Pbx1 and Pbx3 cooperatively regulate intermediate progenitor genesis and corticogenesis in the mouse neocortex

Intermediate progenitor cells (IPCs) are key amplifying neuronal precursors that generate the majority of glutamatergic projection neurons during neocortical development. Despite their central role in corticogenesis, the transcriptional mechanisms controlling IPC proliferation and lineage progression remain incompletely defined. Here we combined single-nucleus transcriptomics with bulk RNA sequencing of purified IPCs to identify the TALE homeodomain transcription factors Pbx1 and Pbx3 as prominent regulators in cortical progenitor populations. Single-nucleus RNA-seq of Tbr2⁺ IPCs reveals broad expression of Pbx1 across IPC states, with Pbx3 selectively enriched in proliferative IPCs. Conditional dual deletion of Pbx1/Pbx3 in the dorsal telencephalon using Emx1-Cre resulted in a marked reduction of proliferating IPCs during embryogenesis, while radial glial cell numbers and survival were largely preserved. At postnatal stages, Pbx1/Pbx3 double conditional mutants displayed microcephaly with reduced cortical size, disrupted laminar organization, increased numbers of deep-layer neurons, and a selective depletion of upper-layer neurons. These defects were accompanied by severe abnormalities in forebrain connectivity, including complete loss of the anterior commissure and partial agenesis of the corpus callosum. Integrated bulk-RNA-seq and CUT&Tag profiling of IPCs identified a core set of direct Pbx1/Pbx3 transcriptional targets implicated in IPC identity and lineage progression, including Cux2, Insm1, Lhx2, Myt1l, and Trnp1. Together, our findings establish Pbx1 and Pbx3 as essential transcriptional regulators of IPC proliferation and differentiation, thereby ensuring proper cortical neuron production and forebrain morphogenesis.