Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

In the paper

“although FSC-A can be influenced by cell-size-independent factors as well (Tzur et al., 2011).”

What the sources say

• Supported

  The Introduction explicitly says that FSC-A measurements are influenced not only by particle size but also by refractive-index differences, absorbing substances, and the optical design of the measurement system. This directly supports the citing sentence's qualified claim. (LLM, conf=0.95)

  cites Tzur et al., 2011 source

In the paper

“Fish lacking TH have gross defects in pigment cell numbers and pattern with ~two fold the normal complement of melanophores and the simultaneous absence of visible xanthophores (McMenamin et al., 2014).”

What the sources say

• Partly supported

  The paper’s thyroid-ablation passage (section labeled Abstract; page unavailable) reports excess melanophores and a lack of adult body xanthophores in juvenile fish. It also says some xanthophores developed later, and the passages do not establish the claimed approximately twofold melanophore count. (LLM, conf=0.86)

  cites McMenamin et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“fish lacking TH have about twice the normal number of melanophores and lack visible xanthophores (Figure 1A) (McMenamin et al., 2014).”

What the sources say

• Partly supported

  The passage labeled Abstract (no page supplied), describing Fig. 1, reports that fish thyroid-ablated early in development lacked adult body xanthophores and had excess melanophores. It does not quantify the excess as about twice the normal number. It also notes that some xanthophores developed later, so their absence is not permanent. (LLM, conf=0.86)

  cites McMenamin et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“Prior analyses indicated that melanophores of euthyroid fish fail to divide whereas those of hypothyroid fish continue to do so (McMenamin et al., 2014).”

What the sources say

• Partly supported

  The indexed passage labeled Abstract reports that melanophores in euthyroid larvae rarely divided, while those in hypothyroid larvae divided frequently. It supports the comparison, but “fail to divide” overstates the finding that division was rare, not absent. (LLM, conf=0.88)

  cites McMenamin et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“Multipotent progenitors that give rise to adult melanophores, some xanthophores, and iridophores are established in the embryo and reside within peripheral nerves as development progresses (Budi et al., 2011; Budi et al., 2008; Camargo-Sosa et al., 2019; Dooley et al., 2013a; Singh et al., 2016).”

What the sources say

• Partly supported

  The Discussion (page unavailable) describes embryonically established precursors to adult melanophores and iridophores that subsequently associate with peripheral nerves and ganglia. It does not include xanthophores among their descendants; the Figure 12 discussion instead says metamorphic xanthophores presumably arise from a different precursor population. (LLM, conf=0.90)

  cites Budi et al., 2011 sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites Budi et al., 2008 source
• Partly supported

  The Introduction says adult pigment stem cells are set aside from neural crest cells during embryogenesis, with at least some occupying a niche within dorsal root ganglia. The Discussion says these cells produce adult melanophores and iridophores, with a small contribution to xanthophores. The claim overstates their location: the paper places at least some in ganglia and links others to the peripheral nervous system, but does not establish that the progenitors generally reside within peripheral nerves. (LLM, conf=0.84)

  cites Camargo-Sosa et al., 2019 sourceExperimental — accuracy not yet measured

In the paper

“In zebrafish, TH has opposite effects on neural crest derived pigment cells of the adult stripe pattern, limiting melanophore population expansion, yet increasing yellow/orange xanthophore numbers.”

• Also stated in: § Introduction · hedged

  “Remarkably, TH has seemingly opposite effects on two classes of NC-derived pigment cells, curtailing the population of black melanophores yet promoting development of yellow/orange xanthophores; fish lacking TH have about twice the normal number of melanophores and lack visible xanthophores (Figure 1A) (McMenamin et al., 2014).”

What the sources say

• Supported

  The paper’s Abstract states that thyroid hormone represses melanophore numbers and promotes xanthophore development in the adult pigment pattern. It also reports that thyroid-ablated fish had excess melanophores and lacked adult body xanthophores, supporting the opposite effects described in the claim. (LLM, conf=0.94)

  cites McMenamin et al., 2014 source

In the paper

“Melanoblasts derived from peripheral-nerve associated progenitors are proliferative during adult pigment pattern formation yet this activity largely ceases as the cells differentiate (Budi et al., 2011; McMenamin et al., 2014).”

