Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.
Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.
The overgrowth of a preneoplastic cell progeny within an otherwise normal epithelial sheet strongly depends on an interaction with its microenvironment. The interaction with its host tissue is likely to be the major determinant of whether a preneoplastic cell is able to establish a clonal niche to further develop into a full blown cancer. Despite its importance in development, this initial phase of cancer progression is inaccessible in most commonly used model systems, in vivo.
The zebrafish, Danio rerio, is a well-established model organism for live imaging studies because of its transparency throughout development, the possibility for genetic manipulation, and accessibility for water-soluble drugs. Recent studies using a larval zebrafish model, overexpressing the human oncogene HRASG12V to transform epithelial cells, showed that host cell derived signals, in particular H2O2 lead to the recruitment of innate immune cells. Interestingly, the recruited immune cells, neutrophils and macrophages, were shown to have a trophic role in supporting preneoplastic cell proliferation2,3.
In order to understand the early events of tumor initiation and progression as well as the contribution of an inflammatory response, one needs to be able to image these events from the earliest time point onwards. Therefore it is necessary to control cell transformations in a temporal and tissue specific manner. We utilize the Gal4/UAS system that has been successfully adapted from Drosophila4 to conditionally express the human oncogene HRASG12V under the control of the skin specific promoter keratin 4 (krt4)5. The modified version of the transcriptional activator Gal4, namely KalTA46, enables expression of HRASG12V under the control of the Upstream Activating Sequence (UAS). To conditionally induce the transcriptional activator expression, KalTA4 has been fused to the mutant ligand-binding domain of the human estrogen receptor α (ERT2)7 which binds specifically 4-Hydroxytamoxifen (4-OHT)1. In the absence of 4-OHT the ERT2 is bound in the cytoplasm by heat-shock proteins. Upon 4-OHT binding the heat-shock proteins dissociate, allowing a nuclear translocation of KalTA4-ERT2 and subsequent activation of the UAS controlled gene of interest8 (Figure 1C, b). As this expression system depends on the presence of 4-OHT, the KalTA4 controlled expression of a gene of interest is reversible. In contrast to the Cre-ERT2/Lox system, which leads to an irreversible recombination event, the induction of transcriptional activation by KalTA4-ERT2 is reversible by addition or removal of 4-OHT.
The following protocol allows a conditional transformation of skin cells in zebrafish larvae and a subsequent monitoring of the interaction with innate immune cells by fluorescent protein expression. The basic methodologies employed in this protocol are similar to some commonly used techniques within the zebrafish community9–13.
The generation and combinatory use of transgenic lines together with the microinjection of transgenic constructs enables a transient approach to only transform single cells in a mosaic manner. The oxygen permeability of the embryonic and larval zebrafish skin raises the possibility for time-lapse live imaging studies by high-resolution microscopy from hours to days. Furthermore, its accessibility to water-soluble drugs will allow future mechanistic studies and the monitoring of cell biological events in vivo that could lead to a better understanding of the earliest stages of cancer development.
This 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 other than skin.
The following experimental procedures were conducted in strict accordance with the Animals (Scientific Procedures) Act 1986.
1. Preparation of Plasmid DNA for Microinjection
2. Microinjection of Plasmid DNA for Transient Gene Expression
3. Conditional Skin Cell Transformation by 4-Hydroxytamoxifen (4-OHT)
4. Mounting and Live Imaging of Zebrafish Larvae
Zygotes of a cross between Tg(UAS:eGFP-HRASG12V)io006 and Tg(lysC:dsRed2)nz50Tg were collected 10-15 min post fertilization (Figure 1A) to obtain 1 cell-stage embryos. After injection the embryos were raised until 3 dpf, before induction with 4-OHT and mounting for live imaging (Figure 1B). 2-6 hr post induction the mounted embryos were placed either under an inverted fluorescent microscope (Figure 2A) or an inverted confocal microscope (Figure 2B-C) and imaged for 2-3 hr with 1.5 min intervals. Preneoplastic superficial skin cells can be identified by their more rounded morphology compared to normal keratinocytes, as well as the green fluorescent emission of the membrane localized eGFP-HRASG12V. Areas of imaging were selected for co-localization of preneoplastic skin cells and innate immune cells (Figure 2A arrowhead). Due to the fast migration process of the neutrophils the time-lapse intervals were chosen to be between 1-1.5 min with Z stack step sizes of 0.7-1.7 μm to obtain maximum intensity projections (Figure 2, Video 1). There is a wide range of behaviors that can be observed while live imaging: neutrophils migrate next to and under preneoplastic cells (Figure 2C, Video 1), form direct contacts to cells (Figure 2B and C), remove remnants of preneoplastic cells that undergo apoptosis (Video 1), or actively engulf parts of preneoplastic cells (not shown). To capture the full spectrum of interactions, it is advisable to follow their behaviors by time-lapse live imaging.
