Embryonic exposure to Smoothened Agonist disrupts tongue development in mice

The tongue is an important muscular organ for chewing and speech, and its development is regulated by multiple molecular signaling pathways, such as the hedgehog and TGF pathways. These pathways are particularly crucial in muscle patterning, which determines the structural and functional integrity of the tongue. Proper hedgehog signaling is essential for craniofacial tissue development, influencing muscle arrangement and differentiation critical for normal tongue morphology. In this study, we administered the Smoothened Agonist (SAG) 25 mg/kg to pregnant mice via intraperitoneal injection at E10.5 to further investigate the regulatory role of overexpressed hedgehog signaling in the early developmental process of the tongue. The intraperitoneal injection of SAG at E10.5 resulted in reduced tongue height and abnormal muscle development. Consequently, these alterations led to a midline cleft. Detection of cell proliferation using PHH3 and Ki67 showed that cell proliferation was significantly inhibited in the experimental group, while apoptosis showed no significant difference compared to the control group. At E11.5, the expression levels of downstream markers of hedgehog signaling, including Gli1, Ptch1, Foxf1, and Foxf2, were significantly elevated in the experimental group compared to the control group, whereas TGF-β2 mRNA expression was significantly downregulated(P < 0.05). Thus, overexpression of hedgehog signaling, induced by SAG, disrupts normal cellular processes by inhibiting proliferation and downregulating TGF-β2, ultimately leading to cleft tongue malformations.