Elucidation of cellular reprogramming processes that drive lens regeneration in axolotl as a basis for future therapeutic approaches

火蜥蜴，如和Axolotls，在受伤时可以再生眼睛组织。这是可能的，因为 他们的色素上皮细胞重新编程到视网膜和晶状体的能力。这项研究的目标是 解开重新编程的机制，并将其用于再生医学应用。主 研究的重点是虹膜色素上皮(IPE)细胞激活，增殖， 重新编程，并最终在完全拆卸后重新生成晶状体。在Newts中，这个过程是 仅限于位于虹膜背侧的IPE细胞，而腹侧IPE细胞被认为不参与 细胞水平。相比之下，幼年的Axolotl不能再生晶状体。这些再生的差异 能够使用能够提供对分子的重要见解的实验范例 IPE重新编程的机制。在具体目标1中，结合比较转录学、FLOW 将使用细胞学分类和谱系追踪来识别和表征IPE亚群 触发一种再生反应。这些方法还将与一种新的转基因水貂配对。 能够跟踪重编程期间眼睛转录因子动态的变化。这将使我们能够 以更好地了解再生过程中分子和细胞变化之间的联系。在……里面 具体目标2，将进行晶状体再生的功能表征。为了成功 为了实现这一点，将实施禽类RCAS/TVA系统，以提供空间和时间控制 目的基因在着色上皮细胞中的表达。禽类的TVA受体将无处不在 在眼组织、背侧IPE细胞或腹侧IPE细胞中表达，使它们对RCAS易感 逆转录病毒。利用这个系统，将测试基因和通路的影响，以确定它们诱导 腹侧再生，或抑制背侧IPE细胞再生。这种方法 通过进行必要性和充分性实验，提供了功能读数。在具体目标3中， 我们将探讨衰老、DNA损伤和再生潜力之间的关系。蝾螈可以 在整个生命过程中，他们的晶状体从IPE细胞中再生，这种能力似乎不受老化和 反复受伤。首先，将开发一个重复损伤模型，并将其与以下分子特征相结合 衰老和DNA损伤。然后，将使用药理学和遗传学的方法来研究 增加的遗传毒性应激对色素上皮重新编程到眼组织的能力。

Salamanders, like newts and axolotls, can regenerate ocular tissues when injured. This is possible due to the ability of their pigmented epithelial cells to reprogram to retina and lens. The goal of this research is to unravel the reprogramming mechanism and leverage it for regenerative medicine applications. The main focus of the study is the ability of iris pigmented epithelial (IPE) cells to get activated, proliferate, reprogram, and ultimately regenerate the lens upon its complete removal. In newts, this process is restricted to IPE cells residing in the dorsal iris while ventral IPE cells are thought to not participate at the cellular level. In contrast, juvenile axolotls cannot regenerate their lens. These regenerative discrepancies enable the use of experimental paradigms that could provide important insights into the molecular mechanism of IPE reprogramming. In Specific Aim 1, a combination of comparative transcriptomics, flow cytometry sorting and lineage tracing will be used to identify and characterize IPE subpopulations capable of triggering a regenerative response. These approaches will also be paired with a novel transgenic newt capable of tracking changes in eye transcriptional factor dynamics during reprogramming. This will allow us to better understand the association between molecular and cellular changes during regeneration. In Specific Aim 2, functional characterization of lens regeneration will be performed. To successfully accomplish that, the avian RCAS/TVA system will be implemented to provide spatial and temporal control of target gene expression in the pigmented epithelium. The avian TVA receptor will be ubiquitously expressed in eye tissues, in dorsal IPE cells, or in ventral IPE cells making them susceptible to the RCAS retrovirus. Using this system, the effects of genes and pathways will be tested for their ability to induce regeneration from the ventral, or inhibit regeneration from the dorsal IPE cells, respectively. This approach provides a functional readout by performing necessity and sufficiency experiments. In Specific Aim 3, the relationship between aging, DNA damage, and regeneration potential will be explored. Newts can regenerate their lens from IPE cells throughout life and the ability appears unaffected by aging and repeated injury. First, a repeated injury model will be developed and coupled with molecular hallmarks of aging and DNA damage. Then, pharmacological and genetic methods will be used to study the effects of elevated genotoxic stress on the ability of the pigmented epithelium to reprogram to eye tissues.