A systems biology approach uncovers the core gene regulatory network governing iridophore fate choice from the neural crest

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

mitfa is expressed initially in almost all neural crest cells.

“with mitfa being expressed initially in almost all NC cells [32]”

• cites [32] K. Curran, 2009, Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf. sourceDisputed

  The Introduction (page unavailable) says that, in wild-type zebrafish, mitfa is expressed only in a subset of neural crest cells, not almost all of them. The Results section on migratory neural crest cells (page unavailable) also reports that mitfa and Foxd3 are largely mutually exclusive even at the earliest stages of reporter expression. (LLM, conf=0.87)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

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

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].”

• cites [48] C. Higdon, 2013, Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin. sourceDisputed

  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

May combine two claims.

tfec is expressed in early neural crest cells and later in a pattern reminiscent of iridophores, making it a strong candidate master regulator of iridophore development.

“tfec, is expressed in early NCCs, but later in a pattern strikingly reminiscent of iridophores, and is a strong candidate for a master regulator of iridophore development ([29] and Petratou et al., in prep.).”

• cites [29] J. Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec. sourcePartly supported

  The Results and Discussion section (‘tfec’; page unavailable) reports tfec expression in early neural crest cells and a later pattern strongly suggestive of iridophore cells. It does not call tfec a candidate master regulator of iridophore development; the Summary and Conclusions section says functional studies remain to be done. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Petratou et al., in prep.Not matched

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

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

A core gene regulatory network for melanocyte fate specification has been identified.

“we have identified a core GRN for melanocyte fate specification [22].”

• cites [22] E. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development sourceSupported

Classified as probably not a substantial claim.

Constitutively activated Ltk signalling drives neural crest cells to adopt an iridophore fate.

“constitutively activated Ltk signalling driving NCCs to adopt an iridophore fate [9], [27], [28].”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourcePartly supported

  The Discussion (Figure 8; page unavailable) proposes that Ltk signalling, together with Sox10, initiates iridoblast specification from neural crest cells. The paper supports a role for Ltk in that fate choice, but the passages do not show that constitutively activated Ltk signalling drives it. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [27] F. Rodrigues, 2012, A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors sourceSupported
• cites [28] A. Fadeev, 2016, Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival. sourceNot checked — no full text of the source

May combine two claims.

Sox10 and the receptor tyrosine kinase Ltk play crucial roles in iridophore specification.

“In the case of iridophores, as well as Sox10, the receptor tyrosine kinase Leukocyte tyrosine kinase (Ltk) plays a crucial role”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourceSupported
• cites [27] F. Rodrigues, 2012, A Simple, Highly Visual in Vivo Screen for Anaplastic Lymphoma Kinase Inhibitors sourcePartly supported

  The Introduction (page unavailable) states that Ltk acts cell-autonomously in zebrafish iridophore fate specification and that ltk mutants lack iridophores. The Results and Discussion and Figure 2 legend (pages unavailable) use a sox10 promoter to drive expression in neural crest cells, but do not establish that Sox10 itself is crucial for iridophore specification. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [28] A. Fadeev, 2016, Zebrafish Leucocyte tyrosine kinase controls iridophore establishment, proliferation and survival. sourceNot checked — no full text of the source

May combine two claims.

ltk expression shows two phases: one in early neural crest development representing a multipotent chromatoblast-like progenitor, and a second in the definitive iridophore lineage.

“We have shown that ltk expression appears to show two phases, one in early NC development which we propose represents a multipotent, chromatoblast-like progenitor, and a second in the definitive iridophore lineage [9].”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourceSupported

May combine two claims.

In the WT context, Sox10 and Tfec stay upregulated in mature pigment cells, Ltk and Pnp4a increase and reach positive steady-state, and Mitfa initially rises rapidly then drops to a lower level at differentiation stages.

“Specifically, in the WT context, it was crucial that Sox10 and Tfec stay upregulated in mature pigment cells, i.e. reach a positive steady-state. Similarly, Ltk and Pnp4a concentrations should increase and reach a positive steady-state. Mitfa levels should initially rise rapidly, reflecting the widespread expression of mitfa in all pigment cell progenitors [32], but should then drop to a distinct”

• cites [32] K. Curran, 2009, Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf. sourcePartly supported

  The Introduction says Mitf is among the earliest genes expressed in melanoblast precursors, and the Results section on the mitfa:gfp line documents early mitfa expression in cells identified as melanoblasts (no page numbers supplied). These passages support early expression, but not the cited phrase’s claim that mitfa is expressed in all pigment-cell progenitors. The citation is attached to that phrase, not to the surrounding steady-state predictions. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

For all three versions of model A, simulation of loss of Sox10 function predicted maintenance of tfec and ltk, consistent with observations that tfec+;ltk+ progenitors remain trapped in the dorsal trunk and tail.

