Single-Nucleus RNA Sequencing of Developing and Mature Superior Colliculus Identifies Neuronal Diversity and Candidate Mediators of Circuit Assembly

The superior colliculus (SC) is a sensorimotor structure in the midbrain that integrates inputs from multiple sensory modalities to initiate motor commands. It undergoes well-characterized steps of circuit assembly during development, rendering the mouse SC a popular model to study establishment and refinement of neural connectivity. Here we performed single nucleus RNA-sequencing analysis of the mouse SC isolated at various developmental time points. Our study provides a transcriptomic landscape of the cell-types that comprise the SC across murine development with particular emphasis on neuronal heterogeneity. We used these data to identify Pax7 as a marker for an anatomically homogeneous population of GABAergic neurons. Lastly, we report a repertoire of genes differentially expressed across the different postnatal ages, many of which are known for regulating axon guidance and synapse formation. Our data provide a valuable resource for interrogating the mechanisms of circuit development, and identifying markers for manipulating specific neuronal populations and circuits in the SC. Overall design: Superior colliculus was microdissected from embryonic day 19 (E10), post-natal day 4 (P4), P8, or P21 C57BL/6J mice. Tissue was pooled from multiple animals and homogenized inRNAase-free lysis buffer (0.32 M sucrose, 3 mM CaCl2, 3 mM MgAc2, 0.1 mM EDTA, 10 mM Tris-HCl,1 mM DTT, 0.1% Triton X-100 in DEPC-treated water) using glass dounce homogenizer on ice. The homogenate was loaded into a polycarbonate ultracentrifuge tube containing sucrose solution (1.8 M sucrose, 3 mM MgAc2, 1 mM DTT, 10 mM Tris-HCl in DEPC-treated water) in the bottom and centrifuged at 107,000 g for 2.5 hours at 4°C. Supernatant was aspirated, and the nuclei containing pellet was incubated in RNAse-free 1x PBS, 0.04% BSA, 0.2 U/µl RNAse Inhibitor on ice before resuspending the pellet. The nuclear suspension was filtered twice through a 30 µm cell strainer RNAase-free. Single nucleus suspension was processed and sequenced using 10X Genomics 3' v3 snRNA-seq platform with a target capture of 2000 nuclei per sample. Libraries were sequenced on NovaSeq SP 100 (200,000 reads/nucleus). After sequencing, Illumina output was processed using CellRanger v3.0.2. Base call files for each sample were demultiplexed. A pre-mRNA reference was generated with cellranger mkref using the mm10 mouse genome. Each sample was aligned to the custom mm10 mouse reference genome using CellRanger.