The Gal4-UAS system

The Gal4-UAS-system originates in yeast and has been established in various vertebrate and non-vertebrate model organisms since it was first described in the 1980s (Brand & Perrimon, 1993; Giniger et al., 1985; Kakidani & Ptashne, 1988; Scheer & Campos-Ortega, 1999;

Webster et al., 1988). Its mechanism is based on Gal4, a transcriptional activator, binding to a specific upstream activating sequence (UAS) and thereby inducing the expression of any downstream located gene of interest. To use this feature in order to achieve a tissue-specific gene expression, the generation of two stable transgenic lines is required. The driver line expresses Gal4 under the control of a tissue specific enhancer or promoter and the effector line contains any gene of interest under the control of UAS (Fig. 7A) (Asakawa & Kawakami, 2008). The development of the Tol2 transposon system was therefore an important milestone in the implementation of the Gal4-UAS system to facilitate the production of transgenes (Asakawa & Kawakami, 2008; Kawakami, 2007).

The minimal region of Gal4 needed for DNA binding is the Gal4 DNA binding domain (DBD) consisting of the N-terminal 74 amino acids (Keegan et al., 1986). Based on this, diverse constructs were generated to optimize the handling, functionality and efficiency of the Gal4-UAS system. First, the Gal4 DBD was fused to the strong transcriptional activation domain from the VP16 protein isolated from the Herpes simplex virus, in order to enhance the transcriptional activator potential, which is now even exceeding the original (Köster &

Fraser, 2001; Sadowski et al., 1988).

To enable temporal regulation of transgene expression in addition to a local restriction, inducible Gal4 variants were designed (Akerberg et al., 2014; Gerety et al., 2013; Wang et al., 2012). In one variant, the Gal4-VP16 was fused to the hormone-binding domain of the human estrogen receptor 2 (ERT2), which has an especially high affinity for the estrogen receptor modulator 4-hydroxy-tamoxifen (4-OHT) (Akerberg et al., 2014; Gerety et al., 2013).

After the addition of 4-OHT, ERT2-Gal4-VP16 enters the cell nucleus, binds to UAS and activates target gene expression, whereas in absence of 4-OHT, no UAS binding takes place, which ensures rapid reversibility of this system upon drug washout. The level of expression can furthermore be controlled via the dose of 4-OHT (Akerberg et al., 2014; Gerety et al., 2013) (Fig. 7B).

A second variant, GAVPO, is a light-inducible version of Gal4, consisting of the Gal4 DBD connected to the smallest light-oxygen-voltage (LOV) domain Vivid (vvd) from N. crassa and the p65 transactivation domain (AD) isolated from human cells (Wang et al., 2012). The Vivid domain includes a flavin co-factor that forms a cystein-flavin adduct with Cystein(108) upon blue-light activation. This in turn leads to a conformational change that results in dimerization of GAVPO and its binding to UAS (Fig. 7C). This process is reversible as soon as illumination is switched off (Wang et al., 2012; Zoltowski et al., 2007). Using GAVPO in driver lines circumvents the treatment with 4-OHT, which always must be handled carefully, because of its toxicity and its instability upon light-exposure.

Further modifications were introduced to adapt the Gal4-UAS system to certain model organisms. To be named is the variant KalTA4, which is a Gal4 version optimized for the use in zebrafish. KalTA4 consists of a Kozak sequence and a codon usage optimized for zebrafish, thus significantly enhancing transcriptional efficiency in this species (Distel et al., 2009). In addition, only the minimal, but potent TA4 core region from the VP16 transactivation domain was integrated in this construct. Due to the modification of the transcriptional activator, KalTA4 is still able to activate UAS in the same manner as Gal4, but is less toxic to the organism. Toxicity of Gal4 is a consequence of a squelching effect, meaning that the activation regions of Gal4 interact with the intrinsic transcriptional machinery of the organism, although UAS are absent, resulting in the inhibition of numerous genes (Gill &

Ptashne, 1988). Similar to Gal4, the combination of KalTA4 with ERT2 has already been proven to be effective (Kajita et al., 2014) (Fig. 7B).

The effector lines posses multiple upstream activating sequences as a rising level of activation has been demonstrated with increasing number of sequences, up to a certain threshold of consecutive UAS, until a plateau is reached (Distel et al., 2009). In this work, five repetitive UAS (5xUAS) as well as four non-repetitive UAS (4xnrUAS) were used (Akitake et al., 2011; Goll et al., 2009). The latter exhibit a difference in their sequence of 50 % and are said to be less susceptible to methylation and subsequent transcriptional silencing while the activation of downstream genes is just as good. This was verified in comparison to the frequently used 14xUAS construct, composed of repetitive sequences (Akitake et al., 2011).

