Dopamine (DA) neurons located in the Substantia nigra pars compacta (SNc) are involved in motor control, and their degeneration is associated with disorders such as Parkinson’s disease (PD), the second most frequent neurodegenerative disease in the US and practically incurable. Recent studies have revealed that DA neurons are molecularly, anatomically, and functionally heterogeneous. In fact, certain DA neuron subtypes are selectively vulnerable in animal PD models. Hence, recognizing this diversity will have a significant impact on the study of PD pathophysiology and the development of treatments. This proposal aims to shed light on early molecular events, particularly involving transcription factor SOX6, that govern DA neuron fate and diversification. All midbrain DA neurons originate from progenitors in the embryonic mesencephalic floor plate. Sox6 is expressed in progenitors of the most medial domain of the floor plate. It is widely accepted that Sox6-expressing progenitors give rise to the entire SNc DA neuron population, however, existing publications challenge this model suggesting that Sox6- progenitors, located in the lateral domain of the floor plate, also contribute to the adult SNc. Yet, none of these studies performed lineage analysis of Sox6. A proper Sox6 progenitor fate map would reconcile these results, however, existing lineage tracing tools are inadequate to resolve this progenitor-progeny relationship since Sox6 is expressed in progenitors and in postmitotic neurons. We have developed a new lineage tracing tool, a progenitor-restricted intersectional fate-mapping strategy (PRISM), that circumvents the limitations of recombinase-based strategies by anchoring the labeling to progenitors. With PRISM, we will unambiguously reveal the contribution of Sox6+ progenitors to specific DA neuron subtypes, particularly those found selectively vulnerable in PD models. Additionally, we will combine PRISM with a single-cell resolution analysis for the first time, to further refine the molecular landscape of the Sox6 progeny. Our lab has highlighted Sox6 expression as the distinctive variable in adult DA neuron classification. Still, more evidence is needed to establish whether SOX6 regulates genes that define specific neuronal subtypes. Exploring the role of Sox6 in development will help understand if progenitor pools are restricted to give rise to certain subtypes of the SNc. Additionally, elucidating the molecular targets of SOX6 will deepen our understanding of the determination of DA neuron fate. To this end, we will pair a Sox6 knockout model with RNA sequencing to determine the necessity of this gene in the diversification of DA neurons. We will also apply ChIP- Seq to find the main target DNA sequences of this transcription factor. The proposed aims will provide insights into the role of Sox6 in DA neuron development and diversification, with the hope of contributing to the optimization of in vitro protocols for regenerative medici...