The zebrafish thyroid gland shows a unique pattern of growth as a differentiated endocrine gland. Here, we analyze the onset of differentiation, the contribution of lineages, and the mode of growth of this gland. The expression of genes involved in hormone production and the establishment of epithelial polarity show that differentiation into a first thyroid follicle takes place early during embryonic development. Thyroid follicular tissue then grows along the pharyngeal midline, initially independently of thyroid stimulating hormone. Lineage analysis reveals that thyroid follicle cells are exclusively recruited from the pharyngeal endoderm. The ultimobranchial bodies that merge with the thyroid in mammals form separate glands in zebrafish as visualized by calcitonin precursor gene expression. Mosaic analysis suggests that the first thyroid follicle differentiating at 55 hours postfertilization corresponds later to the most anterior follicle and that new follicles are added caudally.

Thyroid hormone (T4) can be detected in thyroid follicles in wild-type zebrafish larvae from 3 days of development, when the thyroid has differentiated. In contrast, embryos or larvae treated with goitrogens (substances such as methimazole, potassium percholorate, and 6-n-propyl-2-thiouracil) are devoid of thyroid hormone immunoreactivity. Phenythiourea (PTurea; also commonly known as PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. PTurea contains a thiocarbamide group that is responsible for goitrogenic activity in methimazole and 6-n-propyl-2-thiouracil. In the present study, we show that commonly used doses of 0.003% PTurea abolish T4 immunoreactivity of the thyroid follicles of zebrafish larvae. As development of the thyroid gland is not affected, these data suggest that PTurea blocks thyroid hormone production. Like other goitrogens, PTurea causes delayed hatching, retardation and malformation of embryos or larvae with increasing doses. At doses of 0.003% PTurea, however, toxic side effects seem to be at a minimum, and the maternal contribution of the hormone might compensate for compromised thyroid function during the first days of development.

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

During vertebrate development, the thyroid gland undergoes a unique relocalisation from its site of induction to a distant speciesspecific position in the cervical mesenchyme. We have analysed thyroid morphogenesis in wild-type and mutant zebrafish and mice, and find that localisation of growing thyroid tissue along the anteroposterior axis in zebrafish is linked to the development of the ventral aorta. In grafting experiments, ectopic vascular cells influence the localisation of thyroid tissue cell non-autonomously, showing that vessels provide guidance cues in zebrafish thyroid morphogenesis. In mouse thyroid development, the midline primordium bifurcates and two lobes relocalise cranially along the bilateral pair of carotid arteries. In hedgehog-deficient mice, thyroid tissue always develops along the ectopically and asymmetrically positioned carotid arteries, suggesting that, in mice (as in zebrafish), co-developing major arteries define the position of the thyroid. The similarity between zebrafish and mouse mutant phenotypes further indicates that thyroid relocalisation involves two morphogenetic phases, and that variation in the second phase accounts for species-specific differences in thyroid morphology. Moreover, the involvement of vessels in thyroid relocalisation sheds new light on the interpretation of congenital thyroid defects in humans.KEY WORDS: Thyroid, Zebrafish, Mouse, Arteries, Vegf, Scl, Hedgehog Development 133, 3797-3804 (2006) DEVELOPMENT MATERIALS AND METHODS AnimalsZebrafish work was carried out according to standard procedures and staging in hours post fertilisation (hpf) refers to development at 28.5 to 29°C. The term 'larva' is used in the text for fry that have hatched from the chorion and generally refers to an age older than 72 hpf. The mutant zebrafish line kdr y17 (Covassin et al., 2006) is allelic to the kdr (flk1) lines described previously (Habeck et al., 2002) and develops heart oedema owing to compromised circulation or other specific defects at stages older than 40 hours, so that determination of mutant phenotypes was possible based on morphology. Homozygous cloche mutant embryos (clo s5 ) (Stainier et al., 1995) were also identified before fixation according to their phenotype at 24 hpf. Phenotypic details described in this study were always evident in all homozygous specimens (more than 10 in each experiment).For analysis of short digits (Dsh/Dsh) (Niedermaier et al., 2005) and Xt J /Xt J mutant mouse embryos (Persson et al., 2002) timed matings of mutants were generated. The homozygous phenotypes are distinguishable from wild type owing to severe morphological defects, and in the case of Dsh-Xt J matings we determined the genotype by PCR from extra-embryonic membranes (primer information available upon request). Preparation of specimensIn situ hybridisation on zebrafish was carried out according to standard procedures, using nk2.1a (Rohr and Concha, 2000) as molecular marker for the thyroid primordium. Whole-mount immunohistochemistry with antibodies against thyroid h...

The first occurrence of slender sunfish Ranzania laevis from the eastern coast of Libya (Southern Mediterranean) is reported herewith. The present finding appears to be the first record from the Libyan waters.