Smad4 Regulates the Developmental Coordination of Cortical Neurons via the CXCL12-CXCR4 Axis

Background: The functional assembly of neural circuits during cortical development relies on the precise spatiotemporal coordination of excitatory projection neurons and inhibitory interneurons. Here, we show that SMAD family member 4 (SMAD4) is required for this developmental coordination. Methods: We performed conditional deletion of Smad4 in pallial progenitors and evaluated the spatial distribution and number of Glutamic acid decarboxylase 67-green fluorescent protein positive (Gad67-GFP+) interneurons in the embryonic and adult cortex. Single-cell RNA sequencing (scRNA-seq) was utilized to assess transcriptomic changes in the Smad4-deficient cortex. Further in vivo expression validation and functional assessments were conducted using a C-X-C motif chemokine ligand 12 (Cxcl12)-Discosoma red fluorescent protein (DsRed) reporter line and Cxcl12 loss-of-function mutants, respectively. Results: Conditional deletion of Smad4 in pallial progenitors disrupts the spatial distribution and overall number of Gad67-GFP+ interneurons in both the embryonic and adult cortex. scRNA-seq revealed a marked reduction in Cxcl12 expression within the mutant cortex, while its receptor, C-X-C chemokine receptor type 4 (Cxcr4), remained normally expressed in interneurons. We confirmed this downregulation in vivo using the Cxcl12-DsRed reporter line. Functionally, Cxcl12 loss-of-function mutants phenocopy the interneuron migration deficits seen in Smad4 mutants. Conclusions: Our results uncover a projection neuron-derived SMAD4-CXCL12 signaling cascade that regulates CXCR4-dependent interneuron migration, providing a molecular basis for the cross-talk between these two distinct neuronal lineages.