A Single-Cell Atlas of an Early Mongolian Sheep Embryo

SIMPLE SUMMARY: This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep (the day of mating was defined as day 0, and embryo samples were taken on the 16th day). TBXT mutations are related to tailless or short-tailed phenotypes in vertebrates. In our previous study, we detected the TBXT expression level at a different embryo stage in sheep and found that TBXT exhibited the highest expression at E16. We believe that TBXT plays an important role in E16; thus, we chose E16 as the focus of this study. This comprehensive single-cell map reveals previously unrecognized signaling pathways that will improve our understanding of the mechanism of short-tailed sheep formation. ABSTRACT: Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.