Figure 1: Schematic for conditional skin cell transformation in zebrafish larvae. (A) An image of a zygote of Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) zebrafish at 10 min post fertilization (mpf). The fertilized egg is surrounded by the chorion (arrowhead). The site for injection is the blastodisc (asterisk), formed by cytoplasmic streams from the yolk (arrow) that defines the animal pole of the embryo. (B) Mounting of zebrafish larvae. 60 mm Petri dish with an 18-20 mm hole (arrowhead) that is sealed by a 25 mm cover glass. The zebrafish larvae are immobilized and mounted onto the cover glass by 1% LMP agarose. 0.3x Danieau solution (arrow) containing 5 μM 4-OHT is used to cover the LMP agarose. (C) 4-OHT induced transformations of superficial skin cells. (a) Schematic cross sectional profile of larval skin. The embryonic and larval skin is composed of two epithelial cell layers, a superficial cell layer and a basal cell layer of keratinocytes that are connected to the underlying basement membrane (BM). (b) Transient expression system to induce a preneoplastic cell clone amongst superficial skin cells. KalTA4-ERT2 is exclusively expressed in the outermost skin layer driven by the krt4 promoter (yellow). Mosaic expression of KalTA4-ERT2 activates UAS controlled (blue) eGFP-HRASG12V expression in superficial cells, leading to a clone of preneoplastic cells (green). (D) Schematic of the transient expression system. (a) Tol2 cassette of the pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP expression vector used transiently. (b) Conditional induction of the transcriptional activator expression. KalTA4 is fused to the mutant ligand-binding domain of the human estrogen receptor α (ERT2) which binds specifically 4-Hydroxytamoxifen (4-OHT). In the absence of 4-OHT the ERT2 is bound in the cytoplasm by heat-shock proteins (Hsp90). Upon 4-OHT binding Hsp90 dissociates, allowing a nuclear translocation of KalTA4-ERT2 and subsequent activation of the UAS controlled eGFP-HRASG12V expression. Scale bar A = 500 μm. Please click here to view a larger version of the figure.
Figure 2: Live imaging of preneoplastic and innate immune cell interaction. (A-C) Images of a 4 dpf Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) embryo, which has been injected with pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP and transposase mRNA at one cell stage. Images were taken after 6 hours of 4-OHT induction. (A) Lateral view of the tailfin region at 6 hours post treatment, showing patches of eGFP-HRASG12V expressing skin cells (arrow), and lysC:dsRed2+ neutrophils (arrowhead). (B-C) Representative images taken from a confocal time-lapse movie, showing neutrophil (red) interactions with eGFP-HRASG12V expressing skin cells (green). (B) A preneoplastic cell forms a short filopodial extension with the contacting neutrophil (arrowhead). (C) Neutrophils (red) is in close contact to a clone of preneoplastic skin cells (green). Scale bar in A = 100 μm; B/C = 50 μm. Please click here to view a larger version of the figure.
Video 1: Time-lapse live imaging of preneoplastic and innate immune cell interaction. Time-lapse of a 4 dpf Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) embryo, injected with pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP and transposase mRNA at one cell stage. Confocal time-lapse movie from 6-9 hours after 4-OHT induction showing lysC:dsRed2+ neutrophil (red) interactions with eGFP-HRASG12V expressing skin cells (green). Neutrophils migrate next to and under preneoplastic cells and remove remnants of a preneoplastic cell that undergoes apoptosis. Time-lapse intervals of 1.5 min over 3 hr with Z stack step sizes of 1.3 μm.
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 techniques9–13, and our protocol can be easily adapted and modified.
The krt4 promoter5 used here drives KalTA4-ERT2 expression specifically in the outermost skin layer (Figure 1C, b). However, the modular MultiSite Gateway cloning strategy, used to generate the krt4:KalTA4-ERT2construct (Figure 1D, a), provides the possibility to clone any promoter of interest in front of KalTA4-ERT2 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-ERT2 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-ERT2/UAS expression system.
