Intra-retinal axon guidance involves a coordinated expression of transcription factors, axon guidance genes, and secretory molecules within the retina. Pax6, the master regulator gene, has a spatio-temporal expression typically restricted till neurogenesis and fate-specification. However, our observation of persistent expression of Pax6 in mature RGCs led us to hypothesize that Pax6 could play a major role in axon guidance after fate specification. Here, we found significant alteration in intra-retinal axon guidance and fasciculation upon knocking out of Pax6 in E15.5 retina. Through unbiased transcriptome profiling between Pax6^fl/fl and Pax6^−/− retinas, we revealed the mechanistic insight of its role in axon guidance. Our results showed a significant increase in the expression of extracellular matrix molecules and decreased expression of retinal fate specification and neuron projection guidance molecules. Additionally, we found that EphB1 and Sema5B are directly regulated by Pax6 owing to the guidance defects and improper fasciculation of axons. We conclude that Pax6 expression post fate specification of RGCs is necessary for regulating the expression of axon guidance genes and most importantly for maintaining a conducive ECM through which the nascent axons get guided and fasciculate to reach the optic disc.

视网膜内轴突导向涉及转录因子、轴突导向基因和视网膜内分泌分子的协调表达。Pax6 是主调节基因，其时空表达通常仅限于神经发生和命运规范。然而，我们对成熟 RGC 中 Pax6 持续表达的观察使我们假设 Pax6 可能在命运指定后的轴突引导中发挥重要作用。在这里，我们发现敲除 E15.5 视网膜中的 Pax6 后，视网膜内轴突引导和束颤发生显着改变。通过 Pax6 ^fl/fl和 Pax6之间的无偏转录组分析^-/-视网膜，我们揭示了其在轴突引导中作用的机械洞察力。我们的结果显示细胞外基质分子的表达显着增加，而视网膜命运规范和神经元投射指导分子的表达减少。此外，我们发现 EphB1 和 Sema5B 由于轴突的引导缺陷和不正确的束状作用而直接受 Pax6 调节。我们得出结论，RGC 的 Pax6 表达后命运规范对于调节轴突引导基因的表达是必要的，最重要的是对于维持有利的 ECM，新生轴突通过该 ECM 被引导和束状到达视盘。