HOXD13 may play a role in idiopathic congenital clubfoot by regulating the expression of FHL1

Idiopathic congenital clubfoot (CCF) is a type of congenital foot malformation, and previous studies using the extended transmission disequilibrium testing (ETDT) analysis have confirmed the HOXD13 gene as the susceptible gene of CCF. This study aimed to verify whether Hoxd13 directly regulates skeletal muscle LIM protein 1 (Fhl1) expression during limb development in rat embryo, which will serve as a basis for comprehending etiological research of idiopathic CCF. Immunofluorescence staining was utilized to detect Hoxd13 and Fhl1 expression in 12.5d rat embryo, while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were used to confirm the interaction between the two genes. Both Hoxd13 and Fhl1 were expressed in the interdigital tissues of E12.5 rat embryo. Luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with Hoxd13. ChIP of the Hoxd13-Fhl1 promoter complex from the developing limb confirmed that endogenous Hoxd13 interacts with this region. Thus, Hoxd13 directly regulates Fhl1 expression in rat embryo. These findings suggest that HOXD13 may regulate the expression of FHL1 in the development of idiopathic CCF.