Lu Chen

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.

Background: Since both fetal and postnatal development is determined by early embryonic development and the molecular mechanism and organogenesis process of early mouse embryos have been thoroughly studied. However, the specific development process of early embryos in Mongolian sheep remains poorly understood. Methods: The 16-day and 25-day embryo samples of Inner Mongolia Hulunbuir short-tailed sheep were evaluated by transcriptome sequencing technique and the genes with significant differences were screened and the gene function was enriched by gene ontology (GO) database and Kyoto Encyclopedia of Gene and Genome (KEGG) database. Result: A total of 4348 differentially expressed genes (DEGs) were analyzed by sequencing and especially significant DEGs included the genes such as COL6A2, COL4A4, LAMA4, ITGA8, FN1 involved in ECM-receptor interaction, focal adhesion, PI3K-Akt signaling and protein digestion and absorption pathways; YAP1, FZD4, TGFB1, WNT2, BMP5, BMP2, WNT11 involved in Hippo signaling pathway. In pathway analysis, those pathways mainly related to cell interaction and organogenesis were highlighted at the E16 stage. They are critical for transcriptional control in early embryonic development, especially during organ building. The genes that make organs and body structures more complex and detailed and those related to the development of brain, liver and immune system are enriched for the E25 stage. Therefore, this study provides important supplementary and exploratory reference data for the further research on early embryonic development in sheep.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.