For dense labeling with green fluorescent protein (GFP; flat-mount preparations, population studies of axonal outgrowth and immunohistochemistry for postnatal brains), , 4–5 ng/μL; Mizuno et al.

2014), which label cells sparsely but more strongly, were used.

This output system is primarily formed by excitatory cortical neurons, the major neuronal type in the cerebral cortex.This labeling method permits the examination of morphological changes in developing excitatory cortical neurons at the single-cell level.Using this method, we showed that neurons labeled at embryonic day (E)12.5 initiated directed axonal outgrowth in the intermediate zone (IZ) before migrating into the cortical plate (CP; Hatanaka and Yamauchi 2013).We also investigated the molecular identity, laminar fate, and birthdates of these neurons.Timed-pregnant Institute of Cancer Research mice were purchased from Japan SLC, Inc. Embryonic day (E)0.5 was defined as noon on the day of vaginal plug observation.

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