Shinozaki et al., 2004; Shen et al., 2006; Osumi et al., 2008), thus providing the rationale for using PROX1 as a reliable marker to identify our target neuronal subtype. Furthermore, the temporal expression patterns of these transcription factors during the our differentiation protocol mimic that of the hippocampal neurogenesis process observed in vivo, providing additional confidence to the identity of these PROX1+ neurons as DG granule neurons. Second, recent studies have also demonstrated the critical role for PROX1 in defining the granule neuron identity during hippocampal development. The sustained presence of PROX1 is necessary to specify the granule neuron identity relative to the CA3 pyramidal fate in the hippocampus, and the loss of Prox1 in immature DG neurons results in the manifestation of CA3 pyramidal neuronal properties (Lavado et al., 2010; Iwano et al., 2012). Our data show sustained levels of PROX1 expression from week 3 of differentiation and onward as well as double labeling of PROX1 with MAP2AB, a postmitotic neuronal marker. Finally, we also developed a new tool (lentiviral Prox1-EGFP reporter) that can be readily used to visualize and sort granule neurons in culture. Prox1-EGFP-positive neurons are electrophysiologically active, demonstrating that our protocol is indeed producing functional DG
