In vivo tracking of KCC2b expression during early brain development

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.