XB-IMG-175699
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Fig. 1. Sema3A and 3F are co-expressed with Sdf1 and restrict NC migration in vivo. a In situ hybridisation for neural crest markers (st17, slug; st21–28, twist), Sdf1, Semaphorin-3A and 3F. NC cells migrate as streams numbered 1–4, anterior to posterior. Asterisk marks the eye. b, c Diagrams depicting the relative distribution of Sdf1 (grey), Sema3A (red) and Sema3F (blue) with respect to NC cells (green) at stage 17 in wholemount (b) or transversal section (c). d Loss-of-function with antisense Morpholinos for Sema3A and 3F analysed by in situ hybridisation for twist, embryos st25. Arrows indicate cells from stream 1 that did not reach the area ventral to the eye. Black arrowheads indicate shorter streams. Red arrowheads, cells accumulated in dorsal region. Asterisks mark the eye. Note cells migrating over the eye on the injected sides. e Anterior view of a representative embryo injected with both MOs against Sema3A and 3F. Dotted line marks the midline. f Loss-of-function with CRISPR/Cas9 and gRNAs for Sema3A and 3F analysed by in situ hybridisation for twist, embryos st25. g Anterior view of a representative embryo injected with gRNAs against Sema3A and 3F together with dCas9. h Percentages of embryos with symmetrical migration along the dorso-ventral axis on non-injected and injected side. i Percentages of embryos with NC cells in ectopic locations (over the eye, in between streams, caudal expansion, between midline and NC streams). Proportions for each experimental conditions were compared to control uninjected embryos. 327 embryos were used obtained from three independent experiments, n of embryos per conditions are indicated on the figure. Comparisons of proportions were made using contingency tables60. Null hypothesis rejected if T > 3.841 (*α = 5%); T > 6.635 (**α = 1%); T > 10.83 (***α = 0.1%). Note that normal NC migration in control embryos displays some level of randomness. Around 15% of non-injected embryos had noticeable differences between their left and right sides. The front of migration was more ventral on one side than the other. Also, about 20% of non-injected embryos had some cells that would be counted as ectopic in experimental embryos, mainly cells located dorsally to the streams, seemingly late. Scale bars are 500 μ, except for e and g, 50 μ. Source data are provided as a Source Data file
Image published in: Bajanca F et al. (2019) © The Author(s) 2019. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |