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.
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.
Tonoli
H
H
INSERM, U-369, Faculté de Médecine Lyon-RTH Laennec, Marseille, France.
Flachon
V
V
Audebet
C
C
Callé
A
A
Jarry-Guichard
T
T
Statuto
M
M
Rousset
B
B
Munari-Silem
Y
Y
D000818:Animals; D002450:Cell Communication / Q000502:physiology*; D002460:Cell Line / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology*; D018031:Connexin 43 / Q000235:genetics / Q000502:physiology; D017630:Connexins / Q000235:genetics / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D051381:Rats; D011916:Rats, Inbred F344; D013961:Thyroid Gland / Q000166:cytology / Q000502:physiology*; D014162:Transfection; D000097002:Gap Junction beta-1 Protein
D018031:Connexin 43; D017630:Connexins; D018076:DNA, Complementary; D000097002:Gap Junction beta-1 Protein