“For all three versions of model A, simulation of loss of Sox10 function appropriately predicted maintenance of tfec (Fig 3B) and of ltk [9], consistent with observations that tfec+;ltk+ progenitors remain trapped in the dorsal trunk and tail.”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourceSupported

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.

May combine two claims.

Tfec is expressed much earlier than mitfa and ltk in neural crest development, being detected widely in early NCCs.

“However, Tfec is expressed much earlier than mitfa and ltk in NC development, being detected widely in early NCCs [29]”

• cites [29] J. Lister, 2011, Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec. sourcePartly supported

  The Results and Discussion section on tfec reports expression in a few cranial neural crest cells at the 8-somite stage, expanding to include trunk neural crest by 10–12 somites. This supports early, spreading neural crest expression, but the paper does not establish that tfec appears much earlier than mitfa or ltk, and describes only a few neural crest cells at its first detection. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

In melanocytes, Sox10 acts together with Wnt signalling to establish mitfa expression, but sox10 is then strongly downregulated; maintenance of expression is thought to promote multipotency and delay differentiation.

“In melanocytes, Sox10 acts together with Wnt signalling to establish mitfa expression, but sox10 is then strongly downregulated, and indeed maintenance of expression is thought to promote multipotency and delay differentiation [22].”

• cites [22] E. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development sourcePartly supported

  The Results report that Sox10 induces mitfa and is rapidly downregulated in differentiating zebrafish melanocytes. The Discussion proposes that continued Sox10-mediated repression may delay differentiation and maintain precursor multipotency. The Results discuss Wnt-related factors as possible regulators of sox10, but do not establish that Wnt signalling acts together with Sox10 to initiate mitfa expression in melanocytes. Page numbers are unavailable in our copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

May combine two claims.

The tfecba6 allele is a recessive loss-of-function allele generated by CRISPR/Cas9, with a 6-nucleotide deletion in exon 7 predicted to interrupt critical spacing in the second alpha-helix of the dimerization domain.

“The tfecba6 allele shows recessive loss of function, and was generated via CRISPR/Cas9 directed mutagenesis. It corresponds to the deletion of 6 nucleotides from the 7th exon of the gene. This deletion of two amino acids is predicted to interrupt critical spacing in the second alpha-helix of the dimerization domain [35].”

• cites [35] V. Pogenberg, 2012, Restricted leucine zipper dimerization and specificity of DNA recognition of the melanocyte master regulator MITF sourcePartly supported

  The section ‘An unusual out-of-register leucine zipper restricts MITF assembly partners’ reports that a three-residue shift in MITF’s leucine zipper is crucial to its dimerization specificity. This supports the structural rationale for the cited prediction, but the paper does not examine the tfecba6 allele or predict the effect of its two-amino-acid deletion. It also does not establish the allele’s recessive loss of function, CRISPR/Cas9 origin, or exon 7 location. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

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

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”

• cites [48] C. Higdon, 2013, Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin. sourcePartly supported

  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

Classified as probably not a substantial claim.

Previously published experimental data show that maintenance of mitfa is restricted to melanocytes.

“These predictions are in contrast to previously published experimental data, showing that maintenance of mitfa is restricted to melanocytes [21], [22], although lower level mitfa expression has been detected by RNA-seq in differentiated iridophores [48].”

• cites [21] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development. sourceNot checked — no full text of the source
• cites [22] E. Greenhill, 2011, An Iterative Genetic and Dynamical Modelling Approach Identifies Novel Features of the Gene Regulatory Network Underlying Melanocyte Development sourcePartly supported

  The Discussion (page unavailable) reports evidence that Mitfa helps maintain mitfa expression during melanocyte differentiation. It does not establish that maintained expression is restricted to melanocytes; its discussion of iridophores concerns cells that initially turn on mitfa, not whether expression persists in them. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

mitfa and ltk are activated widely by 22 hpf, with pnp4a following soon after.

