애벌레 Zebrafish의 피부에 유도 GAL4 / UAS 식 시스템을 사용하여 선천성 면역과 종양 전 세포 상호 작용의 라이브 영상

During embryogenesis and larval development, the embryonic zebrafish skin is composed of two epithelial layers, the superficial layer called periderm and a layer of basal keratinocytes that are attached to the underlying basement membrane¹⁹ (Figure 1C, a). The protocol presented here describes a straightforward method to conditionally induce preneoplastic-cell transformation in the superficial skin layer of zebrafish larvae, and allows for subsequent interaction analysis with innate immune cells by live imaging. The basic methodologies in our protocol follow well-established zebrafish techniques^9–13, and our protocol can be easily adapted and modified.

The krt4 promoter⁵ used here drives KalTA4-ER^T2 expression specifically in the outermost skin layer (Figure 1C, b). However, the modular MultiSite Gateway cloning strategy, used to generate the krt4:KalTA4-ER^T2construct (Figure 1D, a), provides the possibility to clone any promoter of interest in front of KalTA4-ER^T2 in a single cloning step. An adaptation of the method presented herein is therefore possible for most tissues during embryogenesis and larval stages. The availability of promoter sequences is hereby the limiting factor. Furthermore, almost any gene of interest cloned behind a UAS can be conditionally overexpressed at different developmental stages by use of the spatial and temporally controlled KalTA4-ER^T2 expression. Therefore, our protocol can be adapted to perform conditional gene expression in any tissue of interest in zebrafish larvae, in a mosaic manner. One can also optimize the microscope setting in order to live image deeper tissues such as liver or pancreas.

We have described one application using the protocol here, similarly, one can overexpress other inflammation modulators using this protocol to study the regulation of inflammatory responses, as one can easily live image neutrophil and macrophage behavioral changes following the induction and arrest in the expression of a candidate inflammatory modulator. Furthermore, the adapted protocol would also be beneficial for those who want to trace cell movement and behavior change during different stages of tissue morphogenesis and organ formation and, finally live image the interaction of innate immune cell interactions with other specific cell lineages that can be targeted by the inducible KalTA4-ER^T2/UAS expression system.

We used the pDestTol2CG vector from the zebrafish Tol2kit^20–23 that contains a cmlc2:eGFP-pA cassette (Figure 1D, a) which allows subsequent selection of embryos by green fluorescent positive hearts as a selection marker²⁰ that simplifies the F0 screening process. A stable insertion of the transposon flanked gene of interest into the genome is facilitated by co-injection of the plasmid DNA together with transposase mRNA. The preparation of plasmid DNA and the transposase mRNA for microinjection follows standard protocols. However, a successful integration of the construct into the genome depends on the Tol2-based transposition¹⁴. This highlights the particular attention to be paid to work on ice under sterile conditions to avoid contaminations and degradations by RNases and DNases. Microinjection into one-cell stage embryos is a robust and well-established technique for gain and loss of function studies in zebrafish^9,10. Although a well-trained person can easily inject into the yolk of over 1,000 embryos in 1 hr, the injection into the blastodisc (Figure 1A, asterisk) requires more experience and training. Critical during this procedure is the handling of the needle. The needle has to be very thin to penetrate the cell without damage and therefore has to be broken only on its very tip. It is important to regularly control and measure the drop size during the injection to guarantee a uniform injection into each embryo. Carefully handled, the needle can be used to rotate the embryo. This is often needed to find the blastodisc and the optimal penetration angle.

