Opposing activity of SOX9 and Wnt in the developing otocyst ensures SOX10 control of endolymphatic development

Sensorineural deafness can occur in campomelic dysplasia (CD). In a mouse model of the SOX9Y440X/+ CD mutation, SOX10 downregulation was previously shown to be associated with impaired development of the endolymphatic system at mid-gestation. The underlying molecular causes are unknown. Here we found at E10.5 when the otocyst lacks overt inner ear structures, Sox10 was downregulated in the dorsal and medial aspects of the Sox9Y440X/+ otic epithelium, regions associated with specification of the endolymphatic sac and duct. Single-cell transcriptomic profiling revealed differential elevation of Wnt signaling pathway genes associated with the domain-specific Sox10 reduction. Otocyst-specific forced elevation of Wnt signaling by expression of stabilized -catenin suppressed Sox10 expression. Similarly, WNT exerted an inhibitory effect on SOX10 expression in human inner ear organoids derived from pluripotent stem cells. We propose a conserved regulatory model of SOX9-WNT antagonism governing SOX10 action in the otocyst underlies the specification of endolymphatic cell fate, with important implications for understanding human deafness syndromes. E10.5 wild type and Sox9Y440X/+ (CD) otic vesicles (OVs) were isolated and dissociated for single-cell profiling. One additional replicate was included for the wild type.感音神经性耳聋可并发于坎佩尔软骨发育不全（Campomelic Dysplasia，CD）。此前针对SOX9Y440X/+ CD突变小鼠模型的研究显示，SOX10表达下调与妊娠中期内淋巴系统发育受损相关，但其潜在分子机制尚未阐明。本研究于胚胎第10.5天（E10.5）——此时耳泡尚未形成明显内耳结构——发现，在Sox9Y440X/+小鼠的耳上皮背侧与内侧区域（该区域与内淋巴囊及导管的特化过程密切相关），Sox10的表达出现显著下调。单细胞转录组测序（single-cell transcriptomic profiling）结果显示，与该区域特异性Sox10表达下调相关的Wnt信号通路（Wnt signaling pathway）基因呈现差异性上调。通过在耳泡中过表达稳定化β-连环蛋白（stabilized β-catenin）以强制激活Wnt信号，可有效抑制Sox10的表达。同样地，在多能干细胞（pluripotent stem cells）诱导构建的人内耳类器官（inner ear organoids）中，WNT信号同样对SOX10的表达产生抑制作用。我们提出一项保守调控模型：SOX9与WNT的拮抗作用在耳泡中调控SOX10的功能，进而决定内淋巴系细胞的命运，该发现对理解人类耳聋综合征具有重要意义。本研究分离并解离了胚胎第10.5天的野生型与Sox9Y440X/+（CD）小鼠耳泡（otic vesicles，OVs），用于单细胞转录组分析；野生型样本额外增设了1次生物学重复。