Author response: Thyroid hormone regulates distinct paths to maturation in pigment cell lineages (2019)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Thyroid hormone (TH) regulates diverse developmental events and can drive disparate cellular outcomes. 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. To learn how TH elicits seemingly opposite responses in cells having a common embryological origin, we analyzed individual transcriptomes from thousands of neural crest-derived cells, reconstructed developmental trajectories, identified pigment cell-lineage specific responses to TH, and assessed roles for TH receptors. We show that TH promotes maturation of both cell types but in distinct ways. In melanophores, TH drives terminal differentiation, limiting final cell numbers. In xanthophores, TH promotes accumulation of orange carotenoids, making the cells visible. TH receptors act primarily to repress these programs when TH is limiting. Our findings show how a single endocrine factor integrates very different cellular activities during the generation of adult form. https://doi.org/10.7554/eLife.45181.001 eLife digest Hormones control the development of animals from embryos all the way into adulthood. For example, thyroid hormone is needed to transform a tadpole into an adult frog, and it is essential for developing the nervous system and regulating metabolism in countless other adult animals. However, it remains unclear how a single hormone can control such a diverse range of outcomes. One way to learn more about the effects of thyroid hormone during development is to study zebrafish pigmentation. Pigment cells arise from a group of stem cells in the embryo called the neural crest. Two of these pigment cells respond to thyroid hormone in different ways: it causes orange pigment cells called xanthophores to expand in number, and at the same time limits the number of black pigment cells called melanophores. To investigate how thyroid hormone effects the numbers of these pigment cells Saunders et al. mapped the active genes of individual cells derived from the neural crest. Further experiments were then performed on the fish themselves based on these gene activity maps. Comparing fish with and without thyroid hormone showed the hormone actually helps both orange and black pigment cells to mature, but in very different ways. For the orange xanthophores, thyroid hormone drives cells already poised to change into their adult form to acquire orange pigments. For the black melanophores, it causes them to mature into their final non-dividing adult state. This results in xanthophores becoming visible just as the number of melanophores is forced to curtail. Saunders et al. also found the receptor for thyroid hormone acts like a brake for both pigment cells, making sure neither cell type matures in the absence of the hormone. These experiments show how one hormone can independently regulate different cell types as they mature into their adult form. The finding that thyroid hormone limits the growth of melanocytes may explain why people who produce too little thyroid hormone are at greater risk of melanoma – a form of skin cancer that starts in the melanocytes. But more studies are needed to see if thyroid hormone has the same limiting effect on melanocytes in humans. https://doi.org/10.7554/eLife.45181.002 Introduction Mechanisms that synchronize developmental signals and integrate them across cell types and organ systems remain poorly defined but are fundamentally important to both development and evolution of adult form (Atchley and Hall, 1991; Ebisuya and Briscoe, 2018). A powerful system for elucidating how organisms coordinate fate specification and differentiation with morphogenesis is the array of cell types that arise from embryonic neural crest (NC), a key innovation of vertebrates (Gans and Northcutt, 1983). NC cells disperse throughout the body, contributing peripheral neurons and glia, osteoblasts and chondrocytes, pigment cells and other derivatives. Differences in the patterning of these cells underlie much of vertebrate diversification. Thyroid hormone (TH) coordinates post-embryonic development of NC and other derivatives through mechanisms that are incompletely characterized (Brent, 2012; Brown and Cai, 2007; Sachs and Buchholz, 2017; Shi, 1999). During the abrupt metamorphosis of amphibians, TH drives outcomes as disparate as tail resorption and limb outgrowth. In the more protracted post-embryonic development of zebrafish—which has similarities to fetal and neonatal development of mammals (Parichy et al., 2009)—TH coordinates modifications to several traits including pigmentation. 