Single-cell transcriptomics illuminates anterior-posterior patterning of the mesoderm in Drosophila embryos at gastrulation

Single-cell technology was used to investigate dynamic transcriptional changes in the blastula and gastrula of Drosophila embryos. Cells from embryo of stages 5 to 9 were isolated and subjected to library construction. Mesoderm cells were labeled with mCD8-GFP utilizing the GAL/UAS system. PCA identified 12 unique cell clusters that represent the blastoderm, mesoderm, endoderm and ectoderm anlage, primordia, and a sampling of differentiating cell types. In the cellular blastoderm, we found that the major wave of zygotic gene activation consists of two steps, which is supported by sub-clustering analysis. Trajectory analysis of stage 6-8 mesoderm cells suggested that transcription factors, previously linked to ectodermal segment polarity, also control mesoderm segmentation, patterning and differentiation. With the cells originating from the posterior ventral region of the blastoderm, trajectory analysis identified a group of cells that are multipotent progenitors giving rise to visceral muscles, Malpighian tubules as well as a new cell type. In summary, this single-cell study of gastrulation demonstrates early patterning involves trunk- versus pole-centered programs, the mesoderm is segmentally patterned, and a new cell type arises from the caudal mesoderm. Embryos with mesoderm cells marked with twi(2xPE)>mCD8-GFP were collected for 1.5 hours and aged for 3 hours 15min at 22°C. A single-cell suspension was prepared through manual dissociation and immediately subjected to library construction without fixation.