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Method Article
RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.
RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing.
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral Förster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.
RASopathies are genetic syndromes that impair normal development and affect various organs and tissues. These conditions are often caused by germline gain-of-function (GoF) mutations in the key genes and players involved in RAS/MAK signaling, resulting in a hyperactivation (increased phosphorylation) of the extracellular signal-regulated kinase (ERK). ERK regulates some fundamental processes important during development-tissue growth-by translocating to the nucleus1,2. Somatic mutations in genes involved in the RAS-MAPK pathway are the most common events leading to cancer3. Thus, not surprisingly, cancer predisposition is also observed in RASopathies. Noonan syndrome (NS), characterized by developmental delay, short stature, cognitive deficits with variable severity, and cardiomyopathy, is the most common form of RASopathy2. In most cases, the disease is caused by GoF mutations in PTPN11, the first RASopathy gene to be discovered in early 20004 encoding for the protein tyrosine phosphatase SHP2, which acts as a positive regulator of the pathway.
Since then, thanks to the exponential use of exome sequencing approaches in undiagnosed patients, potentially pathogenic variants affecting factors involved in the RAS-MAPK, and likely linked to various forms of RASopathies, continue to be discovered and await functional characterization for efficient patients' stratification2. To achieve this goal, experimental protocols that guarantee fast and informative functional validation at the organismal level are required. Employing classical and standardized mammalian models to test variants with unknown significance would be costly, extremely time-consuming, and require invasive methods in non-transparent large animals. Such a strategy is clearly not compatible with the requirement for fast testing, given the societal burden represented by poor or undiagnosed RASopathy patients, currently without management or treatment. Protocols for quantitative assessment of key phenotypic traits and molecular correlates in entire organisms would also serve to accelerate the possible clinical translation of drugs possibly available to RASopathy patients by repurposing/repositioning.
Zebrafish is an ideal vertebrate model to study diseases that affect early development. As a start, zebrafish share a high level of genetic homology with humans. The high fecundity of adult fish results in a large production of embryos that are small and develop fast. Embryos are transparent at early stages, such that major developmental processes-epiboly, gastrulation, axes, and body plan formation-can be visualized effortlessly using standard microscopy. In addition, the availability of transgenic lines that can be used to track specific cellular behavior and dynamic molecular events in space and time during development, in conjunction with advanced techniques to generate genetic models, is unbeatable. Furthermore, phenotypic readouts can be assessed at multiple levels in zebrafish (from organismal to cellular defects), and dedicated assays are already established for several diseases, including RASopathies5. Moreover, relatively simple bath-immersion methods for drug administration during the early stages, at least for water-soluble compounds, permit high-throughput drug screening in vivo in a 96-well format.
From a molecular point of view, studies using standard approaches, such as immunohistochemistry and immunoblot, robustly demonstrate the correlation between ERK activation and RASopathy-associated developmental defects in fish embryos6,7. The recently developed EKAR-type FRET biosensor in zebrafish (Tg[ef1a:ERK biosensor-nes], Teen) provides a reliable in vivo tool to register ERK activation during embryogenesis in a spatiotemporally resolved manner. Hence, it could be valuable for better assessment of dynamic ERK alterations and pharmacological modulations in RASopathy fish models.
In the Teen sensor, a specific ERK substrate in the reporter is phosphorylated upon ERK activation, triggering a conformational change that brings in close vicinity the fluorescent CFP donor (D) and the fluorescent Ypet (improved YFP) acceptor (A). If the D emission spectrum overlaps considerably with the absorption spectrum of the A, FRET can occur (energy absorption from D to A). This is proportional to the distance between D and A and, therefore, in Teen, to the ERK activation status. Different imaging protocols can be set up using both standard and advanced imaging modules of standard or confocal microscopes in both live and fixed samples. Upon D excitation, the acquisition of multispectral scans along a defined spectrum of emission (λ) from CFP to YFP followed by spectral "unmixing" algorithms is among the most reliable methods to register and quantify FRET data8. It can be applied also to live zebrafish specimens to record in vivo tissue dynamics.
Following previous reports6,9 and our recent application7, here, we detail the step-by-step workflow using Teen fish to assess ERK activation in cells at the margin of the animal pole of NS models at the beginning of gastrulation and correlate it with characteristic body axes defects visible only later in development. We show how to obtain and examine quantitative FRET data from live NS gastrulae before and after treatment with an available MEKi and how to cross-validate the results via standard immunohistochemistry against phosphorylated (active) ERK or perform correlative morphometric analysis of embryo elongation defects.
The workflow could be applied to boost the functional test of emerging variants and disease genes putatively associated with RASopathies and to get insights into the correlation of ERK activation dynamics spatially and temporally during vertebrate development and the morphological defects in embryos. We show that this protocol can also be used to test the efficacy of candidate drugs acting to modulate ERK activation.
All experimental procedures involving animals' housing and breeding were conducted according to ARRIVE guidelines for the use of zebrafish in animal research and authorized by the Italian Ministry of Health (Direzione Generale della Sanità Animale e dei Farmaci veterinari - DGSAF). All the DNA/RNA reactions and imaging sessions may be scaled up or down as desired, depending on the final material required or the number of genes and variants tested.