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). Figure 1 Download asset Open asset TH-dependent phenotypes and models for TH action. (A) Euthyroid and hypothyroid zebrafish [stage 10 (mm) standardized standard length (SSL) (Parichy et al., 2009); ~21 d post-fertilization, dpf]. Insets, yellow/orange xanthophores of euthyroid fish and absence of these cells in hypothyroid fish. (B) Models for TH effects on alternative cell types derived from a common progenitor (P), by regulating: (i) cell fate specification; or (ii) amplification and restraint of committed cell-types by differential effects on morphogenesis or differentiation. https://doi.org/10.7554/eLife.45181.003 We asked how a single endocrine factor can have such different effects on cells sharing a common embryonic origin. Using transcriptomic analyses of individual cell states, we comprehensively defined the context for TH activities by identifying populations and subpopulations of adult NC derivatives. We then assessed the consequences of TH status for lineage maturation across pigment cell classes. 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. These mechanisms reflect different developmental histories of melanophores and xanthophores and yield different cell-type abundances when TH is absent. Our findings provide insights into post-embryonic NC lineages, contribute resources for studying adult pigment cells and other NC-derived cell types, and illustrate how a circulating endocrine factor influences local cell behaviors to coordinate adult trait development. Results Post-embryonic NC-derived subpopulations revealed by single-cell RNA sequencing To explain the pigment cell imbalance of hypothyroid fish, we envisaged two models for TH activity during normal development (Figure 1B). In the first model, TH influences states of specification, directing multipotent cells away from one fate and toward the other, or preventing the transdifferentiation of cells already committed to a particular fate. In the second model, TH influences cells that are already committed and remain committed, to their fates. In this scenario, discordant effects across lineages might be observed if TH promotes a cellular process in one lineage that amplifies its population, while simultaneously inhibiting the same process in the other lineage to restrain its population. To evaluate the applicability of these models to TH-dependent regulation of pigment cell populations, we sought to capture the range of intermediate states through which these cells transit during normal and hypothyroid development. Accordingly, we sequenced transcriptomes of thousands of individual NC-derived cells isolated from trunks of euthyroid and hypothyroid fish (Figure 2—figure supplements 1 and 2). Dimensionality reduction (Becht et al., 2018; Cao et al., 2019) followed by unsupervised clustering identified melanophores, xanthophores and a third class of NC-derived pigment cells, iridescent iridophores (Figure 2A and B; Supplementary file 1). A cluster likely corresponding to multipotent pigment cell progenitors (Budi et al., 2011; Singh et al., 2016) was marked by genes encoding pigment cell transcription factors, general markers of mesenchymal NC and factors associated with proliferation and migration but not pigment synthesis (Figure 2—figure supplement 3; Supplementary file 2—Table 1). Some cells within this cluster also expressed the zebrafish-specific embryonic NC marker crestin, which is generally down-regulated at later developmental stages but is still expressed in a subset of presumptive progenitor cells (Budi et al., 2011). Figure 2 with 5 supplements see all Download asset Open asset Single-cell transcriptomic identification of post-embryonic NC-derived cell types. (A) Cell-type assignments for clusters of cells (n = 16,150) from euthyroid and hypothyroid fish. Cell types known to be of non-NC derivation are not shown (Figure 2—figure supplement 2D and E). (B) Known cell-type marker genes and new candidate markers (for cluster-specific genes, see Supplementary file 2—Table 1). https://doi.org/10.7554/eLife.45181.004 Beyond pigment cells and their presumptive precursors, other clusters were identifiable as neurons, Schwann cells, other glia, and chromaffin cells. 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. Bioinformatic comparisons across all clusters revealed distinct expression profiles of genes encoding ligands and receptors, cell adhesion molecules, and products likely to have diverged in function after the teleost-specific whole-genome duplication (Figure 2—figure supplement 4). The larva-to-adult transformation of zebrafish entails changes in a variety of traits including NC derivatives (Parichy et al., 2009). In some instances, cell types at different stages that are superficially similar (e.g. larval vs. adult melanophores) can be distinguished by different genetic requirements (Budi et al., 2008; Larson et al., 2010; Parichy et al., 1999), raising the possibility that distinct gene expression programs regulate early larval and adult populations. If so, we predicted that NC-derived cells isolated from middle larval–juvenile stages, during development of the adult phenotype (i.e. Figure 2a), should form clusters distinct from cells that developed during embryonic stages to form the embryonic–early larval ('EL') phenotype. To test this idea, we isolated EL NC-derived cells, which clustered in identifiable cell types similar to those of middle-larval juvenile stages (Figure 2—figure supplement 5A). 