“However, mitfa and ltk are activated widely by 22 hpf [9], [19], with pnp4a following soon after [15].”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourceSupported
• cites [19] J. Lister, 1999, nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. sourceNot checked — no full text of the source
• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

In the sox10 mutant context, Sox10, Tfec and Ltk acquire positive values, but Mitfa and Pnp4a do not become upregulated.

“In the sox10 mutant context we asked that Sox10, Tfec and Ltk acquire positive values, as expression has been identified in trapped chromatoblasts ([9], [26] and this work), however at no point do either Mitfa or Pnp4a become upregulated.”

• cites [9] S. Lopes, 2008, Leukocyte Tyrosine Kinase Functions in Pigment Cell Development sourceSupported
• cites [26] K. Dutton, 2001, Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. sourceNot checked — no full text of the source

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

NC-specific expression of sox9a, sox9b and sox10 depends on both foxd3 and tfap2a function in zebrafish embryos.

“NC-specific expression of sox9a, sox9b and sox10 has been shown to depend on both foxd3 and tfap2a function in zebrafish embryos [49].”

• cites [49] B. Arduini, 2009, Genetic ablation of neural crest cell diversification. sourceSupported

May combine two claims.

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].”

• cites [48] C. Higdon, 2013, Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin. sourcePartly supported

  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

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

Previous analyses using a foxd3:gfp transgenic line suggested that Foxd3 is expressed in mature iridophores.

“Previous analyses using a foxd3:gfp transgenic line have suggested that Foxd3 is expressed in mature iridophores [32].”

• cites [32] K. Curran, 2009, Foxd3 controls melanophore specification in the zebrafish neural crest by regulation of Mitf. sourceSupported

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

A recent study suggests FoxD3 has a new role as a pioneer factor in neural crest development.

“a recent study suggests a new role for FoxD3, namely as a pioneer factor in neural crest development [56]”

• cites [56] D. Gavriouchkina, 2017, From pioneer to repressor: Bimodal foxd3 activity dynamically remodels neural crest regulatory landscape in vivo sourceSupported

May combine two claims.

Neither mutant Mitfa nor mutant Tfec alters pnp4a transcript levels compared to GFP overexpression.

“As expected, neither mutant Mitfa nor mutant Tfec altered pnp4a transcript levels, compared to overexpression of GFP.”

May combine two claims.

Compared to models A1-A3, a broader range of model B trials achieved high scores, with absolute values higher than those attainable through models A1-A3.

“Specifically, compared to models A1-A3, a broader range of model B trials achieved high scores, with absolute values higher than those attainable through models A1-A3 (Fig 5; Fig 6).”

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.
• AssumptionThe authors state this as an assumption: Factor R is absent in multipotent progenitors of the ARPT at t = 18 hours.

May combine two claims.

At 48 hpf, tfec and mitfa show discrete expression, confirming that tfec definitively marks differentiating iridophores.

“Co-expression analyses using RNAscope showed discrete expression of tfec and mitfa (Fig 1I and 1I’; S3 Table), confirming that tfec expression at this stage definitively marked differentiating iridophores.”

May combine two claims.

In ltk mutants at 18 and 24 hpf, tfec expression in the premigratory NCC domain, specified iridoblasts in the posterior dorsal trunk, and the medial migration pathway is unaffected.

“Specifically, the premigratory NCC domain as well as specified iridoblasts in the posterior dorsal trunk and on the medial migration pathway of the trunk were unaffected in all examined embryos.”

May combine two claims.

In sox10 mutants, pnp4a is largely absent, with weak expression persisting in a few premigratory NCCs at 30 hpf and in rare escaper cells in iridophore positions at 48 hpf.

“In sox10 mutants, pnp4a is largely absent, although weak expression persists in a few premigratory NCCs (K, arrowheads) at 30 hpf, and in rare escaper cells in iridophore positions at 48 hpf (O, asterisks).”

May combine two claims.

tfec mutants display a 35% decrease in tfec-expressing cells along the migratory pathways and a 45% decrease in the developing lateral patches compared to WT siblings.

“Specifically, tfec mutants displayed a 35% and a 45% decrease in the number of tfec-expressing cells along the migratory pathways and in the developing lateral patches respectively, compared to WT siblings.”

May combine two claims.

By 30 hpf, pnp4a transcripts are detectable in differentiating melanocytes clustered behind the otic vesicle and in melanised cells of the head, in a pattern similar to mitfa but not tfec.

“By 30 hpf, this pattern is still detectable, and indeed now pnp4a transcripts are detectable in differentiating melanocytes clustered behind the otic vesicle, as well as in melanised cells of the head, in a pattern similar to mitfa, but not tfec, expression (Fig 4B, 4E and 4H and insets).”

May combine two claims.

At 24 hpf, prominent tfec expression is retained in a small subset of precursors scattered over the spinal cord and in presumed iridoblasts migrating through the medial pathway.

“prominent tfec expression was retained in a small subset of precursors scattered over the spinal cord (Fig 1D), as well as in presumed iridoblasts migrating through the medial pathway (Fig 1Ei).”

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

tfecba6 homozygotes show nearly complete loss of iridophores and failure to inflate the swim-bladder, identical to frameshift mutations in the DNA binding domain.

“Phenotypically, homozygotes show a phenotype (nearly complete loss of iridophores and failure to inflate the swim-bladder) identical to those resulting from frameshift mutations in the DNA binding domain (Petratou et al., in prep.).”

• cites Petratou et al., in prep.Not matched

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

May combine two claims.

By 48 hpf, the pattern of pnp4a strikingly resembles that of tfec, with both transcripts detected in iph positions, whereas mitfa is expressed in melanised cells of the head and dorsal, lateral and ventral stripes.

“By 48 hpf, the pattern of pnp4a strikingly resembled that of tfec, with both transcripts detected in iph positions, consistent with previously reported data [15], whereas mitfa was expressed in melanised cells of the head and of the dorsal, lateral and ventral stripes (Fig 4C, 4F and 4I).”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

Neither WT nor mutant Tfec is sufficient to drive pnp4a expression at 6 hours post-injection.

“Interestingly, neither WT nor mutant Tfec is sufficient to drive pnp4a expression at this stage.”

May combine two claims.

Functional Sox10, but not null Sox10, ectopically activates expression of endogenous tfec.

“Our data clearly showed that functional Sox10, but not the null version of Sox10, ectopically activated expression of endogenous tfec (Fig 3O).”

• Also stated in: § sox10 maintains tfec expression in iridoblasts undergoing specification · plain

  “(O) Overexpression of WT sox10 mRNA results in an increased number of tfec transcripts, compared to overexpression of null sox10 mRNA.”

May combine two claims.

In models A2 and A3, ltk expression was correctly predicted to remain undetectable throughout iridophore specification and differentiation, while tfec and pnp4a were gradually diminished.

“In models A2 and A3, ltk expression was correctly predicted to remain undetectable throughout iridophore specification and differentiation, while tfec and pnp4a were gradually diminished.”

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.

May combine two claims.

sox10 expression is readily detectable by RNAscope in all stages of specified iridoblasts and by WISH in mature iridophores.

“with expression of the gene being readily detectable by RNAscope in all stages of specified iridoblasts and by WISH in mature iridophores.”

May combine two claims.

At 30 hpf, the pattern of tfec and pnp4a expression in the dorsal posterior trunk and overlying the RPE shows strong similarities, while mitfa transcript is absent from the latter region.

“In addition, at this stage, the pattern of tfec and pnp4a expression in both the dorsal posterior trunk as well as overlying the RPE showed strong similarities; mitfa transcript is absent from the latter region (Fig 4B, 4E and 4H).”

May combine two claims.

At 24 hpf, clusters of cells just posterior to the otic vesicle and numerous cells on the medial migration pathway are positive for both mitfa and pnp4a, but only sparsely positive for tfec.

“Specifically, we see clusters of cells just posterior to the otic vesicle and numerous cells on the medial migration pathway in the expression patterns of both mitfa and pnp4a, although both these regions are only sparsely positive for tfec (Fig 4A, 4D and 4G and insets).”

May combine two claims.

This partial reduction in pnp4a-positive cells in tfec mutants was also observed at 24 hpf and principally affected cells in the ventral trunk and premigratory NC.

“This partial reduction was also observed at 24 hpf (Petratou et al., in prep.), and principally affected cells in the ventral trunk and premigratory NC.”

• cites Petratou et al., in prep.Not matched

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

May combine two claims.

