Background: GLIS3 (GLI-Similar protein 3) is a transcription factor involved in several cellular processes. Homozygous mutations in the GLIS3 gene have been typically associated with neonatal diabetes and congenital hypothyroidism (CH) in a syndrome called NDH. NDH patients present developmental abnormalities including endocrine pancreas defects and a spectrum of thyroid abnormalities, mainly including thyroid dysgenesis (TD). The mouse models revealed a key role of Glis3 in pancreatic islets but not in early thyroid development, as Glis3 was described to retain a role in regulating thyroid hormone synthesis downstream the thyrotropin (TSH)/TSHR signaling pathway and in postnatal follicle proliferation. Hence, in this study, we have been taking advantage of the zebrafish model to gain insights on the Glis3 activity during thyroid organogenesis. Methods: Transient glis3-knockdown zebrafish embryos (called glis3 morphants) were generated by the microinjection of specific glis3 morpholinos at one- to two-cell stage to analyze the thyroid phenotype in vivo. Several additional analyses (in situ hybridization, immunohistochemistry, and pharmacological treatments) were performed for further molecular characterization. Results: The analysis of thyroid embryonic development revealed that Glis3 is involved in early steps of thyroid specification. glis3 morphants exhibited a reduced expression of the early transcription factors nkx2.4 and pax2a at the thyroid primordium level, which is not caused by changes in proliferation or apoptosis of the pharyngeal endoderm. As a result, the differentiated thyroid tissue in morphants appeared reduced in size with decreased expression of tg and slc5a5, a low number of thyroxine (T4)-producing follicles, associated with an elevation of tshba (homologous of the human TSHβ), thus resembling the clinical and biochemical manifestations of patients with TD. Interestingly, glis3 morphants have pancreatic β-cell defects, but not liver defects. In vitro and in vivo data also demonstrated that Glis3 is an effector of the Sonic Hedgehog (SHH) pathway. Molecular and pharmacological inhibition of SHH reproduced the thyroid defects observed in glis3 morphant. Conclusions: Our results demonstrate that glis3, within the SHH pathway, appears to determine the number of endodermal cells committed to a thyroid fate. This is the first evidence of the involvement of Glis3 in TD, thereby expanding the understanding of the genetic basis of thyroid development and CH.
Keywords: Sonic Hedgehog; congenital hypothyroidism; endoderm; thyroid development; thyroid dysgenesis; zebrafish.