June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Growth factors regulate early events in adult zebrafish extraocular muscle (EOM) regeneration
Author Affiliations & Notes
  • Alfonso Saera Vila
    Ophthalmology and Visual Sciences Department, University of Michigan, Ann Arbor, MI
  • Alon Kahana
    Ophthalmology and Visual Sciences Department, University of Michigan, Ann Arbor, MI
  • Footnotes
    Commercial Relationships Alfonso Saera Vila, None; Alon Kahana, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 561. doi:
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      Alfonso Saera Vila, Alon Kahana; Growth factors regulate early events in adult zebrafish extraocular muscle (EOM) regeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):561.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Zebrafish are able to regenerate complex tissues, whereas in mammals this ability is limited. Our laboratory uses EOMs as an experimental model to study regeneration in sexually mature adult zebrafish. Fibroblast growth factors (FGFs) have been shown to promote tissue regeneration so we hypothesize that FGFs regulate EOM regeneration in zebrafish.

Methods: A myectomy surgery that excised about 50% of the lateral rectus (LR) muscle was performed to sexually mature adult α-actin:EGFP zebrafish. At the end of the experiment, fish treated with DMSO or the FGF receptor inhibitor SU5402 (17 μM) were craniectomized and imaged. Muscle regeneration was calculated using the uninjured contralateral muscle as a reference (100%). Additionally, a-actin:EGFP or hsp70:dnfgfr1a-EGFP (a line that expresses a dominant negative FGF receptor under control of the heat shock protein 70 promoter) zebrafish were myectomized and analyzed as described. Cell proliferation was analyzed injecting fish with EdU to label cells in the S-phase of the cell cycle. The involved cell signaling pathway was analyzed by testing the effect of the MEK inhibitor U0126 (25 μM) in EOM regeneration and proliferation as described. The activation of ERK was analyzed by immunofluorescence and western blot.

Results: LR myectomy resulted in loss of abduction and, therefore, the optokinetic response. Within 7 days, a new and functional LR muscle regenerated. Both pharmacologic and genetic approaches were used to analyze the role of FGFs. Treating myectomized fish with the inhibitor SU5402 for 3 days reduced LR regeneration from 70% to 58% (p<0.05). FGF role was confirmed using hsp70:dnfgfr1a-EGFP in a time course experiment showing a significant regeneration delay at 3 (13%, p<0.05), 5 (15%, p<0.05), 9 (36%, p<0.05), and 14 days (25%, p<0.01). Cell proliferation was decreased in hsp70:dnfgfr1a-EGFP fish compared to WT fish (19% and 27%, respectively; p<0.05). U0126 treated fish showed a decrease in LR regeneration (71% vs 57%, p<0.05) and cell proliferation (17% vs 9%, p<0.05). Immunofluorescence and western blot confirmed the activation of ERK in the regenerating LR.

Conclusions: Our results show that FGFs, and most likely other growth factors, play a significant role in the regeneration of EOMs in zebrafish. Additional studies will be required to further uncover the molecular mechanisms and target genes involved.

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