‘Mi is a member of a discrete family of b-HLH-ZIP factors’ section reports that Mi and TFEC form stable DNA-binding heterodimers, supporting the potential for this type of interaction. The paper tests Mi and TFEC in vitro, however, rather than directly testing the specifically named Mitfa and Tfec proteins. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [58] V. Pogenberg, 2012, Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF, Genes and Development sourceSupported

May combine two claims.

sox10 mutants still strongly express tfec (as well as ltk) within trapped premigratory progenitors, yet fail to produce Ib(sp).

“In contrast, sox10 mutants still strongly express tfec (as well as ltk) within trapped premigratory progenitors, yet fail to produce Ib(sp) [14].”

• cites [14] K. Petratou, 2018, A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest, PLoS Genetics sourcePartly supported

  The Results section (“sox10 maintains tfec expression in iridoblasts undergoing specification”; page unavailable) reports prominent tfec expression in premigratory progenitors of sox10 mutants, but no tfec expression in migrating iridoblasts, including ib(sp). It cites earlier ltk studies as evidence that progenitors remain trapped and iridophore specification fails, but our copy does not establish that ltk itself is strongly expressed in those trapped progenitors. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

Work in mice has demonstrated at least partial functional redundancy between the two genes in the mammalian RPE and a cooperative relationship between mitfa and tfec in the context of eye melanisation and choroid fissure closure.

“Work in mice has demonstrated at least partial functional redundancy between the two genes in the mammalian RPE [47] and a cooperative relationship between mitfa and tfec has also been recently indicated in the context of eye melanisation and choroid fissure closure [48].”

• cites [47] K. Bharti, 2012, A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development, PLoS Genetics sourceSupported
• cites [48] K. L. Sinagoga, Mitf-family transcription factor function is required within cranial neural crest cells to promote choroid fissure closure, DevelopmentNot matched

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

May combine two claims.

Melanocyte development recovers by 4 dpf; mutant embryos present with the same number of melanised cells along their trunk as WT or heterozygous siblings.

“Melanocyte development recovers, and by 4 dpf mutant embryos present with the same number of melanised cells along their trunk as their WT or heterozygous siblings (Fig 4J–4L and S1 Table).”

May combine two claims.

No changes were detected in the number of ventral stripe iridophores between sox10:tfec-injected and uninjected embryos, nor in ubi:tfec-injected embryos.

“Supporting our conclusion that these alterations are not biologically meaningful, we detect changes neither in the number of ventral stripe iridophores between sox10:tfec-injected and uninjected embryos, nor in ubi:tfec-injected embryos.”

May combine two claims.

tfec is a consistent marker of mature iridophores, but not melanocytes nor xanthophores.

“In conclusion, at the detection threshold of our expression studies, tfec is a consistent marker of mature iridophores, but not of melanocytes nor xanthophores.”

• Also stated in: § tfec is a candidate for the iridophore master regulator · plain

  “tfec is a marker of differentiated iridophores, but not xanthophores.”

May combine two claims.

Our mutants presented with delayed differentiation of both NC-derived and RPE melanocytes, as well as of xanthophores.

“In addition to a complete absence of iridoblasts and mature iridophores our mutants also, surprisingly, presented with delayed differentiation of both NC-derived and RPE melanocytes, as well as of xanthophores.”

May combine two claims.

In tfec mutants, mitfa expression was restricted to premigratory late Cbls, whereas mitfa-positive melanoblasts occupied the medial and lateral migratory pathways in WT and heterozygous siblings.

“Strikingly, mitfa expression was restricted to premigratory late Cbls in tfec mutants, whereas mitfa-positive melanoblasts occupied the medial and lateral migratory pathways in WT and heterozygous siblings (Fig 5M and 5N).”

May combine two claims.

Developing oligodendrocytes in the CNS appear unaffected in their numbers and distribution in tfec mutants.

“Likewise, developing oligodendrocytes in the CNS appear unaffected in their numbers and distribution.”

May combine two claims.

At 30 hpf, the trunk of tfec mutants was occupied by a relatively small number of mitfa-positive NC derivatives, whereas in the tail cells had still not entered the migratory pathways.

“The trunk was occupied by a relatively small number of mitfa-positive NC derivatives, whereas in the tail of mutants cells had still not entered the migratory pathways (Fig 5O and 5P).”

May combine two claims.

