A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration
A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration
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DOI：
10.1242/dev.199637
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发表时间：
2022-03-01
期刊：
影响因子：
4.6
通讯作者：
Monaghan, James R.
中科院分区：
文献类型：
--
作者：
Duerr, Timothy J.;Jeon, Eun Kyung;Monaghan, James R.
细胞周期进程的调节对损伤后再生过程中的细胞增殖至关重要。在切除附肢后，美西螈（Ambystoma mexicanum）通过被称为胚基的增殖细胞的积累来再生缺失的结构。为了研究胚芽生长过程中的细胞分裂，我们培育了一种蝾螈转基因系，该系普遍表达基于荧光泛素化的细胞周期指示器（FUCCI）的双电子版本。我们证实了在发育和成年组织中几乎无处不在的FUCCI表达，并通过DNA合成和有丝分裂期标记物验证了这些表达模式。我们展示了FUCCI在活体和全载成像中的应用，显示了肢体和尾巴再生过程中细胞的主要局部贡献。我们还表明，脊髓截肢会导致离损伤部位至少5mm的增生增加。最后，我们在单个FUCCI组织切片上结合荧光原位杂交、EdU点击化学和免疫组织化学，使用多模态染色为循环细胞提供细胞类型信息。这一新的动物系列将有助于研究细胞周期动力学，使用原位终点分析和体内成像在发育和再生动物。
Regulation of cell cycle progression is essential for cell proliferation during regeneration following injury. After appendage amputation, the axolotl (Ambystoma mexicanum) regenerates missing structures through an accumulation of proliferating cells known as the blastema. To study cell division during blastema growth, we generated a transgenic line of axolotls that ubiquitously expresses a bicistronic version of the fluorescent ubiquitination-based cell-cycle indicator (FUCCI). We demonstrate near-ubiquitous FUCCI expression in developing and adult tissues, and validate these expression patterns with DNA synthesis and mitosis phase markers. We demonstrate the utility of FUCCI for live and whole-mount imaging, showing the predominantly local contribution of cells during limb and tail regeneration. We also show that spinal cord amputation results in increased proliferation at least 5 mm from the site of injury. Finally, we use multimodal staining to provide cell type information for cycling cells by combining fluorescence in situ hybridization, EdU click-chemistry and immunohistochemistry on a single FUCCI tissue section. This new line of animals will be useful for studying cell cycle dynamics using in situ endpoint assays and in vivo imaging in developing and regenerating animals.