A constitutively expressed fluorescent ubiquitination-based cell-cycle indicator (FUCCI) in axolotls for studying tissue regeneration

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.

Development of a model system to study the regulation of patterning competency in regenerating axolotl limbs

“…1c), we tested whether the cell cycle was activated prior to competency induction using the new FUCCI transgenic axolotl line, which allows us to visualize the activation of the cell cycle in real-time in vivo. 35 We observed a detectable increase in mitotic cells in the blastema mesenchyme between 5 and 6 days postsurgery (Fig. 2d), indicating that the acquisition of patterning competency correlates with the activation of the cell cycle.…”

“…This analysis also identi ed pathways related to the cell cycle and DNA replication as targets downstream of FGF/BMP signaling. Using the FUCCI transgenic axolotl, 35 we found that the activation of the cell cycle in the blastema mesenchyme correlates with the window of patterning competency establishment (Fig. 2).…”

“…As the blastema develops, the cell cycle slows in the region closest to the stump where blastema cells begin to redifferentiate. 35,36 Therefore, we next tested whether patterning competency was retained in the blastema as it progressed from the early bud (EB) stage (composed of undifferentiated cells) to the late bud (LB) stage (composed of spatially restricted undifferentiated and redifferentiating cells). We performed qRT-PCR to detect differences in limb patterning gene expression following a 24-hour RA treatment of mature tissue, EB, and LB that was divided into apical (undifferentiated) and basal (redifferentiated) halves (Fig.…”

Meeting report: Salamander models in cross‐disciplinary biological research meeting

“…Salamanders are excellent vertebrate models to apply novel tools to study cell biological aspects of regeneration. For example, the recent development of axolotl Fucci lines independently by both Duerr et al, as well as Cura Costa et al, allows for an unprecedented level of detail to understand cell cycle dynamics during axolotl tissue regeneration, 3,4 particularly in combination with either live imaging or the recently developed optical clearing protocols for salamander systems. [5][6][7][8] Leo Otsuki (IMP, Austria) provided an overview of how these novel imaging tools can be used to study cell cycle kinetics during axolotl spinal cord regeneration.…”

The amazing and anomalous axolotls as scientific models

“…These experiments are key to support comparative developmental studies and identify conserved and divergent regulatory modules controlling axolotl developmental and regenerative programs. So much can be done with these animals, such as gain and loss of function, 8‐10 CRISPR‐mediated transgenesis, 10,11 single‐cell characterization, 12‐16 live imaging, 17‐20 transplants and cell lineage tracing, 20‐27 and the use of ex vivo explants 28 to compare the developmental processes of this unique salamander to the robust body of avian, frog, zebrafish, and mouse developmental research. For example, axolotl gills can regenerate, yet they are one of three modes of respiration at the organism's disposal, and their full role remains unknown 29 …”