Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC176

Poster Communications

Genetic dissection of potassium chloride cotransporter (KCC) functions in epithelial development and carcinogenesis in Drosophila

Y. Chen1,2, M. Shen1,2, R. J. Wilkins3, J. C. Ellory3, C. Wilson3

1. Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 2. Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan. 3. Physiology, Anatomy & Genetics, University of Oxford, Oxford, United Kingdom.

Electroneutral potassium chloride cotransporters (KCC) belong to the superfamily of electroneutral cation-chloride cotransporters and are encoded by four genes in vertebrates (KCC1 through KCC4) (1). The activity of KCC plays an important role in variety of physiological and pathological cellular functions, including cell volume regulation, epithelial ion transport, and osmotic homeostasis. Our previous studies have highlighted important roles of KCC in tumor progression. Malignant transformation of cervical epithelial cells is associated with the differential expression of volume-sensitive KCC activities resulting from the up-regulation of mRNA transcripts for KCC1, KCC3 and KCC4 isoforms (2). Loss-of-function KCC mutant cervical cancer cells exhibit inhibited cell growth accompanied by decreased activity of the cell cycle proteins retinoblastoma and cdc2 kinase (3). In this study, we aim to investigate the in vivo functions of KCC in epithelial development and carcinogenesis using Drosophila melanogaster as the model. There is a single KCC homologue in Drosophila, but its functional properties remain largely unknown. Hypomorphic mutations in the Drosophila KCC homologue, CG5594, render flies susceptible to epileptic-like seizures associated with excitatory GABAergic signaling, consistent with the function of mammalian KCC2 (4). However, strong loss-of-function alleles are lethal, suggesting other critical developmental roles. Using the GAL4/UAS (Upstream Activation Sequence) expression, we have overexpressed CG5594 in multiple tissues (5). Crosses were performed at 25°C, and 1-2 day old adult progeny were anesthetized with CO2 for eye and wing analyses. Overexpression of CG5594 in differentiating cells of the eye using the GMR-GAL4 driver had no significant effect on the size of fly eye but produced a mild disarrangement of the ommatidial array (Fig. 1A). Additionally, CG5594 overexpression in the posterior compartment of the developing wing, under the control of engrailed-GAL4, resulted in drastic size reduction in the region of overexpression with associated reduction in cell size (Fig. 1B). We conclude that KCC can regulate cell growth and cell size of fly eyes and wings. Analyses of mutant clones in the fly eye and wing will provide an excellent assay system in which to investigate how modulation of KCC function influences epithelial development and how overexpression might lead to metastasis in an appropriate genetic background.

Where applicable, experiments conform with Society ethical requirements