Cytokine Signaling Plays Central Roles in the Initiation of Limb Regeneration

Abstract

Newts and salamanders possess the unique ability amongst vertebrates to regenerate their limbs. While mammals respond to limb amputations with a scarring response, salamanders instead form an undifferentiated cellular structure distal to the amputation plane known as the blastema, which eventually differentiates into a new limb. Formation of the blastema is a complex process requiring coordinated migration and proliferation of progenitors derived from many tissues (muscle, bone, connective tissue etc.) and the exact molecular mechanisms underlying the initiation of this process remain unknown. Here, we have elucidated the distinct transcriptional programs that are active in blastemal progenitors as well as the surrounding tissues during early stages of regeneration. These analyses suggested that dividing progenitors selectively establish an autocrine TGF-beta signaling network and further revealed the enriched expression of several cytokines, indicating progenitors may play an immunomodulatory role. Functional analyses of the role of one such cytokine, interleukin-8 (il-8), indeed led us to discover that blastemal progenitors are immunomodulators during early stages of regeneration through interleukin-8/cxcr-1/2 signaling. We also examined how these gene expression programs change when we prevented the formation of the wound epidermis, an important epithelial support structure for blastema formation. Our data suggest that the early wound epidermis plays a large role in modulating inflammation, ECM degradation, and tissue histolysis. These analyses further identified the decreased expression of one growth factor cytokine, midkine (mk), in the absence of the wound epidermis. Gain- and loss-of-function analyses of mk during limb regeneration revealed that it is a critical regulator of wound epidermis maturation during blastema formation. Altogether, these studies emphasize the importance of cytokine signaling in the initiation of limb regeneration and provide the field with resources that may lead to the discovery of more putative regulators of blastema formation.