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METHODS
published: 25 September 2018
doi: 10.3389/fcell.2018.00111
Frontiers in Cell and Developmental Biology | www.frontiersin.org 1September 2018 | Volume 6 | Article 111
Edited by:
Eirini Trompouki,
Max-Planck-Institut für Immunbiologie
und Epigenetik, Germany
Reviewed by:
Heinz-Georg Belting,
Universität Basel, Switzerland
Teresa Venezia Bowman,
Albert Einstein College of Medicine,
United States
*Correspondence:
Martin Distel
martin.distel@ccri.at
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Cell Growth and Division,
a section of the journal
Frontiers in Cell and Developmental
Biology
Received: 26 June 2018
Accepted: 20 August 2018
Published: 25 September 2018
Citation:
Mayr V, Sturtzel C, Stadler M,
Grissenberger S and Distel M (2018)
Fast Dynamic in vivo Monitoring of Erk
Activity at Single Cell Resolution in
DREKA Zebrafish.
Front. Cell Dev. Biol. 6:111.
doi: 10.3389/fcell.2018.00111
Fast Dynamic in vivo Monitoring of
Erk Activity at Single Cell Resolution
in DREKA Zebrafish
Vanessa Mayr †, Caterina Sturtzel †, Manuela Stadler, Sarah Grissenberger and
Martin Distel*
Innovative Cancer Models, St. Anna Kinderkrebsforschung, Children’s Cancer Research Institute, Vienna, Austria
Precise regulation of signaling pathways in single cells underlies tissue development,
maintenance and repair in multicellular organisms, but our ability to monitor signaling
dynamics in living vertebrates is currently limited. We implemented kinase translocation
reporter (KTR) technology to create DREKA (“dynamic reporter of Erk activity”) zebrafish,
which allow one to observe Erk activity in vivo at single cell level with high temporal
resolution. DREKA zebrafish faithfully reported Erk activity after muscle cell wounding
and revealed the kinetics of small compound uptake. Our results promise that kinase
translocation reporters can be adapted for further applications in developmental biology,
disease modeling, and in vivo pharmacology in zebrafish.
Keywords: Zebrafish (Danio rerio), signaling pathway activation, ERK activity dynamics, in vivo imaging, in vivo
pharmacology, wounding
INTRODUCTION
Signaling pathways underlie cellular behavior during development, repair and disease, but fully
understanding the function of any one pathway requires one to follow its dynamic activity
within the context of single cells in tissues of a whole living organism. An essential pathway
for cell proliferation and differentiation is the evolutionarily conserved mitogen activated
protein kinase (MAPK) pathway, consisting of a three kinase phosphorylation relay cascade
(e.g., RAF/MEK/ERK) (Krens et al., 2006). Dysregulation of the MAPK pathway can lead to
severe developmental abnormalities and diseases (Kim and Choi, 2010; Rauen, 2013; Burotto
et al., 2014). Hyperactivating mutations in the RAS/MAPK pathway underlie a group of
developmental disorders like Costello or Noonan Syndrome, commonly termed RASopathies,
and also occur in many types of cancer, including melanoma (BRAFV600E mutation) and
colon cancer (K-RASG12D/G12V) (Forrester et al., 1987; Davies et al., 2002; Rauen, 2013). The
RAS/RAF/MEK/ERK signaling cascade has therefore become a major drug target in various cancers
and inhibitors for RAF, MEK and ERK are available (Girotti et al., 2015).
The need to understand MAPK signaling activation in normal and disease states has led to
the development of live reporters for visualization of kinase activity. Sensors based on Förster
resonance energy transfer (FRET) provide great insights by visualizing ERK activity in cultured
cells and more recent also in mice and zebrafish, but are difficult to implement and fail to accurately
report the downregulation of activity (Vandame et al., 2013; Regot et al., 2014; Depry et al., 2015;
Hirata et al., 2015; Hiratsuka et al., 2015; Sari et al., 2018). Regot et al. recently introduced an
alternative kinase activity reporter termed kinase translocation reporter (KTR) and demonstrated
its high sensitivity in vitro (Regot et al., 2014). This technology translates a phosphorylation event
into a nucleo-cytoplasmic shuttling event of the synthetic reporter, which can easily be observed
Mayr et al. Live Monitoring of Erk Activity in Zebrafish
by fluorescence microscopy. We reasoned that transferring KTR
technology to zebrafish would result in novel vertebrate kinase
activity reporters with unprecedented temporal resolution and
sensitivity. Due to its transparency and external development,
zebrafish is ideally suited for in vivo fluorescence microscopy
investigations. Current zebrafish pathway reporters like the FGF
reporter Tg(Dusp6:d2EGFP)pt6strain are based on expression of
destabilized fluorescent proteins with a half-life of ∼2 h (Molina
et al., 2007). Still, fast and dynamic changes in signaling activity
cannot be visualized by such reporters. Here, we generated a
KTR-based Erk activity reporter zebrafish strain (DREKA) and
successfully demonstrated its ability to visualize fast Erk signaling
dynamics in a wound response and its possible application for in
vivo pharmacology.
MATERIALS AND METHODS
Maintenance of Fish
Zebrafish (Danio rerio) were maintained at standard
conditions (Kimmel et al., 1995; Westerfield, 2000) according
to the guidelines of the local authorities under licenses
GZ:565304/2014/6 and GZ:534619/2014/4.
Plasmid Construction
The DREKA transgenesis vector #260 (pDESTubi:ERK-KTR-
CloverpATol2) was generated by Gateway R
recombination using
p5‘ubiquitin, pSR1835 containing (pENTR)ERK-KTR-Clover
(addgene #59138), p3‘pA and pDestTol2pA4 vectors.