The concentration of DNA and transposase mRNA used for injection almost certainly influences the expression efficiency. Higher doses can lead to an increased proportion of cells expressing the transgene but with higher concentrations of DNA (>50 ng/μl) and transposase mRNA (>100 ng/μl) also the potential for toxic effects amongst injected embryos does arise. We favor the use of 10 ng/μl DNA and 20 ng/μl transposase mRNA for injection, which corresponds to 50 pg of plasmid DNA and 100 pg of RNA per injected embryo. 100% of embryos injected with these concentrations do develop normally, and between 40-70% show eGFP-HRAS^G12V expression, after 4-OHT induction, depending on the injection efficiency into the single cell. Amongst positive embryos we normally find approximately 30% that have 1-10 clones, 40% that have 10-50 clones and 30% having more than 50 clones. Due to our research aims, we favor the use of embryos with fewer clones expressing eGFP-HRAS^G12V. We select embryos with 10-50 clones for our transient experiments. For the generation of transgenic founder fish, we recommend to only select embryos with both the eGFP positive cardiac marker and a large number of eGFP-HRAS^G12V positive clones in their epidermis.

(Z)-4-Hydroxytamoxifen undergoes a cis-trans (E-Z) interconversion when exposed to light²⁴. It was found that the cis isomer (E) is 100x less anti-estrogenic than its trans counterpart^25,26. Exposure to light therefore has to be avoided. The 4-OHT stock solution dissolved in ethanol can be stored at -20 °C in the dark over a long time period. We recommend the use of a box to protect the Petri dishes from light during target gene induction within the incubator and transport. Although the area of imaging is very small and exposure of 4-OHT to UV light is reduced to a minimum, a constant exchange of induction solution every 2 hours while imaging over longer time periods is recommended to maintain maximum expression levels. 4-OHT effectively permeabilizes through the chorion and the outermost skin layers during zebrafish embryogenesis, therefore it can induce gene expression very rapidly. We can readily observe eGFP emission after 2 hr of induction and it has been reported that first signs of target-gene expression can be observed after 1 hour and plateau after 3 hr of treatment⁸. The differences might be due to the different promoter used in our study. As has been previously reported that 4-OHT treatment is dose dependent^8,27, we chose to use the highest reported concentration of 5 μM to achieve robust and reproducible expression levels in our transient approach. This should guarantee that all cells carrying the transgene will induce target gene expression.

It has to be taken into account that the transient approach presented here could lead to varying expression levels due to the possibility of multiple and random genome insertion events. Furthermore, the use of the transposase mRNA in the Tol2 transgenic background of Tg(UAS:eGFP-HRAS^G12V)io006 can lead to potential transpositions of the UAS transgene that could result in gene silencing or disruption. However, as the method presented here represents a transient approach, the pre-selection of embryos subsequent to injection is mandatory. Many labs have found that UAS sequences tend to be silenced in the transgenic offspring, hence injecting the pTol2-krt4:KalTA4-ER^T2;cmlc2:eGFP construct into Tg(UAS:eGFP-HRAS^G12V)io006 embryos might not be as efficient as injecting a UAS construct into Tg(krt4:KalTA4-ER^T2;cmlc2:GFP) embryos. The use of the stable transgenic line Tg(krt4:KalTA4-ER^T2) crossed with Tg(UAS:eGFP-HRAS^G12V)io006 will allow a precise monitoring of the on/off kinetics of the time-dosage dependent 4-OHT gene activation.

A critical step during the mounting of larvae is the transfer into the LMP agarose and onto the cover glass (Figure 1B). There is only a short time frame for liquid LMP agarose temperature that allows a safe transfer and orientation of the preselected larvae without harming the fish by either heat shock or hardening of the gel. The larvae should be orientated directly onto the cover glass to reduce the working distance for the subsequent microscopic analysis. As this can be a limiting factor during microscopy the choice of appropriate objectives is mandatory. Once mounted, the larva can be maintained from hours to days.

The conditionality of the model system presented herein allows for repeated transformation by 4-OHT activation of the transgene. The expression system depends on the presence of 4-OHT and therefore the KalTA4 controlled expression of a gene of interest is reversible (Figure 1D, b). In contrast to the Cre-ER^T2/Lox system, which facilitates an irreversible genomic recombination event²⁸, KalTA4-ER^T2/UAS can be activated and deactivated upon addition or removal of 4-OHT and this potential for repeated induction is an advantage of the technique over the Cre-ER^T2/Lox system. However, the requirement for constant levels of 4-OHT is a limitation if the experiment has to be prolonged from days to weeks.