The zebrafish model to identify new genes involved in thyroid development

Abstract

Thyroid organogenesis is a very complex developmental process that leads to the formation of an organ able to produce specific hormones through specification, migration, structural organization and functional differentiation of thyroid precursor cells. In mammals, alteration of these mechanisms might be responsible for congenital hypothyroidism (CH), the most frequent inherited endocrine disease. Thyroid development in fish is comparable to mammals at both ontogenetic and molecular levels: the thyroid develops from the endodermal tissue, at the midline of the pharyngeal floor, and its formation is subdivided into main successive steps shared with mammals. The genetic program involved in fish thyroid differentiation, including the activity of transcription factors such as Nkx2.1a, Pax8 and Hhex, is conserved with respect to expression patterns and functions. To gain further insights on the genetic machinery implicated in normal thyroid development, and therefore in CH disease, an oligonucleotide microarray analysis was performed in mouse with the aim to identify genes differentially expressed during gland organogenesis. This approach highlighted a list of genes enriched in embryonic mouse thyroid. In the present work, the zebrafish model was used to perform functional analysis on fish counterparts of the murine thyroid enriched genes. Looking at the expression territories of the zebrafish orthologs, a very low correlation in thyroid expression profile between mouse and zebrafish was found: indeed, very few mouse thyroid expressed genes have their zebrafish ortholog being expressed in thyroid primordium. This observation strongly suggests that some changes concerning thyroid development occurred from lower to higher vertebrates; during evolution, the thyroid of higher vertebrates probably recruited new genetic pathways/functions by means of mutations occurred in cis-regulatory elements. Despite the low correlation in expression profiles, a new conserved function relevant for thyroid development was identified with this comparative approach; an anti-apoptotic function, represented by Bcl2 in mouse and bcl2l in zebrafish, was conserved during thyroid evolution. Knock-down experiments conducted by means of morpholinos injection, have revealed that bcl2l is regulated by the thyroid transcription factors and plays a relevant role in normal thyroid development. Indeed, bcl2l thyroid expression was lost in embryos deprived of the thyroid transcription factors, leading to thyroid degeneration via apoptosis. It is feasible to hypothesize that a conserved antiapoptotic function during thyroid evolution could counteract a putative proapoptotic function, ready to intervene if something fails during normal gland morphogenesis.