Optimized Gal4 genetics for permanent gene expression mapping in zebrafish

Abstract

Combinatorial genetics for conditional transgene activation allows studying gene function with temporal and tissue specific control like the Gal4-UAS system, which has enabled sophisticated genetic studies in Drosophila. Recently this system was adapted for zebrafish and promising applications have been introduced. Here, we report a systematic optimization of zebrafish Gal4-UAS genetics by establishing an optimized Gal4-activator (KalTA4). We provide quantitative data for KalTA4-mediated transgene activation in dependence of UAS copy numbers to allow for studying dosage effects of transgene expression. Employing a Tol2 transposon-mediated KalTA4 enhancer trap screen biased for central nervous system expression, we present a collection of self-reporting red fluorescent KalTA4 activator strains. These strains reliably transactivate UAS-dependent transgenes and can be rendered homozygous. Furthermore, we have characterized the transactivation kinetics of tissue-specific KalTA4 activation, which led to the development of a self-maintaining effector strain "Kaloop." This strain relates transient KalTA4 expression during embryogenesis via a KalTA4-mediated autoregulatory mechanism to live adult structures. We demonstrate its use by showing that the secondary octaval nucleus in the adult hindbrain is likely derived from egr2b-expressing cells in rhombomere 5 during stages of early embryogenesis. These data demonstrate prolonged and maintained expression by Kalooping, a technique that can be used for permanent spatiotemporal genetic fate mapping and targeted transgene expression in zebrafish.

Fig. 1.

Optimization of the Gal4 system in zebrafish. (A) Schematic representation of construct tub-GVP-Uunc (10) and transgenic F1 zebrafish embryos. (B) Comparison of Gal4VP16 (set as 100%) and GalTA4 activity in luciferase assays (n = 3, Pac2 fibroblasts) using a 5xUAS:luciferase construct. (C) Schematic representation of the TG5xR construct and transgenic F₁ embryo. Inset: mRNA in situ hybridization of GalTA4 expression throughout the notochord. (D) Activation potentials of differently modified Gal4 activators as determined by luciferase assays (n = 3). The activity of GalTA4 was set as 1. (E) Effects of different numbers of Gal4 DNA binding sites using pCSKalTA4GI and UAS-luciferase constructs (ratio 1:1 blue columns or 10:1 black columns) in luciferase assays (n = 3, 1xUAS:luciferase set as 1). (F) Schematic representation of the TK5xC construct and transgenic F₁ embryo. Data are presented as mean ± SEM. Embryos in C and F are counterstained with green Bodipy Ceramide.

Fig. 2.

KalTA4GI enhancer trapping. (A) Schematic representation of the TK1xC enhancer trapping construct. (B) Examples of transgenic Gal4 enhancer trap lines at 50 hpf (C) kalTA4 mRNA expression (asterisk: olfactory bulb, arrow olfactory epithelium) in line olf:KalTA4. (D) Transactivation of lynGFP in Ulyn (10) injected olf:KalTA4 embryo. (E) KalTA4-mediated transactivation of GFP expression in offspring (26 hpf) from crosses of heterozygous olf:KalTA4 and 4xUAS-KGFPGI carriers. (F) Different expression patterns in F₁ embryos (50 hpf, lateral view) derived from the same P₀ founder fish. (G) Offspring of a rh3/5:KalTA4 homozygous carrier crossed to a wild-type fish. Note that all embryos display the characteristic mCherry fluorescence in rhombomeres 3 and 5. Embryos in B and F are counterstained with green Bodipy Ceramide.

Fig. 3.

Maintenance of rhombomeres 3/5 labeling in Kaloop fish. (A–E) Expression of GFP in rhombomeres 3/5 in offspring from a cross between rh3/5:KalTA4 and 4xUAS-KGFPGI or (F-J) rh3/5:KalTA4 and 4xKaloop carriers (schematic representation of effector constructs shown). (A–C, F–H) Expression of mCherry and transactivated GFP at 26 hpf. (D) At 11 hpf, GFP fluorescence (red ovals) is diminished in 4xUAS-KGFP carriers and (E) lost in the adult brain, while in 4xKaloop carriers (I, J) it is maintained in 2 clusters in the hindbrain until adulthood (white arrows). (E, J) Sagittal sections at 100 μm, abbr.: see Fig. 4.

Fig. 4.

Rh3/5-Kalooping identifies the secondary octaval nucleus (SON) as rhombomere 5-derived. Immunhistochemistry for GFP (green) and Cholinacetyltransferase (ChAT; red) on brain vibratome sections of adult zebrafish derived from crosses of (A) rh3/5:KalTA4 × 4xKGFP and (B–F) rh3/5:KalTA4 × 4xKaloop. All nuclei were counterstained with DAPI (blue). (A and B) Sagittal sections of the hindbrain. (C) Transverse section through the caudal GFP-expressing hindbrain region (r5) in B. (midline marked by dashed line). (D) Sagittal section (rostral is left) showing GFP-positive dendrites in the crista cerebellaris (white arrow). (E) Axons of GFP-positive neurons project through the midbrain into the (F) torus semicircularis. Note typical periventricular cholinergic cells in optic tectum. Abbr.: cb: corpus cerebelli, cc: crista cerebellaris, llf: lateral longitudinal fascicle, nVI: rostral and caudal abducens nuclei, OEN: octavolateralis efferent neurons, r: rhombomere, SGVN: secondary gustatory/viscerosensory nucleus, ts: torus semicircularis, tec: optic tectum, va: valvula cerebelli.