Previous transcriptomic profiling studies have typically focused on separately analyzing mRNA expression, alternative splicing and alternative polyadenylation differences between cell and tissue types. However, the relative contribution of these three transcriptomic regulatory layers to cell type specification is poorly understood. This question is particularly relevant to neurons, given their extensive heterogeneity associated with brain location, morphology and function. In the present study, we generated profiles for the three regulatory layers from developmentally and regionally distinct subpopulations of neurons from the mouse hippocampus and broader nervous system. Multi-omics factor analyses revealed differing contributions of each transcriptomic layer in the discrimination of neurons based on their stage of development, region, and function. Importantly, profiles of differential alternative splicing and polyadenylation better discriminated specific neuronal subtype populations than gene expression patterns. These results provide evidence for differential relative contributions of coordinated gene regulatory layers in the specification of neuronal subtypes.