The Superior Colliculus (SC) is a highly conserved midbrain organ implicated in sensorimotor integration. Pitx2on neurons are a recently identified population of glutamatergic neurons in the intermediate SC, whose activation leads to spatially oriented head movements. Viral tracing of these output neurons revealed a plethora of input and output regions. Particularly, while Pitx2on neurons project to tecto-spinal systems, traditionally associated with the control of head and eye movements, they also target the mesocorticolimbic system, suggesting a possible role of these neurons going beyond motor control. This project aims to unravel the molecular underpinnings of the apparent heterogeneity of the Pitx2on population. We aim to understand whether these diverse outputs are indicative of genetic heterogeneity within the population and, in turn, whether such heterogeneity supports diverse functional roles of the Pitx2on population beyond motor control. We used single nucleus RNAseq to interrogate the molecular profiles of Pitx2on neurons and utilised unsupervised clustering for an unbiased overview of the heterogeneity present. Expression suggests there are different classes within the Pitx2on population, cemented by in situ hybridisation data. To understand the relationship between these molecularly identified Pitx2on subclasses and their connectivity patterns, we used a self-inactivating rabies virus (SiR) to trace their outputs and then obtained bulk RNAseq from the projection-specific subclasses. By integrating these datasets we aim to define the relationship between molecular identity and connectivity for these subpopulations. Furthermore, in order to understand the functional role of the identified subclasses, we are investigating their role in both motor and cognitive control.