Northeastern University, Department of Biology, Boston, MA 02115, USA.
University of Massachusetts Boston, Department of Biology, Boston, MA 02125, USA.
Development. 2022 Mar 15;149(6). doi: 10.1242/dev.199637. Epub 2022 Mar 17.
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.
细胞周期进程的调控对于损伤后再生过程中的细胞增殖至关重要。在附肢切除后，蝾螈（Ambystoma mexicanum）通过积累称为芽基的增殖细胞来再生缺失的结构。为了研究芽基生长过程中的细胞分裂，我们生成了一种转基因蝾螈系，该系在整个细胞中表达荧光泛素化细胞周期指示剂（FUCCI）的双顺反子版本。我们证明了在发育中和成年组织中几乎普遍存在 FUCCI 表达，并通过 DNA 合成和有丝分裂阶段标志物验证了这些表达模式。我们展示了 FUCCI 在活体和整体成像中的实用性，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，脊髓切除导致损伤部位至少 5mm 处的增殖增加。最后，我们通过在单个 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。这种新型动物将有助于使用原位终点测定和发育中和再生动物的体内成像研究细胞周期动力学。
