Formation of the eye lens depends on the continuous differentiation of lens epithelial cells into lens fiber cells. To attain their mature structure and transparent function, nascent lens fiber cells must complete a precise cellular remodeling program hallmarked by the complete elimination of organelles to form the core lens organelle-free zone (OFZ). Lacking a blood supply, the lens resides in a hypoxic environment that results in a decreasing oxygen concentration from the lens surface to the lens core. This oxygen gradient results in a hypoxic microenvironment in the region of the lens where immature lens fiber cells initiate loss of organelles to form the core OFZ. These features of the lens suggest a potential role for low lens oxygen levels in the regulation of organelle degradation and other events critical for mature lens fiber cell formation. Hypoxia activates the master regulator of the hypoxic response, hypoxia-inducible factor 1a (HIF1a) that regulates hypoxia-responsive genes. To identify a potential role for hypoxia and HIF1a in the elimination of organelles during lens fiber cell maturation, we tested the requirement for hypoxia in the degradation of non-nuclear organelles in ex vivo cultured embryonic chick lenses by monitoring the degradation of mitochondria (MT), Golgi apparatus (GA) and endoplasmic reticulum (ER) under conditions of low (1% O₂) and high (21% O₂) oxygen. We also examined the requirement for HIF1a activation for elimination of these organelles under the same conditions using a specific HIF1a activator (DMOG) and a specific HIF1a inhibitor (chetomin) and examined the requirements for hypoxia and HIF1a for regulating transcription of BNIP3L that we previously showed to be required for elimination of non-nuclear lens organelles. We used ChIP-qPCR to confirm direct binding of HIF1a to the 5' untranslated region of the BNIP3L gene. Finally, we examined the effects of expressing an oxygen insensitive mutant form of HIF1a (P402A/P565A) and BNIP3L on non-nuclear organelle degradation. Our data demonstrate that hypoxia and HIF1a are required for the degradation of non-nuclear organelles during lens fiber cell formation and that they regulate this process by governing BNIP3L transcription. Our results also provide evidence that hypoxia and HIF1a are essential for achieving mature lens structure.

眼睛晶状体的形成取决于晶状体上皮细胞不断分化为晶状体纤维细胞。为了获得成熟的结构和透明功能，新生晶状体纤维细胞必须完成精确的细胞重塑程序，其标志是完全消除细胞器以形成核心晶状体无细胞器区 (OFZ)。由于缺乏血液供应，晶状体处于缺氧环境中，导致从晶状体表面到晶状体核心的氧气浓度降低。这种氧梯度导致晶状体区域的缺氧微环境，在该区域，未成熟的晶状体纤维细胞开始丢失细胞器以形成核心 OFZ。晶状体的这些特征表明，低晶状体氧水平在调节细胞器降解和其他对成熟晶状体纤维细胞形成至关重要的事件中具有潜在作用。缺氧会激活缺氧反应的主要调节因子，即调节缺氧反应基因的缺氧诱导因子 1a (HIF1a)。为了确定缺氧和 HIF1a 在晶状体纤维细胞成熟过程中消除细胞器中的潜在作用，我们测试了在非核细胞器降解过程中缺氧的要求。通过在低 (1% O ₂ ) 和高 (21% O ₂ ) 条件下监测线粒体 (MT)、高尔基体 (GA) 和内质网 (ER) 的降解，离体培养的鸡胚晶状体) 氧气。我们还使用特定的 HIF1a 激活剂 (DMOG) 和特定的 HIF1a 抑制剂 (chetomin) 在相同条件下检查了 HIF1a 激活以消除这些细胞器的要求，并检查了缺氧和 HIF1a 对调节 BNIP3L 转录的要求，我们之前展示过为消除非核晶状体细胞器所必需。我们使用 ChIP-qPCR 来确认 HIF1a 与 BNIP3L 基因的 5' 非翻译区的直接结合。最后，我们检查了表达 HIF1a (P402A/P565A) 和 BNIP3L 的氧不敏感突变形式对非核细胞器降解的影响。我们的数据表明缺氧和 HIF1a 是晶状体纤维细胞形成过程中非核细胞器降解所必需的，并且它们通过控制 BNIP3L 转录来调节这一过程。