SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms

The embryonic endolymphatic sac mediates fluid resorption for which anion exchangers such as SLC26A4, and its transcriptional activator FOXI1, are required. Apart from FOXI1, little is known about transcriptional regulators of this fluid balance. Altered fluid homeostasis in the inner ear is the leading cause of congenital hearing loss. Some patients with campomelic dysplasia (CD) caused by heterozygous mutations in SOX9, in addition to skeletal malformation, are deaf, with an unknown etiology. In a mouse model of CD caused by the SOX9Y440X mutation, which results in a truncated protein lacking the transactivation domain, we show severe endolymphatic dysfunction leading to deafness and vestibular problems. Adult heterozygous Sox9Y440X mice that express the mutation in the developing inner ear display sensorineural deafness combined with endolymphatic hydrops and lack of endocochlear potential. The mutant inner ear is enlarged from E15 and fewer Slc26a4-expressing cells are present in the endolymphatic epithelium. By single cell RNA sequencing of mutant and wild-type endolymphatic sacs we find marked reduction in genes important for ion transport such as Slc24a4 and Ttyh1 and gap-junctions such as Gjb2 and up-regulation of Wnt signaling and Igfbp2 that promotes progenitor proliferation. The cochlea overexpresses Aqp3, which encodes a water channel. We find by biochemical and cell-based transactivation assays and genetic interaction tests that SOX9 and SOX10 normally work co-operatively to repress Aqp3 transcription: SOX9Y440X blocks this repression by dominant interference. Our study reveals the key roles of SOXE transcription factors in the development of endolymphatic sac function, and highlight a molecular mechanism whereby the CD mutation exerts both a dominant negative and haploinsufficient mechanisms in different compartments of the inner ear to maintain fluid homeostasis. In the cochlea, SOX9 and SOX10 together repress aquaporin expression whereas in the endolymphatic sac, SOX9 controls SOX10 expression and genes for ion transport and gap-junctions. We postulate that in addition to loss of control of cell proliferation results in the exhaustion of progenitors which in turn leads to a deficit in mitochondria-rich cells. These results highlight a multifaceted mechanism for maintaining the proper fluid balance for hearing. We sequenced 120 cells from mouse wild-type and Sox9Y440X/+ endolymphatic sacs by Smart-seq2 protocol胚胎期内淋巴囊（embryonic endolymphatic sac）介导液体重吸收，该过程依赖于SLC26A4等阴离子交换蛋白及其转录激活因子FOXI1。除FOXI1外，目前对该液体平衡的转录调控因子所知甚少。内耳液体稳态失衡是先天性听力损失的首要诱因。部分因SOX9杂合突变导致的躯干发育异常（campomelic dysplasia, CD）患者除骨骼畸形外，还会出现听力损失，但其发病机制尚未明确。在由SOX9Y440X突变（该突变会产生缺失转录激活结构域的截短蛋白）构建的CD小鼠模型中，我们发现其存在严重的内淋巴功能障碍，进而引发听力损失与前庭功能异常。发育阶段内耳表达该突变的成年杂合Sox9Y440X小鼠，表现出感音神经性听力损失，伴随内淋巴积水以及耳蜗内电位缺失。突变型小鼠的内耳自胚胎期15天（E15）起便出现膨大，且内淋巴上皮中表达Slc26a4的细胞数量减少。通过对突变型与野生型内淋巴囊进行单细胞RNA测序（single cell RNA sequencing），我们发现：离子转运相关基因（如Slc24a4、Ttyh1）以及缝隙连接相关基因（如Gjb2）的表达显著下调，而促进祖细胞增殖的Wnt信号通路与Igfbp2的表达则出现上调。耳蜗中Aqp3的表达出现上调，该基因编码水通道蛋白。我们通过生化实验、细胞水平转录激活实验以及遗传互作实验发现：SOX9与SOX10通常可协同抑制Aqp3的转录，而SOX9Y440X突变蛋白会通过显性负效应阻断这一抑制过程。本研究揭示了SOXE转录因子（SOXE transcription factors）在内淋巴囊功能发育中的关键作用，并阐明了CD突变通过显性负效应与单倍剂量不足两种机制，作用于内耳不同区域以破坏液体稳态的分子机制。在耳蜗中，SOX9与SOX10可共同抑制水通道蛋白的表达；而在内淋巴囊中，SOX9则调控SOX10的表达以及离子转运与缝隙连接相关基因的表达。我们推测：细胞增殖调控失控会导致祖细胞耗竭，进而造成富线粒体细胞数量不足。上述结果揭示了维持听力相关液体稳态的多维度调控机制。本研究通过Smart-seq2技术对野生型与Sox9Y440X/+小鼠的内淋巴囊共计120个细胞进行了测序。