The T55L/T62L reporter was created using Gibson assembly
(NEBuilder Hifi DNA assembly cloning kit, E5520, New
England BioLabs GmbH, Frankfurt, Germany) of PCR fragments
amplified from #260 using the following primer pairs:
639_ubiERK-TtoL1R: ACGTGGCTTCTTCGATGGcagCGC
TG/
513: CATTTGGACAATTTTGCTGCAGGTAAAATGC and
640_ubiERK-TtoL2-F: tgCCATCGAAGAAGCCACGTctg
CCATC/
516: TCGCCCTTGCTCACCATACTAGTGGA and ligated
into #260 opened by PstI/SpeI digest (New England BioLabs
GmbH, Frankfurt, Germany).
The T55V/T62V reporter was created using Gibson assembly
(NEBuilder Hifi DNA assembly cloning kit, E5520) of PCR
fragments amplified from #260 using the following primer pairs:
641_ubiERK-TtoV1-R: ACGTGGCTTCTTCGATGGcac
CGCTG/
513: CATTTGGACAATTTTGCTGCAGGTAAAATGC and
642_ubiERK-TtoV2-F: tgCCATCGAAGAAGCCACGTgtg
CCATC/
516: TCGCCCTTGCTCACCATACTAGTGGA
and ligated into #260 opened by PstI/SpeI digest.
The T55D/T62D reporter was created using Gibson assembly
(NEBuilder Hifi DNA assembly cloning kit, E5520) of PCR
fragments amplified from #260 using the following primer pairs:
517:ubiERK-TtoD1-R: ACGTGGCTTCTTCGATGGGTC
CGCTG/513: CATTTGGACAATTTTGCTGCAGGTAAAATG
C and
518: ubiERK-TtoD2-F: acCCATCGAAGAAGCCACGTgac
CCATC/
516: TCGCCCTTGCTCACCATACTAGTGGA and ligated
into #260 opened by PstI/SpeI digest.
In vitro Transcription of RNA
RNA for microinjection was transcribed in vitro using the
InvitrogenTM mMessage mMachineTM SP6 transcription kit
according to the manufacturer’s recommendations (Ambion,
AM1340, Waltham, MA, USA).
Microinjection for Transient Assays and
Generation of Transgenic Strains
DNA/RNA injection was performed using injection capillaries
(glass capillaries GB100F-10, with filament, Science Products
GmbH, Hofheim, Germany) pulled with a needle puller
(P-97, Sutter Instruments, Novato, USA) mounted onto a
micromanipulator (M3301R, World Precision Instruments Inc.,
Berlin, Germany) and connected to a microinjector (FemtoJet 4i,
Eppendorf, Hamburg, Germany).
For transient assays, fertilized Sanger AB Tübingen (SAT) eggs
were injected with 25 pg pDESTubi:ERK-KTR-CloverpATol2
and 20 pg H2B-CFP mRNA.
MAPK pathway activation experiments were carried out by
co-injecting KalTA4 mRNA (20 pg), H2B-CFP:UAS:HRASG12V
(20 pg), and pDESTubi:ERK-KTR-CloverpATol2 (25 pg) at the
one cell stage.
To create transgenic zebrafish, 20 pg Tol2 mRNA and 25 pg
pDESTubi:ERK-KTR-CloverpATol2 were injected into fertilized
SAT eggs at the one cell stage.
mClover expressing embryos were raised to adulthood and
screened for germline transmission.
Chemical Inhibition
Transiently ERK-KTR-Clover expressing SAT or DREKA
zebrafish embryos were dechorionated and incubated in the
following compounds from 29 to 48 hpf: PD98059 (30 µM),
vemurafenib (10 µM), PD0325901 (5 µM), trametinib (10 µM),
and ulixertinib (1 µM). Stock solutions of compounds were
in DMSO and control experiments were carried out in 0.1%
DMSO. All compounds were purchased via MedChemTronica,
Stockholm, Sweden with the respective ordering numbers
HY-12028, HY-12057, HY-10254, HY-10999, HY-15816. Images
were recorded at 48 hpf and Erk activity status was analyzed
(n=5 embryos each condition, except for vemurafenib n=4
embryos).
Leptomycin B (Cat No. L2913, Sigma Aldrich, Saint Louis,
USA) treatment was carried out from 26 hpf for 24 h at 92 µM.
Leptomycin B stock solution was in 70% methanol and control
experiments were carried out in 0.7% methanol/E3.
Imaging
Zebrafish embryos were prepared for imaging as described
previously. (Distel and Köster, 2007). In brief, zebrafish embryos
were dechorionated, anesthetized using 1x tricaine in E3 medium
(0.16 g/l tricaine (Cat No. E1052110G, Sigma-Aldrich Chemie
GmbH, Steinheim, Germany), adjusted to pH 7 with 1M
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Mayr et al. Live Monitoring of Erk Activity in Zebrafish
Tris pH 9.5, in E3), and embedded in 1.2% ultra-low gelling
agarose (Cat. No. A2576-25G, Sigma-Aldrich Chemie GmbH,
Steinheim, Germany) in a glass bottom imaging dish (D35-14-
1.5-NJ, Cellvis, Mountain View, CA, USA). Images and time-
lapse movies were recorded on a Leica SP8 X WLL confocal
microscope system.
Image and Movie Rendering
Images were rendered using Photoshop CS6 (Adobe), Leica LAS
X software, Quicktime Pro and Fiji.
