Background: Proper tongue development is important to related structures in the craniofacial region; normal contractions of the tongue and other facial muscles control the forward growth of bone, cartilage growth and facial muscle bulk.Embryonic tongue muscles have unique characteristics that are distinct from other skeletal muscles such as limb and trunk.Although early tongue (somite-derived muscles) specification is mediated by the intrinsic molecular factor Pax3, core myogenic regulators including MyoD, Myf5, myogenin and MRF4 are required for its differentiation.In this context, the molecular details of muscle development in tongue remain poorly understood.Retinoic acid (RA), a metabolite of vitamin A, is required for growth and development in chordate animals, including mice.Concurrently, reduced RA synthesis, loss of Wnt5a or Tbx1 (a member of T-box transcription factors) led to similar phenotypes with cardiac abnormalities, i.e., severe hypoplasia of second heart field (SHF)-derived heart.Increased Wnt5a expression was also found in perioptic mesenchyme of the eyes of RA-deficient mice.Wnt5a also participates in myogenesis during embryonic development and activates myogenic determination in paraxial mesoderm of mouse embryos.It is highly relevant that Wnt5a also plays a role in tongue size, fungiform papilla patterning and development through interaction with the Ror2 receptor.However, the aberrant morphogenesis and the molecular mechanisms that regulate myogenic development in the tongues of developing mice exposed to high-dose RA remain unclear.Objective: To study the aberrant morphogenesis and underlying mechanism of RA induced tongue malformation in fetal mice.Study design: Exogenous RA was used to induce tongue malformation.The histological changes of tongue were characterized by HE stainingand the ultrastructural changes of tongue body and genioglossus in mouse fetus were analyzed by transmission electron microscopy.Proliferation of tongue was detected by BrdU stainning, myogenic differentiation was measured by immunohistochemical assays of Myf5, MyoD and myosin, and qRT-PCR of Myf5 and MyoD.The signaling genes such as Wnt5a, CaMK2d, Tbx1, Ror2 and PKCδ were evaluated by qRT-PCR.Results: The pregnant mice treated with retinoic acid (+RA) produce embryos with tongue malformation and a cleft palate.Histological analyses revealed that at perinatal stage E14.5, the tongues of +RA fetuses failed to descend and flatten.Ultrastructural analysis showed that at E18.5, the myofibrils failed to form normal structures of sarcomeres, and that muscle fibers were disordered in the genioglossus.The proliferation rates, and levels of myogenic determination markers (Myf5 and MyoD) and myosin in the genioglossus were profoundly reduced.The down-regulation of Wnt5a was positively correlated with Camk2d and inversely correlated with Tbx1, Ror2, and PKCδ in the tongues of +RA fetuses.Conclusion: Our work shows that excess RA induces tongue malformation in fetal mice.At E14.5, tongues of fetal mice from pregnant females treated with RA failed to descend and flatten.Myofibrils of the genioglossus failed to form normal structures of sarcomeres, resulting in disorderly arranged muscle fibers.RA exposure also caused hypoplasia and derangement of the genioglossus and tongue body.RA also impaired proliferation and differentiation of the genioglossus cells.Wnt5a/CaMKⅡ signaling plays a potentially important role in the normal development of mouse tongue.This study indicates a crosstalk between Wnt5a, Camk2d, and Ror2 in myogenic development of the tongue in response to RA.Wnt5a/ CaMKⅡ pathway is involved in RA-induced abnormal myogenic development of tongue in fetal mice.While the potential negative correlation of Wnt5a with Tbx1 and PKCδ in tongue malformation following RA exposure needs further investigation, our study provides novel insights into RA-mediated birth defects and tongue-related diseases.