At 30 hpf, mitfa mutants retain a distinct group of dorsally located cells patterned in an ib(df)-like manner along the posterior trunk and anterior tail, while medially migrating cells are almost absent.

“Whereas in the former stage very few pnp4a expressing cells persisted along the posterior trunk of homozygous mutants (S1A–S1B’ Fig), in the latter a distinct group of dorsally located cells patterned in a ib(df)-like manner along the posterior trunk and anterior tail region were retained. In contrast, medially migrating cells were almost absent (Fig 4M).”

May combine two claims.

At 30 hpf in the ARPT, tfec and mitfa expression is non-overlapping in the majority of cells (76.9%), a statistically significant drop in co-expression.

“co-expression analyses via RNAscope revealed that expression of tfec and mitfa has now resolved to be non-overlapping in the majority of cells (76.9%) (Fig 1I and 1I’; S3 Table), which corresponds to a statistically significant drop in the proportion of cells co-expressing the two genes.”

May combine two claims.

At 18 hpf, in premigratory NCCs of the dorsal ARPT, sox10 and tfec transcripts are detectable, but ltk, mitfa and pnp4a are not.

“Using chromogenic WISH, sox10 and tfec, but not ltk, mitfa or pnp4a transcripts were detectable in this population of cells (Fig 1B), allowing us to approximate the initial conditions for our simulations.”

May combine two claims.

At 30 hpf, mitfa and tfec mutants retain only a subset of WT pnp4a expression; remaining cells in mitfa mutants display an ib(df) pattern and in tfec mutants a melanoblast pattern.

“At 30 hpf, mitfa (M) and tfec (L) mutants retain only a subset of the WT pnp4a expression; remaining cells in the former display a ib(df) pattern and in the latter a melanoblast pattern (M, L, arrows).”

May combine two claims.

Co-expression of mitfa with tfec occurs at 24 hpf, but is not detectable at 30 hpf and 48 hpf.

“Even with this technique, notably more sensitive than conventional chromogenic WISH, we confirmed that co-expression of mitfa with the iridoblast marker, tfec, occurred at 24 hpf, but such overlap was not detectable at 30 hpf and at 48 hpf (Fig 1I).”

May combine two claims.

The lack of ltk expression in tfec mutants remains identifiable at 30 hpf and persists until at least 48 hpf.

“This phenotype remained clearly identifiable at 30 hpf and persisted until at least 48 hpf (Fig 3I–3L).”

May combine two claims.

Model A2 was able to predict those features correctly, although only limited subsets of parameters achieved admissible outputs.

“Model A2 was found able to predict those features correctly, although only limited subsets of parameters achieved admissible outputs (Fig 5H).”

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.

May combine two claims.

Models A2 and A3, but not A1, predict that dorsally trapped progenitors continue to express sox10 upon loss of Sox10 function.

“However, it has been previously shown that dorsally trapped progenitors continue to express sox10 upon loss of Sox10 function [26], a feature predicted successfully by models A2 and A3, but not by A1.”

• cites [26] K. Dutton, 2001, Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. sourceNot checked — no full text of the source

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.

May combine two claims.

At 30 hpf, sox10 mutants lack tfec expression in medially migrating iridoblasts and show an anteriorly expanded multipotent progenitor domain.

“At 30 hpf, sox10 mutants (B) lack tfec expression in medially migrating iridoblasts (A, arrows) and instead show a striking anteriorly expanded multipotent progenitor domain (B, arrowheads).”

May combine two claims.

Overexpression of WT Mitfa results in ectopic activation of pnp4a at 6 hours post-injection, whereas mutant Mitfa does not.

“Overexpression of WT Mitfa results in ectopic activation of pnp4a in injected embryos at 6 hours post-injection, whereas mutant Mitfa (null) does not.”

May combine two claims.

The Cbl stage has two phases: early Cbl cells initially express tfec but not ltk, mitfa nor pnp4a; late Cbl cells rapidly turn on all these genes.

“We further distinguish two phases to this Cbl stage, with cells initially expressing tfec, but not ltk, mitfa nor pnp4a (which we designate early Cbl cells), before rapidly turning on all these genes (becoming late Cbl cells; Fig 7).”

May combine two claims.

In sox10 mutants at 24 and 30 hpf, tfec expression is retained in the premigratory NCC domain, which extends anteriorly throughout the embryo unlike in WT siblings.

