Fig 2: Transcriptional activation leads to increased Smn levels in motoneurons.(a) Single molecule FISH on pmnx1:eGFP embryos. DNA, GFP and smn mRNA signals are shown in Z-projected confocal sections. Scale bars: 10 μm for low and 5 μm for high magnification. (b) Quantification of smn mRNA signal from one representative experiment. Exact values are (mean ± SD): 127 ± 42 (GFP−) and 218 ± 61 (GFP+), p = 8.06 × 10−8 with Wilcoxon Sum Rank Test. (c) Average increase of smn mRNA levels in motoneurons versus control cells. The exact value of enrichment is 1.73 ± 0.09 (mean ± SD). N = 3 experiments, n = number of analyzed cells. (d) Putative smn promoter in zebrafish. Genomic situation (top) and mCherry construct (bottom). Arrows denote start of transcription. (e) scIF on pmnx1:eGFP × psmn:mCherry embryos. GFP and mCherry signals are shown. Scale bars: 10 μm for low and 5 μm for high magnification. (f) Quantification of mCherry signal from one representative experiment. Exact values are (mean ± SD): 8.9 ± 4 (GFP−) and 15 ± 4 (GFP+), p = 9.37 × 10−5 with Wilcoxon Sum Rank Test. (g) Average increase of mCherry levels in motoneurons versus control cells. The exact value of enrichment is 1.87 ± 0.27 (mean ± SD). N = 2 experiments, n = number of analyzed cells.

Fig 3: Etv5b activates the smn promoter leading to increased Smn levels in motoneurons.(a–d) Whole-mount in situ hybridization of etv5b expression in 24 hpf embryos. Expression pattern in the entire embryo (a), in magnified lateral (b) and dorsal (c) views as well as in a transverse section (d) of the trunk. In the latter case, yellow arrowhead denote signal in the motoneuron region. Scale bars: 100 μm (a) and 50 μm (b–d). nc, notochord. (e) RT-PCR in control (C - only psmn:mCherry) versus etv5b overexpressing (OE - etv5b mRNA and psmn:mCherry) embryos at early gastrula stage. (f,g) scIF of control (f) and etv5b MO (g) cells from pmnx1:eGFP embryos. H2B, GFP and Smn signals are shown in Z-projected confocal sections. GFP+ cells marked by white rectangles are magnified on the right. Scale bars: 10 μm for low and 5 μm for high magnification. (h) Relative Smn signal in control and in etv5b MO cells from one representative experiment. Exact values are (mean ± SD): 0.99 ± 0.25 (control) and 0.7 ± 0.14 (etvb5 MO), p = 0.003 with Wilcoxon Sum Rank Test. (i) Average relative Smn levels in control and in etv5b MO motoneurons. The exact value of decrease is 0.75 ± 0.04 (mean ± SD). N = 3 experiments, n = number of analyzed cells.

Fig 4: Model.Under healthy conditions in motoneurons, basal smn transcription (orange circle) is enhanced by Etv5b (green oval) acting together with other unknown factor(s) (yellow diamond) leading to more Smn protein (purple) in those cells. More Smn could be needed in motoneurons to meet an increased demand for the splicing machinery and/or for axonal mRNA localization. In the event of reduced Smn levels (that is in SMA), SMA-insusceptible cells are able to tolerate such a reduction, while motoneurons suffer from the cumulative reduction of multiple processes, thereby rendering them more vulnerable.

Fig 5: Motoneurons exhibit elevated Smn levels.(a–c) Single-cell immunofluorescent (scIF) experiments. 24 hpf pmnx1:eGFP embryos were dissociated and immunostained for Smn ((a) see also Methods). DIC image of cells following 3-hour (b) and 24-hour (c) incubation are shown with GFP in green. Note that the GFP signal in the cell body is oversaturated so that the weaker signal in the axon becomes visible. In (c) DNA is in blue and the motoneuron marker Znp1 (synaptotagmin) is in red; the axon growth cone is magnified in the corner of the images. Scale bars are 100 μm (a), 10 μm (low magnification in (b,c)) and 2 μm (high magnification in (c)). (d) scIF on pmnx1:eGFP embryos. Histone 2B (H2B), GFP and Smn signals are shown in Z-projected confocal sections. Cells marked by white rectangles are magnified on the right. Scale bars: 10 μm for low and 5 μm for high magnification. (e) To account for potential variability in the immunostaining, Smn levels were quantified relative to H2B. Diamonds denote GFP negative (GFP−) and GFP positive (GFP+) cells from one representative experiment. Blue bars indicate mean ± SD with significance values of *p < 0.05 and **p < 0.01. Exact values are (mean ± SD) 0.51 ± 0.14 (GFP−) and 0.87 ± 0.17 (GFP+), p = 0.001 with Wilcoxon Sum Rank Test. For more details, see Materials and Methods and Supplementary Table S1. (f) Average increase of Smn levels in motoneurons versus control cells. The exact value of enrichment is (mean ± SD): 1.67 ± 0.14. N = 3 experiments, n = number of analyzed cells.