Tsukuba scientists solve Spallanzani's dilemma

March 30, 2016

Imagine losing an eye, an arm or even your spinal cord. When we are wounded, our bodies, and those of other mammals, generally respond by sealing the wound with scar tissue. The newt, however, has evolved unique strategies that allow it to repeatedly regenerate lost tissues, even as an adult.

Newts are the masters of regeneration. No other animal can match their regenerative abilities in body parts including the limbs, the tail and spinal cord, parts of the eye (such as the retina and the lens), the brain, the heart and the jaws. What happens when a newt loses, for example, a leg? A mass of cells, called a blastema, is generated at the stump, from which a new, fully functional leg is eventually regenerated.

The newt is unique in having this ability even as an adult. Other amphibians with regenerative potential, such as axolotls, lose this ability once they metamorphose from a larva to a juvenile. The brilliant 18th century Italian physiologist, Lazzaro Spallanzani, was one of the first scientists to investigate this dilemma in great detail.

Recent research led by Chikafumi Chiba at the University of Tsukuba, Japan and Panagiotis Tsonis at the University of Dayton, Ohio, and published in the current volume of the prestigious journal Nature Communications, has shed some light on the newt's exceptional regenerative ability and may provide further insight into regeneration in other species, including mammals.

The researchers made the exciting discovery that the mechanism for regeneration in the larval newt is different to the one used after metamorphosis. This discovery was made using transgenic newts, the use of which has only recently been made possible. One of the researchers on the team, Martin Casco-Robles, from the Faculty of Life and Environmental Sciences, University of Tsukuba, is a pioneer in developing techniques for the creation of transgenic newts.

Using transgenic Japanese fire bellied newts, the team were able to track different types of muscle cells during limb regeneration in both larval and metamorphosed animals. It had been suggested previously that either skeletal muscle fiber cells (SMFCs) or muscle stem/progenitor cells (MPCs) contribute to new muscle in regenerated limbs of newts. SMFCs make up skeletal muscles, which are one of the three major types of muscles. MPCs are the dormant predecessors of muscle fiber cells and are located within muscle fibers. They can be triggered to proliferate for both self-renewal and specialization into muscle fiber cells.

The researchers inserted a gene known to be active in SMFCs into single-celled newt embryos. The transgenic newt embryos were then reared until the swimming larval stage, at 3 months of age, or the metamorphosed juvenile stage, at 16 months. The gene was linked to a red fluorescent protein which could be switched on and off at precise times with the addition of a particular chemical to the rearing solution.Selected transgenic newts had a limb removed under anesthesia. The fluorescence of the tissues in the living newts was monitored under a microscope during development and limb regeneration. In addition, tissue samples were collected for further MPC cell-specific staining. Lead author, Hibiki Tanaka, of the Graduate School of Life and Environmental Sciences, University of Tsukuba, explains that "we found that larval newts did not require muscle fiber cells to regenerate their amputated limbs."

These experiments showed that the new muscle in larval newt regeneration tissue is primarily derived from muscle stem/progenitor cells, not skeletal muscle fiber cells. In contrast, after metamorphosis, the team found that the skeletal muscle fiber cells in the stump temporarily regress to a more primitive state, that is, they become dedifferentiated. The cells then re-enter the cell cycle and proliferate to produce more muscle cells. Hibiki Tanaka says "larval newts use stem/progenitor cells for new muscle in a regenerated limb while metamorphosed newts recruit muscle fiber cells in the stump for the same purpose."

Next the researchers looked at whether or not the tissues in the limb strictly regenerated the same tissue types using reporter-gene expressing tissue transplantation experiments. The principal tissues of the adult limb, skin, bone, muscle and nerve tissues, were obtained from transgenic newts and grafted onto or into the corresponding regions of normal newts. These newts were then used in regeneration experiments. The team discovered that skin, bone, muscle and nerve tissues faithfully regenerated themselves.

Chikafumi Chiba explains these remarkable discoveries, saying "the newt switches the cellular mechanism for limb regeneration from a stem/progenitor-based mechanism (larval mode) to a dedifferentiation-based one (adult mode) as it transits beyond metamorphosis". He says "delineating the mechanisms of these strategies will undoubtedly provide clues for regeneration in other species including mammals".

Thus, while we may never have the incredible regenerative powers of the newt, it is likely that this little amphibian will continue to provide us with insights into mammalian tissue regeneration, wound healing and repair.

University of Tsukuba

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