Watch an animal's limb regrow
Image: Regenerating crustacean leg.
France-based researchers have, for the first time, recorded how cells of the epidermis behave during the regrowth of adult limbs after amputation.
Michalis Averof, Director of Research at the Institute of Functional Genomics in Lyon and colleagues carried out continuous live imaging of a regenerating leg in the crustacean Parhyale hawaiensis, a close relative of the common sand hopper.
"Parhyale hawaiensis is well suited for imaging limb regrowth," explains Averof.
"The animals have relatively rapid limb regeneration, requiring as little as one week for young adults to fully regrow their legs," he says. "Also, their tiny limbs enable us to image the regeneration process in unprecedented cell-by-cell detail through their entire thickness."
Live imaging reveals the progenitors and cell dynamics of limb regeneration [Alwes et al]
To record the regrowth of the Parhyale hawaiensis leg, the team used transgenes that expressed fluorescent proteins to the limb's epidermal cells.
They then turned to confocal microscopy to continuously image the limb over the first days of regrowth.
"The transgenes allow us to track the position of each cell and to detect mitoses and apoptotic events throughout the leg, with single cell resolution," says Averof, "[But] a major challenge for achieving this was finding a way to immobilize regenerating legs under the microscope, while keeping the animals alive."
Cells dividing at the tip of a regenerating Parhyale leg. [Alwes et al]
"We chose to immobilize individual thoracic legs for imaging by glueing them to the surface of a coverslip, using surgical glue," he explains. "This approach allowed us to continuously image the immobilized leg, through the coverslip, while allowing other parts of the animal to move freely."
According to the researcher, approximately 80% of animals survive this procedure and release themselves from the coverslip with fully regenerated legs during their subsequent moult.
Using this method, the researchers were able to identify a specific sequence of events and cell behaviours that unfolded during limb regrowth, including wound closure, followed by a quiet period when the epidermal cells migrate slowly towards the site of the wound.
"[This stage was followed by] extensive cell division and movement as the new leg started to develop its shape," highlights Averof's colleague, Frederike Alwes. "We were surprised to see that there was a sharp transition between the last two stages, which suggests the cells were coordinated by a common signal."
The study also showed for the first time that there are no specialised stem cells for regenerating the epidermis in new limbs.
Instead, most of the leg stump's epidermal cells divide and re-arrange, contributing to new segments in the leg.
Regenerating crustacean leg; distinct cell behaviours underpin different phases of leg regeneration [Frederike Alwes]
As Alwes points out: "Traditionally, insight into cell behaviour during limb regrowth has been gathered by imaging fixed samples and attempting to fill in the missing pieces, due to the difficulties in tracking cells during regeneration in active adult animals."
"But with the ability to track the movements and behaviour of single cells individually through time, we now have the means to understand the cellular dynamics of the regeneration process, which could not have been reconstructed from fixed material," he says.
The researchers focused on the behaviour of epidermal cells, but now plan to extend this work to include all the different cell types that are involved in limb regrowth.
"The ultimate aim of our research is to explore how some animals can respond to a severe injury by regenerating an entire body part that was lost," says Alwes.
Research is published in eLife.