哺乳动物新皮层具有标志性的六层结构，六层结构精密排列执行不同的生理学功能，同一层次中又包含多种类型的神经元，从而形成更为细致的亚层结构，行使更为精细的功能，这种高度复杂的结构形成源于紧密协调的发育程序。
新皮层第V层（内锥体细胞层）主要由锥体神经元组成，其中包括投射至同侧或对侧皮质区域，参与联合信息处理的端脑内神经元与投射至皮质下结构（如丘脑、脑干、脊髓），调控运动输出的锥体束神经元1，以其轴突投射目标加以区分，并在解剖学上分层为不同的亚层：Va层和Vb层。然而，神经元多样化的机制及其决定性的分子标记尚未完全阐明。本研究针对新皮层第V层，结合新鉴定的分子标志物与单细胞转录组学（scRNA-seq），进一步探究新皮层第V层层次细分与神经元的多样性。本研究发现两个新的皮层层次分子标记物：Pcp4（浦肯野细胞蛋白4，又称 Pep19）和 FoxO1（叉头盒蛋白 O1），它们具有严格的时空特异性表达，并可以将第V层细分为 Va 层（以 FoxO1 阳性和 Ctip2 阴性神经元为特征）和 Vb 层（以 Pcp4 和 Ctip2 阳性神经元为特征）；为了进一步验证Pcp4、FoxO1与经典的第V层层次标志物Ctip2、Satb2的表达关系，本研究在单细胞转录组数据中对新皮层兴奋性神经元进行基因表达与神经元亚类的分析；结合实验室前期研究发现， microRNA（miRNA）会=在有丝分裂后层面对层次形成具有显著影响，本研究采用了两种 Dicer 条件性基因敲除（D6-cre-Dicer cKO，hGFAP-cre-Dicer cKO）小鼠模型，在皮层亚层角度进一步验证miRNA对层次形成的作用，研究发现miRNA缺失会导致大脑皮层亚层形成紊乱以及 Pcp4 和 FoxO1 的表达缺失，从而证明了miRNA在神经元亚型形成中的重要作用。
综上所述，本研究鉴定两种第V层亚层神经元标志物，并进一步加深了我们对miRNA对皮层层次形成的调控认识，并且为第V层神经元亚型多样化及皮层发育轨迹提供新的理解。

The mammalian neocortex has a signature six-layered structure, which is precisely arranged to carry out different physiological functions, and contains multiple types of neurons in the same layer, resulting in more detailed sublayers that carry out more refined functions. This highly complex structure results from a tightly coordinated developmental program. The layer V of the neocortex (inner pyramidal cell layer) consists mainly of pyramidal neurons, including telencephalic intrinsic neurons, which project to ipsilateral or contralateral cortical areas and are involved in joint information processing, and pyramidal fasciculus neurons, which project to subcortical structures (e.g., thalamus, brainstem, and spinal cord) and modulate motor output.1 These neurons are differentiated by their axon projection targets, and are anatomically stratified into different sublaminae: layers Va and Vb. However, the mechanisms underlying neuronal diversification and its defining molecular markers have not been fully elucidated. In this study, we targeted layer V of the neocortex, combining newly identified molecular markers with single-cell transcriptomics (scRNA-seq) to further investigate the hierarchical subdivision of layer V of the neocortex and neuronal diversity. In this study, two new molecular markers of cortical layers were identified: Pcp4 (Purkinje cell protein 4, also known as Pep19) and FoxO1 (Forkhead box protein O1), which have strict spatiotemporal specific expression and allow subdividing layer V into layer Va (characterized by FoxO1-positive and Ctip2-negative neurons) and layer Vb (characterized by Pcp4- and Ctip2- positive neurons). characterized); in order to further verify the relationship between the expression of Pcp4 and FoxO1 and the classical layer V hierarchical markers Ctip2 and Satb2, this study analyzed gene expression and neuronal subclasses of neocortical excitatory neurons in single-cell transcriptomic data; in conjunction with the laboratory's previous findings that microRNAs (miRNAs) would = have a significant effect on hierarchical formation, two Dicer conditional knockout (D6-cre-Dicer cKO, hGFAP-cre-Dicer cKO) mouse models were used in this study to further validate the role of miRNAs on hierarchical formation from the perspective of cortical sublayers, and it was found that miRNA deletion resulted in disrupted cortical sublayer formation as well as deficient expression of Pcp4 and FoxO1 expression deficiency, thus demonstrating the important role of miRNAs in neuronal subtype formation. In summary, this study identifies two layer V sublayer neuronal markers and further deepens our understanding of miRNA regulation of cortical hierarchy formation and provides new insights into layer V neuronal subtype diversification and cortical developmental trajectories