SoxC transcription factors shape the epigenetic landscape to establish competence for sensory differentiation in the mammalian organ of Corti

Understanding the molecular basis of competence acquisition by the lineage-specific progenitor cells can provide mechanistic insights into tissue development and regeneration. The sensory epithelium of the inner ear represents a convenient model to study this process, as only two cell types - the mechanosensory hair cells and their associated supporting cells - are specified from a single pool of progenitors in this lineage. Here we show that competence to respond to Atoh1, a transcriptional master regulator both necessary and sufficient for induction of mechanosensory hair cells, is established in the organ of Corti progenitor cells between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11 - the two homologous family members transiently expressed in the inner ear at the time of competence establishment - blocks the ability of sensory progenitors to differentiate into hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 itself and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear. 1. Cochlear duct epithelia from E13.5 wildtype and SoxC conditional double knockout animals were collected and analyzed using scRNA sequencing (10x); 2. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) for cochlear duct epithelia from E13.5 wildtype and SoxC conditional double knockout animals; 3. Cut & Run ChIP-seq (Chromatin immunoprecipitation DNA-sequencing) for Sox4 transcription factor in FACS sorted E13.5 p27kip1 positive cochlea progenitor cells解析谱系特异性祖细胞获得感受态（competence）的分子基础，可为组织发育与再生研究提供机理层面的深入见解。内耳感觉上皮是研究该过程的理想模型：该谱系中仅存在两种细胞类型——机械感觉毛细胞及其配套的支持细胞，二者均由单一祖细胞池分化而来。本研究证实，介导机械感觉毛细胞生成所必需且充分的转录主调控因子Atoh1的应答感受态，于E12.0至E13.5期间在耳蜗器（organ of Corti）祖细胞中建立。向感受态的转变过程极为迅速，且伴随由SoxC家族转录因子调控的表观遗传景观的广泛重塑。在感受态建立阶段于内耳中瞬时表达的两个同源家族成员Sox4与Sox11，其条件性敲除会阻断感觉祖细胞向毛细胞分化的能力。从机制层面来看，Sox4可结合早期感觉谱系特异性调控元件并开放其染色质可及性，这些元件包括与Atoh1自身及其直接下游靶标相关的序列。与上述结果一致，在发育层面的感受态建立前过表达Sox4或Sox11，可提前诱导耳蜗祖细胞向毛细胞分化。此外，重新恢复Sox4或Sox11的表达，可在体外与体内环境中恢复出生后支持细胞向毛细胞分化的能力。本研究结果证实，SoxC家族成员作为介导内耳感觉受体分化所需的表观遗传与转录调控变化的核心因子，在感受态建立过程中发挥关键作用。 1. 收集E13.5野生型与SoxC条件性双敲除小鼠的耳蜗导管上皮，采用搭载10x平台的单细胞RNA测序（scRNA sequencing）进行分析； 2. 针对E13.5野生型与SoxC条件性双敲除小鼠的耳蜗导管上皮开展ATAC-seq（转座酶可及性染色质测序，Assay for Transposase-Accessible Chromatin using sequencing）； 3. 对经荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）得到的E13.5 p27kip1阳性耳蜗祖细胞，开展Sox4转录因子的Cut & Run ChIP-seq（染色质免疫沉淀测序，Chromatin immunoprecipitation DNA-sequencing）