Technical Reference #1843

1843.

The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation* Marcelo F. Santiago; Pepe Alcami; Katharine M. Striedinger; David C. Spray; and Eliana Scemes, Albert Einstein College of Medicine, Journal of Biological Chemistry, 285(1843), (2010)
Link To Paper

Abstract:
Connexin43 (Cx43) is widely expressed in embryonic brain; and its expression becomes restricted mainly to astrocytes as the central nervous system matures. Recent studies have indicated that Cx43 plays important; nonchannel; roles during central nervous system development by affecting neuronal cell migration. Here; we evaluated the effects of Cx43 on neuronal differentiation. For that we used an in vitro model of neural cell development (neurospheres) to evaluate; through immunocytochemistry;electrophysiology;andmolecular biology; the degree of neuronal maturation from neurospheres derived from wild-type (WT) and Cx43-null mice. Our results indicate that Cx43 is a negative modulator of neuronal differentiation. The percent neurospheres containing differentiated neurons and the number of cells displaying inward currents were significantly higher in Cx43-null than in WT littermate neurospheres. Knockdown of Cx43 with small interfering RNA increased the number of WT neurospheres generating differentiated neurons. Blockade of gap junctional communication with carbenoxolone did not induce neuronal differentiation in WT neurospheres. Transfection of Cx43-null neurospheres with Cx43 mutants revealed that Cx43 carboxyl terminus prevents neuronal maturation. In agreement with these in vitro data; in situ analysis of embryonic day 16 brains revealed increased -III-tubulin expression in germinal zones of Cx43-null compared with that of WT littermates. These results indicate that Cx43; and specifically its carboxyl terminus; is crucial for signaling mechanisms preventing premature neuronal differentiation during embryonic brain development.

Keywords:
Connexin; Development; Gap Junctions; Neural Stem Cell; Neuroprogenitor Cell; Connexin43; Development; Neural Progenitor; Neuronal Differentiation; Neurosphere

Materials & Methods:
Neurosphere Cultures—Neurospheres were prepared as described previously (20 21). Briefly neural progenitor cells were obtained by aspiration of forebrain tissues of E14 wildtype and Cx43-null C57BL/6J mouse embryos and mechanically dissociated into single cells in ice-cold Hanks’ balanced solution (Ca2 - and Mg2 -free). Cells were transferred to tissue culture dishes containing Dulbecco’s modified Eagle’s medium nutrient mixture F12 (DMEM-F12; Invitrogen) supplemented with 5% B27 (Invitrogen) 1% antibiotics and 20 ng/ml human recombinant epidermal growth factor (Sigma) and allowed to grow into floating neurospheres. Culture medium was changed twice a week and neurospheres were mechanically dissociated into smaller neurospheres once a week. Neurosphere cultures were maintained for no longer than 2 months. For in vitro cell differentiation floating neurospheres were plated on glass bottom microwells (MatTek Co. Ashland MA) coated with poly-D-lysine- (10 g/ml; Sigma) and fibronectin (10 g/ml; Sigma-Aldrich) and bathed in DMEM-F12 in the absence of epidermal growth factor. About 10 –15 neurospheres were seeded/MatTek dish.

Microscopic Technique
Confocal inverted microscopy

Cell Type(s)
Neurosphere cultures