Zebrafish embryo: A new model for studying thyroid morphogenesis

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Highlights

  • Animal models are essential for the study of developmental biology.

  • Main signalling pathways of thyroid organogenesis are conserved in all vertebrates.

  • Zebrafish has numerous advantages over other animal models, especially translucency.

  • CRISPR-Cas9 coupled to easy genome editing greatly facilitate studies in this model.

Abstract

Thyroid development anomalies, known as thyroid dysgenesis, are the commonest cause of congenital hypothyroidism. Most of the cases remain unexplained, although the occurrence of familial cases strongly suggests a genetic contribution. To validate a genetic implication in thyroid development, animal models are required. Recently, zebrafish has become extensively used due to its numerous advantages. Moreover, thyroid development is similar in zebrafish and in human. However, caution is advised when analyzing results from zebrafish. It is important to keep in mind that phenotype is often divergent from the human when knocking out/down a gene. Furthermore, when a phenotype is observed, it is often accompanied by a vascular phenotype, so maybe it is the vascular anomalies that lead to thyroid disorganization.

Introduction

Congenital hypothyroidism (CH) is estimated to affect one in 2500 live newborns among Caucasians, making it the most common neonatal endocrine disease [1]. CH can lead to irreversible intellectual disabilities if left untreated. Fortunately, neonatal screening for thyroid function, established four decades ago, has had a dramatic impact on intellectual prognosis. Nonetheless, even with early treatment, a slight reduction of the intellectual quotient (but within the normal range) is still observed in some patients [2]. Defects in thyroid hormone synthesis (thyroid dyshormonogenesis) are seen in up to 15% of the cases and are usually explained by mutations in genes implicated in thyroid hormone production such as NIS, DUOX2, TPO, TG or DEHAL [3]. A defect in thyroid development during embryogenesis, referred to as thyroid dysgenesis (CHTD), is the commonest etiology of CH. A round, oval, or dumbbell-shaped ectopic sublingual gland is most frequently observed in patients with CHTD 4, 5. Less common are complete absence of thyroid (athyreosis), hypoplasia of a normally located gland or hemiagenesis. The cause of CHDT is unknown in most cases. The main pathways and key steps of thyroid morphogenesis are known, but the molecular mechanisms driving thyroid migration and the implication of intrinsic and/or extrinsic factors in this process remain incompletely understood to date 6, ∗∗7.

Most discoveries on thyroid morphogenesis were made in mice. For instance, crosstalk between Hhex, Pax8, Foxe1, and Nkx2.1 during thyroid embryogenesis was first shown in mice [8]. However, despite their important roles, mutations in these transcription factors explained only a small percentage of CHDT cases (about 3%) [9]. Furthermore, these mutation-carrying patients usually have athyreosis or orthotopic hypoplasia but not ectopy, the most common phenotype observed in the clinic 10, ∗11. Thus, the reasons why the thyroid does not reach its final destination in about one in 4000 humans remains unknown in the vast majority of cases. The migration defect may be due to multiple germline and/or somatic mutations combined with epigenetic factors. Considering the prevalence of 1% in first-degree relatives (a 40-fold increase in relative risk), the different prevalence between ethnic groups (one in 30,000 newborns of Black-African descent compared to one in 4,000 in Caucasians), and the link with well-defined syndromes, a genetic predisposition to CHTD is clear 10, 12, 13, 14.

Zebrafish emerged in the 2000s as a good model for studying developmental biology [15]. Accordingly, it is now also increasingly used for studying thyroid development and diseases 16, 17, 18, 19, ∗20. Easy genome manipulation, simple housing and breeding, and embryo transparency are some of the advantages of zebrafish. In this review, we highlight the similarities in thyroid morphogenesis between human and zebrafish and we describe several genetic tools used in this model and their limitations. Moreover, we underscore the latest discoveries regarding thyroid development using zebrafish.

Section snippets

Thyroid morphogenesis

Thyroid is the first glandular tissue to appear during embryogenesis [21]. In mammals, such as human and mice, it originates from two endodermal regions; the thyroid diverticulum and the ultimobranchial bodies (UBB) ∗∗7, 22. The diverticulum originates from the midline of the pharyngeal floor and the UBB, the most important source of calcitonin-producing cells (C cells), derives from the lateral thyroid anlagen. Both fuse to form a composite gland in higher vertebrates ∗∗7, 23. In non-mammalian

Animal models used to study thyroid development

Mice have been most widely used to study thyroid development. The mouse thyroid is mostly similar to its human counterpart ∗∗7, 30. On the other hand, zebrafish have been proved to be a good model to study developmental biology [15]. Even though there are some important morphologic differences between zebrafish and human thyroid, key steps and main signalling pathways implicated in thyroid morphogenesis are conserved between mammals and fish [25]. For instance, pax2a, nkx2.4b, and hhex have the

Genetic tools

One of the most widely used techniques to disrupt gene expression in zebrafish is morpholinos (MO) [42∗∗]. These synthetic oligonucleotides hybridize to RNA transcripts and knockdown gene expression by blocking translation. The morpholino ring is not sensitive to nucleases, so MO are very stable [43]. Off-target effects are one of the main disadvantages of MO, making it sometimes hard to determine if the results are due to inhibition of the gene of interest or to a non-specific effect [44].

Limitations of the zebrafish model

Some limitations need to be considered when using this model. For instance, zebrafish thyroid anatomy is different compare to the human gland, as mentioned earlier. Therefore, some pathways cannot be studied with zebrafish like follicle organization in a tight gland. Regarding the genetic tools used in zebrafish, off-target effects, discrepancy between knockout and knockdown model results, and genetic compensation have been observed. Concerns regarding the use of morpholinos, one of the main

Conclusion

Mice have been the main model used to study thyroid development. Despite the fact that it remains an important model, first because its thyroid is very similar to the human gland, and second because it is a higher vertebrate, a new interesting model has emerged as a powerful tool to study thyroid morphogenesis, the zebrafish.

There are many genetic tools and transgenic lines available for the zebrafish that greatly facilitate gene study. The many advantages of this model explain why it is now

Disclosure

The authors have no disclosure relevant to this study.

Funding

Research in pediatric thyroid diseases at the CHU Ste-Justine is supported by private donations to JD (Girafonds, CHU Ste-Justine Foundation).

Acknowledgments

We thank Dr. Guy Van Vliet and Dr Pierre Drapeau for the revision of the manuscript. We thank the support of the Girafonds/Fondation du CHU Sainte-Justine.

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