Needle Induced Wounding
Zebrafish embryos were anesthetized in 1x tricaine/E3,
embedded in 1.2% ultra-low gelling agarose in an imaging
dish and manually wounded by introducing a small puncture
using an injection needle.
Laser Induced Wounding
Zebrafish embryos were embedded for imaging as described
above. A laser-inflicted wound was introduced using the Leica
SP8 X FRAP module and laser lines 405, 458, 476, and 488 nm
simultaneously at ∼75% laser power. A region of interest was
selected manually and was illuminated for 80 to 90 s. After ∼40 s
a wound started to appear.
Compound Kinetics Experiments
To investigate compound uptake kinetics, zebrafish embryos
(55 hpf) were embedded in 1.2% ultra-low gelling agarose
containing 10 µM trametinib. Embedded embryos were covered
with 10 µM trametinib in 1x tricaine /E3/PTU (Cat. No.
P762925G, Sigma- Aldrich GmbH, Steinheim, Germany) and
imaged continuously on a Leica SP8 X WLL system.
Quantification of ERK Signaling Activity
To quantify Erk signaling activity, Clover intensity was measured
in the nucleus and in the cytoplasm of cells by selecting
the respective region using the intensity v time monitor tool
of the time series analyzer plugin for Fiji (J. Balaji, UCLA).
The cytoplasmic to nuclear intensity ratio was calculated using
Microsoft Excel.
Immunofluorescence
Wounded and control zebrafish embryos (48 hpf) were fixed
in 4% paraformaldehyde/PBS (Cat. No 15710-S, Electron
Microscopy S ciences, Hatfield, PA, USA) for 4 h at room-
temperature. Afterwards zebrafish embryos were transferred into
100% methanol and were incubated at −20◦C overnight. Then
embryos were transferred into acetone (7 min at −20◦C) and
incubated in H2O (room-temperature for 1 h). After washing
with PBST (PBS with 0.1%Tween20) embryos were incubated
in 150 mM TrisHCl (pH 9) (70◦C for 15 min). After washing
in PBST embryos were blocked in 10% normal goat serum
(NGS) in PBST. Samples were incubated in p-ERK antibody
(Cell Signaling technology, Cat. No. #4370) at 1:400 in 10% NGS
in PBST overnight at 4◦C. The primary antibody was removed
and embryos were washed in PBST (6 ×15 min). A secondary
antibody, Alexa Fluor 568 goat anti-rabbit antibody (Cat. No. A-
21069, Invitrogen) was used at 1:2,000 together with DAPI in
10% NGS in PBST for overnight incubation at 4◦C. After this
incubation step embryos were washed in PBST (6 x 15 min) and
imaged.
RESULTS
In order to generate a highly dynamic reporter for Erk activity,
we aimed to adapt KTR technology for its application in
zebrafish. The ERK reporter ERK-KTR-Clover consists of an
ERK docking site fused to nuclear localization (NLS) and nuclear
export signals (NES) and the fluorescent protein mClover.
Upon phosphorylation of phospho-acceptor sites within the NLS
the export signal overrides the import signal and the green
fluorescent reporter localizes from the nucleus to the cytoplasm,
hereby visualizing ERK activity (see Figure 1B;Regot et al.,
2014).
We first investigated in silico, if zebrafish Erk1/2 would
be able to bind to the reporter construct, which carries the
minimal ERK specific docking site (F-site) of mouse Elk1 (FQFP),
which is also present in Danio rerio Elk1 (Jacobs et al., 1999).
ERK1/2 is generally well conserved between human, mouse and
zebrafish and the F-site recruitment site (FRS), which binds to
the F-site, is present in zebrafish, suggesting that the synthetic
KTR construct will likely be able to report ERK activity in
zebrafish (Figure S1) (Roskoski, 2012; Busca et al., 2016). To
test this, we next placed ERK-KTR-Clover under control of
the zebrafish ubiquitin promoter and transiently expressed the
reporter in zebrafish embryos (Figure 1A;Mosimann et al.,
2011; Regot et al., 2014). In cells of these embryos, ERK-KTR-
Clover was found either in the cytoplasm sparing the nucleus, in
the cytoplasm and nucleus or predominantly in the nucleus as
confirmed by co-expression with the nuclear marker histone2B–
CFP (H2B-CFP), indicating various degrees of Erk activity (Distel
et al., 2010). We also observed cell type specific differences in
reporter localization, e.g., skin epithelial cells showed dynamic
and generally higher reporter intensity in the cytoplasm, whereas
muscle cells showed stronger reporter signal in the nucleus at 48
hpf (n=385 cells, 10 embryos) (Figures 1C,C′). Control reporter
constructs, where threonines within the NLS were replaced by
either leucine (T55L/T62L) or valine (T55V/T62V) were found
in nuclei of all cell types investigated and a phosphomimetic
reporter variant T55D/T62D localized to the cytoplasm (n=
132 muscle cells, 3 embryos), indicating that the localization of
the reporter is indeed regulated by phosphorylation in zebrafish
(Figure S2).
We next tested if the ERK-KTR reporter responds to
inactivation and activation of the MAPK pathway in skin
epithelial and muscle cells. Applying a MEK inhibitor (30 µM
PD98059) for 17 h resulted in nuclear localization of the reporter
in the majority of cells at 48 hpf (n=300 cells, 5 embryos)
(Figures 1D,D′). In contrast, stimulation of Erk signaling by co-
expression of a constitutively active RAS (H-RASG12V) shifted
reporter localization to the cytoplasm of muscle cells at 48
hpf (n=235 cells, 5 embryos) (Figures 1E,E′). These results
suggested that ERK-KTR-Clover faithfully reports Erk activity in
skin epithelial and muscle cells in living zebrafish embryos.
