Cell‐cell interactions in the process of differentiation of thyroid epithelial cells into follicles: A study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles

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Abstract
Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36–48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell‐cell contacts involved in (a) the formation of the thyroid follicular lumen and (b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC‐Dextran and Cascade Blue conjugated to bovine serum albumin: CB‐BSA). LY microinjected into IL of 2–9‐day‐old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 μm. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC‐Dextran or CB‐BSA microinjected into IL behaved as LY; the probes were restrained into the iumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15–30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2+‐dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FiTC‐Dextran or CB‐BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell‐to‐cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell‐to‐cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.