Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl

Abstract

BackgroundTo gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb.ResultsComparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema.ConclusionsWe have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb.

Mechanisms of urodele limb regeneration

“…Changes in the proteome during blastema formation in regenerating axolotl, newt, and developing and adult Xenopus limbs have been investigated by Rao et al. (2009, 2014), Looso et al. (2013), and King, Mescher, and Neff (2009).…”

“…Consistent with the scarring of denervated and amputated newt limbs, collagen fibrillogenesis begins prematurely (Bryant, Fyfe, & Singer, 1971; Vanrapenbush & Lasalle, 1989). The effect of denervation on a wide array of genes and proteins revealed by global genomic and proteomic analysis (Looso et al., 2013; Monaghan et al., 2009; Rao et al., 2009, 2014; Voss et al., 2015) is now wide open for investigation.…”

“…Research on urodele limb regeneration will continue to inform this challenge, especially through comparative studies between urodele and regeneration‐deficient anuran limbs (Rao et al., 2014) and between position‐specific differences in the regenerative ability of mammalian appendages (Simkin, Sammarco, & Dawson, 2015). Why amputated urodele appendages regenerate and amputated anuran and mammalian appendages regenerate hypomorphically or not at all has been a long‐standing evolutionary question.…”

“…Dedifferentiation was associated with autonomously controlled autophagy that promoted cytoplasmic remodeling, mitochondrial regression, and a bioenergetic shift from oxidative phosphorylation to anaerobic metabolism. This bioenergetic shift has been previously observed in proteomic studies of regenerating urodele and anuran limbs (Rao et al., 2009, 2014). The role of metabolism in regeneration‐competent versus regeneration‐deficient limbs has been largely neglected since publication of the histochemical studies of Schmidt (1968) on newt limb regeneration.…”

Temporal Expression Pattern of Peptides in the Regenerating Caudal Fin of Teleost Fish Poecilia latipinna With Special Emphasis on krt15 and myl-1

“…It includes the regeneration in axololtl [20,21], Xenopus laevis froglet limb-bud [22,23] and zebrafish caudal fin regeneration [24]. Herein, in P. latipinna, twodimensional gel electrophoresis was used to find out the overall expression pattern of peptides across the stages of epimorphic regeneration.…”