In vivo tracking of KCC2b expression during early brain development

Abstract

The neuronal chloride (Cl−) exporter, KCC2, regulates neuron excitability and development and undergoes a stereotypical pattern of delayed upregulation as neurons mature. KCC2 upregulation favors neural inhibition by establishing a negative Cl− gradient, ensuring GABA‐induced Cl− currents are inward and inhibitory. We developed a zebrafish fluorescent reporter line, KCC2b:mCitrine, to track KCC2 expression in vivo during early brain development. KCC2b:mCitrine was first detected at 16 h postfertilization and by day 6 labeled most central and peripheral neurons and processes. At 20 h, expression was greatest in the soma‐dense basal neuroepithelium but largely absent in apical and mantle zones where differentiation and migration primarily occur, and time lapse imaging at this stage supports a postmigration upregulation of KCC2b. Central dopamine neurons showed low KCC2b expression as observed in other species. KCC2b:mCitrine fluorescence was stable over minutes in most neurons, but brightness transients observed in single cells fit our expectation for real‐time tracking of KCC2b upregulation in new neurons. To further assess whether fluorescence brightness tracks KCC2b expression, zebrafish embryos were exposed to bisphenol‐A (BPA), which is known to suppress KCC2 expression. Fluorescence decreased after 6 days of BPA exposure but not after 2 or 4 days, suggesting that it is an accurate but delayed indicator of KCC2b expression. KCC2b:mCitrine zebrafish present a new method for visualizing KCC2b's complex dynamics during brain development, and potentially screening compounds aimed at modulating KCC2 expression.