Stem Cell Signal Redirects Macrophages to Promote Regeneration of Tadpole Tails

Researchers at the University of Tokyo studying tissue regeneration in tadpoles of the frog Xenopus laevis used techniques including single cell RNA sequencing and gene knockdown (KD) experiments to discover that a secreted protein, c1qtnf3, is expressed in putative muscle stem cells and shifts macrophages from immune to regenerative functions in the animals’ regenerating tails. The discovery, reported by Sumika Kato, PhD, Takeo Kubo, PhD, and Taro Fukazawa, PhD, at the University’s Department of Biological Sciences, offers a crucial insight into the regenerative capabilities of certain animals and paves the way for further research into potential applications in mammals.

Their findings are reported in Proceedings of the National Academy of Sciences (PNAS), in a paper titled “Putative muscle stem cells promote Xenopus tail regeneration by modifying macrophage function via c1qtnf3.” In their report the scientists stated “Through identification and subsequent analysis of genes affected by c1qtnf3 KD, we revealed that c1qtnf3 regulates the accumulation of macrophage-like cells at the amputation site and is required for normal tail regeneration.”

Animals have evolved various strategies to cope with injuries and diseases, including the ability to replace lost tissue, appendages or organs, by regeneration, the authors wrote. Yet while regeneration is a superpower that would be useful to any living creature, it is possessed by only a special few. One of these, the tadpole of the African clawed frog, Xenopus laevis, can regrow a fully functioning tail, including spinal cord and muscles. “As an anuran amphibian, Xenopus laevis tadpoles can regenerate their whole tails with all the tissue components, such as muscle, notochord, epidermis, spinal cord, and other tissues, within a week after amputation,” the authors explained.

For this to happen, stem cells need to jump into action. The cellular origin of regenerated tissues varies significantly among species, organs/appendages, and animal age, but there are two main sources from which regenerative tissue is derived, the team further explained. These are mature differentiated cells, and resident stem/undifferentiated cells. “… in zebrafish and X. laevis tadpole, tail regeneration tissue stem cells and progenitor cells are reported to provide cells for regenerating tissue via activation and proliferation,” they noted.

“A major focus in tissue regeneration research is identifying the factors that make regeneration possible: the cells that give rise to new tissues and their regulatory mechanisms during regeneration.” However, studying these early steps of the regenerative process has been challenging because stem cells are present only in small numbers, making it difficult to observe them. And there is a lack of reliable methods for enriching these extremely rare tissue stem cells. “Despite their importance, however, the molecular mechanisms and behavioral dynamics of the activation of these tissue stem cells after amputation and subsequent differentiation into the lineages required for regeneration remain unclear,” the authors reported.

One method for concentrating tissue stem cells is known as the side population (SP method). “We previously reported that multiple tissue stem/progenitor cells can be efficiently enriched from regeneration buds using the side population (SP) method,” the authors reported. They had the idea of using single-cell transcriptomics of SP cells from regenerating tails to better understand gene expression during tail regeneration. “We have previously established a method for efficiently enriching tissue stem cells,” said Fukazawa. “Building on this technique, we planned to clarify the behavior of tissue stem cells during tail regeneration by examining genes specifically expressed in the tissue stem cells.”

For their newly reported study the researchers performed single cell RNA-sequencing to determine which genes were actively expressed in each cell. Through this process, they identified various cell types and chose to focus on putative muscle stem cells. They found that these putative muscle stem cells expressed complement c1q tumor necrosis factor-related protein 3 (c1qtnf3) more than did other cell types. To understand the function of the gene in the regeneration process, the researchers then performed CRISPR/Cas9 knockdown (KD) experiments in which they blocked the gene to infer its function.

“The knockdown of c1qtnf3 resulted in impaired tail regeneration,” explained Kato, “indicating that the function of c1qtnf3 is essential for successful tail regeneration. We also found that the number of macrophages at the tail stump was reduced in knocked-down tadpoles, suggesting that macrophage function may be impaired.”

This finding led to the hypothesis that macrophages played a role in regeneration facilitated by muscle stem cells via c1qtnf3, but more evidence was needed. The researchers then “restarted” macrophages using another gene and molecular route; neutrophil cytosolic factor 1, a gene involved in macrophage function. “… we found that the impaired tail regeneration by c1qtnf3 KD was accompanied by abrogation of macrophage-like cell accumulation at the amputation site,” they stated. Kato added, “When I found that forced expression of the gene, and consequently rising macrophage numbers, rescued tail regeneration in tadpoles, it felt like the dots connected.”

The authors proposed a mechanism of tadpole tail regeneration in which putative muscle stem cells secrete c1qtnf3, which leads to accumulation of macrophages in the tail stub, promoting tail regeneration. In summary, the authors wrote, “We found that complement c1q tumor necrosis factor-related protein 3 (c1qtnf3) is specifically expressed in putative muscle stem cells (MSC) and, using knockdown (KD; CRISPR/Cas9-based F0 crispants) experiments, demonstrated that c1qtnf3 is necessary for tail regeneration.”

They say their findings “provide clues” to the molecular mechanisms of tail regeneration, in which tissue stem cells indirectly promote tissue regeneration by modulating immune responses. “Understanding such stem cell–immune cell interactions in highly regenerative organisms is an important step toward elucidating the fundamental principles of tissue regeneration across vertebrate species.”

In the future, the researchers aim to uncover how macrophages promote regeneration under the influence of c1qtnf3 and the precise cellular and molecular mechanisms at work within regenerating tissues. “Uncovering the regenerative molecular mechanism by which stem cells themselves recruit immune cells in X. laevis, which has high tissue regeneration potential, will be an important step in elucidating the complex interactions between tissue stem cells and immune cells that enable tissue regeneration,” the authors commented.