What the sources say

• Supported

  The Results section on nerve-associated precursors reports that melanophore precursors proliferate during pigment pattern metamorphosis. The Results section on morphogenesis and differentiation contrasts proliferation of unmelanized cells with rare division of differentiated melanophores in wild-type fish (0.1%). No page number is available in our copy. (LLM, conf=0.94)

  cites Budi et al., 2011 source
• Partly supported

  The passage labeled Abstract says adult melanophores arise from peripheral nerve-associated precursors. It also reports that melanophores rarely divided in euthyroid larvae, but it does not establish that their precursors proliferate or that proliferation ceases as they differentiate; melanophores divided frequently in hypothyroid larvae. (LLM, conf=0.85)

  cites McMenamin et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“for example mitfa, encoding a transcription factor required for melanophore fate specification (Lister et al., 1999) was expressed in melanophores and progenitors, but also xanthophores (Figure 3C), consistent with prior reports (Eom et al., 2012; Parichy et al., 2000b).”

What the sources say

• Partly supported

  In the Results section on igsf11 expression (Figure 5C,E), mitfa:GFP marks pigment-cell precursors, and the paper also uses a mitfa-driven transgene in melanophores (Figure 4). It does not establish mitfa expression in xanthophores: Figure 5F measures igsf11, not mitfa, in xanthophore-enriched cells. Page numbers were not provided in our copy. (LLM, conf=0.84)

  cites Eom et al., 2012 sourceExperimental — accuracy not yet measured
• ?Not matched

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

  cites Parichy et al., 2000b

In the paper

“Ablated thyroid glands fail to regenerate (McMenamin et al., 2014) and absence of regeneration in this study was confirmed by continued absence of nVenus expression.”

What the sources say

• Partly supported

  The paper’s section labeled Abstract (page unavailable) reports that fish thyroid-ablated at 4 days post-fertilization still lacked tg mRNA and T4 as juveniles, supporting persistent loss of thyroid function. It does not establish that the glands themselves cannot regenerate. The nVenus observation is described as a finding of the citing study, not attributed to McMenamin et al. (LLM, conf=0.78)

  cites McMenamin et al., 2014 sourceExperimental — accuracy not yet measured

In the paper

“The yellow-orange color of xanthophores can depend on pteridine pigments, carotenoid pigments, or both”

What the sources say

• ?Not checked — no full text of the sourcecites Bagnara and Matsumoto, 2006 source
• Supported

  The Results section (‘Plin6 is expressed in xanthophores and traffics with carotenoid droplets’) describes xanthophores as concentrating yellow/orange pigment and says teleost xanthophores contain yellow pteridines and red/orange carotenoids. Figure 6 also describes yellow carotenoid pigmentation, supporting the claim that either pigment type can contribute to yellow-orange color. (LLM, conf=0.90)

  cites Granneman et al., 2017 source
• ?Not checked — no full text of the sourcecites Lister, 2019 source

In the paper

“Measurements of forward scatter (FSC-A) also suggested that melanophores from juvenile euthyroid fish were larger than melanophores from hypothyroid fish (Figure 5—figure supplement 1D), although FSC-A can be influenced by cell-size-independent factors as well (Tzur et al., 2011).”

In the paper

“In stage-matched comparisons for TH status, ~70% of melanophores from euthyroid fish were binucleated, whereas only ~25% of melanophores from hypothyroid fish were in this state (Figure 5D and E).”

In the paper

“By contrast, pteridine pathway genes tended to be expressed similarly across stages regardless of TH status and were even moderately upregulated in hypothyroid xanthophores (Figure 6C, Figure 6—figure supplement 4C).”

In the paper

“For example, cells of subcluster melanophore 2 exhibited low levels of transcriptional activity and expressed fewer genes, suggesting a more advanced state of differentiation, as compared to cells of melanophore 1 (Figure 3—figure supplement 1).”

In the paper

“These data do not indicate markedly different transcriptomic programs of NC-derived cell types across life stages, despite the existence of some stage-specific requirements for particular genes and pathways.”