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). 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. 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. Pigment cell sub-classes and gene expression dynamics across differentiation To understand the gene expression context in which TH impacts each pigment cell type, we compared pigment cells and progenitors, the lineages of which have been described (Budi et al., 2011; Mahalwar et al., 2014; McMenamin et al., 2014; Patterson and Parichy, 2019; Singh et al., 2016) (Figure 3A). These analyses revealed subsets of melanophores and xanthophores (Figure 3B), consistent with differences in states of differentiation and morphogenetic behaviors (Eom et al., 2015; Parichy et al., 2000b; Parichy and Spiewak, 2015). 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). 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). Figure 3 with 5 supplements see all Download asset Open asset Pigment cell subpopulations and dynamics of gene expression across pigment cell lineages. (A) Established lineage relationships of embryonic (e) and post-embryonic pigment cells. Multipotent pigment cell progenitors (P) in the peripheral nervous system generate adult iridophores (I), melanophores (M) and some xanthophores (X). A few embryonic melanophores (Me) persist, whereas embryonic xanthophores (Xe) proliferate and lose their pigment to enter a cryptic phase (Xc), and then reacquire pigment late in pattern formation to form most adult xanthophores (McMenamin et al., 2014). (B) Sub-clusters of melanophores and xanthophores with distinct gene expression signatures. (C) Pigment cell clusters defined by markers for each cell-type. (D–E) Pseudotemporal ordering (D) and BEAM (E) revealed dynamics of gene expression over pseudotime for each pigment cell branch [q < 6.0E-11 for all genes except pax3a, starred, q = 0.03), expressed as anticipated during early pseudotime in each branch]. https://doi.org/10.7554/eLife.45181.010 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). Expression of other genes was broader than might be expected from mutational or other analyses (Figure 3—figure supplement 4); 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). To characterize transcriptional dynamics through lineage maturation, we pseudotemporally ordered cells (Qiu et al., 2017a; Qiu et al., 2017b; Trapnell et al., 2014), yielding a differentiation trajectory with each pigment cell type arising from a common progenitor (Figure 3D). 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). Branch expression analysis modeling (BEAM) (Qiu et al., 2017a) confirmed that genes with functions in specification (e.g. mitfa in melanophores) were expressed early in pseudotime whereas genes associated with differentiation [e.g. dct, encoding a melanin synthesis enzyme (Kelsh et al., 2000b)] were expressed late (Figure 3E; Figure 3—figure supplement 5A). These analyses revealed dynamics of dozens of genes potentially identifying discrete processes in lineage-specific maturation (Supplementary file 2—Table 2) as well as broader trends. 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). TH-independence of pigment cell fate specification and absence of lineage-specific restraints on developmental progress Resolution of pigment cell states through their development allowed us to test if TH functions in fate specification (Figure 1B–i). If so, the excess melanophores and missing xanthophores of hypothyroid fish should reflect biases on specification of multipotent progenitors, or the transdifferentiation (Lewis et al., 2019; Niu, 1954) of initially specified cells. Such alterations should be evident in reduced-dimension transcriptomic space as strong skew in the apportionment of cells between branches or abnormal paths in the cellular trajectory, respectively. Yet, euthyroid and hypothyroid trajectories were topologically equivalent. Moreover, pigment cell progenitors were not depleted in hypothyroid fish as might occur were these cells being allocated inappropriately as melanophores (Figure 4A–D). Figure 4 Download asset Open asset TH biased pigment cell lineages toward later steps of pseudotime. (A) UMAP dimensionality reduction of euthyroid and hypothyroid pigment cells and pigment progenitors. Sub-clustering revealed two xanthophore and two melanophore clusters (indicated by different shades of yellow and gray, respectively). (B) Pigment cells colored by TH status. Euthyroid and hypothyroid cells were generally intermixed with some biases apparent within melanophore and iridophore clusters. (C) Percentages of each pigment cell class by TH-status. Colors are consistent with other pigment cell plots. Of cells captured, a higher proportion of pigment cells from euthyroid fish were xanthophores and iridophores compared to those from hypothyroid fish. (D) Trajectories for euthyroid and hypothyroid pigment cells. Broad differences in trajectory topologies were not apparent between the two conditions. (E) Distributions of each pigment cell-type across pseudotime by condition. For each trajectory branch (mel, xan, irid), hypoTH cells were biased toward early pseudotime (Wilcoxon signed-rank tests, mel: Z = −6.54, p<0.0001, xan: Z = −4.54, p<0.0001, irid: Z = −13.55, p<0.0001). Median is indicated by red arrowhead and different colors demarcate quartiles over pseudotime. https://doi.org/10.7554/eLife.45181.016 Through a second model—lineage discordance—TH could have opposite effects on cells already committed to particular fates, selectively amplifying one cell type while simultaneously repressing amplification of the other (Figure 1B–ii). For example, TH could promote differentiation of xanthoblasts to xanthophores, but prevent differentiation of melanoblasts to melanophores. Alternatively, TH could be a survival factor in the xanthophore lineage but a pruning factor in the melanophore lineage. Terminal phenotypes of both hypothyroid and hyperthyroid mutant fish are consistent with such a mechanism (McMenamin et al., 2014). If TH has discordant effects between lineages, we predicted that hypothyroid fish should exhibit a strong depletion of xanthophores from the end of their branch of the trajectory, whereas melanophores should be strongly over-represented near the tip of their branch. Yet, empirical distributions of pigment cell states in hypothyroid fish were all biased towards earlier steps in pseudotime, sometimes severely (Figure 4E). Indeed, prior analyses showed that addition of exogenous TH to hypothyroid cells ex vivo can promote differentiation of unpigmented melanoblasts to melanophores (McMenamin et al., 2014), contrary to the idea that TH specifically blocks melanophore development. Together these findings allow us to reject a model in which TH regulation of pigment cell abundance in the adult fish occurs through discordant effects on specific cellular processes across lineages. TH promotes a melanophore maturation program Having rejected both of our initial models (Figure 1B), we considered a third possibility, that TH promotes the maturation of both lineages, but in distinct ways. For melanophores, inspection of transcriptomic states and cellular phenotypes supported a role for TH in promoting maturation of this lineage. Genes expressed during terminal differentiation of melanophores from euthyroid fish (e.g. tfap2a, tyrp1b) were expressed at lower levels in melanophores of hypothyroid fish, suggesting an impediment to maturation in the absence of TH (Figure 5A). Figure 5 with 1 supplement see all Download asset Open asset TH promoted melanophore maturation by measures of transcriptomic state and cellular phenotype. (A) Gene expression differences between melanophores over pseudotime by TH-status (q < 1E-7, genes expressed in >10% of melanophores). Heatmap is hierarchically clustered by row (method, Ward D2). The largest cluster (#3) contains 41% of the genes and represents loci expressed late in pseudotime of euthyroid melanophores but downregulated in hypothyroid melanophores (e.g. tfap2a and tyrp1b), identifying novel candidate genes for roles in melanophore maturation (see Supplementary file 2—Table 6, in which published melanophore-related genes are highlighted) (Baxter et al., 2019). (B) Euthyroid melanophores tended to be highly melanized and stellate, whereas hypothyroid melanophores were variably melanized and often dendritic. (C) Quantification of melanin contents per cell, as estimated by area of pixels (px) having melanin following contraction of melanin granules in response to epinephrine (e.g. Figure 1A). Melanophores of euthyroid fish contained more melanin than those of hypothyroid fish (F1,2710=271.2, p<0.0001), after controlling for individual variation among fish within TH conditions (F10,2710=8.5, p<0.0001; sample sizes: n = 1180 cells from five euthyroid fish, n = 1542 cells from five hypothyroid fish). If planar areas of concentrated melanin granules are assumed spherical, then euthyroid melanophores had on