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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 - BMC Dev. Biol. (2014)

Bottom Line: 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.These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, and Center for Developmental and Regenerative Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA. dstocum@iupui.edu.

ABSTRACT

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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Related in: MedlinePlus

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.
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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.

Mentions: We fluorescently immunostained longitudinal cryosections of Xenopus limb tissue at each dpa to validate FC data obtained by LC/MS/MS and quantitated the intensity of fluorescence by densitometry (Figure 3). Three proteins were validated: β1 integrin, vimentin, and β2 dystroglycan. The immunofluorescent imaging and densitometry data were in good agreement with the mass spectrometry data.


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 - BMC Dev. Biol. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4222900&req=5

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.
Mentions: We fluorescently immunostained longitudinal cryosections of Xenopus limb tissue at each dpa to validate FC data obtained by LC/MS/MS and quantitated the intensity of fluorescence by densitometry (Figure 3). Three proteins were validated: β1 integrin, vimentin, and β2 dystroglycan. The immunofluorescent imaging and densitometry data were in good agreement with the mass spectrometry data.

Bottom Line: 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.These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, and Center for Developmental and Regenerative Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA. dstocum@iupui.edu.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus