Developmental emergence of first- and higher-order thalamic neuron molecular identities

The thalamus is organized into nuclei that have distinct input and output connectivities with the cortex. While first-order (FO) nuclei also called core nuclei relay input from sensory organs on the body surface and project to primary sensory areas, higher-order (HO) nuclei matrix nuclei instead receive their driver input from the cortex and project to secondary and associative areas within cortico-thalamo-cortical loops. Input-dependent processes have been shown to play a critical role in the emergence of FO thalamic neuron identity from a ground state HO neuron identity, yet how this identity emerges during development remains unknown. Here, using single-nucleus RNA sequencing of the developing embryonic thalamus we show that FO thalamic identity emerges after HO identity, and that peripheral input is critical for the maturation of excitatory, but not inhibitory FO-type neurons. Our findings reveal that subsets of HO neurons are developmentally co-opted into FO-type neurons, providing a mechanistic framework for the diversification of thalamic neuron types during development and evolution. Diencephalon of the embryos were collected in ice-cold Hank's Balanced Salt Solution (HBSS) and segments were microdissected under a stereomicroscope at different time points. Thalamic chunks were frozen and stored at -80C until further processing for the nuc seq samples, or immediately processed for the single-cell ones.