At 4 dpf, the number of trunk and tail melanocytes is not significantly altered along the dorsal, ventral or lateral stripes, but there is a statistically significant increase in the number of melanocytes on the dorsal head.

“In embryos treated with melanin-concentrating hormone (MCH) to facilitate their quantitation, the number of trunk and tail melanocytes is not significantly altered along the dorsal, ventral or lateral stripes (J,K, red region; quantitated in L), however there is a statistically significant increase in the number of melanocytes located on the dorsal head (J,K, green region; quantitated in L).”

May combine two claims.

Approximately 45% of mutants displayed eye iridophore rescue, 3% showed rescue in the trunk only and 6% showed rescue both in the eyes and trunk; iridophores were never observed in uninjected tfecvc60 sibling larvae.

“Approximately 45% of mutants displayed eye iridophore rescue, 3% showed rescue in the trunk only and 6% showed rescue both in the eyes and trunk (n = 62). By contrast, iridophores were never observed in uninjected tfecvc60 sibling larvae (n = 58).”

May combine two claims.

Gain of function experiments in WT embryos suggest that the iridophore number is not altered upon supplementation of tfec, whether ubiquitously in the embryo using the ubi promoter, or specifically in NCCs using the sox10 promoter.

“Our additional gain of function experiments in WT embryos suggest that the iridophore number is not altered upon supplementation of tfec, whether ubiquitously in the embryo using the ubi promoter, or specifically in NCCs using the sox10 promoter.”

May combine two claims.

At 24 hpf, laterally migrating Xbl(sp) are restricted to more anterior regions and precursors located in the head are reduced in tfec mutants compared to their WT siblings.

“At 24 hpf, laterally migrating Xbl(sp) (arrows) are restricted to more anterior regions and precursors located in the head are reduced in tfec mutants (V,X) compared to their WT siblings (U,W).”

• AssumptionRests on an assumption: gch2 and aox5 are xanthophore lineage markers.

May combine two claims.

The effect of mitfa mutation on tfec expression persists at 30 hpf and is also observed by RNAscope; persistence of tfec expression is seen in cells migrating through the lateral pathway.

“This effect persists at 30 hpf and is also observed by a complementary, and more sensitive fluorescent transcript detection technique, RNAscope; we see persistence of tfec expression in cells migrating through the lateral pathway (Fig 8E–8F’).”

May combine two claims.

At 30 hpf, the numbers and distributions of mitfa-positive late Cbls are indistinguishable between tfec mutants and WT or heterozygous siblings, but the delay in Mb(sp) migration remains distinguishable towards the tail.

“At 30 hpf, the numbers and distributions of mitfa-positive late Cbls (arrowheads) are indistinguishable between tfec mutants and WT or heterozygous siblings (O,P), but the delay in Mb(sp) migration (arrows) remains distinguishable towards the tail.”

May combine two claims.

Injection of wild-type tfec cDNA under its own promoter could rescue, albeit only partially, the iridophore phenotype in tfec mutants, with rescued iridophores in the eye observed more often than in the trunk.

“Injection of wild-type tfec cDNA under its own promoter could rescue, albeit only partially, the iridophore phenotype in tfec mutants, with rescued iridophores in the eye observed more often than in the trunk.”

May combine two claims.

This effect was not observed in genotyped siblings, heterozygous for one or both alleles nor those homozygous for a single mutant allele.

“This effect was not observed in genotyped siblings, heterozygous for one or both alleles nor those homozygous for a single mutant allele.”

May combine two claims.

At 24 hpf, mitfa marker expression is restricted to an increased number of premigratory Cbls and is undetectable in migrating Mb(sp) in tfec mutants.

“At 24 hpf, mitfa marker expression is restricted to an increased number of premigratory Cbls (arrowheads) and is undetectable in migrating Mb(sp) (arrows) in tfec mutants (M,N).”

May combine two claims.

Loss of sox9b alone caused an apparent delay in induction of tfec expression in posterior NC yet, despite this alteration in the pattern, expression remained strongly present in the progenitor population.

“Notably, loss of sox9b alone caused an apparent delay in induction of tfec expression in posterior NC yet, despite this alteration in the pattern, expression remained strongly present in the progenitor population.”

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

The increase in sox10:dsRed control-injected compared to uninjected embryos is likely biologically irrelevant.

“which we attribute to a likely biologically irrelevant increase in the cell numbers of sox10:dsRed control-injected compared to uninjected embryos.”

