Cre/loxP-regulated transgenic zebrafish model for neural progenitor-specific oncogenic Kras expression

Abstract

Ras proteins regulate signaling pathways that control many cellular responses, such as proliferation, survival, and differentiation. However, there are intriguing questions about the relationship between the developmental timing of specific mutations and the resultant phenotypes in individual cells. In this study, we used the Cre/loxP system for maintaining transgenic zebrafish lines harboring oncogenic KrasV12 under the nestin promoter, and investigated the developmental effects of Ras activation in neural progenitor cells. Activated human KrasV12 was induced within pDSNesLCherryLEGFPKRasV12 transgenic fish by Cre mRNA injection. Cre-mediated gene excision was confirmed by polymerase chain reaction, and the injected embryos were investigated for KrasV12 effects using the hemotoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay, and in situ hybridization. pDSNesLCherryLEGFPKRasV12 transgenic embryos normally expressed mCherry in their central nervous system throughout the developmental stage. However, Cre mRNA injection efficiently excised the flanking stop sequence, and the injected embryos expressed enhanced green fluorescent protein in their brain with severe edema. Brain histology showed that neuronal cell differentiation could occur in spite of oncogenic KrasV12 overexpression, but massive apoptosis and brain edema caused early embryonal death. In summary, the overexpression of KrasV12 induces extensive apoptosis of neural progenitor cells followed by severe edema of the brain. However, some neural progenitor cells are resistant to KrasV12 and can retain their ability to differentiate into neurons. Finally, our transgenic model demonstrates the inability of KrasV12 alone to induce brain tumors at the early stage of development. (Cancer Sci 2009).