Figure 1.
Modeling early-stage carcinoma in Xenopus laevis
(A) Schematic of the microinjection protocol. Xenopus embryos were injected with Cherry-histone-H2B and BFP-CAAX mRNA at the 2-cell stage. At the 32-cell stage, a single cell was injected with GFP, GFP-kRasV12, or GFP-cMYC mRNA. Embryos were developed to early gastrula stage 10 and imaged.
(B–D) Confocal microscopy images of Xenopus embryos developed to early gastrula stage 10, following injection of a single cell at the 32-cell stage with (B) GFP, (C) GFP-kRasV12, or (D) GFP-cMYC mRNA. Scale bars represent 100 μm.
(E) Western blot showing phosphorylated ERK, unphosphorylated ERK, and α-tubulin expression in uninjected control embryos and embryos injected with GFP, GFP-kRasV12, or GFP-cMYC mRNA.
(F) Bar chart showing the average percentage of cells that divided per minute of time lapse, in either GFP, GFP-kRasV12, or GFP-cMYC overexpression clusters (∗p < 0.05; Kruskal-Wallis test: n = 7 GFP, 8 GFP-kRasV12, and 9 GFP-cMYC embryos). Also displayed is the proportion of cell divisions that occurred out of the epithelial plane (shaded portion of the bar). Error bars show SEM.
(G) Stills from a confocal microscopy time lapse of a representative embryo with a GFP-kRasV12 cell cluster at stage 10. White arrows highlight cells observed to be lost basally over the course of the time lapse.
(H) Dot plot showing average percentage of cells that extruded basally from GFP, GFP-kRasV12, or GFP-cMYC cell clusters (∗p < 0.05; Kruskal-Wallis test: n = 7 GFP, 9 GFP-kRasV12, and 5 GFP-cMYC embryos). Error bars are SEM.
(I and J) Microscopy images of representative embryos at stage 38 that had a (I) GFP- or (J) GFP-kRasV12-expressing cluster at stage 10. Arrow indicates an induced tumor-like structure (ITLS). Anterior is toward the left; scale bars represent 500 μm.
(K) Quantification of ITLS formation at stage 38 in embryos that had GFP or GFP-kRasV12 clusters at stage 10 (∗∗p < 0.01; Mann-Whitney test; n = 5 independent experiments; a total of 120 GFP and 97 GFP-kRasV12 embryos were assessed). Error bars are SEM.