Expression of CD45 by mouse and human IACs.

<p>Transverse sections of AGM region were made from ICR mouse embryos at 9.5 and 10.5 dpc and from human embryos at 32 day-old, according to the Carnegie classification, stained with antibodies and observed by confocal microscopy. Arrowheads indicate IACs. (<b>A</b>) Mouse IACs in the omphalomesenteric artery (OMA) at 9.5 dpc expressed c-Kit, but not CD45. CD45 (green) and c-Kit (red). Magnified view of IACs is shown at right upper panel in Merge panel. Original magnification is 40x. (<b>B-D</b>) Mouse IACs in the dorsal aorta (DA) (B), OMA (C) and umbilical artery (UA) (D) at 10.5 dpc expressed c-Kit, and some expressed CD45. CD45 (green) and c-Kit (red). Original magnification is 40x. (<b>E</b>) All human IACs in the DA expressed CD34, and some expressed CD45. CD34 (green), CD45 (red) and TOTO-3 (blue). NT (Neural Tube); Ao (Aorta); Mn (Mesonephros). Original magnification is 20x.</p

Confocal images of IACs expressing CD31/CD34/c-Kit in the AGM region.

<p>Transverse sections of AGM region from ICR mouse embryos at 9.0 and 10.5 dpc were stained with antibodies and observed by confocal microscopy. (<b>A</b>) IACs were observed in the omphalomesenteric artery (OMA) at 9.0 dpc (left; magnified view of IACs in upper right panel) and in the OMA, dorsal aorta (DA) and umbilical artery (UA) at 10.5 dpc (right). CD31 (red), c-Kit (green), and TOTO-3 (blue). Arrows indicate IACs. Original magnification is 20x. (<b>B-D</b>) IACs were observed in the DA (B), OMA (C) and UA (D) at 10.5 dpc. Left panel shows staining for CD31 (red), c-Kit (green), and TOTO-3 (blue), and right panel shows staining for CD34 (red), c-Kit (green), and TOTO-3 (blue) staining. Images were taken at 40x and zoom was used to show a detail at right lower panel. Another IAC in the DA is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s001" target="_blank">Figure S1</a>. (<b>E</b>) IACs expressing Ki-67, a marker of proliferation, were observed in the DA (left), OMA (middle) and UA (right). Ki-67 (red), c-Kit (green), and TOTO-3 (blue). Images were taken at 40x and zoom was used to show a detail.</p

Gene expression analysis in CD31⁺/CD34⁺/c-Kit⁺ AGM cells separated by CD45 expression.

<p>(<b>A</b>) Single cell suspensions of the caudal portion of embryos containing the AGM region at 10.5 dpc were prepared and analyzed by flow cytometry. Cells expressing CD31 and CD34, IAC markers, were first gated. The profile shows expression of c-Kit (x-axis) and CD45 (y-axis) in CD31⁺/CD34⁺ AGM cells (left). Based on intensity of CD45 expression, CD31⁺/CD34⁺/c-Kit⁺ AGM cells were separated into three fractions, CD45-negative (under 10² of CD45-fluorescence, same as negative control), -low positive (from 10².⁵ to 10³.⁵ of CD45-fluorescence), and -high positive (approximately over 10⁴ of CD45-fluorescence). Isotype control and compensation samples of flow cytometric analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s004" target="_blank">Figure S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035763#pone.0035763.s001" target="_blank">S5</a>. (<b>B</b>) The percentage of CD45-negative, -low positive, and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells was calculated both at 9.5 dpc (white bars) and 10.5 dpc (black bars). (<b>C-H</b>) Gene expression of <i>CD45</i> (C), <i>Runx1</i> (D), <i>c-Myb</i> (E), <i>Evi-1</i> (F), <i>SCL</i> (G) and <i>Gata2</i> (H) was analyzed in sorted CD45-negative, -low positive and -high positive c-Kit⁺/CD31⁺/CD34⁺ AGM cells. Expression levels of <i>CD45</i> mRNA are up-regulated as c-Kit⁺/CD31⁺/CD34⁺ cells express CD45 surface protein. Expression levels of <i>Runx1</i>, <i>c-Myb</i>, <i>Evi-1</i>, <i>SCL</i> and <i>Gata2</i> were highest in CD45-low positive c-Kit⁺/CD31⁺/CD34⁺ cells, whereas that of <i>Evi-1</i> was highest in CD45-negative c-Kit⁺/CD31⁺/CD34⁺ cells. RQ represents relative quantity of template in the original sample.</p

Punctuate staining with SG marker eIF3e was commonly found adjacent to or surrounding FUS-GFP SGs.

<p>Scale bars  = 10 µm; Insets  = 1 µm.</p

Mislocalization of mutant FUS-GFP was also found in motor neurons.

<p>(<b>A</b>) FUS-R521C-GFP was similarly mislocalized to the cytosol in motor neurons (labeled with 39.4D5 for islet1 and islet2 homeodomain marker). (<b>B</b>) Quantification of FUS-GFP signal in nucleus vs. cytosol in 39.4D5 marked motor neurons demonstrated that FUS-R521C-GFP was significantly more cytosolic compared to FUS-WT-GFP (post-hoc Tukey HSD. ** = P<0.01, N.S. = >0.05, n = 40 for all samples. Error bars represent SE. Scale bars  = 20 µm.</p

Whole mount and cell cultures of FUS-GFP transgenic zebrafish.

