PROJECT SUMMARY/ABSTRACT Cell fate diversification is a critical step in producing the many neuronal subtypes required for functional circuitry in the vertebrate brain. We know a considerable amount about how neural progenitors diversify fates to form distinct daughter neurons. However, much less is known about later processes that diversity the fates of postmitotic neurons. One reason for this knowledge gap is that in most vertebrate models it is impossible to recognize and study precisely the same neurons in separate individuals, limiting predictive and statistical power. We can overcome these limitations in zebrafish by studying two adjacent, individually identifiable, spinal motoneurons. We discovered that these neurons are initially equivalent, and that interactions between them, in addition to interactions with an identified set of muscle fibers, breaks the equivalence between the two neurons and causes them to adopt distinct fates. Moreover, one of these neurons then typically dies, an important fate for sculpting brain architecture and circuitry. This is a unique situation in vertebrates, in which we can observe fate diversification as it is occurring in living embryos and predict that a neuron will die well before the process occurs, yet we still do not know the underlying molecular events. We will overcome this barrier using a variety of tools that enable us to manipulate the fates of these two neurons and single cell RNA sequencing at defined stages during the developmental process. This combination with enable us to discover genes involved in neuronal cell fate diversification as well as genes that predict neuronal cell death and survival. We plan to validate these genes using quantitative RNA in situ hybridization techniques. We will then test validated candidates using an innovative F0 CRISPR screen. Our proposed studies will reveal genes previously unknown to function during neuronal cell fate diversification, survival, and death. If successful, they will uncover genetic mechanisms of neuronal cell fate diversification with unprecedented precision, and thus will open new avenues of inquiry and deepen our understanding of neurodevelopmental mechanisms.