Abstract

Frizzled 4 (FZD4), a member of the frizzled family, is involved in various cancers and neurological disorders because of its abnormal expression. However, its regulatory role in skeletal development and bone formation, especially in livestock and poultry, remains poorly characterized. RNA sequencing (RNA-seq) analysis of bone tissue samples collected during physiological bone remodeling in chickens identified FZD4 as a differentially expressed gene. To investigate the function of FZD4 in the osteogenic differentiation of chicken bone marrow mesenchymal stem cells (BMSCs), we assessed the expression of osteogenic marker genes and canonical Wnt signaling pathway factors at the mRNA and protein levels. Additionally, we evaluated differentiation and mineralization potential via alkaline phosphatase (ALP) and alizarin red S (ARS) staining. The results demonstrated that FZD4 expression was significantly upregulated during early differentiation. Furthermore, FZD4 overexpression increased the expression of early osteogenic markers, whereas FZD4 knockdown suppressed this expression. Although FZD4 acts as a receptor for the canonical Wnt pathway, its role in promoting osteogenesis in chickens through this pathway remains unclear. Importantly, FZD4 overexpression upregulated key canonical Wnt signaling factors at both the mRNA and protein levels, whereas FZD4 knockdown had the opposite effect. These findings indicate that FZD4 participates in chicken bone formation by regulating osteogenic marker expression, likely through activation of the canonical Wnt signaling pathway. This study elucidates the specific regulatory role of FZD4 in avian skeletal development, providing novel insights and theoretical support for understanding bone development and skeletal health in poultry and livestock.