GAVPO

In the next project, the light-inducible Gal4 derivate GAVPO was investigated (Fig. 33A).

Again, an universal driver plasmid was cloned first, which contained GAVPO under the control of the ubiquitously expressed ubiquitin promoter (ubi) (Mosimann et al., 2011). As backbone, one of the 'zero background' Tol2 plasmids designed by David Richter (University of Bayreuth) was used, the one possessing the α-crystallin:Citrine marker gene (Fig. S15A). In addition to the toxic ccdB gene (Couturier et al., 1998), the 4xnrUAS sequences were removed as well in this case. The two fragments to be inserted - the ubi promoter and GAVPO - were PCR-amplified from the vectors pENTR5'_ubi (p334; Mosimann et al., 2011) and pGAVPO (p427; Wang et al., 2012), respectively, and ligated with the linearized pTol2 backbone by means of Gibson Assembly to obtain pTol2_ubi:GAVPO (Fig. 33B).

In order to test whether GAVPO is working in combination with the 4xnrUAS effector constructs, the driver plasmid pTol2_ubi:GAVPO was co-injected with the effector plasmid pTol2_4xnrUAS:dnRarα2a-IRES-eGFP (Schmidt, 2017) (each 12,5 ng/µl) in zebrafish eggs of the Casper strain at the one-cell stage. At 50 % epiboly, the embryos were illuminated with blue LED light until 24 hpf under the exclusion of further external light exposure (Mayer, 2020). This treatment resulted in eGFP expression in 16 % of the embryos that were kept under blue-light (21/126), while only 1 % of the control embryos that were kept in the dark during the whole time showed a weak fluorescence signal (2/149) (Fig. 33G-I) (Mayer, 2020).

This indicates, that GAVPO is in fact able to bind to the 4xnrUAS and activate downstream gene expression. It also demonstrates the blue-light inducibility and the low background activity of GAVPO, since the control groups that were kept in the dark barely showed any transgene expression. In most eGFP expressing embryos, the fluorescence was found in the yolk (Fig. 33H), however, some also exhibited expression in distinct body cells (Fig. 33I) (Mayer, 2020). Apart from eGFP fluorescence, many embryos showed several malformations like a shortened tail or reduced eyes, but did not represent the RA deficiency phenotype known from the nls mutant (Begemann et al., 2001). This is in conformity with the expectations, since plasmid injections always results only in mosaic expression of the transgene, which means that no holistic effect can be achieved in the entire embryo.

However, the observed side effects indicate that GAVPO seems to be quite toxic to the embryos. This probably could be improved by lowering the injected plasmid concentration

Results

Fig. 33 Schematic representations of GAVPO driver plasmids. A: Gene architecture of the light-inducible, Gal4-derived construct GAVPO, consisting of the Gal4 DNA binding domain (DBD), the smallest light-oxygen-voltage (LOV) domain Vivid (VVD) and the p65 transactivation domain (AD). B: Driver plasmid containing GAVPO under the control of the ubi promoter. C: Driver plasmid containing GAVPO driven by different fin specific enhancers, either present in a single copy (Prrx1a/Prrx1b1/Pel2.5kb) or in four tandem repeats (Prrx1ax4/Prrx1b1x4). The marker genes α-crystallin:Citrine or α-crystallin:mRFP facilitate the identification of transgenic zebrafish. D-I: GAVPO is able to bind to 4xnrUAS and initiate gene expression upon blue-light activation. Zebrafish eggs of the Casper strain were co-injected with pTol2_ubi:GAVPO and pTol2_4xnrUAS:dnRarα2a-IRES-eGFP (12,5 ng/µl each) at the one-cell stage and illuminated with blue LED light from 50 % epiboly onwards. eGFP fluorescence was observed almost exclusively in embryos that were exposed to blue-light, mostly located in the yolk (H), but also in distinct body cells (I) (arrowheads). Embryos are shown in lateral view with anterior to the left. J-M: GAVPO is expressed under the control of fin specific enhancers. Driver plasmids containing GAVPO and either Prrx1a, Prrx1ax4, Prrx1b1, or Prrx1b1x4 enhancer (C) were co-injected with Tol2 mRNA (40 ng/µl each) in zebrafish eggs of the Casper strain at the one-cell stage. WISH against GAVPO mRNA demonstrates its expression in pectoral fin buds at 48 hpf (arrowheads). Embryos are shown in dorsal view with anterior to the left. Scale bars = 500 µm. Pictures D-M by Anna-Maria Mayer; taken and modified from Mayer, 2020.

