Extended Data Fig. 7: Detection of Vsp1 in Xenopus cranial neural crest and knockdown specificity and effectiveness controls.
From: Stretch-induced endogenous electric fields drive directed collective cell migration in vivo
a, Scheme and workflow of RNA-seq experiments from a single neural crest explant dissected at pre-migratory stages (~st17) and processed for SMART-seq2 RNA-seq. b, Heatmap showing relative expression levels. Note that neural crest markers are enriched (warm colors) in our libraries, unlike surrounding tissues markers (cold colors), confirming library purity (N stands for independent experiments). c, List of represented ion channels found by RNA-seq after TMM (Trimmed Mean of M-values). The voltage-sensitive phosphatase vsp1 (tpte2.L) is highlighted in red. d, Vsp1-GFP locates in the membranes of neural crest cells ex vivo. e, Representative bands of a PCR against vsp1 or sox8 (a neural crest marker) and embryo hybridized with a probe against vsp1. One side of the embryo injected with Vsp1-Cas13, showing a decreased vsp1 signal. f–k, Lateral views of embryos displaying the result of in situ hybridization against sox8 (a neural crest marker). Scale bar, 200 μm. l, Percentage of embryos displaying streams. Bars represent mean and error bars standard deviation. Two-tailed Fisher’s exact test, ****p < 0.0001 and **pVsp1-SSMO vs. Vsp1-SSMO + Vsp1-GFP = 0.0011, nControl = 25, nXl-Vsp1-C301S = 24, nControl = 35, nVsp1-SSMO = 35, nVsp1-SSMO + Vsp1-GFP = 22, nControl Cas13 = 24, nVsp1-Cas13 = 37 and nVsp1-Cas13 + Vsp1-GFP = 47 embryos. m, Representative gel showing that Vsp1-SSMO specifically targets the splicing site of vsp1. Primers spam a splicing site; hence, the presence of a band means that Vsp1-SSMO precluded splicing, and in turn knocking down vsp1. n, Representative gel showing that Vsp1-Cas13 effectively downregulates vsp1 transcripts. o, Normalized ratio intensity of vsp1/ef1α. Bars represent mean and error bars standard deviation. Two-tailed Mann Whitney U-test, ***p = 0.0001; nControl = 7, nVsp1-Cas13 = 10 gel bands. p–r, Representative confocal projections of immunofluorescence against Vsp1, showing that Vsp1-SSMO and Vsp1-Cas13 downregulates Vsp1 protein in the membranes of neural crest cells ex vivo. s, Quantification of the fluorescence intensity profile of lines overlaying cell membranes. Solid lines represent mean and shades the 95% confidence interval. Brown-Forsythe and Welch ANOVA with Dunnett’s multiple comparison against control, ****p < 0.0001 for both comparisons, n = 18 cell membranes. d,e,f–k,m,n,p–r, Representative examples from three independent experiments; CI = 95%.