average ~1.7 x the total melanin content of hypothyroid melanophores. (Data in supplementary file Figure 5—source data 1) (D) Fully differentiated melanophores of zebrafish are often binucleate (Usui et al., 2018). Left panel shows a binucleate stripe melanophore in a euthyroid fish (12 SSL). Right panel shows a mononucleate melanophore in a hypothyroid fish at the same stage. Magenta, membrane labeling of melanophores by tyrp1b:palm-mCherry. Blue, nuclei revealed by tuba8l3:nEosFP. (E) Euthyroid fish had proportionally more binucleate melanophores than hypothyroid fish (χ2=230.3, d.f. = 1, p<0.0001) after controlling for a higher incidence of binucleation in more advanced fish = d.f. = 1, indicate of binucleate melanophores observed in and which not sample sizes: n = melanophores in euthyroid fish, n = melanophores in hypothyroid fish). (Data in supplementary file Figure 5—source data 2). Figure 5—source data 1 content data corresponding to Figure Download Figure 5—source data 2 binucleation incidence data corresponding to Figure Download To test test the idea that TH promotes the maturation of melanophores, we additional cellular Melanophores of juvenile euthyroid fish tended to be and stellate, whereas melanophores of juvenile hypothyroid fish were variably melanized and (Figure of earlier stages of melanophore development in (Eom et al., 2012; Parichy and Quantification of melanin content within individual cells confirmed that melanophores of euthyroid fish are more melanized than those of hypothyroid fish (Figure analyses indicated that melanophores of euthyroid fish to whereas those of hypothyroid fish to do (McMenamin et al., 2014). These findings the possibility that melanophores of euthyroid fish might exhibit of cellular or other of proliferative not observed in melanophores of hypothyroid fish. and melanophores of melanoma exhibit or phenotypes and can be et al., 2015; et al., 2008; et al., 2019; Accordingly, we asked similar were evident for zebrafish stripe melanophores. ex some stripe melanophores exhibited activity (Figure supplement although we were to such comparisons across TH conditions. results from activity and both activity and number in cells et al., et al., We labeling (Figure supplement and of melanophores by cell melanophores. contents of melanophores from euthyroid fish were greater than melanophores from hypothyroid fish (Figure supplement of also that melanophores from juvenile euthyroid fish were than melanophores from hypothyroid fish (Figure supplement although can be by factors as well et al., 2011). we a to cell survival and 2015; et al., 2018). In euthyroid fish, of melanophores were binucleate near the of adult melanophore differentiation but were binucleate by juvenile stages, an in binucleation with and melanophore (Figure supplement In comparisons for TH of melanophores from euthyroid fish were whereas of melanophores from hypothyroid fish were in this state (Figure and E). our and those of McMenamin et al. a model in which TH drives melanophores into a differentiated state of and and proliferative TH promotes carotenoid-dependent xanthophore during adult development We TH functions specific to the xanthophore lineage. adult xanthophores from EL xanthophores that lose their pigment and then reacquire it late in adult pattern formation (Figure 3A) (McMenamin et al., 2014). xanthophores of hypothyroid fish persist, in a cryptic we predicted that TH effects should be in these cells than in melanophores that from transit amplifying cells from multipotent progenitors. Indeed, fewer genes were expressed between TH in xanthophore than melanophore lineages vs. Figure among these were several loci or associated the of yellow/orange (Figure and Figure supplement derived that contribute to xanthophore et al., et al., Figure with 4 supplements see all Download asset Open asset TH promotes xanthophore maturation (A) of expressed genes in euthyroid and hypothyroid cells across pseudotime expressed fewer TH-dependent genes than melanophores gene = of are genes with q < and change Of xanthophore and melanophore were found to be (B) TH-dependent expression of genes to in xanthophores. q < (C) gene expression was higher in xanthophore lineage cells of euthyroid fish compared to hypothyroid fish gene expression was lower in cells from euthyroid fish represent across and lower (D) were by in skin xanthophores of euthyroid but not hypothyroid fish SSL). (E) expression in euthyroid and hypothyroid zebrafish SSL). mature, yellow xanthophores (12 SSL). Differences in gene expression a corresponding in xanthophores of hypothyroid fish that we confirmed by and (Figure Figure supplement 2). genes, receptor a receptor essential for accumulation in and et al., et al., and we found it to be required in zebrafish for