Neuronal cell culture from transgenic zebrafish models of neurodegenerative disease

Acosta, J.R., Watchon, M., Yuan, K.C., Fifita, J., Svahn, A.J., Don, E.K., Blair, I.P., Nicholson, G.A., Cole, N.J., Goldsbury, C., Laird, A.S.
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Optimization of the zebrafish primary neural cell culture. (A) Images of cell cultures derived from 24 hpf and 48 hpf-aged embryos. Motor neurons in both cultures exhibited outgrowth of long processes (arrows). (B) No difference in motor neuron survival rate was evident for cells incubated at 37°C or 28°C after 1 day (cultures from 24 hpf larvae). (C) Motor neurons in cultures derived from de-yolked embryos exhibited shorter neurites compared to those derived from whole embryos. Note however that by 2 div, almost 100% of cells from the de-yolked cultures were non-viable (not shown). Scale bar: 10 µm.

Images of cultured 24 hpf Islet1:GFP zebrafish embryos stained with zebrafish-specific neuronal markers to confirm that the cell cultures contain various types of neurons. (A) An Islet1:GFP motor neuron within the cultures is positively stained (red) for the neuronal marker 39.4D5 (islet1 and islet2 homeobox). (B) Another Islet1:GFP motor neuron, and nearby islet1:GFP negative cells, are stained positively (red) for the neuronal cell surface marker Zn12, indicating the inclusion of other types of neurons in addition to motor neurons. Scale bar: 10 µm.

Cultured cells derived from transgenic zebrafish larvae expressing neurodegenerative disease associated proteins FUS or ataxin-3. (A) In cells cultured from mutant human FUS-GFP (FUS-R521C) zebrafish the FUS-GFP protein was mislocalized to the cytosol, whereas it remained predominantly nuclear in cells cultured from wild-type FUS-GFP zebrafish. (B) Cells cultured from double transgenic zebrafish expressing mCherry (red) and EGFP-ataxin-3-23Q/84Q (green) showed no obvious difference in fluorescent protein distribution in cells expressing non-pathogenic EGFP-ataxin-3-23Q and pathogenic EGFP-ataxin-3-84Q. Aggregates of mCherry-positive protein (arrows) were present in some neurons (Jakobs et al., 2000). (C) Immunolabeling cell cultures with anti-polyQ (pale blue) demonstrated cytosolic distribution of the ataxin-3 protein in cells expressing either EGFP-ataxin-3-23Q or pathogenic EGFP-ataxin-3-84Q. Scale bars: 10 µm. (D) Cross-sections of the spinal cord of 3 dpf transgenic SCA3 zebrafish revealed a similar expression pattern of EGFP-ataxin-3 and mCherry to that seen in the cell cultures. Scale bars: 5 µm.

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