Characterization of Cajal-Retzius Neuron Populations in Ebf2-GFP Mice

Cajal-Retzius (CR) cells, which are amongst the earliest born neurons in the brain, are located in the marginal zone of the cerebral cortex. They secrete the glycoprotein Reelin, which acts as an extracellular cue that guides the migration of cortical neurons to their correct layers during embryonic development. Establishing their exact birthplace has been challenging due to the lack of a specific marker of these neurons. Recently, however, we showed that transgenic mice expressing green fluorescent protein (GFP) under the early B-cell factor 2 (Ebf2) promoter selectively label all Reelin⁺ CR neurons, which are born between embryonic day (E) 10 and E12. Here, we wanted to investigate the exact time of birth and place of origin of CR neurons. We injected pregnant Ebf2-GFP mice with bromodeoxyuridine (BrdU), to label recently born cells, at E9, E10, E11 and E12, and then fixed the brains of their pups at postnatal day (P) 0, P6, P14 and P21. Next, we used standard fluorescence immunohistochemistry in fixed brain slices to process the tissues and visualize CR neurons using immunofluorescence microscopy. Our results will allow us to characterize the precise time-window of origin for Ebf2⁺ CR neurons as well as the survival of the different age subsets. By harvesting embryos a few hours after injection and analyzing the proximity of double-labeled cells (GFP and BrdU) to the proposed origin sites, we will also be able to quantify the proportion of CR neurons born from each site.