Ex vivo model for elucidating the functional and structural differentiation of the embryonic mouse thyroid.

Terminal thyroid gland differentiation - the last developmental step needed to enable thyroid hormone (T4) synthesis - involves profound structural and biochemical changes in the thyroid follicular cells (TFCs). We aimed to develop an ex vivo thyroid model of embryonic mouse thyroid that would replicate the in vivo TFC differentiation program. E13.5 thyroid explants were cultured ex vivo in chemically defined medium for 7 days. Immunostaining and qPCR of thyroid explants showed thyroglobulin production onset, follicle formation, and T4 synthesis onset in 1-, 3-, and 5-day-old cultures, respectively. Differentiation was maintained and follicular growth continued throughout the 7-day culture period. Pharmacological approaches to culture inhibition were performed successfully in the ex vivo thyroids. Our robust and well described ex vivo thyroid culture model replicates the sequence of thyroid differentiation to T4 synthesis seen in vivo. This model can be used to test the effects of pharmacological inhibitors on thyroid hormone production.

TUBB1 mutations cause thyroid dysgenesis associated with abnormal platelet physiology.

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel TUBB1 gene mutations that co-segregated with TD in three distinct families leading to 1.1% of TUBB1 mutations in TD study cohort. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the ß-tubulin protein family. TUBB1 gene is expressed in the developing and adult thyroid in humans and mice. All three TUBB1 mutations lead to non-functional α/ß-tubulin dimers that cannot be incorporated into microtubules. In mice, Tubb1 knock-out disrupted microtubule integrity by preventing ß1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for ß1-tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin-coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis.

Cell Growth Dynamics in Embryonic and Adult Mouse Thyroid Revealed by a Novel Approach to Detect Thyroid Gland Subpopulations.

BACKGROUND: The thyroid is composed of endocrine epithelial cells, blood vessels, and mesenchyme. However, no data exist thus far on absolute cell numbers, relative distribution, and proliferation of the different cell populations in the developing and mature thyroid. The aim of this study was therefore to establish a flow cytometry protocol that allows detection and quantification of discrete cell populations in embryonic and adult murine thyroid tissues. METHODS: Cell-type anti-mouse specific antibodies were used for erythroid cells (Ter119), hematopoietic cells (CD45), epithelial cells (EpCam/CD326, E-cadherin/CD324), thyroid follicular cells and C-cells (Nkx2-1), endothelial cells (Pecam/CD31, Icam-1/CD54), and fibroblasts (PDGFRa/CD140a). Proliferating cells were detected after labeling with 5-bromo-2'-deoxyuridine (BrdU). For flow cytometry analyses, micro-dissected embryonic (E) and adult thyroids were pooled (E13.5, n = 25; E15.5, n = 15; E17.5, n = 15; adult, n = 4) in one sample. RESULTS: The absolute parenchymal cell numbers per mouse thyroid (M ± SD), excluding the large number of CD45(+) and Ter119(+) cells, increased from 7425 ± 1338 at E13.5 to 271,561 ± 22,325 in adult tissues. As expected, Nkx2-1(+) cells represented the largest cell population in adult tissues (61.2 ± 1.1%). Surprisingly, at all three embryonic stages analyzed, thyroid follicular cells and C-cells accounted only for a small percentage of the total thyroid cell mass (between 4.7 ± 0.4% and 9.4 ± 1.6%). In contrast, the largest cell population at all three embryonic stages was identified as PDGFRa/CD140a(+) fibroblasts (61.4 ± 0.4% to 77.3 ± 1.1%). However, these cells represented the smallest population in adult tissues (5.2 ± 0.8%). Pecam/CD31(+) endothelial cells increased from E13.5 to E15.5 from 3.7 ± 0.8% to 8.5 ± 3.0%, then remained stable at E17.5 and adult tissues. Proliferation rates were sizable during the entire organogenesis but differed between cell populations, with distinct proliferative peaks at E13.5 in epithelial cells (32.7 ± 0.6% BrdU(+) cells), and at E15.5 in endothelial cells (22.4 ± 2.4% BrdU(+) cells). Fibroblasts showed a constant proliferation rate in embryonic tissues. In adult tissues, BrdU(+) cells were between 0.1% and 0.4% in all cell types. CONCLUSIONS: Using a novel flow cytometry-based method, a previously unobserved highly dynamic growth pattern of thyroid cell populations during embryogenesis was uncovered. This approach will provide a useful new tool for cell function analyses in murine thyroid disease models.