Comparative iTRAQ proteomics revealed proteins associated with lobed fin regeneration in Bichirs

Abstract

Background: Polypterus senegalus can fully regenerate its pectoral lobed fins, including a complex endoskeleton, with remarkable precision. However, despite the enormous potential of this species for use in medical research, its regeneration mechanisms remain largely unknown.

Methods: To identify the differentially expressed proteins (DEPs) during the early stages of lobed fin regeneration in P. senegalus, we performed a differential proteomic analysis using isobaric tag for relative and absolute quantitation (iTRAQ) approach based quantitative proteome from the pectoral lobed fins at 3 time points. Furthermore, we validated the changes in protein expression with multiple-reaction monitoring (MRM) analysis.

Results: The experiment yielded a total of 3177 proteins and 15,091 unique peptides including 1006 non-redundant (nr) DEPs. Of these, 592 were upregulated while 349 were downregulated after lobed fin amputation when compared to the original tissue. Bioinformatics analyses showed that the DEPs were mainly associated with Ribosome and RNA transport, metabolic, ECM-receptor interaction, Golgi and endoplasmic reticulum, DNA replication, and Regulation of actin cytoskeleton.

Conclusions: To our knowledge, this is the first proteomic research to investigate alterations in protein levels and affected pathways in bichirs' lobe-fin/limb regeneration. In addition, our study demonstrated a highly dynamic regulation during lobed fin regeneration in P. senegalus. These results not only provide a comprehensive dataset on differentially expressed proteins during the early stages of lobe-fin/limb regeneration but also advance our understanding of the molecular mechanisms underlying lobe-fin/limb regeneration.

Keywords: Blastema; Limb; Polypterus senegalus; Quantitative proteome; Regrowth.

Fig. 1

Study sampling and experimental scheme. a The overall workflow of this study. b A photo of a live specimen of P. senegalus with a homemade fish-holder. c Growth curve of P. senegalus pectoral lobed fin after amputation. The sampling time points of 0 dpa (0D_L and 0D_R), 4 dpa (4D_B) and 12 dpa (12D_D) are labeled. d P. senegalus lobed fins at different regeneration stages. e Tissues of three time points were shown by Alcian blue- and alizarin red- staining, and feulgen staining on paraffin tissue sections. The position of the section was shown on the X-ray photo. Scale bars: 0.5 mm. dpa: days post amputation

Fig. 2

Enrichment analysis of the differentially expressed proteins in 4D_B and 12D_D. GO terms and KEGG pathways enriched in the differentially expressed proteins at 4 and 12 days post amputation. 12D_D VS 0D_L was shown in red. 4D_B VS 0D_L was shown in green

Fig. 3

Protein-protein interaction network of DEPs in cluster 7 by STRING and MCODE. Proteins that are associated with each other are linked by an edge. The up-regulated DEPs (4D_B/0D_L) in 4D_B sample were in red, and the down-regulated DEPs were in green. Integrins were displayed in circles. The main pathway descriptions of these DEPs were from STRING

Fig. 4

Validation of some proteins. Correlation between MRM and iTRAQ data of 30 proteins. 4D/0D, 4 days post amputation / 0 day post amputation

Fig. 5

Proposed model depicting the molecular mechanism based on the P. senegalus proteome. The combination of all of these mechanisms regulates the expression of hundreds of proteins and promotes cell migration, dedifferentiation and regrowth, and scar suppression