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Mayr et al. Live Monitoring of Erk Activity in Zebrafish
FIGURE 1 | Characterization of ubi:ERK-KTR-Clover in zebrafish embryos. (A) Schematic illustration of the DREKA transgenesis vector
pDESTubi:ERK-KTR-CloverpATol2. (Tol2, Tol2 recombinase recognition sites; ubiquitin, ubiquitin promoter; Elk1312−356, ERK docking sites from Elk1; bNLS, bipartite
nuclear localization signal; P-sites, phosphorylation sites; NES, nuclear export signal; modified after Regot et al., 2014)(B) Schematic depiction of the ERK-KTR
reporter principle. ERK signaling inactive: reporter is mainly localized in the nucleus; ERK signaling active: reporter is localized in the cytoplasm sparing the nucleus.
(C) pDESTubi:ERK-KTR-CloverpATol2 and H2B-CFP mRNA co-injected wildtype zebrafish embryos express ERK-KTR-Clover in a mosaic manner. mClover
fluorescence is mainly found in the cytoplasm of skin epithelial cells, indicating active Erk signaling (upper panel) and in the nucleus of muscle cells indicating absence
of Erk signaling activity (lower panel). (C′)Quantification of ERK-KTR localization in skin epithelial cells and muscle cells at 48 hpf. Green, mClover fluorescence in the
nucleus; Red, mClover fluorescence distributed throughout the cell; Blue, mClover fluorescence in the cytoplasm sparing the nucleus (n=385 cells, 10 embryos at 48
hpf). (D) Skin epithelial cells of co-injected embryos incubated with MEK1/2 inhibitor PD98059 (30 µM) overnight show mClover fluorescence in the nucleus at 48 hpf
indicating the reporter responds to Mek inhibition. (D′)Quantification of mClover localization shows reporter shuttling to the nucleus after MEK1/2 inhibition (n=300
cells, 5 embryos at 48 hpf). (E) Muscle cells expressing constitutively active HRAS (zebrafish injected with pDESTubi:ERK-KTR-CloverpATol2,
H2B-CFP:UAS:HRASG12V, and KalTA4 mRNA Distel et al., 2009) show active Erk signaling at 48 hpf. (E′)Quantification of mClover localization shows constitutively
active HRAS induced reporter shuttling to the cytoplasm (n=235 cells, 5 embryos at 48 hpf). (F) Mitotic skin epithelial cells of pDESTubi:ERK-KTR-CloverpATol2
injected zebrafish at 24 hpf. Dividing skin epithelial cells (white arrowheads) show dynamic Erk signaling with a sudden change from cytoplasmic to nuclear reporter
localization before cytokinesis [compare time points 11:24 min/13:41 min (left arrowhead) and 13:41 min/21:40 min (right arrowhead)]. After cytokinesis the reporter
remains in the nuclei of both daughter cells (arrowheads at 1:00:28 h). Images taken from a time-lapse movie (Movie 2) are maximum projections of several planes. All
scale bars are 25 µm. All images were recorded on a Leica SP8 X WLL confocal microscope and rendered using Photoshop CS6.
Frontiers in Cell and Developmental Biology | www.frontiersin.org 4September 2018 | Volume 6 | Article 111
Mayr et al. Live Monitoring of Erk Activity in Zebrafish
Finally, we probed the temporal resolution of the reporter
by recording time-lapse movies of ERK-KTR-Clover injected
embryos. This revealed changes in Erk activity within minutes in
skin epithelial cells over time (Movie 1). Interestingly, dividing
cells showed a stereotypical pattern of the reporter shuttling to
the nucleus right before cytokinesis and staying in the nuclei of
both daughter cells afterwards, indicating low Erk activity after
cell division (Figure 1F and Movie 2).
With these transient assays being successful, we next generated
transgenic zebrafish [Tg(ubi:ERK-KTR-Clover)vi1], expressing
ERK-KTR-Clover under control of the rather weak, but
ubiquitous ubiquitin promoter in order to be able to investigate
Erk signaling over longer periods of time in a non-mosaic
manner (Mosimann et al., 2011). We named Tg(ubi:ERK-KTR-
Clover)vi1“DREKA” for “dynamic reporter of Erk activity.”
DREKA zebrafish were viable, showed no obvious morphological
defects and were fertile (now in F4), indicating that the
reporter does not negatively affect endogenous Erk signaling.
F2 DREKA were confirmed to report changes in Erk activity
by applying different inhibitors of the MAPK pathway. As
expected MEK inhibitors trametinib (10 µM) or PD0325901
(5 µM) and ERK inhibitor ulixertinib (1 µM) decreased Erk
signaling activity in skin epithelial cells of DREKA embryos,
but vemurafenib (10 µM) a type I BRAF inhibitor specific for
mutant BRAF (BRAFV600E) did not (Figure S3). These results
confirmed that DREKA report changes in Erk activity upon
external manipulation of the MAPK pathway.
In cells of DREKA zebrafish, changes in reporter localization
lead to changes in mClover fluorescence intensity in the
cytoplasm (C) and the nucleus (N). Calculating the C/N intensity
ratio is thus a way to visualize and quantify relative changes
in Erk activity over time on the single cell level. In muscle
and skin epithelial cells, the C/N ratio ranged approximately
from 0.6 to 1.5 in untreated DREKA zebrafish. Shuttling of the
reporter to the cytoplasm by nuclear export is believed to be
Exportin dependent. Indeed, inhibiting Exportin 1 by leptomycin
B treatment (92 µM for 24 h) led to accumulation of the reporter
in the nucleus reaching C/N intensity ratios of up to 0.23–0.27
in muscle and skin cells (typically 0.6–0.7 in untreated zebrafish)
(Figure S4;Kudo et al., 1999). This shows that the reporter
concentration is not reaching saturation in the nucleus in these
cell types in untreated DREKA fish.
