Inhibition of Pi4kb activity causes malformation of vestibular apparatus in zebrafish by downregulating hey1

Phosphatidylinositol 4 kinase-β (PI4KB) plays critical roles in human genetic diseases. In zebrafish, Pi4kb is strongly expressed in hair cells (HCs), which are necessary for detecting sound vibrations, head movements, and water motion. However, the role of PI4KB in HC or semicircular canal development is unclear. Herein, we report that pi4kb morphants exhibit insensitivity to sound stimulation and abnormal morphological vestibular organs, including cilium loss in HCs of the cristae and semicircular canal malformation. As bone morphogenetic protein (BMP) signaling is associated with HC and semicircular canal development, we analyzed the expression of BMP-related genes; the phosphorylated Smad1/5/9 (p-Smad1/5/9) expression was markedly reduced in otic HCs. RNA-sequencing data indicated that the transcriptional levels of BMP membrane receptor 2 (bmpr2a and bmpr2b) and hes-related family of bHLH transcription factors with YRPW motif 1 (hey1), a direct downstream target gene of p-Smad, were significantly reduced in the pi4kb morphants, as verified using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. Co-injection of hey1 mRNA and pi4kb morpholino notably recovered vestibular apparatus development, including the number and length of cilia in HCs of the cristae and semicircular canal formation. Collectively, these results suggest that Pi4kb is involved in vestibular apparatus development in zebrafish by regulating BMP membrane receptor 2 and p-Smad1/5/9 levels, thereby affecting the transcriptional activation of the target gene hey1. This study sheds light on the interaction between Pi4kb and the BMP-Hey1 signaling axis, which is critical for HC and semicircular canal formation.