Most early-born subplate neurons persist as Layer 6b neurons in the adult mouse neocortex

Subplate neurons (SpNs) are among the earliest-born neurons in the mammalian neocortex and play key roles in radial migration and transient circuit formation. It has long been assumed that most SpNs undergo extensive postnatal cell death, leaving only a small remnant population that contributes to layer 6b (L6b) in adulthood. However, the extent to which SpNs actually persist as L6b neurons has remained unresolved, partly because previous studies lacked quantitative, whole-cortex analyses that account for postnatal cortical expansion. Here, we performed a comprehensive birthdating analysis using multiple EdU injections spanning the entire neurogenic window of SpNs in mice, combined with whole-neocortex 3D tissue clearing to measure subplate and L6b volumes. This approach allowed us to directly estimate the total number and distribution of SpN-derived neurons in adulthood. We found that most early-born SpNs persist as L6b neurons, and that the apparent postnatal reduction in SpN density reflects tangential cortical expansion rather than neuronal loss. Moreover, surviving SpNs comprise diverse neuronal subtypes reminiscent of those present at early postnatal stages. Together, these findings demonstrate that, in rodents, the majority of adult L6b neurons originate from SpNs, revising the long-held view that the subplate is a largely transient neuronal population.