We next aimed to apply DREKA to assess the temporal
dynamics of Erk signaling in a biological process under
experimental conditions with control over an external stimulus
eliciting Erk signaling and turned to a wounding assay.
Upregulation and correct temporal orchestration of ERK
activity is necessary for proper wound healing across species.
In Xenopus embryos, two temporally distinct phases of wound
healing have been observed: an early and fast phase with high Erk
activity and a second slow phase with high PI3K activity (Li et al.,
2013).
We asked if Erk signaling dynamics are similar in zebrafish
embryos after wounding muscle cells. To verify that wounding
activates Erk signaling, we punctured muscle tissue of 48
hpf DREKA embryos using a glass needle. Indeed, muscle
cells close to the wound showed Erk signaling activation
whereas muscle cells further away remained unaltered (n=
6 embryos; Figures 2A,A′). Erk signaling activity in muscle
cells surrounding the wound was independently confirmed by
immunofluorescence for phosphorylated Erk (n=10 embryos;
Figures 2B,B′). We next switched to a laser-induced wounding
assay, which enabled us to follow Erk signaling changes
continuously from the moment the wound was introduced.
We detected an almost immediate response in neighboring
cells after wounding of zebrafish muscle by high power
laser illumination for 80–90 s (Figures 2C,D). The intensity
of mClover in the cytoplasm increased steadily with the
cytoplasmic/nuclear intensity ratio becoming >1 between 2
and 3 min and peaking around 4min after wounding in fast
responding cells (n=7 cells, 3 embryos) (Figures 2C,D and
Movie 3). Directly neighboring cells (Figure 2C, red arrow)
responded first and subsequently Erk signaling activity also
increased in cells further away (Figure 2C, purple arrow) from
the wound. Spreading of Erk activity within one cell type
(epithelial cells) was reported previously in mouse (Hiratsuka
et al., 2015). Here, we observed two cell types, muscle cells
and skin epithelial cells, relaying Erk signaling (Figures 2C,E
and Movie 3,4). After ∼45 min muscle cells more distant to
the wound started to become inactive for Erk signaling again
and cells adjacent to the wound followed around 1 h after
wounding (n=6 embryos; Figures 2E,F). Intriguingly, we
observed wounds, which spontaneously ruptured a second time
after Erk signaling had already decreased, inducing another rapid,
and simultaneous activation of Erk signaling in surrounding
cells (Figure 2F and Movie 4). In such cases, live monitoring
of Erk activity is of tremendous advantage as second rupture
events would have likely been missed with current methods (e.g.,
immunofluorescence or Western Blotting), complicating the
interpretation of the Erk signaling pattern (Movie 4). Wounding
muscle cells in the presence of MEK1/2 inhibitors trametinib
(10 µM, Movie 5) or TAK-733 (10 µM, Movie 6) did not elicit a
translocation of the reporter as observable in 0.1% DMSO control
experiments (Movie 7) (n=4 embryos each), confirming that
changes in reporter localization are MAPK dependent in our
muscle wounding assay.
We next assessed the use of DREKA for in vivo pharmacology.
Zebrafish is a popular model organism for small compound
screening due to the ease of compound administration to the
water. However, the kinetics of compound uptake, although
of great importance are often unknown. To reveal uptake
kinetics DREKA fish were exposed to 10 µM trametinib (MEK
1/2 inhibitor) and Erk activity was continuously monitored in
skin epithelial cells, which showed predominantly active Erk
signaling in their unperturbed state (Figure 3A). Ten minutes
after treatment Erk signaling activity was still strong, however
already after ∼20 min a significant reduction in activity was
detected with full inhibition being visible after around 50–60 min
(n=6 embryos) (see Figure 3 and Movie 8). Only mitotic skin
cells maintained active Erk signaling, a finding that is consistent
with previous research describing the insensitivity of mitotic cells
to Mek inhibition (Hiratsuka et al., 2015). This indicates the
potential of DREKA zebrafish and KTR technology for in vivo
pharmacology at cellular resolution.
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Mayr et al. Live Monitoring of Erk Activity in Zebrafish
FIGURE 2 | Monitoring dynamic Erk activity in DREKA zebrafish (A) Brightfield image and fluorescence image (A′)of a DREKA embryo wounded with a glass needle
at 48 hpf. Images were taken 30 min after wounding. Active Erk signaling was observed in muscle cells around the wound (n=6 embryos). (B) Immunofluorescence
staining for phosphorylated Erk (red) in muscle cells of control and zebrafish embryos wounded with a glass needle 10 min post-wounding (B′). DAPI staining is
shown in blue. Arrows mark some of the cells with phosphorylated Erk being visible in the nucleus surrounding the wound (circle). (C) Erk signaling activation in
muscle and skin epithelial cells after laser induced wounding in 54 hpf DREKA. Four time points (0, 3, 8, and 14 min) taken from time-lapse Movie 3 show the fast
activation of Erk signaling. (D) Quantification of Erk activity in 5 cells as depicted in (C) starting ∼65 s post-wound appearance. Erk activity is shown as
cytoplasmic/nuclear ratio of mClover intensity over time (seconds). Cells close to the wound [#1(red) and #2(green)] show a fast response with higher reporter
concentrations in the cytoplasm compared to the nucleus around 145 s (cell #1) and 191 s (cell #2) post-wound appearance and peaking after ∼233 s (cell #1) and
248 s (cell #2), respectively. Cells further away respond later [#3(purple)] or remain inactive [#5(yellow)]. In addition to muscle cells, Erk signaling activation was also
observed in skin epithelial cells [#4(blue)]. (E) Erk signaling activity in response to laser induced wounding of muscle cells in 72 hpf DREKA. mClover fluorescence
(shown in gray) at 0 and 26 min after the wound (circle) has been introduced (see Movie 4). After wounding mClover starts to localize from the nucleus to the
cytoplasm in cells directly adjacent to the wound, indicating Erk signaling activation (cell #1). Muscle cells further away from the wound become active for Erk signaling
at later time points (cell #2) or remain inactive (cell #3). (F) Quantification of Erk activity in 3 cells as shown in (E) over 2.5 h starting ∼40 s post-wound appearance (see
Movie 4). Erk signaling activity is shown as cytoplasmic to nuclear mClover intensity over time (minutes) for one muscle cell close to the wound (#1 red), one muscle
cell further away (#2 green), and one non-responding muscle cell (3# purple) (colored arrows in E). Note the second rupture event leading to a rapid activation of Erk
signaling. All scale bars are 25 µm. All images were recorded on a Leica SP8 X WLL confocal microscope and rendered using Photoshop CS6.
