Optical Control of Tumor Induction in the Zebrafish

Abstract

The zebrafish has become an increasingly popular and valuable cancer model over the past few decades. While most zebrafish cancer models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression in live zebrafish. We utilize this technique to transiently or constitutively activate a typical human oncogene, kRASG12V, in zebrafish embryos and investigate the developmental and tumorigenic phenotypes. We demonstrate the spatiotemporal control of oncogene expression in live zebrafish, and characterize the different tumorigenic probabilities when kRASG12V is expressed transiently or constitutively at different developmental stages. Moreover, we show that light can be used to activate oncogene expression in selected tissues and single cells without tissue-specific promoters. Our work presents a novel approach to initiate and study cancer in zebrafish, and the high spatiotemporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells.

Control of Protein Activity and Gene Expression by Cyclofen‐OH Uncaging

“…We decided to use our optogenetics approach to address that issue. Carcinogenesis was investigated in zebrafish through the initiation of the expression of a single oncogene (kRasG121V) in two‐ to three‐day‐old embryos . We showed that transient activation of the oncogene (Figure A–C) did not result in cancer (Figure A–C), whereas permanent activation (Figure D–F) led in some cases to tumors (Figure D–F), albeit with a very low probability (ca.…”

“…To achieve transient genetic activation, we used a fusion between the ERT domain and a transcription factor (Gal4) that can similarly be activated by cyclofen‐OH uncaging . Diffusion of the transcription factor to the nucleus activates genes under control of a UAS promoter.…”

“…F) This approach can be used to achieve permanent activation of a gene of interest. Adapted from Feng et al …”

“…In the context of development, permanent activation of Fgf8 yields an increase in the concentration of the protein and its fluorescent marker (CFP) during somitogenesis with G) observable phenotypic consequences and, in particular, H) a decrease in the somite size. Adapted from Feng et al …”

Cell‐Lineage Tracing in Zebrafish Embryos with an Expanded Genetic Code

“…Examples of spatial control include the use of tissue-specific promotors, [11] and examples of temporal control include using heat-shock-induced Cre recombinase expression [12] or the addition of 4-hydroxy tamoxifen to translocate a Cre–estrogen receptor fusion protein (Cre–ER) to the nucleus. [13] Both spatial and temporal control were achieved by using a photocaged 4-hydroxy tamoxifen analogue [14] with a Cre–ER-expressing fish line. [15] However, Cre–ER fusions have shown activation in the absence of 4-hydroxytamoxifen, and although this is reduced by certain mutants of ER, such as Cre–ER T2 , some background activity is still possible.…”