“In sox10 mutants, both at 24 hpf and at 30 hpf (Petratou et al., in prep; Fig 3A and 3B), tfec expression is prominently retained in the premigratory NCC domain, which unlike in WT siblings extends anteriorly throughout the embryo.”

May combine two claims.

At 30 hpf, tfec mutants display partial loss of pnp4a-positive cells, with decreased numbers along the dorsal trunk and few cells on the migration pathway and ventral trunk.

“In contrast, tfec mutants displayed partial loss of pnp4a-positive cells (Fig 4L). Specifically, compared to WT siblings, tfec mutants showed decreased numbers of cells (expressing relatively low levels of pnp4a) along the dorsal trunk, and have comparatively few cells both on the migration pathway and in the ventral trunk, mostly more anterior.”

May combine two claims.

Model A3 performed similarly to Model A2, except that it generated predictions broadly consistent with the known biology for a wider range of parameter combinations.

“Model A3 performed similarly to Model A2, except that it generated predictions broadly consistent with the known biology for a wider range of parameter combinations (Fig 5I).”

• AssumptionThe authors state this as an assumption: The model assumes that each gene's output can be captured as average protein product in a homogeneous group of cells, with direct correlation between protein product and transcript production.

May combine two claims.

In sox10 mutant embryos, pnp4a is completely absent and remains inactive throughout the investigated developmental time-course.

“Loss of function studies using sox10 mutant embryos revealed that pnp4a was completely absent (Fig 4K), and that the gene remained inactive throughout the investigated developmental time-course (Fig 4O).”

May combine two claims.

mitfa mutants at 30 hpf maintain the same or increased numbers of pnp4a positive cells along the dorsal trunk but show a consistent lack of cells in the migratory pathways compared to WT siblings.

“mitfa mutants at 30 hpf appear to maintain the same, or even increased numbers of pnp4a positive cells along the dorsal trunk, but to show a consistent lack of cells in the migratory pathways compared to their WT siblings”

Classified as probably not a substantial claim.

Neural crest cells are a multipotent embryonic cell type that generate diverse derivatives including peripheral neurons, glia, skeletogenic cells and pigment cells.

“Neural crest cells (NCCs) are a multipotent embryonic cell-type, sharing many properties with stem cells and indeed being retained as adult neural crest stem cells in various niches [1].”

• cites [1] O. Shakhova, 2008Not matched

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

Classified as probably not a substantial claim.

The increase in iridophores in mitfa mutants reveals a shared bipotent progenitor, the melanoiridoblast.

“interpreted as revealing a shared bipotent progenitor, a melanoiridoblast [15], [19].”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

• cites [19] J. Lister, 1999, nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate. sourceNot checked — no full text of the source

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

Foxd3 has been proposed to repress mitfa expression, biasing bipotent melanoiridoblast progenitors towards an iridophore fate.

“the Forkhead box D3 transcription factor (Foxd3) has been proposed to repress mitfa expression, consistent with the suggested role of FOXD3 in repressing MITF in other models [30], [31], thus biasing bipotent melanoiridoblast progenitors towards an iridophore fate [15], [32].”

• cites [30] R. Kos, 2001, The winged-helix transcription factor FoxD3 is important for establishing the neural crest lineage and repressing melanogenesis in avian embryos. sourceNot checked — no full text of the source
• cites [31] A. Thomas, 2009, FOXD3 regulates the lineage switch between neural crest-derived glial cells and pigment cells by repressing MITF through a non-canonical mechanism. sourceNot checked — no full text of the source
• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

pnp4a is expressed widely in NCCs and long before iridophores differentiate.

“The gene pnp4a has been defined as an iridophore lineage marker, although it is expressed rather widely in NCCs and long before iridophores differentiate [15].”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

Cells on the medial pathway that are mitfa-positive and tfec-weak or tfec-negative have lost or are losing iridophore potential and are likely definitive melanoblasts.

“We interpret these as having lost, or being on the way towards losing, iridophore potential, and are likely definitive melanoblasts.”

May combine two claims.

mitfa is an important regulator of pnp4a in premigratory and migrating NC cells, but pnp4a activation in ib(df) is not affected.

“These results indicated that mitfa is an important regulator of pnp4a in premigratory and migrating NC cells, but that pnp4a activation in ib(df) is not affected.”

