Zebra-Fishing to obtain new insights into vertebrate thyroid development

The thyroid follicular cells originate from the ventral floor of the anterior foregut and, during development, follow some conserved morphogenetic steps such as their relocalization in the anterior neck region, the organization into follicular structures and the proliferation and functional differentiation. Alterations in these initial events lead to congenital hypothyroidism (CH), a frequent endocrine disorder that, in 85% of the cases, is the result of disturbed thyroid organogenesis, a condition known as thyroid dysgenesis (TD). The etiology of TD is still poorly understood, as only few cases are associated with mutations of known thyroidal transcription factors, suggesting that other unknown mechanisms play a role during thyroid organogenesis.A comparative gene expression analysis allowed us to identify new TSH-sensitive genes. Several genes, belonging to Notch pathway, which is involved in multi-organ development, were down-regulated by TSH treatment. Therefore, we focused our attention on these genes as novel candidates that might contribute to thyroid organogenesis. To functionally characterize the role of these novel candidates, we took advantage of the vertebrate model zebrafish. We have recapitulated the main steps of zebrafish embryonic thyroid development by whole mount in situ hybridization (nkx2.1a, pax2a, tg and slc5a5) or immunohistochemistry techniques to assess zebrafish thyroid hormone production (T4). We have then analyzed the expression of our candidate genes in combination with thyroid specific markers at different time-points during development. In case of a co-expression in the thyroid primordium, we have further investigated their role analyzing the thyroid phenotype with different approaches: morpholino knock-down or over-expression strategies, and, when available, analysis of mutant zebrafish lines. In some of these conditions, thyroid development was severely impaired. The combination of these different techniques allowed us to dissect the contributions of Notch-related genes to thyroid development.