Abstract
In this study, we describe the adaptation of the split Gal4 system for zebrafish. The Gal4-UAS system is widely used for expression of genes-of-interest by crossing driver lines expressing the transcription factor Gal4 (under the control of the promoter of interest) with reporter lines where upstream activating sequence (UAS) repeats (recognized by Gal4) drive expression of the genes-of-interest. In the Split Gal4 system, hemi-drivers separately encode the DNA-binding domain (DBD) and the activation domain (AD) of Gal4. When encoded under two different promoters, only those cells in the intersection of the promoters' expression pattern and in which both promoters are active reconstitute a functional Gal4 and activate expression from a UAS-driven transgene. We split the zebrafish-optimized version of Gal4, KalTA4, and generated a hemi-driver encoding the KalTA4 DBD and a hemi-driver encoding KalTA4's AD. We show that split KalTA4 domains can assemble in vivo and transactivate a UAS reporter transgene and that each hemi-driver alone cannot transactivate the reporter. Also, transactivation can happen in several cell types, with similar efficiency to intact KalTA4. Finally, in transient mosaic expression assays, we show that when hemi-drivers are preceded by two distinct promoters, they restrict the expression of an UAS-driven reporter from a broader pattern (sox10) to its constituent smaller neuronal pattern. The Split KalTA4 system should be useful for expression of genes-of-interest in an intersectional manner, allowing for more refined manipulations of cell populations in zebrafish.
Highlights
Ectopic expression of genes-of-interest (GOI) in cells is a fundamental method used to understand the function of genes and the biology of cells
We describe an adaptation of the Split Gal[4] system for use in zebrafish, which we term Split KalTA4, since we based it on the optimized version of Gal[4], KalTA4.30 Compared to the original Gal[4] constructs, KalTA4 shows robust zebrafish expression due to a number of optimal modifications: (1) it includes a strong Kozak sequence and a rabbit b-globin intronic sequence that increase expression levels; (2) codon usage is optimized throughout for translation in zebrafish; (3) the entire VP16 activation domain (AD) is replaced with attenuated repeats of the VP16 core sequence, TA4, which is potent in transgene activation, but less toxic
To generate a conditional expression system in zebrafish where GOI are expressed in the intersection of expression patterns, we adapted the split Gal[4] system developed for Drosophila,[21] where the DNA-binding domain (DBD) and the AD of the transcription factor Gal[4] are encoded in separate hemi-drivers
Summary
Ectopic expression of genes-of-interest (GOI) in cells is a fundamental method used to understand the function of genes and the biology of cells. Existing Drosophila or zebrafish Gal[4] driver lines, whether enhancer-trapped or genetically engineered to drive Gal[4] expression by a known promoter, rarely confine expression to a single cell type.[19,20] This may preclude the analysis of the cell autonomy of the effects of gene expression, for instance, or may not enable visualization of a particular cell type with the necessary specificity, if related or nearby cell types express a reporter protein To overcome this limitation, ternary systems such as the Split Gal[4] system have been developed (Fig. 1B).[21,22] This system takes advantage of the modularity of Gal[4] domains, whereby the DNA-binding domain (DBD) can be encoded separately from the activation domain (AD) in so called hemi-drivers. We design KalTA4-based hemi-drivers and confirm that they work in the developing zebrafish, and we provide an example where the use of split KalTA4 allowed the dissection of a complex expression pattern by restricting reporter expression to a subset of cells in that pattern
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