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Mayr et al. Live Monitoring of Erk Activity in Zebrafish
FIGURE 3 | Monitoring small compound uptake kinetics in DREKA zebrafish (A) 55 hpf DREKA embryos were embedded in agarose containing 10 µM trametinib to
investigate compound uptake kinetics in skin epithelial cells. After 10 min skin epithelial cells still show active Erk signaling. Around 20 min Erk activity is visibly
decreased and by 50–60 min cells show full Erk signaling inhibition. (B) Quantification of Erk activity over time. Cytoplasmic to nuclear mClover intensity over time in
three representative skin epithelial cells in DREKA zebrafish treated with 10µM trametinib at 55 hpf. All scale bars are 25 µm. All images were recorded on a Leica
SP8 X WLL confocal microscope and rendered using Photoshop CS6.
DISCUSSION
Recently, KTRs were introduced to report kinase activity with
higher sensitivity and higher temporal resolution than commonly
used kinase fusion or FRET based reporters in vitro (Regot et al.,
2014). KTRs were also successfully applied in C. elegans (de La
Cova et al., 2017). Here, we demonstrate that KTR technology
can be transferred to a vertebrate model organism for real-time
kinase activity monitoring. We created the zebrafish Erk activity
reporter tg(ubi:ERK-KTR-Clover)vi28 (DREKA), containing an
ERK-KTR, reported to not cross-react with p38 or JNK (Regot
et al., 2014). DREKA zebrafish are viable, show no morphological
abnormalities and are fertile (now in F4), indicating that ERK-
KTR does not have adverse effects on zebrafish development
when expressed under the ubiquitin promoter. We show that
ERK-KTR faithfully reports changes in Erk activity in zebrafish
skin epithelial cells and muscle cells by chemical and genetic
perturbation as well as using phosphomimetic and “phospho-
dead” reporter constructs. The dynamic range of this KTR
appears well suited for measurements in muscle and skin cells
with a relatively large cytoplasm. The cytoplasmic to nuclear
(C/N) reporter intensity ratio, used as readout for Erk activity,
typically ranges from 0.6 to 1.5, allowing one to monitor a
broad range of Erk activity without saturation in skin and
muscle cells. Whether this holds true for all cell types in the
developing zebrafish needs further investigation as observed
minimal and maximal C/N intensities vary between different
cell types. In fact, in contrast to C. elegans, in zebrafish neural
cells within the CNS, the reporter localized predominantly
to the cytoplasm at all investigated time points. However,
(T55L/T62L) and (T55V/T62V) reporter variants were present
in nuclei of neural cells within the CNS (Figure S2). This
suggests that either due to its high sensitivity ERK-KTR is
already reporting very weak Erk activity in neural cells or that
the C/N baseline ratio of reporter localization is shifted in
this cell type. Multiple factors including cellular concentrations
of nuclear import/export machinery proteins (e.g., importins,
exportins, and Ran proteins) as well as phosphatases and cell
morphology (cytoplasmic to nuclear volume ratio) can influence
the localization and the relative intensity of the reporter in
the respective compartment and thus the C/N ratio. Therefore,
optimizing the NLS for reporter usage in neural cells in zebrafish,
similar to modification attempts for C. elegans could improve
the ERK activity reporter for this cell type (de La Cova et al.,
2017). In addition, second generation constructs including
a nuclear marker being co-expressed with the KTR can be
used to solely measure changes in nuclear reporter intensity
(de La Cova et al., 2017).
Frontiers in Cell and Developmental Biology | www.frontiersin.org 7September 2018 | Volume 6 | Article 111
Mayr et al. Live Monitoring of Erk Activity in Zebrafish
Although no absolute values of Erk activity can currently be
stated, the generated transgenic Erk reporter strain (DREKA)
offers unprecedented temporal resolution for monitoring
changes in Erk activity in specific zebrafish cell types. State-of-
the-art reporter strains are based on expression of destabilized
fluorescent proteins. Thus, they suffer from delays reporting
the onset and offset of signaling activity and are not capable
of reporting fast dynamic signaling processes. In contrast, a
readout based on nucleo-cytoplasmic shuttling of the fluorescent
KTR reporter is not subject to limitations imposed by protein
expression and stability rates.
