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The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non‐thyrocyte cells surrounding the follicle, we developed a single‐cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249‐cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9‐based pax2a knock‐in line that monitors pax2a expression in the thyrocytes. A population of pax2a‐low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a‐high and pax2a‐low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.

Background: Defects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns, but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification, and subsequent morphogenetic processes in thyroid development. Methods: To begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small-molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. Results: Systematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding, as well as hypoplastic, enlarged, or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified bone morphogenetic protein and fibroblast growth factor signaling as key factors for thyroid specification and early thyroid organogenesis, highlighted the importance of low Wnt activities during early development for thyroid specification, and implicated drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. Conclusions: By integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken, and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases., info:eu-repo/semantics/published

Summary The thyroid plays an essential role in homeostasis and development, but the extrinsic regulation of its embryonic development remains poorly understood. Recently, we have identified the FGF and BMP pathways to be crucial for thyroid specification and have confirmed the hypothesis that the cardiac mesoderm provides the FGF and BMP ligands to regulate this process. However, it is not clear how these ligands control thyroid specification. To study the molecular mechanisms underlying early thyroid development, we combined a pharmacological approach in zebrafish embryos with genetic models, to modulate the activity of the FGF and BMP pathways at different embryonic stages. We first characterized the expression of the transcription factors pax2a and nkx2.4b - the two main early thyroid markers - in the anterior foregut endoderm and observed that pax2a was expressed from 18 hours post fertilization (hpf) and marked a large endodermal cell population while nkx2.4b was expressed from 24 hpf and marked only a subset of the pax2a-positive endodermal cells. Interestingly, the activity profiles of FGF and BMP coincided with the detection of pax2a and nkx2.4b expression, respectively. Brief modulations of the FGF and/or BMP pathways support the hypothesis that the FGF pathway regulates the expression of pax2a and the BMP pathway regulates the expression of nkx2.4b. Furthermore, inhibition of the BMP pathway during early segmentation has dramatic effects on thyroid specification, probably via the FGF pathway. Together with our previous observations, we propose here, an updated model of early thyroid development in which the foregut endoderm receives several synchronized waves of FGF and BMP signals from the cardiac mesoderm, which result in sequential activation of pax2a and nkx2.4b gene expression and subsequent thyroid specification.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover genetic heterogeneity in the thyrocyte population, and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells and fibroblasts. Further, the thyrocytes could be split into two sub-populations with unique transcriptional signature, including differential expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line, which demonstrated specific pax2a expression in the thyrocytes. However, a population of pax2a-low mature thyrocytes interspersed within individual follicles could be distinguished, corroborating heterogeneity within the thyrocyte population. Our results identify and validate transcriptional differences within the nominally homogenous thyrocyte population., info:eu-repo/semantics/published

BackgroundCongenital hypothyroidism (CH) due to thyroid dysgenesis is a frequent congenital endocrine disorder for which the molecular mechanisms remain unresolved in the far majority of cases. This situation reflects in part our still limited knowledge about the mechanisms involved in the early steps of thyroid specification from the endoderm, in particular the extrinsic signaling cues that regulate foregut endoderm patterning. In this study, we used small molecules and genetic zebrafish models to characterize the role of various signaling pathways in thyroid specification.MethodsWe treated zebrafish embryos during different developmental periods with small molecule compounds known to modulate the activity of Wnt signaling pathway and observed effects in thyroid, endoderm and cardiovascular development using whole mountin situhybridization and transgenic fluorescent reporter models. We used an antisense morpholino to create a zebrafish acardiac model. For thyroid rescue experiments, BMP pathway induction in zebrafish embryos was obtained by using heatshock inducible transgenic lines.ResultsInterestingly, combined analyses of thyroid and cardiovascular development revealed that overactivation of Wnt signaling during early development leads to impaired thyroid specification concurrent with severe defects in the cardiac specification. When using a model of morpholino-induced blockage of cardiomyocyte differentiation, a similar correlation was observed, suggesting that defective signaling between cardiac mesoderm and endodermal thyroid precursors contributes to thyroid specification impairment. Rescue experiments through transient overactivation of BMP signaling could partially restore thyroid specification in models with defective cardiac development.ConclusionCollectively, our results indicate that BMP signaling is critically required for thyroid cell specification and identify cardiac mesoderm as a likely source of BMP signals.

BackgroundDefects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification and subsequent morphogenetic processes in thyroid development.MethodsTo begin to address this problem in a systematic way, we used zebrafish embryos to perform a series ofin vivophenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm and cardiovascular development using whole mountin situhybridization and transgenic fluorescent reporter models.ResultsSystematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding as well as hypoplastic, enlarged or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified BMP and FGF signaling as key factors for thyroid specification and early thyroid organogenesis, highlight the importance of low Wnt activities during early development for thyroid specification and implicate drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis.ConclusionsBy integrating the outcome of our screening efforts with previously available information from other model organisms includingXenopus, chicken and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues., SCOPUS: ar.j, info:eu-repo/semantics/published