Angela Fan^1,2, Maryam Majeed³, Huihui Qi⁴, Noah Kuehn^1,5, Jack Scott¹, Sabrina Cheng^1,6, Huiqing Zhan⁷, Justus Kebschull⁴, Xiaoyin Chen³, James Bourne¹; ¹National Institutes of Health, ²Monash University, Clayton, Australia, ³Allen Institute, ⁴Johns Hopkins University, ⁵Georgetown University, ⁶University of Washington at Seattle, ⁷Cold Spring Harbour Laboratory

The primate cortex has expanded significantly across evolution, giving rise to primate-specific areas through duplication and specialization. In comparison, all major thalamic nuclei are conserved from rodents to primates and maintain connectivity to the whole cortex. These distinct evolutionary strategies create a paradox: how do the rapidly duplicated and specialized cortical networks remain functionally integrated with subcortical hubs that did not duplicate in parallel? Here we investigate the evolution of thalamocortical wiring rules by integrating single-cell and spatial transcriptomics with high-throughput single-cell connectivity mapping in both the marmoset and mouse thalamus. We sequenced 1.5 million neurons from the marmoset thalamus in-situ, and mapped the projections and transcriptomic identities of 480 thalamocortical neurons in the marmoset, and 1750 thalamocortical neurons in the mouse. In both species, we found continuous gene expression gradients that span across thalamic nuclei, suggesting a gradient-like organization that goes beyond nucleus borders. In contrast to the conserved transcriptomic architecture, projections in the primate thalamus project more focally and topographically compared to their mouse counterparts. Surprisingly, the more precise projections in the marmoset are associated with conserved gene expression axes that are also predictive of projections in the mouse thalamus, suggesting that the increased specificity in primate projections arises through sharpening of ancestral gene-projection relationships. We propose that this refinement of existing connectivity architecture represents an efficient strategy to accommodate projections to evolutionarily expanded cortical areas without requiring reorganization of relatively conserved subcortical hubs.