Fig. 2

HRas^V12 expression in epithelial cells induces cell shape and genetic changes associated with EMT in vivo

(A) Schematic of Tol2 flanked:krt4:RFP-HRas+transposase one-cell stage injection resulting in mosaic expression. (B–C) Fluorescent Z stack projections of HRas^WT and HRas^V12 expressing epithelial cells (magenta) in the trunk region of 3.5 dpf larvae (illustrated in A). (D–E) Lateral fluorescent images, from live imaging, of 3.5 dpf embryos co-expressing GFP-H2B to label the nuclei and either HRas^WT (D) or HRas^V12 (E) at 0, 2 hr and 4 hr time points. Arrows in E indicate cell extensions. (F) Quantification of the 2D area of H-Ras^WT and H-Ras^V12 expressing cells shows a significant decrease in the 2D area of HRas^V12 expressing cells compared to controls. (G) Quantification of the cell roundness of HRas^WT and HRas^V12 expressing cells shows a significant decrease in the cell roundness of HRas^V12 expressing cells compared to controls. (H–I) Double label WMISH with HRas^WT (H) and HRas^V12 (I) transcript labeled in red and mmp9, slug, and vimentin transcript label in blue illustrating that mmp9, slug, and vimentin expression are induced in RFP-HRas^V12 compared to control RFP-HRas^WT expressing larvae. (J) Quantitative RT-PCR (one representative graph shown n = 5) indicates a statistically significant increase in mmp9 and slug transcripts in HRas^V12 transformed cells compared to control HRas^WT expressing cells. hr = hour, dpf = days post fertilization, ** = p<.005, **** = p<.0001 scale bars = 20 microns.