1. Generation and drug treatment of transient zebrafish RASopathy models
NOTE: To monitor the expression of RASopathy-associated variants, specific constructs harboring the desired coding sequence (cds) of the protein of interest in frame with the cds of small non-fluorescent tags (such as myc or similar) can be used. This way, expression levels of the mutant protein can be assessed by standard western blot against the tag. If antibodies against the specific protein of interest are available, tags can be avoided. Immunofluorescence can also be used to assess protein expression within embryo tissue following standard protocols. This type of a control experiment can be useful to correlate mutant protein expression with induced ERK activation levels. The use of fluorescent tags is not advisable in combination of FRET imaging, given the possible fluorescence emission cross-talks during microscopy.
2. Live multispectral FRET imaging of RASopathy zebrafish models at gastrula stage and data analysis
3. IHC validation of the FRET results and correlative morphometric analysis of gastrulation defects
This protocol shows a simple workflow to quickly generate transient RASopathy models in zebrafish embryos and assess ERK fluctuations in early mutants with a standard live FRET imaging method applied to a recently established ERK zebrafish sensor6,9. As recently shown6,7 within the same experimental workflow, FRET results can be cross-validated by standard IHC against phosphorylated and total ERK on ...
Despite decades of research and myriads of mutations leading to highly heterogeneous forms of RASopathies now mapped, genetic variants with unknown significance continue to emerge from sequencing efforts on undiagnosed patients. Indeed, in many cases, diagnosis based solely upon clinical features can be challenging and functional genomic approaches to validate sequencing results remain crucial. Moreover, despite some available anticancer molecules (i.e., MEK inhibitors) being proposed to treat a subset of RASopathie...
We thank Dr. Jeroen den Hertog (Hubrecht Institute, Utrecht, Netherlands) for kindly providing pCS2+_eGFP-2a-Shp2a from which the shp2 full-length CDS was extracted to generate the plasmid template we used7. We thank Nara Institute of Science and Technology (Takaaki Matsui), National Institute of Genetics (NIG/ROIS) (Koichi Kawakami), for providing the transgenic Teen reporter line. This work was supported by the Italian Ministry of Health - Current Research Funds 2021 and Current Research Funds 2024 and Ricerca Finalizzata Giovani Ricercatori GR-2019-12368907 to AL; Current Research Funds 2019, PNRRMR1-2022-12376811, 5x1000 2019, AIRC (IG-21614 and IG-28768) and LazioInnova (A0375-2020-36719) to MT.
Name | Company | Catalog Number | Comments |
Plasticwares | |||
1.7 L Breeding Tank - Beach style Design | Tecniplast | 1.7L SLOPED | Breeding tank |
Capillaries GC100F-10 | Harvard apparatus | 30-0019 | One-cell stage embryo microinjection |
Cell and Tissue Culture Plates - 12 well | BIOFIL | TCP011012 | Embryo collection and treatment |
Cell and Tissue Culture Plates - 6 well | BIOFIL | TCP011006 | Embryo collection and treatment |
Cell Culture Dish | SPL Life Sciences | 20100 | Embryo collection |
Nunc Glass Dishes 12mm | Thermo Fisher | 150680 | Embryo FRET spectral imaging |
Pipette Pasteur | Corning | 357524 | Embryo transfer |
Protein Lobind Tubes 2ml | Eppendorf | 30108450 | IHC assay |
Reagents and others | |||
Caviar 500-800 µm | Rettenmaier Italia | BE2269 (500-800) | Dry fish food |
Great Salt Lake Blue Artemia Cysts | Sanders | 00004727 | Live fish food |
Instant Ocean salt | Tecniplast | XPSIO25R | Dehydrated sea salt for live food preparation |
Tg(EF1a:ERK Biosensor-nes) (Teen) | Contacts for ordering*: National BioResource Project Zebrafish, Support Unit for Animal Resources Development, RRD, RIKEN Center for Brain Science, Japan. https://shigen.nig.ac.jp/zebra/index_en.html *upon MTA signature. | - | Supplier of ERK Reporter zebrafish line. Fish embryos can be obtained upon MTA signature from National BioResource Project of Japan for Zebrafish (RIKEN, Japan). The zebrafish line is deposited by Nara Institute of Science and Technology (Takaaki Matsui) and the National Institute of Genetics (NIG/ROIS) (Koichi Kawakami, patent for Tol2 system) (Wong et al., 2018, Urasaki et al., 2006, Okamoto and Ishioka, 2010). |
6x loading dye | Cell Signaling | B7024S | Gel Elecrophoresis |
100 bp DNA ladder | NEB | N3231S | Gel Elecrophoresis |
