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.

Dkks have inhibitory effects on the Wnt signaling pathway, which is involved in the development of skin and its appendages and the regulation of hair growth. The nucleotide sequences were compared and analyzed to further investigate the relationship between the structure and function of the Dkk gene family and vertebrate epidermal hair. The analysis of the molecular evolution of the Dkk family revealed that the evolution rate of the genes changed significantly after speciation, with the Aves and Reptilia branches showing accelerated evolution. Additionally, positive selection was observed at specific sites. The tertiary structure of the protein was also predicted. The analysis of the functional divergence of the Dkk family revealed that the functional divergence coefficient of each gene was greater than 0, with most of the functional divergence sites were located in the Cys-2 domain and a few in the Cys-1 domain. This suggests that the amino acid and functional divergence sites may play a role in regulating the binding of the Dkk family to LRP5/6, and thus affect the inhibition of Wnt signaling, leading to different functions of Dkk1, Dkk2, and Dkk4 in the development of skin hair follicles. In addition, the Dkk families of Aves and Reptilia may have undergone adaptive evolution and functional divergence.

To investigate the role of long non-coding RNAs (lncRNAs) in the regulation of albendazole resistance in Haemonchus contortus (H. contortus) through competing endogenous RNA (ceRNA) mechanisms, a ceRNA network analysis was performed based on RNA-seq data from albendazole-sensitive and albendazole-resistant strains. Starting with significantly upregulated lncRNAs and incorporating functional enrichment analysis of predicted target genes, a potential regulatory axis—MSTRG.11540.1/novel-m1192-3p/HCON_00073890 (CYP2C70)—was identified from the ceRNA network. Transcriptomic analysis revealed that MSTRG.11540.1 and CYP2C70 were significantly upregulated (fold changes of 1.20 and 4.01, respectively; P < 0.01), while novel-m1192-3p was significantly downregulated (fold change: 5.91; P < 0.05) in resistant strains. This study employed dual-luciferase reporter assays and RNA interference (RNAi) to validate the interactions and functional relevance of the candidate RNA molecules. Furthermore, an egg hatch assay (EHA) was conducted to assess the impact of the lncRNA-mediated ceRNA axis on albendazole resistance. The aim was to provide insights into the molecular mechanisms by which lncRNAs may regulate drug resistance in H. contortus. A dual-luciferase reporter plasmid containing the predicted binding sites was constructed to investigate the candidate ceRNA axis MSTRG.11540.1/novel-m1192-3p/CYP2C70. The interaction assay revealed that novel-m1192-3p binds to MSTRG.11540.1, but no direct binding was observed between novel-m1192-3p and its predicted target gene CYP2C70. However, overexpression of novel-m1192-3p downregulated the expression of both MSTRG.11540.1 (45.71% reduction compared to NC group, P > 0.05) and CYP2C70 (40.74% reduction versus NC group, P < 0.05). Subsequent RNAi experiments confirmed the existence of the MSTRG.11540.1/novel-m1192-3p/CYP2C70 regulatory axis. Moreover, EHA results showed that knockdown of MSTRG.11540.1 significantly decreased the LD50 value of albendazole (52.91 µg/mL to 32.02 µg/mL). This study confirmed that the lncRNA-mediated ceRNA axis MSTRG.11540.1/novel-m1192-3p/CYP2C70 is involved in the regulation of albendazole resistance in H. contortus. These findings provide potential molecular targets for further elucidation of anthelmintic resistance mechanisms and the development of novel therapeutic strategies.