May combine two claims.

tfec was found to be co-expressed with mitfa in specified iridophore (Ib(sp)) cells, proposed to be able to at least give rise to melanocytes and iridophores, but not in mature melanocytes.

“Interestingly, tfec was found to be co-expressed with mitfa in specified iridophore (Ib(sp)) cells, proposed to be able to at least give rise to melanocytes and iridophores, but not in mature melanocytes.”

May combine two claims.

Either Tfec is insufficient to alone drive NSCs to adopt the iridophore fate, or in wild-type embryos any elevated specification of the iridophore lineage is compensated by cell regulation mechanisms.

“This indicates that either Tfec is insufficient to alone drive NSCs to adopt the iridophore fate, or that in wild-type embryos any elevated specification of the iridophore lineage is compensated by cell regulation mechanisms.”

May combine two claims.

The melanocyte specification delay phenotype may help explain observations of tfec-dependent melanocyte rescue upon inducing gain of function in homozygous mitfa mutants, suggesting that tfec can, when overexpressed, functionally substitute for mitfa.

“The melanocyte specification delay phenotype that we describe may also help explain the otherwise rather surprising observations of tfec-dependent melanocyte rescue upon inducing gain of function in homozygous mitfa mutants, suggesting that tfec can, when overexpressed, functionally substitute for mitfa.”

May combine two claims.

The subsequent recovery of normal melanocyte numbers indicates that compensatory factors allow melanoblasts to be specified, albeit with a short delay.

“The subsequent recovery of normal melanocyte numbers makes clear that compensatory factors allow melanoblasts to be specified, albeit with a short delay.”

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

tfec mutants have been generated independently, but that report did not examine NC-related defects, focusing instead on deficiencies in hematopoiesis.

“Although tfec mutants have been generated independently [46], that report did not examine NC-related defects, focusing instead on deficiencies in hematopoiesis.”

• cites [46] C. B. Mahony, 2016, tfec controls the hematopoietic stem cell vascular niche during zebrafish embryogenesis, Blood sourceNot checked — no full text of the source

May combine two claims.

We have shown a Tfec-dependent autoregulatory loop in iridophore development, which may explain why rescue of the iridophore phenotype in tfec mutants is very inefficient.

“We have shown a Tfec-dependent autoregulatory loop in iridophore development, which may explain why rescue of the iridophore phenotype in tfec mutants is very inefficient.”

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

The tfec mutations were independently generated in two laboratories as identical 6 base pair deletions in two different wild-type strains.

“Our two laboratories independently generated identical 6 base pair deletions (the tfecba6 and tfecvc58 alleles) in two different wild-type strains, WIK and NHGRI-1, in addition to frameshifted alleles (Fig 3A).”

May combine two claims.

Premigratory NCCs appear to be present in normal numbers and show unaltered timing of downregulation of sox10 expression in tfec mutants.

“These in situs provide evidence that premigratory NCCs appear to be present in normal numbers and show unaltered timing of downregulation of sox10 expression.”

May combine two claims.

Both injected G0 fish and a single escaper F1 adult carrying biallelic frameshift mutations lack iridophores in patches or whole, indicating an ongoing role in adult iridophores.

“Moreover, both injected (G0) fish raised to adulthood, as well as a single ‘escaper’ surviving F1 adult carrying biallelic frameshift mutations, lack iridophores in patches or in whole, indicating an ongoing role in adult iridophores (S1A–S1C Fig).”

May combine two claims.

Our loss of function data are strongly suggestive of tfec being required for iridophore development, as mitfa is for melanocytes.

“Our loss of function data are strongly suggestive of tfec being required for iridophore development, as mitfa is for melanocytes (Fig 1).”

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

We generated mutations in tfec using CRISPR/Cas9, obtaining several alleles which displayed essentially identical phenotypes.

“We generated mutations in tfec using a CRISPR/Cas9 approach, obtaining several alleles which displayed essentially identical phenotypes.”

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

sox10 and sox9b have been shown to be functionally redundant in DRG sensory neuron development.

“As members of the same SoxE group, it is not surprising that sox10 and sox9b have been shown to be functionally redundant in DRG sensory neuron development [22].”

• cites [22] T. J. Carney, 2006, A direct role for Sox10 in specification of neural crest-derived sensory neurons, Development (Cambridge, England) sourceNot checked — no full text of the source

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

Homozygous mutants of each genotype show highly consistent phenotypes across experimental replicates.

“This persistence differed in severity and duration between different mutants, but homozygous mutants of each genotype show highly consistent phenotypes across experimental replicates.”