In our wounding assay we observed an immediate increase
(within seconds) of the cytoplasmic to nuclear reporter
distribution, indicating Erk signaling activation with a peak
after ∼4 min. Previously, it had been reported that the Ras/Erk
signaling module acts as a high-bandwidth and low-pass filter
with the need for an external stimulus to persist for at least
4 min to activate the Ras/Erk module in vitro (Toettcher et al.,
2013). If the faster response in our assay is due to actual
differences between the in vitro and in vivo situation or if
activation dynamics are cell type or stimuli specific, needs further
investigations. Sensitivity differences of the used ERK reporter
might also play a role. Consistent with possibly faster responses,
Erk activity was found to be active 2 min after wounding in a
Xenopus embryo wound assay based on Western blot analysis of
Erk phosphorylation (Li et al., 2013). In Xenopus, Erk remained
active between 30 and 60 min matching our observation in
DREKA fish. This suggests that Erk activity dynamics might be
similar in response to wounding across different cell types and
developmental time points in Xenopus and zebrafish.
As pilot experiments indicated the feasibility of differential
Erk activity analysis in selected cells during zebrafish
development, we envision that DREKA fish will be useful
for developmental biologists to decipher when and where
Erk activity is needed for proper tissue formation. Here, we
have also generated an UAS:ERK-KTR variant to be readily
combined with available Gal4 zebrafish strains for tissue specific
analysis of Erk activity. Moreover, KTR-enabled monitoring of
Erk signaling activity in various mutant zebrafish and disease
models, including cancer models, will reveal potentially altered
signaling dynamics. Here, the development of software tools for
automated analysis will soon be required due to the large datasets
created by such experiments.
DREKA zebrafish and KTR technology also promise to be
useful for pharmacological applications. For example effects of
single or combination of compounds targeting Erk signaling can
be investigated as we have shown by applying MEK, ERK, or
RAF inhibitors (see Figure 3 and Figure S3). Off target effects
of compounds, which are not primarily directed at the MAPK
pathway will also be revealed. Although zebrafish is widely
used in drug screening, the understanding of pharmacokinetics
in zebrafish is currently lacking behind. A recent study
applied liquid chromatography-mass spectrometry (LC-MS)
based methods to determine paracetamol concentrations in
zebrafish larvae (Kantae et al., 2016). Complementing such rather
laborious approaches, DREKA offer a direct means to determine
the time small compounds added to the water need to accumulate
inside a cell at a concentration able to inhibit Erk activity
as we demonstrated for trametinib (Figure 3). The ability to
measure an effect on single cells within an intact organism in
real time will also be beneficial to understand how drugs work
in vivo and why they might fail. In comparison to conventional
pharmacology approaches at tissue or organ level, single cell
in vivo pharmacology is likely to enhance drug development
(Vinegoni et al., 2015).
Finally, kinase translocation technology can be used to create
reporters for various kinases, including JNK, p38, PKA, or
AKT (Regot et al., 2014; Maryu et al., 2016). Multiplexing
possibilities arise, which will allow one to dissect the interplay
of various signaling pathways in a cell type specific way in vivo.
This capability will likely have impact on our understanding of
vertebrate development and disease.
ETHICS STATEMENT
All procedures involving animals were carried out according
to EU guidelines and Viennese legislation (licenses:
GZ:565304/2014/6 and GZ:534619/2014/4).
AUTHOR CONTRIBUTIONS
VM and CS performed experiments, analyzed data, and wrote the
manuscript. MS performed experiments. SG generated reporter
constructs. MD designed and performed experiments, analyzed
data, and wrote the manuscript.
ACKNOWLEDGMENTS
We would like to thank Niko Popitsch for graphical assistance,
Susana Pascoal for excellent fish care and Stefan Kubicek for
providing us with MAPK pathway inhibitors. We are extremely
thankful to Markus Covert and Sergi Regot for providing
us with KTR constructs. We thank Christian Mosimann for
providing us with the p5‘ubiquitin, Kristen Kwan for p3‘pA and
pDestTol2pA4 Gateway R
cloning vectors and Marina Mione and
Cristina Santoriello for the H2B-CFP:UAS:HRASG12V vector.
We would like to thank Heinrich Kovar, Stefanie Kirchberger,
and Jennifer Hocking for helpful suggestions and proofreading
of the manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fcell.2018.
00111/full#supplementary-material
Figure S1 | In silico cross-species analysis of Erk binding to ERK-KTR. (A)
Alignment of zebrafish (Danio rerio) and mouse (Mus musculus) Elk1. The
conserved FQFP motif of the F-site (ERK docking site) is shown in red. (B)
Alignment of human, mouse and zebrafish Erk2. Amino acids M199, L200, Y233,
L234, L237, and Y263 (human numbering), which form the F-site recruitment site
are shown in red (Roskoski, 2012). Alignments were performed on ensembl.org.
Figure S2 | Localization of ERK-KTR control constructs T55V/T62V and
T55D/T62D. 48 hpf zebrafish embryos expressing either ERK-KTR-mClover or
T55V/T62V and T55D/T62D control reporter constructs. (A) Neural cells within the
Frontiers in Cell and Developmental Biology | www.frontiersin.org 8September 2018 | Volume 6 | Article 111
Mayr et al. Live Monitoring of Erk Activity in Zebrafish
CNS expressing ERK-KTR-mClover. The reporter localizes to the cytoplasm. (B)
The T55V/T62V control reporter is also found in the nucleus of neural cells within
the CNS. (C) Muscle cells expressing ERK-KTR-mClover. (D) The
phosphomimetic T55D/T62D reporter construct localizes predominantly to the
cytoplasm when expressed in muscle cells. All scale bars are 25 µm. Images were
recorded on a Leica Sp8 X WLL confocal system and rendered with Adobe
Photoshop CS6.
