GSE171921
Accelerated evolution analysis uncovers PKNOX2 as a key transcription factor in the mammalian cochlea
The genetic bases underlying the evolution of morphological and functional innovations of the mammalian inner ear are poorly understood. Gene regulatory regions are thought to play an important role in the evolution of form and function. To uncover crucial hearing genes whose regulatory machinery evolved specifically in mammalian lineages, we mapped accelerated noncoding sequences (ACNEs) in inner ear transcription factors (TFs) identifying PKNOX2 as the gene displaying the largest amount of ACNEs. To investigate the function of PKNOX2-ACNEs, we tested them using enhancer assays in transgenic zebrafish and unmasked transcriptional enhancers that acquired novel expression patterns as a consequence of the evolutionary process they underwent. Even though PKNOX2 is one of the most highly expressed genes in cochlear hair cells, its function was unknown. Thus, to explore the role of PKNOX2 in mammalian hearing, we generated Pknox2 null mice using CRISPR/Cas9 technology.  Pknox2-/- mice exhibited reduced distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) thresholds at high frequencies together with an increase in peak 1 amplitude, consistent with a higher number of IHCs-auditory nerve synapsis observed at the cochlear basal region. A comprehensive cochlear transcriptomic analysis of Pknox2-/- and Pknox2+/+ mice revealed that key auditory genes are under Pknox2 control. Hence, we report, for the first time, that PKNOX2 has a critical function regulating cochlear sensitivity at higher frequencies and that its regulatory machinery underwent lineage-specific evolution, providing novel insight into the contribution of this TF to normal auditory function and to the evolution of high-frequency hearing. Overall design: Cochleas were extracted from Pknox2+/+ and Pknox2-/-mice at eight days of age (P8) in 5 independent biological samples