Single-nucleus RNA-sequencing in pre-cellularization Drosophila melanogaster embryos

Our current understanding of the regulation of gene expression in the early Drosophila melanogaster embryo comes from observations of a few genes at a time, as with in situ hybridizations, or observation of gene expression levels without regards to patterning, as with RNA-sequencing. Single-nucleus RNA-sequencing however, has the potential to provide new insights into the regulation of gene expression for many genes at once while simultaneously retaining information regarding the position of each nucleus prior to dissociation based on patterned gene expression. In order to establish the practicality of single-nucleus RNA sequencing in the context of a real biological question, here we look at the difference in gene expression between control and an insulator protein, dCTCF, maternal null embryos during zygotic genome activation at nuclear cycle 14. We find that early embryonic nuclei can be grouped into distinct clusters according to gene expression. From both virtual and published in situ hybridizations, we also find that these clusters correspond to spatial regions of the embryo. Lastly, we present multiple examples of differential gene expression between control and maternal CTCF null nuclei in one or more clusters, but not in bulk when grouping expression across all nuclei. These results highlight the potential for single-nucleus RNA-sequencing to reveal new insights into the regulation of gene expression in the early Drosophila melanogaster embryo. ### Competing Interest Statement The authors have declared no competing interest.