Proceedings of The Physiological Society

Physiology 2012 (Edinburgh) (2012) Proc Physiol Soc 27, PC239

Poster Communications

Ect2, an ortholog of Drosophila pebble, regulates formation of growth cones in primary cortical neurons

T. Tsuji1,2, C. Higashida-Tsuji1,2, H. Higashida1

1. Department of Biophysical Genetics, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640, Japan, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. 2. Neurophysiology, Centre for Integrative Physiology ,University of Edinburgh, Edinburgh, United Kingdom.

To identify genes required for the brain development, we previously performed in vivo RNAi screening in Drosophila embryos. We identified pebble as a gene that disrupts development of the Drosophila nervous system. Although pebble has been shown to implicate in the neuronal development of Drosophila in the several screenings, involvement of Ect2, a mammalian orthologue of pebble, in the mammalian neuronal development has not been addressed. To examine the involvement of Ect2 in the neuronal differentiation, we performed Ect2 RNA interference in the mouse neuroblastoma x rat glioma NG108-15 cell line. Silencing endogenous Ect2 mRNA with double-stranded RNA induced depletion of Ect2 and resulted in increased proportion of binucleate cells and morphological differentiation of NG108-15 cells characterized by the outgrowth of neuritis. These morphological changes were correlated with an increased level of acetylcholine esterase mRNA. In addition, expression of Ect2 was decreased in differentiated NG108-15 cells induced by dibutyryl cyclic AMP. We also confirmed in another adrenergic clone, PC12 cells, that inhibition of Ect2 expression by RNAi stimulated neurite outgrowth. These findings indicate that Ect2 negatively regulates the morphological and functional differentiation in neuronal cell line. Next, the effects of Ect2 depletion were studied in primary cultures of mouse embryonic cortical neurons. In the mouse embryonic cortex, Ect2 was accumulated throughout the ventricular and subventricular zones, which contain neuronal progenitor cells, from immunohistochemistry analysis. Loss of Ect2 did not affect the differentiation stages of neuritogenesis, the number of neurites, or axon length, while the numbers of growth cones and growth cone-like structures were increased. Our results suggest that Ect2 contributes to neuronal morphological differentiation through regulation of growth cone dynamics in mouse embryo neuron. Taken together, our results show that ECT2 may play a role in neuronal differentiation through regulation of cell cycle and cytoslelton reorganization.

Where applicable, experiments conform with Society ethical requirements