SUMMARY Destruction of deposited maternal transcripts is an essential step during early animal embryogenesis. Together with production of new zygotic transcripts, this process is called the maternal-to-zygotic transition (MZT) and enables the handover of developmental control from the maternal genome to the zygotic one. mRNA destruction is triggered by shortening of the 3' poly(A) tail, and control of tail length is an important post-transcriptional regulatory hub. However, we know little about the differences in poly(A)-tail lengths on maternal and zygotic transcripts. We propose a “developmental priming” hypothesis to bridge this conceptual gap: here, maternal transcripts experience oogenesis-specific poly(A)-tail shortening that leads to their destruction during the MZT. Developmental priming thus provides a mechanism whereby maternal and zygotic transcripts can be distinguished to enable the transfer of developmental control to the zygotic genome. In this proposal, we will test and explore this hypothesis using Drosophila as a model system. We will first focus on a potential archetype of developmental priming: the maternal Sex lethal (Sxl) mRNA. Sxl is a master regulator of sex determination and dosage compensation. Sex chromosome number leads to different male and female Sxl transcript isoforms, and the female isoform sets off an auto-regulatory, feed-forward loop that culminates in the maintenance of female identity. Importantly, the maternal (and thus female) Sxl mRNA is deposited in the embryo and must be removed lest it override the zygotic sex chromosome composition and lead to all embryos being specified as “female.” However, the molecular mechanism is unknown. We propose that the removal of maternal Sxl mRNA can be explained by developmental priming. In Aim 1, we will explore this model: during oogenesis the RNA-binding protein Bruno leads to short poly(A) tails on maternal Sxl mRNA and so primes them for destruction. In Aim 2, we will extend our hypothesis transcriptome- wide using a new approach that combines SNP-based allele detection with long-read sequencing to determine the poly(A)-tail lengths on maternal and zygotic transcripts. This second aim is exploratory and perfectly suited to the R21 mechanism. We expect our proposed research to make several significant contributions. First, it will provide an answer to a decades-old mystery about Sxl regulation during embryogenesis. Second, through our experiments in Aim 2, we will create high-quality long-read sequencing datasets spanning the Drosophila MZT. Third, we will develop a new experimental and computational tool that can use long-read sequencing to look at allele-specific regulation, which will enable future mechanistic experiments. Finally, it will provide a new conceptual framework for understanding the specific clearance of maternal transcripts, which will form the basis for future studies and grants. This intellectual framework will likely prove useful for understandin...