We used the pDestTol2CG vector from the zebrafish Tol2kit20–23 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 zebrafish9,10. 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-HRASG12V 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-HRASG12V. 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-HRASG12V 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 counterpart25,26. 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 dependent8,27, 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-HRASG12V)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-ERT2;cmlc2:eGFP construct into Tg(UAS:eGFP-HRASG12V)io006 embryos might not be as efficient as injecting a UAS construct into Tg(krt4:KalTA4-ERT2;cmlc2:GFP) embryos. The use of the stable transgenic line Tg(krt4:KalTA4-ERT2) crossed with Tg(UAS:eGFP-HRASG12V)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-ERT2/Lox system, which facilitates an irreversible genomic recombination event28, KalTA4-ERT2/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-ERT2/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.
The authors have nothing to disclose.
This work was funded by a Wellcome Trust Sir Henry Dale Fellowship to Y. F. 100104/Z/12/Z.
We would like to thank Dr. Carl Tucker and the team of the fish facility at the Queen’s Medical Research Institute, University of Edinburgh. We like to thank Michael Millar of the immunohistology and imaging facility of the MRC Centre for Reproductive Medicine, University of Edinburgh for support.
We thank Prof. Dr. Matthias Hammerschmidt and Dr. Heiko Loehr of the Institute of Developmental Biology, University of Cologne for providing the p5E-krt4 entry vector. We also would like to thank Dr. Dirk Sieger of the Centre of Neuroregeneration, Univerity of Edinburgh for providing the pDestTol2CG destination vector.
Name of Material/Equipment | Company | Catalog Number | Comments/Description |
(Z)-4-Hydroxytamoxifen | Sigma Aldrich | H7904 | dilute in ethanol and store in the dark at -20°C |
20x Objective | Zeiss | 20x/0,5 Ph2 ∞/0,17 | |
40x Objective | Zeiss | 40x/1,2W Korr ∞/0,14-0,18 | |
63x Objective | Zeiss | 63x/1.4 Oil Ph3 ∞/0,17 | |
BamHI | New England Biolabs | R0136S | |
Confocal Microscope | Zeiss | LSM 510 META | |
Cover glass | VWR | 631-0171 | Diameter 25mm |
Dimethylpolysiloxane | Sigma Aldrich | DMPSV | |
Dow Corning high-vacuum silicone grease | Sigma Aldrich | MKBL4135V | |
Flaming/Brown P-97 Micropipette Puller | Sutter Instruments Co. | P-97 | Program: heat 530, pull 200, velocity 80 and time 150 |
Fluorescent stereoscope | Leica | M205 FA | |
Kwik-Fill Brosilicate Glass Capillaries | World Precision Instruments | 1B100F-4 | |
Microinjection mold TU-1 | Adaptive Science Tools | TU-1 | |
Microloader tip | Eppendorf | 930001007 | |
Microscope | Zeiss | Axiovert 200 | |
mMESSAGE mMACHINE Kit | Ambion | AM1348 | |
Nuclease-free water | Invitrogen | AM9937 | |
Pneumatic pico pump | World Precision Instruments | PV820 | |
Pneumatic pico pump | Warner Instruments | PLI-90A | |
pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP | Dr. Thomas Ramezani/Yi Feng Lab/The University of Edinburgh | ||
QIAprep Spin Miniprep Kit | Quiagen | 27106 | |
Rhodamine B isothiocyanate-Dextran | Sigma Aldrich | R8881 | 10mg/μl in water |
Stereoscope | Leica | M80 | |
Tg(lysC:dsRed2)nz50Tg | BMC developmental biology 7, 42, doi:10.1186/1471-213X-7-42 (2007) | ||
Tg(UAS:eGFP-HRASG12V)io006 | PloS one 5 (12), e15170, doi:10.1371/journal.pone.0015170 (2010) | ||
Tricaine/MS-222 | Sigma Aldrich | A5040 | |
UltraPure Agarose | Invitrogen | 16500-100 | |
UltraPure Low Melting Point Agarose | Invitrogen | 16520-050 | |
UltraPure Phenol:Chloroform:Isoamyl Alcohol | Invitrogen | 15593-031 | 25:24:1, v/v |