FGF and BMP derived from dorsal root ganglia regulate blastema induction in limb regeneration in Ambystoma mexicanum

Abstract

Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability.

A brief history of the study of nerve dependent regeneration

“…61 Even more recently, Satoh et al have shown that FGF8 and Bmp7 are expressed at the ends of peripheral nerves, and knockdown of these factors inhibits blastema formation in regenerating limbs. 72 These findings reinforce the validity of the AL model as an analog to the amputated limb while demonstrating the necessity of FGFs and BMPs for nerve dependent limb regeneration.…”

“…61 FGF8 and Bmp7 electroporated into dorsal root ganglia are expressed at the ends of peripheral nerves; knockdown of Fgf8 and Bmp7 blocks blastema formation 72 Neuregulin-1 Present in the newt nervous system and the regenerating blastema; lost upon denervation; increases proliferation in cultured blastemal cells. 80 Expressed in the dorsal root ganglia and peripheral nerves of newts; injections into denervated newt blastemas induced regenerative growth.…”

Mechanisms of urodele limb regeneration

“…Combinations of Fgf8 and bone morphogenetic protein (BMP) have also been tested as neurotrophic factors. Both are expressed in DRG neurons and are detectable in peripheral limb nerve axons in vivo (Satoh, Makanae, Nishimoto, & Mitogawa, 2016). Furthermore, they can substitute for the nerve in the outgrowth of a supernumerary axolotl limb blastema in the Lheureux model (Makanae, Mitogawa, & Satoh, 2014a).…”

Regeneration and Regrowth Potentials of Digit Tips in Amphibians and Mammals

“…The first phase in wound healing involves the contraction of blood vessels and growth of the injured epidermis to cover the remaining limb stump. Blastema cells then accumulate underneath the healed epidermis, which forms a thickened structure at its apex, called the apical epithelial cap (AEC) [21, 22]. The proliferating blastema cells of newts consist of dedifferentiated cells derived from muscle, bone, skin, and other tissues, which serve as progenitors for regenerating the new limb.…”

“…Regeneration occurs by completely different mechanisms between these two different salamander species; thus care must be taken when interpreting results between newts and axolotls. Blastema and AEC formation are dependent on the activation of some unknown signals and several known signals such as ionic fluxes, nitric oxides, MARCKs protein, and trophic factors (e.g., the FGF, TGF, and BMP families) [22, 23] in the wound that consequently promote the formation of the blastema and the AEC. The growth and differentiation phase of the regenerative process includes many features recapitulating embryonic limb development but does exhibit some differences compared to development de novo, for example, the size of the new limb, connection to the existing adult limb, and a nerve requirement [24].…”