<p>(<b>A</b>) Transgenic zebrafish larvae whole mounts showed cytosolic mislocalization of mutant human FUS in FUS-R521C-GFP in comparison to FUS-WT-GFP which was restricted to cell nuclei. (<b>B</b>) FUS-R521C-GFP showed greater cytosolic distribution in comparison to FUS-WT-GFP in zebrafish primary cell cultures. (C) Confocal images of 48 hpf transgenic zebrafish spinal cord further demonstrate mislocalization of mutant FUS-521C-GFP (green) in motor neurons (red) (arrows). Images are maximum projections captured using a Leica SPE5 confocal microscope. Sagittal sections (upper images) of Tg(s1020tGAL4: UASmCherry) (Scott and Baier, 2009) and transverse sections (lower images) of Tg(HB9: mK02caax) membrane localised mk02 expressed in motorneurons by HB9 promoter (Flanagan-Steet et al 2005) with either FUS-WT-GFP or FUS-521C-GFP as indicated. Scale bar  = 20 µm.</p

Ubiquitous FUS-GFP SG assembly in zebrafish cells.

<p>(<b>A</b>) FUS-GFP SGs formed in cultured transgenic zebrafish cells after heat-shock at 40°C for 30 mins (arrows). However, SGs were more abundant in mutant FUS-R521C-GFP cultures (right panels) than in FUS-WT-GFP. (<b>B</b>) Motor neurons (39.4D5-labeled cells) were not particularly susceptible to SG assembly. (<b>C</b>) SGs were reversible when cells were allowed to recover at 37°C for another 30 mins. Some persistent SGs were still present particularly in FUS-R521C-GFP cultures. Motor neurons labeled with 39.4D5 readily reversed SGs. (<b>D</b>) Stress granules (SGs) were also induced by sodium arsenite (Na₃AsO₃; 0.2 mM) treatment. SGs formed in mutant (arrows) but not in the FUS-WT-GFP line after Na₃AsO₃ treatment for 1 hr. Similar to heat-shocked cells, 39.4D5-labeled cells were no more susceptible to chemical-induced SG formation. (<b>E</b>) Chemical-induced FUS-GFP containing SGs were reversible in both lines. Reversibility also occurred readily in 39.4D5 labelled motor neurons.</p

Quantification of SG assembly and reversibility

<p>(<b>A</b>) FUS-R521C-GFP generated SGs in almost double the number of cells compared to FUS-WT-GFP after 40 minutes heat shock. Further, FUS-R521C-GFP cells were less able to reverse SGs compared to FUS-WT-GFP cells. (<b>B</b>) Quantification of the number of SGs per cell in SG containing cells after heat shock and recovery in three experiments showed that FUS-WT-GFP generated ∼5 FUS-containing SGs per cell and recovered to ∼2 SGs per cell. By contrast, FUS-R521C-GFP SG-containing cells remained at ∼12 SGs per cell before and after heat shock recovery despite recovery of many surrounding cells. *P≤0.05 and ***P≤0.005. Error bars indicate SE.</p

Universal cytosolic mislocalization of mutant FUS-GFP protein expressed in zebrafish cells.

<p><b>(A)</b> A band at ∼100 kDa was seen uniquely in transgenic lysates corresponding to full length human FUS (75 kDa) conjugated to GFP (25 kDa) but not non-transgenic lysates. Lower MW bands (∼30–40 kDa) consistent with endogenous FUS were detected in all including non-transgenic zebrafish lysates. Multiple bands for endogenous FUS may indicate some degradation. N.S. denotes non-specific bands. Alpha-tubulin was used as a loading control. (<b>B</b>) Flow cytometric analysis demonstrated GFP expression in dissociated transgenic larvae. Y-axis units are normalized to the number of cells analyzed (5000–10000 cells per sample) and GFP intensity readings are presented in a log scale on the x-axis. (<b>i</b>) Cell suspensions of GFP positive vs. GFP negative larvae siblings derived from human FUS-WT-GFP transgenic fish. Clear separation of cells positive and negative for GFP is shown by the GFP +ve peak on the right and the GFP –ve peak on the left. (<b>ii</b>) Mean GFP fluorescence per cell in fresh cell suspensions from FUS-R521C-GFP and FUS-WT-GFP lines. (<b>C</b>) There was no significant difference in total GFP intensity per cell between lines after plating and culturing cells. Error bars represent SE. (<b>D</b>) Quantification of GFP fluorescence (A.U.) in nucleus and cytosol in individual cells in cultures from each fish line (n = 50–100 cells) demonstrated that there was a significant elevation in cytosolic and reduction in nuclear GFP fluorescence in FUS-R521C-GFP compared to FUS-WT-GFP cells (post-hoc Tukey HSD. *** = P<0.001). (<b>E</b>) To address the question whether elevated levels of FUS fusion protein expression could in itself cause mislocalization, we measured the total GFP fluorescence intensity versus the % nuclear GFP fluorescence in 50 to 100 individual cells of each genotype. Data were collected from 3 independent experiments for each line. There was no significant correlation between level of total GFP expressed in a cell and its subcellular distribution based on the R² values for each line (FUS-WT-GFP: R² = 0.0145 and FUS-R521C-GFP: R² = 0.3458) (n = 79 and 41 respectively).</p