May combine two claims.

A positive feedback loop between Ltk and Tfec is established and drives maintenance of the iridophore specification state.

“A crucial step is then the establishment of a positive feedback loop between Ltk and Tfec, which drives maintenance of the iridophore specification state.”

May combine two claims.

Mitfa, but not Tfec, is sufficient in the ectopic context to upregulate pnp4a.

“Surprisingly, however, overexpression of WT Tfec did not result in a statistically significant ectopic activation of pnp4a (Fig 4Q), suggesting that Mitfa, but not Tfec, is sufficient in this ectopic context to upregulate pnp4a.”

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

Tfec has a similarly central role in iridophore fate specification as Mitfa does in melanocyte development.

“Tfec is a close homologue of Mitfa, so it is intriguing that it seems to have a similarly central role in iridophore fate specification as does Mitfa in melanocyte development.”

• AssumptionRests on an assumption: Tfec is a close homologue of Mitfa.

May combine two claims.

While at later stages pnp4a is a definitive marker of differentiated iridophores, initially it is expressed widely in specified and differentiating melanoblasts.

“Considered together, our data suggest that while at later stages pnp4a is a definitive marker of differentiated iridophores, initially it is expressed widely in specified and differentiating melanoblasts.”

May combine two claims.

Sox10 binds directly to the promoter of mitfa in zebrafish and activates its expression.

“Sox10 has been previously shown to bind directly to the promoter of mitfa in zebrafish, and to activate its expression [21], we thus include that interaction.”

• cites [21] S. Elworthy, 2003, Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development. sourceNot checked — no full text of the source

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

RNAscope results were derived from two independent experimental repeats, with 3 to 5 embryos examined per repeat.

“RNAscope results were derived from two independent experimental repeats. From each repeat, between 3 and 5 embryos were examined.”

May combine two claims.

Foxd3 is unlikely to be the key factor repressing mitfa expression in the iridophore lineage and unlikely to perform the role of factor R in the iridophore GRN.

“making it unlikely to be the key factor repressing mitfa expression in the iridophore lineage.”

• Also stated in: § Repression of mitfa in the iridophore lineage requires an unknown Factor R · hedged

  “we conclude that whilst previous data indicates a role for foxd3 in pigment cell development, it is unlikely to perform the role of factor R in iridophore GRN.”

May combine two claims.

tfec is activated in NCCs and maintained at early stages of iridoblast specification independently of Ltk activity.

“Thus, tfec is activated in NCCs and is maintained at early stages of iridoblast specification, independently of Ltk activity.”

May combine two claims.

pnp4a regulation is more complex than previously assumed and pnp4a should not be considered a definitive marker of the iridophore lineage at early stages.

“More broadly, we conclude that pnp4a regulation is more complex than previously assumed and that it should not be considered a definitive marker of the iridophore lineage at early stages.”

Classified as probably not a substantial claim.

Foxd3 has been previously implicated in repression of mitfa.

“We considered Foxd3 as a candidate for factor R, as the transcriptional regulator has been previously implicated in mitfa repression [15].”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

tfec-expressing cells on the medial pathway are iridophore progenitors that retain at least bipotency.

“leading us to distinguish these as iridophore progenitors which retain at least bipotency.”

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

The Monte Carlo approach is particularly suitable when quantitative data are not available but qualitative behaviours are known.

“We emphasize that our Monte Carlo approach is particularly suitable in all those cases when quantitative data are not available, but rather qualitative behaviours are known.”

May combine two claims.

Sox10 is not essential for initial activation of tfec in early NCCs, but is required for maintenance of tfec expression as multipotent progenitors become specified towards an iridophore fate (iridoblast fate specification).

“Together these data show that Sox10 is not essential for initial activation of tfec in early NCCs, but it is required for maintenance of tfec expression as multipotent progenitors become specified towards an iridophore fate, i.e. for iridoblast fate specification.”

May combine two claims.

Experimental restrictions mean the study can at best assess minimal levels of potency; overlap of expression of key genes for different fates is interpreted as the cell having potential for at least these fates.

“However, the experimental restrictions noted above mean that we can, at best, assess minimal levels of potency: where we see overlap of expression of key genes for different fates, we interpret this as reflecting the cell having potential for at least these fates.”

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

Ltk relies on intracellular cascades and effector transcription factors to activate gene expression, so its input is always indirect.

