Rao N et al. (2014), Proteomic analysis of fibroblastema formation i...

XB-ART-49275

BMC Dev Biol 2014 Jul 25;14:32. doi: 10.1186/1471-213X-14-32.

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

Rao N , Song F , Jhamb D , Wang M , Milner DJ , Price NM , Belecky-Adams TL , Palakal MJ , Cameron JA , Li B , Chen X , Stocum DL .

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BACKGROUND: To 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. RESULTS: Comparison 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. CONCLUSIONS: We 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.

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Species referenced: Xenopus laevis
Genes referenced: acta1 actg1 actn3 aldoa aldob anxa1 anxa2 atp2a3 atp5f1b b3gnt5 bhmt casq1 cct2 cox5a dag1 des.1 des.2 dynll1 eef1a2 eef2.1 eef2.2 eif4a1 eif4b eno1 ezr fgb fgg fkbp10 fn1 fubp1 gdi2 glud1 h2ax hsp90aa1.1 hsp90aa1.2 hsp90b1 krt12.4 krt12.5 krt12.6 krt19 krt55 krt62 macroh2a2 mat2a mat2b mvp mybpc3 myh13 myl11 ocm3 ocm4.2 p4hb pdcd6ip pdia3 pdia6 pgm1 pls3 prdx1 psmb1 pygm rpl12 rpl15 rpl22 rpl23 rpl30 rpl4 rpl7l1 rplp0 rps19 rps6 slc25a24 snrpe tardbp tnnc2 tnnt3 tpi1 tpm2 trim29 tubb4b vim ywhae ywhaz
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	Figure 1. Histological section of regenerating froglet hindlimb at 12 days post-amputation through the mid tibia-fibula. The section is cut through the dorsoventral plane such that the tibia (T) and the accompanying flexor and extensor muscle masses are visible (red). A cartilage collar (CC) has formed around the tibia for some distance proximal to the amputation plane that merges distally with a similar collar surrounding the fibula. A fibroblastema (FB) that will form the cartilage spike is present between the merge point of the cartilage collars and the apical epidermal cap (arrow). Yellow line indicates the plane of tissue harvest.
	Figure 2. Pie chart showing the percentage distribution of the 820 proteins among different biological categories and sub-categories.
	Figure 3. Validation of LC/MS/MS. Immunofluorescent antibody staining and mean densitometric sum for β1 integrin, vimentin, and dystroglycan, comparing control, 5 dpa and 12 dpa sections of regenerating froglet hindlimbs. A-D, β1 integrin; E-H, vimentin; I-L, dystroglycan. The 1, 5, 7 and 12 dpa fold changes for each of these proteins were: β1 integrin-1.05, 1.28, 2.18, 2.80; vimentin—1.07, 1.94, 2.30, 3.15; dystroglycan-1.20, -1.40, -1.49, -2.02. The immunofluorescence and densitometry data thus agree well with the LC/MS/MS proteomic data.
	SFig1A) Global expression intensity maps for the 10 biological process categories at 1, 5, 7 and 12 dpa. A: Signaling, Cytoskeleton, B: .Intracellular Transport, Transcription, Translation; C: Metabolism, Cell Cycle, ECM; D: Cell Protection, Degradation Red = up regulation; green = down regulation. Level of fold change (FC) is indicated by color intensity. Accession numbers to the right of columns. Intensities for some proteins can be 7–10 times the highest and lowest intensities shown.
	SFig1B) Global expression intensity maps for the 10 biological process categories at 1, 5, 7 and 12 dpa. A: Signaling, Cytoskeleton, B: .Intracellular Transport, Transcription, Translation; C: Metabolism, Cell Cycle, ECM; D: Cell Protection, Degradation Red = up regulation; green = down regulation. Level of fold change (FC) is indicated by color intensity. Accession numbers to the right of columns. Intensities for some proteins can be 7–10 times the highest and lowest intensities shown.
	SFig1C) Global expression intensity maps for the 10 biological process categories at 1, 5, 7 and 12 dpa. A: Signaling, Cytoskeleton, B: .Intracellular Transport, Transcription, Translation; C: Metabolism, Cell Cycle, ECM; D: Cell Protection, Degradation Red = up regulation; green = down regulation. Level of fold change (FC) is indicated by color intensity. Accession numbers to the right of columns. Intensities for some proteins can be 7–10 times the highest and lowest intensities shown.
	SFig1D) Global expression intensity maps for the 10 biological process categories at 1, 5, 7 and 12 dpa. A: Signaling, Cytoskeleton, B: .Intracellular Transport, Transcription, Translation; C: Metabolism, Cell Cycle, ECM; D: Cell Protection, Degradation Red = up regulation; green = down regulation. Level of fold change (FC) is indicated by color intensity. Accession numbers to the right of columns. Intensities for some proteins can be 7–10 times the highest and lowest intensities shown.
	SFig2A) Circos representation of differences in protein expression during blastema formationin the axolotl (A) and fibroblastema formation in the Xenopus froglet (B). The outermost circle shows shows protein expression according to biological process. Metabolism is the most over-represented biological process category in the Xenopus data, whereas Cytoskeleton is the most over-represented in the axolotl data. There were no proteins identified in the Transport category in the axolotl compared to 70 such proteins in the Xenopus data. The next circle represents proteins expressed with FC =/>2 (blue) or =/ 4 (pink). The Xenopus data contained a far higher number of proteins with these fold differences, especially in the transcription, cytoskeleton and signaling categories compared to the axolotl data. Progressing inward, the next four circles in Xenopus reflect the fold change difference (red = down regulation; green = up regulation; blue = no change) of proteins at 1 dpa, 5 dpa, 7 dpa, and 12 dpa, respectively. In the axolotl, three circles represent FC in protein expression at 1 dpa, 4 dpa, and 7dpa. The innermost circle represents the connections between the interacting proteins within the Xenopus and axolotl data. A comparison of these interactions indicates that the proteomic composition and protein-protein interactions are much more complex during formation of the fibroblastema in Xenopus.
	SFig2B) Circos representation of differences in protein expression during blastema formationin the axolotl (A) and fibroblastema formation in the Xenopus froglet (B). The outermost circle shows shows protein expression according to biological process. Metabolism is the most over-represented biological process category in the Xenopus data, whereas Cytoskeleton is the most over-represented in the axolotl data. There were no proteins identified in the Transport category in the axolotl compared to 70 such proteins in the Xenopus data. The next circle represents proteins expressed with FC =/>2 (blue) or =/ 4 (pink). The Xenopus data contained a far higher number of proteins with these fold differences, especially in the transcription, cytoskeleton and signaling categories compared to the axolotl data. Progressing inward, the next four circles in Xenopus reflect the fold change difference (red = down regulation; green = up regulation; blue = no change) of proteins at 1 dpa, 5 dpa, 7 dpa, and 12 dpa, respectively. In the axolotl, three circles represent FC in protein expression at 1 dpa, 4 dpa, and 7dpa. The innermost circle represents the connections between the interacting proteins within the Xenopus and axolotl data. A comparison of these interactions indicates that the proteomic composition and protein-protein interactions are much more complex during formation of the fibroblastema in Xenopus.

References [+] :

Abdel-Karim, Mitotic activity in the blastema and stump tissues of regenerating hind limbs of Xenopus laevis larvae after amputation at ankle level. An autoradiographic study. 1990, Pubmed, Xenbase