miniTol3‘

GAVPO

α-crystallin-promoter Citrine

pTol2_ubi driver plasmid

Gal4 DBD VVD p65 AD

A

GAVPO miniTol5‘

miniTol3‘

ubi promoter

GAVPO

α-crystallin-promoter Citrine

pTol2_ubi driver plasmid

Gal4 DBD VVD p65 AD

A

GAVPO

B

miniTol5‘

miniTol3‘

GAVPO

α-crystallin-promoter mRFP

pTol2_GAVPO driver plasmid

fin specific enhancer

β-ac#n basal promoter

C

control (dark) ac#ve GAVPO (light) 24 hpf

eGFP

D E F

G H I

J K L M

48 hpf

Prrx1a:GAVPO Prrx1ax4:GAVPO Prrx1b1:GAVPO Prrx1b1x4:GAVPO

GAVPO

Table 6. Driver constructs for specific expression of GAVPO in zebrafish paired fins. Summary of cloned GAVPO driver constructs using diverse fin specific enhancers. So far, no stable transgenic zebrafish lines have been established yet.

Driver plasmid Stable transgenic zebrafish line

pTol2_ubi:GAVPO_α-crystallin:Citrine - pTol2_Pel2.5kb:GAVPO_α-crystallin:mRFP - pTol2_Prrx1a:GAVPO_α-crystallin:mRFP - pTol2_Prrx1b1:GAVPO_α-crystallin:mRFP - pTol2_Prrx1ax4:GAVPO_α-crystallin:mRFP - pTol2_Prrx1b1x4:GAVPO_α-crystallin:mRFP -

Based on these results, driver plasmids that contain GAVPO under the control of the five available fin specific enhancers - Pel2.5kb, Prrx1a, Prrx1b1, Prrx1ax4 and Prrx1b1x4 - were cloned and tested for activity and tissue specific expression (Table 6; Fig. 33J-M). The cloning process was started on the basis of the pTol2_enhancer:KalTA4-ERT2-GI driver plasmids (Table 5; Fig. 32A), from which the KalTA4-ERT2-GI fragment was excised and replaced with GAVPO. DNA assembly was carried out by means of Gibson Assembly (Mayer, 2020).

Then, the fin specific expression of GAVPO under the control of the respective enhancers was assayed. In parallel, an evaluation of the effectiveness of the four tandem repeats of Prrx1a and Prrx1b1 took place. For this, each of the driver plasmids with the enhancers Prrx1a and Prrx1ax4 as well as Prrx1b1 and Prrx1b1x4 was co-injected with Tol2 mRNA (40 ng/µl each) in the one-cell stage of zebrafish eggs of the Casper strain. The detection of GAVPO transcripts took place via whole-mount in situ hybridisation (WISH) using a GAVPO specific RNA antisense probe (Table 15) (Mayer, 2020). It was found that all four driver constructs mediated pectoral fin specific GAVPO expression at 48 hpf (Fig. 33J-M). The intensity of WISH staining was significantly stronger following injections of Prrx1b1 and Prrx1b1x4 driver plasmids compared to Prrx1a and Prrx1ax4. This was not observed in earlier expression studies in which ERT2-Gal4-VP16 transcripts have been detected (Fig. 30, insets in A'' and E''). Also the expression level of Prrx1a and Prrx1b itself was found to be approximately the same previously (Fig. 13) (Eberlein, 2018a). Therefore the observed variations are most likely attributable to different qualities of the injection process.

Furthermore, no significant differences between the transgene expression mediated by the tandem-copy enhancers compared to their single-copy counterparts were observed (Mayer, 2020).

It is conceivable that this experimental setup is not suitable to detect differences in enhancer activity due to the mosaic distribution of the injected nucleic acids and that stable transgenic lines are needed to carry out more detailed investigations.