Amiodarone-induced disruption of thyroid hormone signaling derails patterning and epithelial-mesenchymal dynamics...

Thyroid hormones (THs) regulate embryonic growth and tissue remodeling, yet their contribution to avian limb morphogenesis remains incompletely defined. We hypothesized that amiodarone-induced inhibition of TH signaling can disrupt chick hindlimb development by altering key patterning pathways and epithelial-mesenchymal dynamics during early limb bud morphogenesis. Fertilized Rhode Island Red eggs received in ovo air-cell injections of phosphate-buffered saline (control) or amiodarone (1 mM) at embryonic day 0 and were analyzed at days 2, 4, and 10, with some embryos allowed to hatch for phenotypic assessment. Hindlimb development was evaluated using morphological, histological, molecular, cellular, and biochemical approaches, including gene and protein expression analyses, apoptosis and cell-cycle profiling, systems-level pathway analyses, and tissue remodeling endpoints. Amiodarone exposure produced consistent limb deformities at hatching and impaired chondrogenic organization at day 10. Molecular profiling revealed elevated SHH/FGF8/WNT3/HOXA10/CASP3 with concomitant suppression of hindlimb identity and mesenchymal/EMT-associated genes (TBX4, PITX1, FGF10, GLI3, SNAIL, WNT7A). Protein and flow cytometry data supported uncoupling of the AER-mesenchyme axis, increased epithelial CDH1-positive populations with reduced SNAIL, and enhanced cleaved Caspase-3 associated apoptosis, corroborated by expanded Nile Blue staining. Proteomic shifts, reduced acetylcholinesterase activity, and decreased hydroxyproline further suggest disrupted differentiation and matrix maturation. Collectively, these results suggest that intact TH signaling might be essential for coordinating patterning cues, epithelial-mesenchymal balance, cell survival, and extracellular matrix maturation during chick hindlimb development, and that amiodarone exposure is associated with disruption of these integrated developmental programs, which may contribute to the observed structural limb defects.