Formation of Three-Dimensional Thyroid Follicle-Like Structures by Polarized FRT Cells Made Communication Competent by Transfection and Stable Expression of the Connexin-32 Gene

• Published in: Endocrinology (Philadelphia) 2000-04, Vol.141 (4), p.1403-1413
• Main Authors: Tonoli, Hélène, Flachon, Virginie, Audebet, Christine, Callé, Aleth, Jarry-Guichard, Therese, Statuto, Massimo, Rousset, Bernard, Munari-Silem, Yvonne
• Format: Article
• Language: English
• Subjects: Animals; Cell Communication - physiology; Cell culture; Cell interactions; Cell Line - physiology; Cell migration; Cell Polarity - physiology; Confocal microscopy; Connexin 32; Connexin 43; Connexin 43 - genetics; Connexin 43 - physiology; Connexins - genetics; Connexins - physiology; DNA, Complementary - genetics; Domes; Epithelial cells; Epithelium; Fluorescent indicators; Follicles; Folliculogenesis; Gap Junction beta-1 Protein; Gap junctions; Gene Expression; Microinjection; Microscopy; Morphogenesis; Multiplication; Phase contrast; Rats; Rats, Inbred F344; Thyrocytes; Thyroid; Thyroid gland; Thyroid Gland - cytology; Thyroid Gland - physiology; Tightness; Transfection
• ISSN: 0013-7227; 1945-7170
• DOI: 10.1210/endo.141.4.7400

Abstract Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.