• Also stated in: § Post-embryonic NC-derived subpopulations revealed by single-cell RNA sequencing · plain

  “Combining profiles for cells at different stages failed to reveal non-contiguous, life-stage-specific clusters, although some EL cells occupied subsets of transcriptomic space relative to their broader cell type (e.g. melanophores) (Figure 2—figure supplement 5B–D).”

In the paper

“When plated ex vivo, some stripe melanophores exhibited senescence-associated β-galactosidase (SA-β-gal) activity (Figure 5—figure supplement 1A), although we were unable to score such staining reliably, precluding comparisons across TH conditions.”

In the paper

“Loss of thrab, on its own or in conjunction with loss of thraa, partially restored the deposition of carotenoids in interstripe xanthophoes; mutation of all three receptors fully rescued the number of carotenoid-containing xanthophores (Figure 7B and C; Figure 7—figure supplement 1E and F).”

In the paper

“In euthyroid fish, ~20% of melanophores were binucleate near the onset of adult melanophore differentiation but >50% were binucleate by juvenile stages, confirming an overall increase in binucleation with somatic stage and melanophore age (Figure 5—figure supplement 1E).”

In the paper

“Carotenoids were undetectable in euthyroid 5 dpf larvae, and carotenoid-related genes were expressed at lower levels in EL xanthophores than adult xanthophores (Figure 6—figure supplement 4B and C).”

In the paper

“For example, transcripts per cell declined in melanophores but not iridophores, consistent with an expectation of reduced RNA abundance as melanophores—but not iridophores—exit the cell cycle with maturation (Figure 3—figure supplement 5B) (Budi et al., 2011; Darzynkiewicz et al., 1980; McMenamin et al., 2014; Spiewak et al., 2018).”

In the paper

“Our collected cell-types, which include immature and mature glia, differentiating pigment cells, and presumptive progenitors of different types identify new candidate genes for promoting—and recognizing—distinct states of differentiation and morphogenetic activities”

In the paper

“Likewise cells of xanthophore 1 had fewer transcripts and expressed fewer genes than cells of xanthophore 2, suggesting they may represent undifferentiated, cryptic xanthophores and actively differentiating populations, respectively (McMenamin et al., 2014).”

In the paper

“An additional cluster expressed markers suggestive of proliferative, non-pigmentary progenitors, and one large cluster (‘unknown’) was not readily assignable to NC-derived populations described previously.”

In the paper

“Overall, our survey captured numerous NC-derived cell types, including abundant pigment cells and progenitors, and revealed substantial variation in gene expression programs among them.”

In the paper

“Genes encoding each of the three zebrafish TRs (thraa, thrab, thrb) were expressed by melanophores and xanthophores, yet presumptive null alleles for each unexpectedly had pigment cell complements and patterns that resembled the wild type (Figure 7A; Figure 7—figure supplement 1A–D).”

In the paper

“Euthyroid melanophores tended to be highly melanized and stellate, whereas hypothyroid melanophores were variably melanized and often dendritic.”

• Also stated in: § TH promotes a melanophore maturation program · hedged

  “Melanophores of juvenile euthyroid fish tended to be uniformly well-melanized and stellate, whereas melanophores of juvenile hypothyroid fish were variably melanized and dendritic (Figure 5B), reminiscent of earlier stages of melanophore development in wild-type (Eom et al., 2012; Parichy and Turner, 2003).”

In the paper

“This topology differed from known lineage relationships (Figure 3A) but was consistent with similarity of EL and mid-larval/juvenile gene expression programs (Figure 2—figure supplement 5D).”

In the paper

“Additional surveys of these data revealed new markers of xanthophore and iridophore lineages (Figure 3—figure supplements 2 and 3), and cell-type-specific expression of some previously identified markers [e.g. tyrp1b, aox5, tfec (Lister et al., 2011; McMenamin et al., 2014) (Figure 3C).”

In the paper

“Among carotenoid genes, scavenger receptor B1 (scarb1) encodes a high-density lipoprotein receptor essential for carotenoid accumulation in birds and invertebrates (Kiefer et al., 2002; Toomey et al., 2017) and we found it to be required in zebrafish for carotenoid deposition, although not cell persistence (Figure 6—figure supplement 3A and B).”