Figure S3 | Effects of different inhibitors on Erk activity in DREKA zebrafish.
Compounds were applied to F2 DREKA embryos at 29 hpf and quantification was
performed on skin epithelial cells at 48 hpf. Percentage of cells with active Erk
signaling shown as box plot from left to right: untreated control; ERK1/2 inhibitor:
ulixertinib (1 µM); MEK1/2 inhibitor: trametinib (10 µM), PD0325901 (5 µM); B-RAF
inhibitor: vemurafenib (10 µM); [each dot represents one embryo; box plot was
created using R (r-project.org)].
Figure S4 | Effects of nuclear export inhibition by leptomycin B. 26 hpf DREKA
zebrafish larvae were treated with the nuclear export inhibiting compound
leptomycin B at 92 µM for 24 h. (A,C,E) Treated DREKA, (B,D,F) untreated
control DREKA. Muscle cells (A,B), neural cells in the hindbrain (C,D), and cranial
ganglion cells (E,F) all show increased reporter signal in the nucleus in treated
embryos compared to untreated siblings. Scale bars in (A,B,D) are 50 µm, in
(C,E,F) 25 µm. Images were recorded on a Leica Sp8 X WLL confocal system
using a 25x objective. Images were rendered using Adobe Photoshop CS6.
Movie 1 | DREKA reveal dynamic Erk signaling in skin epithelial cells during
development. Time-lapse movie of transiently ERK-KTR-Clover (green) expressing
zebrafish embryos from ∼48 to 54 hpf. Dynamic Erk activity can be observed as
nuclear cytoplasmic shuttling of mClover in skin epithelial cells (see arrow). Z-stack
images were recorded approximately every 3.5 min on a Leica SP8 X WLL
confocal microscope using a 40x objective. Z-stacks are shown as maximum
projections.
Movie 2 | Erk activity in dividing cells. Time-lapse movie of transiently
ERK-KTR-Clover (green) expressing zebrafish embryos from ∼24 to 32 hpf.
Dividing cells (arrows) show a stereotypical pattern of Erk signaling activity. Z-stack
images were recorded approximately every 1 min on a Leica SP8 X WLL confocal
microscope using a 40x objective. Z-stacks are shown as maximum projections.
Movie 3 | Fast Erk signaling response in muscle and skin epithelial cells
surrounding a wound. 54 hpf DREKA embryos were wounded by laser
illumination. Erk signaling becomes active in muscle (white arrows) and skin
epithelial cells (yellow arrow) surrounding the wound (circle). Single plane images
were recorded starting ∼65 s after wound appearance every 2.58 s on a Leica
SP8 X WLL confocal microscope using a 40x objective.
Movie 4 | Erk signaling in cells surrounding a wound. 72 hpf DREKA embryos
were wounded by laser illumination (white circle). mClover fluorescence is shown
in gray scale. Erk activity was monitored for 2.5 h after wounding. Spreading of Erk
activity from the wound to muscle cells further away is visible. Note that both,
muscle cells (red and green arrows) and skin cells (yellow arrow) react to
wounding. After ∼30–40 min Erk signaling activity decreases. Around 1 h 54 min
the wound breaks open again activating Erk signaling in many cells
simultaneously. Images were recorded starting ∼40 s after wound appearance
every 2.58 s on a Leica SP8 X WLL confocal microscope using a 40x
objective.
Movie 5 | Wounding response in presence of 10 µM trametinib. 26 hpf DREKA
embryos were treated with 10 µM MEK1/2 inhibitor trametinib for 3 h and then
embedded into ultra-low gelling agarose containing 10 µM trametinib. Larvae
were locally wounded by laser illumination and mClover fluorescence in muscle
cells was recorded. Erk activity was monitored in a time-lapse movie for at least
15 min after wounding. No Erk activation can be observed. Images were recorded
on a Leica SP8 X WLL confocal microscope using a 40x objective.
Movie 6 | Wounding response in presence of 10 µM TAK-733. 26 hpf DREKA
embryos were treated with 10 µM TAK-733 for 3 h and then embedded into
ultra-low gelling agarose containing 10 µM TAK-733. Larvae were locally wounded
by laser illumination and mClover fluorescence in muscle cells was recorded. Erk
activity was monitored in a time-lapse movie for at least 15 min after wounding. No
Erk activation can be observed. Images were recorded on a Leica SP8 X WLL
confocal microscope using a 40x objective.
Movie 7 | Wounding response in presence of DMSO. As control to Movies 5,6
26 hpf DREKA embryos were treated with 0.1% DMSO for 3 h and then
embedded into ultra-low gelling agarose containing 0,1% DMSO. Larvae were
locally wounded by laser illumination and mClover fluorescence in muscle cells
was recorded. Erk activity was monitored for 20min after wounding. After 90 s Erk
activity is visible and spreading to neighboring muscle cells. Images were recorded
on a Leica SP8 X WLL confocal microscope using a 40x objective.
Movie 8 | Small compound uptake kinetics revealed by DREKA. 55 hpf DREKA
embryos were embedded in agarose containing 10 µM trametinib to investigate
compound uptake kinetics in skin epithelial cells. Approximately 10 min later a
time-lapse movie of Erk activity was recorded. Initially, skin epithelial cells are still
active. Around 25 min after compound administration Erk activity is visibly
decreased and by 50 min cells show Erk signaling inhibition. Z-stack images were
recorded approximately every 2 min for 1 h on a Leica SP8 X WLL
confocal microscope using a 40x objective. Z-stacks are shown as maximum
projections.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
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