Agarose | Sigma-Aldrich | 1,01,236 | Gel Elecrophoresis |
Agarose, low gelling temperature | Sigma-Aldrich | A9414-10G | Embryo mounting for FRET spectral imaging and IHC assay |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A8022 | IHC assay |
Calcium chloride | Sigma-Aldrich | 223506 | E3 medium component |
Calcium nitrate | Sigma-Aldrich | 237124 | Danieau stock solution component |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | D8418-100ML | IHC assay |
EDTA | Sigma-Aldrich | E9884 | TBE buffer component for gel preparation |
Ethanol 99%+ | Fisher Scientific | 10048291 | In vitro RNA purification |
Formaldeide 16% | Thermo Fisher | 28908 | Embryo fixation |
Formamide | Sigma-Aldrich | F9037 | Gel Elecrophoresis |
Gel Loading Buffer II (Denaturing PAGE) | Thermo Fisher | AM8546G | In vitro RNA transcription |
Glacial Acetic Acid | Sigma-Aldrich | 695092 | TBE buffer component for gel preparation |
Glycerol | Sigma-Aldrich | G6279-1L | IHC assay |
Goat anti-mouse Alexa Fluor 488 | Thermo Fisher | A11001 | IHC assay |
Goat anti-rabbit Alexa Fluor 633 | Thermo Fisher | A21070 | IHC assay |
HEPES | Sigma-Aldrich | H3375 | Danieau stock solution component |
KpnI - HF (Enzyme + rCutSmart Buffer) | NEB | R3142 | Plasmid linearization |
Magnesium sulfate | Sigma-Aldrich | 230391 | E3 medium component/Danieau stock solution component |
Millennium RNA Markers | Thermo Fisher | AM7150 | Gel Elecrophoresis |
Monarch Genomic DNA purification Kit | NEB | T3010L | Plasmid linearization |
Mouse monoclonal p44/42 MAPK | Cell Signaling | 4696S | IHC assay |
mMACHINE SP6 Transcription Kit | Thermo Fisher | AM1340 | In vitro RNA transcription |
Normal Goat serum (NGS) | Sigma-Aldrich | G9023 | IHC assay |
Nuclease-free water Ambion | Thermo Fisher | AM9937 | In vitro RNA transcription |
PD0325901 | Sigma-Aldrich | PZ0162 | MEK inhibitor |
Phenol Red solution | Sigma-Aldrich | P0290 | Microinjection mix component |
Poly A Tailing Kit | Thermo Fisher | AM1350 | In vitro RNA transcription |
Potassium chloride bioxtra | Sigma-Aldrich | P9333 | E3 medium component/Danieau stock solution component/PBS stock solution component |
Potassium dihydrogen phosphate | Sigma-Aldrich | P0662 | PBS stock solution component |
Proteinase K | Sigma-Aldrich | P2308 | IHC assay |
Rabbit polyclonal phospho-p44/42 MAPK | Cell Signaling | 4695S | IHC assay |
SYBR safe DNA gel staining | Thermo Fisher | S33102 | Gel Elecrophoresis |
Sodium Chloride | Sigma-Aldrich | 31434-M | E3 medium component/Danieau stock solution component/PBS stock solution component |
Sodium phosphate dibasic | Sigma-Aldrich | 71643 | PBS stock solution component |
Trizma base | Sigma-Aldrich | T1503 | TBE buffer component for gel preparation |
Triton X-100 | Sigma-Aldrich | T8787 | PBSTr buffer component |
Equipment | |||
Alliance Mini HD9 | Uvitec | - | Imaging system |
Centrifuge 5430 R | Eppendorf | 5428000205 | Microcentrifuge |
Eppendorf ThermoMixer C | Eppendorf | - | Embryo mounting |
FemtoJet 4x | Eppendorf | - | Microinjection system |
Infinite M Plex | Tecan | - | Multimode plate reader |
Leica M205FA | Leica Microsystems | - | Fluorescence stereo microscope |
Leica TCS-SP8X equipped with incubator (OkoLab) | Leica Microsystems | - | Confocal microscope |
Mini-sub Cell GT Horiziontal Electrophoresis System | Bio-Rad | 1704406 | Gel Elecrophoresis |
PC-100 Vertical puller | Narishige | - | Needle puller |
PowerPac Universal Power Supply | Bio-Rad | 1645070 | Gel Elecrophoresis |
Stellaris 5 | Leica Microsystems | - | Confocal microscope |
Vortex MiniStar silverline | VWR | - | Plasmid preparation |
Softwares | |||
Biorender | Biorender | CC-BY 4.0 license | Cartoon elaboration for Figures |
Excel | Microsoft Office Professional Plus 2019 | - | Data analyses |
Fiji software | ImageJ | 15.3t | Imaging rendering and quantitative analyses (FRET signals measurements, ERK fluorescence intensity in IHC assay, embryo axes lenght) |
GraphPad Prism | GraphPad Software LLC | v. 9 | Statistical data analyses |
iControl spectrophotometer software | Tecan | v. 2.0 | RNA quantification |
Illustrator | Adobe | 26.0.3 (64-bit) | Figure assembling |
LASX software | Leica Microsystems | v. 4.5 (Stellaris 5), v. 3.0 (M205FA), v. 3.5 (TCS-SP8X) | Imaging acquisition for spectral FRET experiments and embryo imaging for axes lenght measurements |
Q9 Mini 18.02-SN software | Uvitec | - | Gel image acquisition |
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