Journal: PLOS Biology
Article Title: Piezo1-mediated spontaneous calcium transients in satellite glia impact dorsal root ganglia development
doi: 10.1371/journal.pbio.3002319
Figure Lengend Snippet: (A) LEFT Depiction of mechanical compression assay where animal is mounted dorsally on inverted spinning disk confocal with a dextran loaded microneedle mounted above the animal. RIGHT image of mechanical compression assay apparatus. (B) Depiction of mechanical compression assay with needle placing force on DRG. (C) Quantification of the percent of DRG responding to mechanical force in animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) (labeled sox10) or expressing Tg(neurod : gal4+myl7); Tg(uas : GCaMP6s) (labeled neurod) and treated with either DMSO or GsMTx4 at both 2 and 3 dpf (sox10 2 dpf DMSO: n = 7 animals, 7 DRG, sox10 2 dpf GsMTx4: n = 5 animals, 5 DRG neurod 2 dpf DMSO: n = 5 animals 5 DRG neurod 2 dpf GsMTx4: n = 4 animals, 4 DRG sox10 3 dpf DMSO: n = 11 animals, 11 DRG sox10 3 dpf GsMTx4: n = 4 animals, 4 DRG neurod 3 dpf DMSO: n = 9 animals, 9 DRG neurod 3 dpf GsMTx4: n = 4 animals, 4 DRG). (D) Confocal image taken of the mechanical compression assay in 2 dpf animal expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) . Images show an inactive time point, a time point with tissue compression, and an active time point in response to tissue compression. Inactive and active DRG marked with an arrow. (E) Quantification of the change in integrated density of fluorescence during each phase of mechanical compression assay of a DRG in a 3 dpf animal treated with either DMSO or GsMTx4. Change in integrated density of fluorescence is scored as time point subtracting the initial time point divided by time point ( Δf / f ). (F) Quantification of the average change in fluorescence during the phases of mechanical compression following treatment with either 2% DMSO or 1 μM GsMTx4 for 30 min (Resting, Compression, and Decompression DMSO: n = 9 DRG, 9 animals, Resting, Compression, and Decompression GsMTx4: n = 4 DRG, 4 animals). (G) Confocal z-projection of peripheral DRG axon in an animal expressing Tg(ngn1 : GFP) at 2 and 3 dpf. Arrow notes the end processes of the peripheral axon. Arrowhead denotes peripheral axons from Rohon beard neurons. (H) Average distance (μM) of DRG displacement needed to elicit a response ( n = 16 animals, 16 DRG). (I) Confocal images of RNAscope- piezo1 and Immunohistochemistry-GFP in Tg(sox10 : meGFP) animals. GFP is shown in magenta and piezo1 is shown in cyan. Arrowheads indicate piezo1 puncta. Arrows indicate autofluorescence. (J) Quantification of DRG at 3 dpf with piezo1 puncta and without piezo1 puncta ( n = 8 animals, 24 DRG). (K) Confocal images of 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) . Red colors indicate a higher intensity of fluorescence and blue colors indicate a lower intensity of fluorescence. Images depicted are of animals either treated with 2% DMSO, 40 μM Jedi2, or 1 μM GsMTx4. Arrows note active cells. (L) Line graphs of z score of integrated density of fluorescence for a 1-h time period in 3 dpf animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 40 μM Jedi2, or 1 μM GsMTx4. A z score greater than 2.58 indicates an active Ca 2+ event. Red scale bar shows a z score of 2.58. (M) Heatmaps of the z score of individual sox10 + cells from animals in G and H during a 1-h period of Ca 2+ imaging. Yellow notes a high z score (2.58 or greater) (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells). (N) Quantification of the average number of Ca 2+ events per sox10 + cell in animals treated with either 2% DMSO, 1 μM GsMTx4, 100 μM Yoda1, or 40 μM Jedi2 (DMSO: n = 8 animals, 17 DRG, 52 cells, Jedi2: n = 7 animals, 18 DRG, 79 cells, GsMTx4: n = 6 animals, 16 DRG, 44 cells, Yoda1: n = 4 animals, 9 DRG, 35 cells). (O) Quantification of the average number of Ca 2+ events per neurod + cell in 3 dpf animals expressing Tg(neurod : gal4+myl7); Tg(uas : GCaMP6s) that were treated with either 2% DMSO, 1 μM GsMTx4, 40 μM Jedi2, or 100 μM Yoda1 (DMSO: n = 10 animals, 24 DRG, 33 cells, Jedi2: n = 5 animals, 18 DRG, 35 cells, GsMTx4: n = 4 animals, 8 DRG, 8 cells, Yoda1: 4 animals, 7 DRG, 8 cells). (P) Quantification of the average number of Ca 2+ events per sox10 + cell following genetic manipulation via injection of uas : cas9mkate-u6 : piezo1gRNA or uas : cas9mkate-u6 : emptygRNA into animals expressing Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) at 3 dpf. Additionally, a group of Tg(sox10 : gal4+myl7); Tg(uas : GCaMP6s) injected with uas : cas9mkate-u6 : piezo1gRNA and treated with 40 μM Jedi2 treatment was also included in the experiment (u6:emptygRNA: n = 6 animals, 16 DRG, 40 cells, u6:piezo1gRNA: n = 4 animals, 13 DRG, 57 cells, u6:piezo1gRNA+Jedi2: n = 4 animals, 4 DRG, 7 cells). Scale bar is 10 μM (D, G, I, K). Statistical tests: unpaired t test (H, N, O), Fisher’s exact (C), multiple unpaired t tests (F). The data underlying this figure can be found in . dpf, days post fertilization; DRG, dorsal root ganglia.
Article Snippet: To create uas : cas9mkate-u6 : piezo1gRNA , we recombined a p5E-UAS (BseRI cleavage site removed), pME-cas9mkate (Addgene 109547), and p3E-pA constructs into a pDestTol2CG2 containing a U6 promoter sequence flanked with BseRI cleavage sites [ , ].
Techniques: Expressing, Labeling, Fluorescence, RNAscope, Immunohistochemistry, Imaging, Injection
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