In the paper

“exogenous TH was sufficient to rescue both expression and carotenoid deposition (Figure 6F; Figure 6—figure supplement 3C).”

In the paper

“q < 6.0E-11 for all genes, except for pax3a”

In the paper

“We showed that TH directly or indirectly regulates carotenoid-associated genes and carotenoid deposition, allowing cryptic xanthophores to reacquire visible pigmentation.”

In the paper

“except for pax3a (starred, q = 0.03)”

In the paper

“In melanophores, TH drives terminal differentiation, limiting final cell numbers.”

In the paper

“We show that TH promotes maturation of both cell types but in distinct ways.”

• Also stated in: § Introduction · plain

  “Our analyses showed that TH drives maturation of cells committed to melanophore and xanthophore fates through different mechanisms, promoting terminal differentiation and proliferative arrest in melanophores, and carotenoid-dependent repigmentation in xanthophores.”

In the paper

“Together these findings demonstrate an essential role for TH in carotenoid pigmentation and suggest that TH modulation of a suite of carotenoid pathway genes is required for cryptic xanthophores to re-pigment during adult pattern formation.”

In the paper

“These findings suggest that repression by unliganded TRs contributes to pigment-associated phenotypes in hypothyroid fish, implying a function for TRs in repressing the repigmentation of xanthophores and terminal differentiation of melanophores until late stages in adult pigment pattern development.”

In the paper

“Collectively, our observations and those of McMenamin et al. (2014) suggest a model in which TH drives melanophores into a terminally differentiated state of increased melanization, larger size and lysosomal content, binucleation, and proliferative cessation.”

In the paper

“TH therefore drives maturation of both xanthophores and melanophores yet has markedly different roles in each lineage.”

In the paper

“human melanoma is associated with hypothyroidism and recurrent TH pathway mutations (Ellerhorst et al., 2003; Shah et al., 2006; Sisley et al., 1993)”

What the sources say

• ?Not checked — no full text of the sourcecites Ellerhorst et al., 2003 source
• ?Not checked — no full text of the sourcecites Shah et al., 2006 source
• ?Not checked — no full text of the sourcecites Sisley et al., 1993 source

In the paper

“Together, these observations imply that TH induces new, carotenoid-based pigmentation, allowing transiently cryptic xanthophores to reacquire coloration during adult pattern development.”

In the paper

“Yet, we found no evidence for early pigmentation of xanthophores in euthyroid fish homozygous for thrab mutation (Figure 7—figure supplement 1G), suggesting essential roles for other factors present only at later stages (Patterson and Parichy, 2013).”

In the paper

“although we were unable to score such staining reliably, precluding comparisons across TH conditions.”

In the paper

“Single transgene controls and wild-type cells were used to adjust voltage and gating.”

In the paper

“Guides were tested for mutagenicity by Sanger sequencing”

In the paper

“That cryptic xanthophores appear poised to re-differentiate likely explains the smaller proportion of genes that were TH-dependent in these cells as compared to melanophores.”

In the paper

“Images were corrected for color balance and adjusted for display levels as necessary with conditions within analyses treated identically.”

In the paper

“For all thyroid ablations, treated individuals were assessed for loss of nuclear-localizing Venus (nVenus) the following day.”

In the paper

“Final cellular barcodes and UMIs were determined using Cell Ranger 2.0.2 (10X Genomics) and cells were filtered to include only high-quality cells.”

In the paper

“We replicated the experiment three times.”

In the paper

“To correct for this bias in aligning reads to the transcriptome, we extended all 3' UTR annotations by 500 bp.”

In the paper

“All procedures involving live animals followed federal, state and local guidelines for humane treatment and protocols approved by Institutional Animal Care and Use Committees of University of Virginia and University of Washington.”

In the paper

“Individual genotypes of fish assessed were confirmed using PCR or Sanger sequencing.”

In the paper

“For parametric analyses, residuals were assessed for normality and homoscedasticity to meet model assumptions and no transformations were found to be warranted.”

In the paper

“We assigned clusters to cell types based on the detection of published marker genes.”

In the paper

“Cells with more than 15,000 UMIs were discarded as possible doublets.”

In the paper

“Batch correction methods were not used between the two groups or across samples because we did not observe sample-specific separation or clustering in UMAP space.”