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

The same study demonstrated the functional compensation of Tfap2a by Tfap2c, since in tfap2a mutants just a single copy of tfap2c was sufficient to maintain tfec expression at WT levels and rescue NC specification.

“The same study demonstrated the functional compensation of Tfap2a by Tfap2c, since in tfap2a mutants just a single copy of tfap2c was sufficient to maintain tfec expression at WT levels and rescue NC specification.”

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

Screening was based on iridophore phenotype and confirmed by a High Resolution Melt Analysis (HRMA) assay for molecular screening.

“Screening was based on iridophore phenotype and confirmed by a High Resolution Melt Analysis (HRMA) assay for molecular screening (see below).”

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

To detect CRISPR/Cas9-mediated mutagenesis, at least 8 WT reference samples were included in the analysis.

“To detect CRISPR/Cas9-mediated mutagenesis, at least 8 WT reference samples were included in the analysis.”

May combine two claims.

Our data provide the first evidence for Tfec function in those early progenitors, since we show it is required to drive early expression of ltk at the Cbl stage, as well as fate specification of the iridophore lineage from these multipotent progenitors.

“Furthermore, our data provide the first evidence for Tfec function in those early progenitors, since we show it is required to drive early expression of ltk at the Cbl stage, as well as fate specification of the iridophore lineage from these multipotent progenitors.”

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

Double mutants for tfap2a;foxd3 have been shown to eliminate induction of NC.

“Double mutants for tfap2a;foxd3 have been shown to eliminate induction of NC [52].”

• cites [52] W. Li, 2007, Redundant activities of Tfap2a and Tfap2c are required for neural crest induction and development of other non-neural ectoderm derivatives in zebrafish embryos, Developmental Biology sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Decades ago, it was proposed that melanocytes share a common origin with iridophores and xanthophores from a pigment-restricted precursor, which we would term a chromatoblast (Cbl(late)).

“Decades ago, it was proposed that melanocytes share a common origin with iridophores and xanthophores from a pigment-restricted precursor [15], which we would term a chromatoblast (Cbl(late)).”

• cites [15] J. T. Bagnara, 1979, Common origin of pigment cells, Science sourceNot checked — no full text of the source

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

Interactions between Pax3 and Mitf have been demonstrated in mammal melanocyte development.

“Intriguingly, interactions between Pax3 and Mitf have been demonstrated in mammal melanocyte development [61].”

• cites [61] D. Lang, 2005, Pax3 functions at a nodal point in melanocyte stem cell differentiation, Nature sourceNot checked — no full text of the source

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

sox10 is required for mitfa expression in late Cbls, and mitfa can promote melanocyte fate in the absence of sox10 if misexpressed.

“Furthermore, this requirement of pigment cells for sox10 manifests itself apparently in different ways; sox10 is required for mitfa expression in late Cbls, and mitfa can promote melanocyte fate in the absence of sox10 if misexpressed [25].”

• cites [25] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development, Development sourceNot checked — no full text of the source

May combine two claims.

Tfec is necessary for both correct and timely chromatoblast specification, either via generation of a tripotent progenitor (Cbl(late)) and/or through independent roles in each chromatophore lineage arising from a multipotent progenitor (Cbl(early)).

“Thus, Tfec is necessary for both correct and timely chromatoblast specification, either via generation of a tripotent progenitor (Cbl(late)) and/or through independent roles in each chromatophore lineage arising from a multipotent progenitor (Cbl(early)).”

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

WIK or NHGRI-1 wild-type embryos served as controls as indicated in each figure.

“WIK or NHGRI-1 wild-type embryos served as controls as indicated in each figure.”

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

At least some zebrafish xanthoblasts express mitfa.

“At least some zebrafish xanthoblasts express mitfa [62] and we show that loss of tfec delays xanthoblast migration, raising the question of whether interplay between Mitfa and Tfec might be important for xanthoblast fate choice.”

• cites [62] D. M. Parichy, 2000, An orthologue of the kit-related gene fms is required for development of neural crest-derived xanthophores and a subpopulation of adult melanocytes in the zebrafish, Danio rerio, Development sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S2 (checkable).

Our study provides additional support for the progressive fate restriction of multipotent stem cells.

“To conclude, our study contributes to deepening our understanding of the molecular basis of NC and pigment cell development in zebrafish, providing additional support for the progressive fate restriction of multipotent stem cells.”