Additional Supporting Information may be found in the online version of this article.

ana23780-sup-0001-SuppFig1.tif16080KSupporting Information Figure 1. DsRed provides a stable and cumulative readout of endogenous hsp70 induction: Whole-mount antibody labelling with hsp70 (green) (A-D) and DsRed (E,F) in non-heat shocked control (A,C,E) and heat shocked (B,D,F) zebrafish embryos at 29 hpf (A,B), 39 hpf (C,D) and 48 hpf (E,F). Magnification: 20X
ana23780-sup-0002-SuppFig2.tif10553KSupporting Information Figure 2. Spinal motor neurons and glial cells do not show induction of the HSR in mutant sod1 transgenic spinal cord at the early larval stages. DsRed (middle panel) did not colocalise with GFP in Sod1-G93Ros10-Sh1 transgenic zebrafish crossed with hb9-GFP (row1), islet-1-GFP (row 2) (markers of differentiating motor neurons) and did not immunolabel with ChAT antibody (row 3)(marker of mature motor neurons). DsRed also did not colocalise with migrating GFP positive oligodendrocytes (arrowhead) in crosses with olig2-GFP fish (row4) or with GFP positive radial glia in crosses with GFAP-GFP zebrafish (row 5). Time 72 hpf. Scale bar 10μm.
ana23780-sup-0003-SuppFig3.tif13077KSupporting Information Figure 3. DsRed colocalises with pax2 positive glycinergic interneurons: Whole-mount antibody labelling with anti-pax 2 antibody (green, Top panel) and DsRed (middle panels) and glycine antibody (green, bottom panel) in non-heat shocked G93Ros10-Sh1 embryos at 48 hpf. Neurons showing colocalization are marked by arrows. Scale bar 10 μm (top panels), 5μm (bottom panels)
ana23780-sup-0004-SuppFig4.tif4099KSupporting Information Figure 4. Zebrafish larvae at 9 dpf do not show induction of stress response in motor neurons. Cross sections of 9 dpf spinal cord immunolabelled with ChAT (column 3, arrow and area in triangle) and DsRed (column 2). Cell nuclei are labelled by Dapi (column 1) and the merged image (column 4) shows no DsRed/ChAT positive motor neurons. DsRed positive neurons are dorsally located in the spinal cord. Scale bar 5μm (top panel), 10μ m (bottom panel)
ana23780-sup-0005-SuppFig5.tif31426KSupporting Information Figure 5. Motor neuron stress is observed in 6 month and 1 year old spinal cords from mutant sod1 transgenic zebrafish. Cross sections of spinal cord from 6 month (top of each group) and 1 year (bottom of each group) old high expressor G93Ros10-Sh1 (top 2 panels) and low expressor G85Ros6-Sh3 (bottom 2 panels) transgenic zebrafish immunolabelled with ChAT (column 3) and DsRed (column 2). The cell nuclei are labelled with Dapi (column 1) and the merged image (column 4) shows DsRed/ChAT positive motor neurons (arrows). Magnification 40X.
ana23780-sup-0006-SuppInfo.doc72KSupporting Information

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