SHH ventralizes the otocyst by maintaining basal PKA activity and regulating GLI3 signaling

During development of the inner ear, secreted morphogens act coordinately to establish otocyst dorsoventral polarity. Among these, Sonic hedgehog (SHH) plays a critical role in determining ventral polarity. However, how this extracellular signal is transduced intracellularly to establish ventral polarity is unknown. In this study, we show that cAMP dependent protein kinase A (PKA) is a key intracellular factor mediating SHH signaling through regulation of GLI3 processing. Gain-of-function experiments using targeted gene transfection by sonoporation or electroporation revealed that SHH signaling inactivates PKA, maintaining a basal level of PKA activity in the ventral otocyst. This, in turn, suppresses partial proteolytic processing of GLI3FL, resulting in a low GLI3R/GLI3FL ratio in the ventral otocyst and the expression of ventral-specific genes required for ventral otocyst morphogenesis. Thus, we identify a molecular mechanism that links extracellular and intracellular signaling, determines early ventral polarity of the inner ear, and has implications for understanding the integration of polarity signals in multiple organ rudiments regulated by gradients of signaling molecules.