Abstract
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.
Original language | English |
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Article number | dev199637 |
Journal | Development (Cambridge) |
Volume | 149 |
Issue number | 6 |
DOIs | |
State | Published - Mar 2022 |
Bibliographical note
Funding Information:The work from this paper was funded by the National Institutes of Health (R01HD099174) and by the National Science Foundation (1558017 and 1656429). Funding for A.V. and E.K.J. was partially provided by the National Science Foundation (REU grant 1757443). Deposited in PMC for release after 12 months.
Funding Information:
The authors thank Jackson Griffiths for assisting with transgenic animal care during the COVID-19 pandemic lockdown, Ester Comellas for assistance with data analysis, both Guoxin Rong and Alex Lovely for imaging assistance, and Malcolm Maden for early conceptual discussions on the project. Images were obtained from the Northeastern University Chemical Imaging of Living Systems core. We thank the Institute for Chemical Imaging of Living Systems at Northeastern University for consultation and imaging support. Non-transgenic animals were obtained from the Ambystoma Genetic Stock Center, which is funded by the National Institutes of Health (P40OD019794). The work from this paper was funded by the National Institutes of Health (R01HD099174) and by the National Science Foundation (1558017 and 1656429). Funding for A.V. and E.K.J. was partially provided by the National Science Foundation (REU grant 1757443). Deposited in PMC for release after 12 months.
Funding Information:
The authors thank Jackson Griffiths for assisting with transgenic animal care during the COVID-19 pandemic lockdown, Ester Comellas for assistance with data analysis, both Guoxin Rong and Alex Lovely for imaging assistance, and Malcolm Maden for early conceptual discussions on the project. Images were obtained from the Northeastern University Chemical Imaging of Living Systems core. We thank the Institute for Chemical Imaging of Living Systems at Northeastern University for consultation and imaging support. Non-transgenic animals were obtained from the Ambystoma Genetic Stock Center, which is funded by the National Institutes of Health (P40OD019794).
Publisher Copyright:
© 2022. Published by The Company of Biologists Ltd.
Keywords
- Axolotl
- Cell cycle
- FUCCI
- Regeneration
ASJC Scopus subject areas
- Molecular Biology
- Developmental Biology