<i>T</i> Gene Mutation Leads to Short Tail in Sheep via Premature AER Degeneration: Single-Cell Evidence from Embryos

Hulunbuir short-tailed sheep (HSTS) and Hu sheep (HS) exhibit distinct tail phenotypes linked to ecological adaptation, with HSTS carrying a loss-of-function mutation (c.G334T) in the <i>T</i> gene while HS retain the wild-type allele. However, the cellular and molecular mechanisms underlying <i>T</i>-mediated tail development remain unclear. Here, we performed single-cell RNA sequencing on HSTS and HS embryos at embryonic days 16 and 19 (E16 and E19), complemented by cross-species validation using a CRISPR/Cas9 mouse model carrying the same mutation. We identified 12 cell types in E16 HSTS and E16 HS embryos, and 15 cell types in E19 HSTS and E19 HS embryos and found that the <i>MDK_ITGA6+ITGB1</i> ligand-receptor pair consistently mediated core intercellular communication. The <i>MDK_ITGA6+ITGB1</i> axis mediates intercellular communication critical for tail bud formation; BMP activation and FGF repression disrupt AER survival, leading to tail shortening. Developmental trajectories showed a shift from early progenitor states at E16 to terminal differentiation at E19. Crucially, HSTS embryos showed transcriptomic signatures consistent with premature AER regression. The <i>T</i> mutation showed transcriptomic signatures of increased BMP pathway activity and reduced <i>FGF8</i> expression, which may disrupt AER survival and contribute to the short-tail phenotype. In the mouse model, mutant <i>T</i> expression was reduced, and expression dynamics of <i>WNT5B</i> and <i>FGF8</i> were perturbed, corroborating the sheep findings; however, homozygous <i>T</i> mutation causes embryonic lethality in mice but not in sheep, indicating species-specific differences. This study provides single-cell transcriptomic evidence linking the <i>T</i> c.G334T mutation to premature AER regression in sheep, complemented by cross-species validation in a CRISPR/Cas9 mouse model, offering new insights into the cellular mechanisms of tail development and may provide a basis for future investigations into tail-related breeding markers, pending experimental validation. These changes are associated with AER maintenance and tail outgrowth.