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. Cochleas were extracted from Pknox2+/+ and Pknox2-/-mice at eight days of age (P8) in 5 independent biological samples目前学界对于哺乳动物内耳形态与功能革新演化背后的遗传基础仍知之甚少。一般认为基因调控区域在生物形态与功能的演化过程中发挥着关键作用。为发掘调控机制在哺乳动物谱系中发生特异性演化的关键听觉基因，我们对内耳转录因子（transcription factors, TFs）的加速非编码序列（accelerated noncoding sequences, ACNEs）开展定位分析，最终确定PKNOX2是携带ACNEs数量最多的基因。为探究PKNOX2相关ACNEs的功能，我们通过转基因斑马鱼增强子实验对其进行验证，揭示出经历演化过程后获得全新表达模式的转录增强子。尽管PKNOX2是耳蜗毛细胞中表达量最高的基因之一，但其具体功能此前尚未明确。因此，为探究PKNOX2在哺乳动物听觉中的作用，我们借助CRISPR/Cas9技术构建了Pknox2基因完全敲除小鼠模型。Pknox2-/-敲除小鼠在高频段表现出畸变产物耳声发射（distortion product otoacoustic emissions, DPOAEs）幅值降低、听觉脑干诱发电位（auditory brainstem response, ABR）阈值升高，同时伴随1波幅值增加，这与耳蜗基底区域观察到的内毛细胞-听觉神经突触数量增多的结果一致。对Pknox2-/-与Pknox2+/+小鼠开展的全面耳蜗转录组分析显示，核心听觉基因的表达均受Pknox2调控。综上，我们首次证实PKNOX2具备调控高频段耳蜗敏感性的关键功能，且其调控机制经历了谱系特异性演化，为阐明该转录因子在正常听觉功能及高频听觉演化中的作用提供了全新视角。本研究从5份独立生物学样本中，提取了出生后8日龄（P8）的Pknox2+/+与Pknox2-/-小鼠的耳蜗组织。