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Formation of Three-Dimensional Thyroid Follicle-Like Structures by Polarized FRT Cells Made Communication Competent by Transfection and Stable Expression of the Connexin-32 Gene¹

INSERM, U-369, Faculté de Médecine Lyon-RTH Laennec (H.T., V.F., C.A., A.C., B.R., Y.M.S.), 69372 Lyon; Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, Campus de Luminy (T.J.G.), 13288 Marseille, France

Address all correspondence and requests for reprints to: Dr. Yvonne Munari-Silem, INSERM U-369, Faculté de Medecine, RTH Laennec, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail: u369{at}laennec.univ-lyon1.fr

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.

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