Multiple cell types guided by neurocytes orchestrate horn bud initiation in dairy goats

Horn development is a key ruminant trait involving multi-cell type coordination via molecular pathways. This study used scRNA-seq to analyze cellular heterogeneity and fate trajectories during early horn bud niche formation, revealing key gene expression profiles. Combining with hematoxylin-eosin (HE) staining and immunohistochemical analysis, we further verified the asynchronous developmental pathways of key cells in the skin tissue of fetal goat horn bud at induction (embryonic day (E) 50; E50), organogenesis (E60), and cytodifferentiation (E70) stages, and demonstrated the signal transmission routes for the development of early horn buds.

We revealed temporal and spatial differences of the main signal transmission of horn bud development combining with existing literatures. We speculated that multiple cell types under the guidance of nerve cells collaborated on horn bud initiation in dairy goats. In detail, neural cells receive initial horn bud signals, stimulating hair follicle cell degeneration and transmitting to dermal cells, which evolve through intermediates, amplify signals to epithelial cells, and differentiate into mesenchymal cells. Nerve cell branches also trigger neural crest cell production/migration, working with chondrocytes to promote keratinocyte differentiation for horn bud formation. In addition, we further identified the early horn bud developmental specific events, including the screening of biological functions, signaling pathways and key candidate genes.

This study employed scRNA-seq to characterize cell fate trajectories and gene expression profiles in goat fetal horn buds. Histological comparisons between hornless and horned fetuses revealed cellular heterogeneity in epithelial, dermal, nerve, and hair follicle cells, with pseudo-time analysis identifying distinct differentiation paths. Dermal and epithelial cell transcriptional dynamics were critical for horn bud initiation (branch 1), supported by immunohistochemistry. Keratinocyte and nerve cell state transitions actively regulated horn development, with asynchronous cell development visualized via immunohistochemistry. Functional enrichment analyses (GO/KEGG) highlighted neural crest development and keratinocyte differentiation pathways, identifying candidate genes (EGR1, ZEB2, SFRP2, KRT10, FMOD, CENPW, LDB1, TWIST1) involved in horn morphogenesis. These findings advance understanding of goat horn development and genetic determinants.