A constitutively expressed fluorescence ubiquitin 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 expression of 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 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.

Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration

“…We also cannot exclude an impact from general housing conditions as the previous experiments were performed in a different animal facility with, for example, a different water supply. We note, however, that the baseline of S/G 2 ependymal cells that we observe in this study is in good agreement with recent results obtained from FUCCI axolotls that were independently generated using zebrafish DNA constructs ( Duerr et al, 2021 ). From the data analysis side, it is important to note that in our AxFUCCI experiments G 0 /G 1 AxFUCCI cells become Transition-AxFUCCI cells and then S/G 2 AxFUCCI cells.…”

Regenerating axolotl retinas regrow diverse cell types with modulation by Notch signaling and reconnect to the brain

“…After a lung injury, a variety of cell types proliferate in the contralateral, uninjured lung (Jensen et al, 2021). In a regenerating tail, proliferating cells can be detected in the spinal cord as far as 5 mm away from the amputation site (Duerr et al, 2021). It remains unclear what drives this spike in proliferation, but the uninjured contralateral eye may have received growth signals from the brain, which connects the two eyes.…”

Local mechanical stimuli shape tissue growth in vertebrate joint morphogenesis

“…Further studies are required to clarify the mechanosignalling mechanisms at play during these stages. Emerging experimental techniques like whole mount staining and imaging provide the opportunity to explore the 3D spatial distribution of mechanosensitive growth regulators [73] involved in cavitation and ossification. Developing such experiments in close association with computational modelling will provide a powerful tool to further explore the mechanobiology of joint formation.…”