Sox9 marks limbal stem cells and is required for asymmetric cell fate switch in the corneal epithelium

Adult tissues with high cellular turnover require a balance between stem cell renewal and differentiation, yet the mechanisms underlying this equilibrium are unclear. The cornea exhibits a polarized lateral flow of progenitors from the peripheral stem cell niche to the center; attributed to differences in cellular fate. To identify genes that are critical for regulating the asymmetric fates of limbal stem cells and their transient amplified progeny in the central cornea, we utilized an in vivo cell cycle reporter to isolate proliferating basal cells across the anterior ocular surface epithelium and perform single-cell transcriptional analysis. This strategy greatly increased the resolution and revealed distinct basal cell identities with unique expression of structural genes and transcription factors. We focused on Sox9; a transcription factor implicated in stem cell regulation across various organs. Sox9 was found to be differentially expressed between limbal stem cells and their central corneal progeny. Lineage tracing analysis confirmed that Sox9 marks long-lived limbal stem cells, which generate progeny that expands centripetally to replenish the cornea. Conditional deletion of Sox9 led to abnormal differentiation and squamous metaplasia in the central cornea, suggesting that it is required for braking cell division symmetry. By inhibiting terminal differentiation of transient amplified progenitors, forcing them into perpetual symmetric divisions, we replicated the Sox9 loss-of-function phenotype. Our findings reveal an essential role for Sox9 in the spatial regulation of asymmetric fate in the corneal epithelium that sustains tissue homeostasis. To investigate the mechanism of stem cell fate regulation in the ocular epithelium we used a reporter mouse model (Cyclin B1-GFP) that marks actively cycling cells in vivo. By fluorescently activated cell sorting we purified GFP-positive cells from freshly excised mouse corneas and performed single-cell RNA sequencing analysis.

高细胞更新速率的成体组织需要维持干细胞自我更新与分化之间的动态平衡，然而该平衡背后的调控机制目前仍不明确。角膜呈现出祖细胞从周边干细胞龛（stem cell niche）向中央区域的极性侧向流动，这一现象源于细胞命运的差异。为了鉴定调控角膜缘干细胞（limbal stem cells）及其中央角膜瞬态扩增祖细胞不对称命运的关键基因，我们利用体内细胞周期报告基因（in vivo cell cycle reporter）分离了眼前表上皮各处的增殖性基底细胞，并开展了单细胞转录组分析。该策略显著提升了检测分辨率，并揭示了不同基底细胞亚群的独特身份——这些亚群具有结构基因与转录因子的特异性表达谱。我们将研究重点聚焦于Sox9，这是一种在多种器官中参与干细胞调控的转录因子。研究发现，Sox9在角膜缘干细胞与其中央角膜祖细胞之间存在差异表达。谱系示踪分析（lineage tracing analysis）证实，Sox9可标记长寿型角膜缘干细胞，这类干细胞产生的子代细胞会向心性扩增以补充角膜组织。条件性敲除Sox9会导致中央角膜出现异常分化与鳞状上皮化生（squamous metaplasia），提示该因子对于抑制细胞对称分裂至关重要。通过抑制瞬态扩增祖细胞的终末分化并强迫其持续进行对称分裂，我们成功复刻了Sox9功能缺失的表型。本研究结果揭示了Sox9在角膜上皮细胞不对称命运的空间调控中发挥的关键作用，该调控过程可维持角膜组织的稳态平衡。为了探究眼表上皮干细胞的命运调控机制，我们使用了可在体内标记活跃增殖细胞的报告基因小鼠模型（Cyclin B1-GFP）。通过荧光激活细胞分选术（fluorescently activated cell sorting），我们从新鲜摘取的小鼠角膜中分离纯化了GFP阳性细胞，并开展了单细胞RNA测序分析。