Fig. 7 The Gal4-UAS system. A: Schematic representation of the binary Gal4-UAS system as applied in the study of the role of RA in pelvic fin development. The driver lines provide expression of an oestrogen- or light-inducible Gal4 derivate under the control of enhancer sequences specific for pelvic and/or pectoral fins. In the effector lines, upstream activating sequences (UAS) control the activity of genes that encode RA signalling inhibitors (RAI) in combination with fluorescing proteins (FP). All used components are listed below the respective bar. B-C: Crossing of driver and effector lines generates double transgenic fish that posses both parts of the system, which enables a spatial disruption of RA signalling exclusively in pelvic and/or pectoral fins. Additionally, a temporal control is mediated by the selection of the starting point of 4-hydroxy-tamoxifen (4-OHT) treatment, in case of ERT2-Gal4-VP16-GI and KalTA4-ERT2-GI (B), or blue-light irradiation, in terms of GAVPO (C). Figure inspired by Akerberg et al., 2014; Mruk et al., 2020;

Wang et al., 2012.

Driver line

Fin specific enhancer inducible Gal4

x ^RAI

Effector line

Fin specific enhancer ERT2-Gal4-VP16-GI RAI

ERT2 -Gal4 4-OHT

-Gal4 ERT2 4-OHT

; UAS

UAS

FP FP

§  ERT2-Gal4-VP16-GI

§  KalTA4-ERT2-GI

§  GAVPO

§  dnRarα2a

§  Cyp26a1 §  eGFP

§  mRFP

§  5xUAS

§  4xnrUAS

§  Prrx1a

§  Prrx1ax4

§  Prrx1b1

§  Prrx1b1x4

§  Pel2.5kb

Fin specific enhancer GAVPO ; UAS RAI ^FP

A

B

C

ERT2-Gal4-GI-VP16/KalTA4-ERT2-GI (+) 4-OHT

GAVPO (+) Blue-light

GAVPO dimer

VP16 VP16

Gal4 vvd p65

To apply this system for the study of the role of RA in pelvic fin development in zebrafish, the genes Cyp26a1 and zfdnRarα2a, whose proteins are associated with the RA signalling pathway, are selected for creating the effector lines. Via overexpression of each of the two genes, a disruption of the RA signalling is intended. Cyp26a1 metabolizes RA to more polar and less biologically active compounds that are subject to further degradation, which ultimately leads to a pronounced RA deficiency in the organism (Niederreither & Dollé, 2008). The gene zfdnRarα2a encodes a dominant negative version of the zebrafish Rarα2a, which is shortened at the C-terminus of the protein after amino acid 403, whereby the activation domain consisting of Helix 12 is missing (Stafford et al., 2006) (Fig. 2). Thus this receptor variant is still able to heterodimerize with Rxr and to bind RAREs, but is unable to activate associated target genes, resulting in an interruption of RA signal transmission. The effectiveness of the dominant-negative action of corresponding receptor variants has already been demonstrated in human cells, Xenopus larvae and zebrafish (Blumberg, 1997;

Damm et al., 1993; Pratt et al., 1990; Sharpe & Goldstone, 1997; Stafford et al., 2006).

For the creation of the driver lines, the enhancers of the limb and fin specific genes Pitx1 and Prrx1 were chosen. The first chosen enhancer is the Pitx1 enhancer Pel2.5kb from the three-spine stickleback (Chan et al., 2010) (Fig. 5). The 2.5 kb enhancer fragment is conserved in zebrafish and other teleost fish and was already used to drive eGFP expression in the pelvic region of sticklebacks as well as in the pelvic fin bud mesenchyme of zebrafish (Chan et al., 2010; Don, 2013). The identification of the corresponding regulatory elements of Pitx1 in the zebrafish genome is however still pending.

In addition, the regulatory elements of the paired-related homeobox gene 1 (Prx1 / Prrx1) were utilized. Prx1 is expressed, among others, in the mesenchymal tissue of the early limb bud and is therefore serving as a marker of the lateral plate and limb bud mesoderm (Cserjesi et al., 1992; Kuratani et al., 1994; Leussink et al., 1995). It has a central role in coordinating the morphogenesis of the handplate and the zeugopod in both, fore- and hindlimbs. This was concluded from the phenotype of mice carrying mutations in Prx1 and its homologue Prx2, which were showing severe disorders in digit number and placement (Lu et al., 1999). The Prx1 limb enhancer was originally identified in mice (Martin & Olson, 2000). Obviously, it also has influence on limb bone growth, which has been visualized in transgenic mice, whose Prx1 enhancer was exchanged with the corresponding regulatory

forelimbs (Cretekos et al., 2008). Zebrafish possess two orthologs of the Prx1 gene, Prrx1a and Prrx1b, whose expressions are regulated by three non-coding enhancer sequences, Prrx1a, Prrx1b1 and Prrx1b2. All three sequences were reported to drive eGFP expression in transgenic zebrafish reporter lines (Hernández-Vega & Minguillón, 2011). For this study, the two enhancers Prrx1a and Prrx1b1 were chosen to generate the Gal4 driver lines.