“Furthermore, Ltk relies on intracellular cascades and effector transcription factors to activate gene expression, thus its input is always indirect.”

May combine two claims.

pnp4a expression switches from Mitfa to Tfec-dependency during the transition from Cbl to ib(df), and is detectable transiently in all melanoblasts and early differentiating melanocytes.

“Thus, pnp4a expression appears to switch from Mitfa to Tfec-dependency during the transition from Cbl to ib(df), and to be detectable transiently in all melanoblasts and early differentiating melanocytes.”

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

ednrba mutants show no discernible embryonic iridophore phenotype but show loss of iridophores in adults.

“ednrba mutants show no discernible embryonic iridophore phenotype, although they do show loss of iridophores in adults [33].”

• cites [33] D. Parichy, 2010, Mutational analysis of endothelin receptor b1 (rose) during neural crest and pigment pattern development in the zebrafish Danio rerio,Not checked yet

  Not checked in the free run.

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

Mutant and wild-type embryos subjected to WISH were morphologically indistinguishable after fixation and were usually processed together.

“We note that mutant and WT embryos subjected to WISH were morphologically indistinguishable after fixation and were usually processed together.”

Classified as probably not a substantial claim.

The exact parameter values were assigned somewhat arbitrarily.

“These parameters were chosen based on ranges indicated from the literature as physiologically relevant, nevertheless the exact values were assigned somewhat arbitrarily.”

May combine two claims.

Tfec activation of ltk in premigratory NCCs is a first step in chromatophore fate restriction, distinguishing the multipotent Cbl from the fully multipotent eNCC.

“we show that one early function of Tfec is to activate ltk expression in premigratory NCCs, in what we consider to be a first step in chromatophore fate restriction (Fig 7), distinguishing the multipotent Cbl from the fully multipotent eNCC.”

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

pnp4a is a useful differentiation marker for the iridophore lineage.

“pnp4a has been identified as a useful differentiation marker for the iridophore lineage [15].”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

May combine two claims.

The remaining tfec-expressing cells in tfec mutants lack both ltk expression and visible reflective platelets, so they cannot correspond to ib(df) or mature iridophores.

“Although the remaining tfec-expressing cells show a distribution consistent with their being iph, we note that they lack both ltk expression and visible reflective platelets and hence cannot correspond to ib(df), which are positive for ltk expression, nor to mature iridophores.”

Classified as probably not a substantial claim.

The study employed unbiased Monte Carlo sampling to assess how significant changes to parameters might be for model output.

“we employed an unbiased Monte Carlo sampling to ask explicitly how significant changes to those parameters might be for the output of the models.”

May combine two claims.

Factor R represses mitfa, and thus melanocyte fate, in iridoblasts.

“namely the factor (factor R) repressing mitfa, and thus melanocyte fate, in the iridoblasts.”

Classified as probably not a substantial claim.

Published experimental observations, including the foxd3 mutant phenotype with partial loss of iridophores partially rescued in foxd3;mitfa double mutants, led to the proposal that FoxD3 might have a role in repressing mitfa in iridoblasts.

“A series of published experimental observations, including the foxd3 mutant phenotype, with partial loss of iridophores, partially rescued in foxd3;mitfa double mutants [15], had led to the proposal that FoxD3 might have such a role.”

• cites [15] K. Curran, 2010, Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.Not matched

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

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

RNAscope is highly sensitive and quantitative, better suited for sensitive detection of co-expression, and rapid mRNA turnover allows more precise assessment of gene expression regulation than GFP.

“This technique is both highly sensitive and quantitative, thus much better suited for sensitive detection of co-expression, with the added advantage that rapid turnover of mRNA (in contrast to slow degradation of GFP) allows for more precise assessment of regulation of gene expression.”

May combine two claims.

The study produced the first core GRN for the zebrafish iridophore, incorporating all known major players.

“In summary, we have produced the first core GRN for the zebrafish iridophore, incorporating all known major players.”

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

There may be two sub-populations of iridophores.

“Our work here, plus that from other groups, provides some indication that there may be two sub-populations of iridophores.”

May combine two claims.

The dorsal trunk pnp4a-positive subgroup is independent of mitfa function, while the migratory pathway subgroup requires mitfa function.

“indicating that the former subgroup is independent of, while the latter requires mitfa function.”