Dual Developmental Origins and Activity-dependent Specification of Mammalian Subplate Neurons

Subplate neurons (SpNs) are among the earliest-generated cortical neurons and are essential for neocortical circuit assembly. Despite this central role, they have long been considered a mammalian innovation, yet their evolutionary origin remains unresolved. Here, using comparative single-cell and spatial transcriptomics across amniotes (mice, chicks, and turtles), we identify two distinct developmental and evolutionary origins of SpNs: atypical SpNs (aSpNs), an Nr4a2-negative population conserved across amniotes and originating from the medial pallium, and mammalian-type SpNs (mSpNs), an Nr4a2-positive population preferentially expanded in mammals and arising from early-born cortical neurons. Cross-species analyses show that early-born pallial neurons in non-mammalian amniotes differentiate into thalamic input neurons, whereas this ancestral program is repurposed in mammals, with early-born neurons transiently adopting a subplate identity. We further show that this fate switch is controlled by Zbtb18 repression linked to thalamic input. Collectively, these findings establish a dual-origin model for SpNs and provide a unifying framework for understanding neocortical evolution.