Exploring the sublayer formation process in layer V of the neocortex through two newly identified molecular markers.

The precisely organized six-layered architecture of the mammalian neocortex containing diverse types of neurons arises from tightly orchestrated developmental programs. Notably, layer V comprises two principal subtypes of excitatory pyramidal neurons distinguished by their axonal projection targets and stratifies into anatomically distinct sublayers: layer Va and layer Vb. Nevertheless, the mechanisms orchestrating neuronal diversification and their defining molecular markers have yet to be fully elucidated. In this study, we identified two novel molecular markers, Pcp4 (Purkinje cell protein 4, also known as Pep19) and FoxO1 (Forkhead box protein O1), which help define layer Va, characterized by FoxO1+ and Satb2+ neurons; and layer Vb, characterized by Pcp4+ and Ctip2+ neurons, and delineate two functionally discrete subpopulations of layer V projection neurons during late differentiation stages. Meanwhile, our findings were validated in single-cell RNA sequencing (scRNA-seq) data. We then employed two Dicer conditional knockout mouse models and found that microRNA (miRNA) deficiency leads to cortical sublayer disorganization and loss of Pcp4 and FoxO1 expression, demonstrating the essential role of miRNAs in neuronal subtype specification. This study advances our understanding of the molecular mechanisms governing the diversification of layer V neuronal subtypes and their developmental trajectories.