FIGURE

Fig. S1

ID

ZDB-FIG-130906-48

Publication

Czopka et al., 2013 - Individual Oligodendrocytes Have Only a Few Hours in which to Generate New Myelin Sheaths In Vivo

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Fig. S1

Generation of Tg(mbp:ca-Fyn) zebrafish.

Relates to Figures 4-6 and Movie S3.

(A) (Top) Amino acid comparison between human, mouse, and zebrafish Fyn showing high degree of overall identity and conservation of the catalytic and key regulatory C terminal tyrosines. (Bottom) Key domain structure of Fyn and indication of the position of the amino acid change induced to generate a constitutively active form of the molecule.
(B) Confocal image of a sox10:mCherry-CAAX, Tg(mbp:EGFP) co-expressing oligodendrocyte showing that the mbp upstream regulatory element is active before the cell initiated formation of myelin sheaths. Scale bar: 10 μm.
(C) Schematic showing transgenic strategy to express Fyn variants in glial cells from the initiation of myelination. The mbp upstream regulatory element is used to drive expression of mCherry and Fyn interspersed with a self-cleaving 2A peptide.
(D) Confocal images of mCherry and EGFP expression in a Tg(mbp:mCherry-2A-wtFyn) animal crossed with a Tg(mbp:EGFP) reporter line. All myelinating oligodendrocytes express the transgene as revealed by 100% overlap in mCherry and EGFP expression. Scale bar: 20μm.
(E) Western blotting of HEK cells transfected with plasmids that drive expression of either GFP alone or GFP-2A-Fyn. The GFP-2A-Fyn construct produces two separate proteins detected with antibodies against GFP (detecting the GFP-2A peptide, which runs slightly higher than GFP alone) and Fyn, respectively.
(F) Western blotting of HEK cells transfected with plasmids that drive mCherry-2A-wt-Fyn or mCherry-2A-ca-Fyn expression. The blot shows no phosphorylation of Tyrosine 531 when the ca-Fyn construct was transfected, which is detectable upon transfection with wt-Fyn. Phosphorylation of the catalytic Tyrosine 420 can be detected in both conditions.
(G) RT-PCR for total and transgenic fyn in wildtype, Tg(mbp:wt-Fyn), and Tg(mbp:ca-Fyn) transgenic zebrafish. Transgenic fyn (the mCherry-Fyn fusion transcript) is only detectable in the transgenic animals but not in wildtype animals, whereas total fyn is detectable in all conditions.
(H) Sequencing traces of PCR amplified total fyn transcripts from Tg(mbp:wt-Fyn) and Tg(mbp:ca-Fyn) transgenic animals. The A/T double peak in the Tg(mbp:ca-Fyn) animal reveals the presence of mutant fyn in a wildtype background.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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Reprinted from Developmental Cell, 25(6), Czopka, T., Ffrench-Constant, C., and Lyons, D.A., Individual Oligodendrocytes Have Only a Few Hours in which to Generate New Myelin Sheaths In Vivo, 599-609, Copyright (2013) with permission from Elsevier. Full text @ Dev. Cell