Name: An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence (Video) | JoVE Journal
Uploaded: 2025-05-02T13:15:00.000+00:00
Duration: 9 min 58 s
Description: Learn live FRET microscopy to assess ERK activity modulation in zebrafish Noonan syndrome models. This protocol enables sensitive detection of pathogenic Ras-MAPK signaling changes during embryogenesis.

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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.

<ol><li>Iroegbu, J. D., Ijomone, O. K., Femi-Akinlosotu, O. M., Ijomone, O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ERK%2FMAPK+signalling+in+the+developing+brain%3A+Perturbations+and+consequences%5BTitle%5D)+AND+%22Neurosci+Biobehav+Rev%22%5BJournal%5D)">ERK/MAPK signalling in the developing brain: Perturbations and consequences.</a> Neurosci Biobehav Rev. 131, 792-805 (2021).</li>
<li>Tartaglia, M., Aoki, Y., Gelb, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+genetics+of+RASopathies%3A+An+update+on+novel+disease+genes+and+new+disorders%5BTitle%5D)+AND+%22Am+J+Med+Genet+C+Semin+Med+Genet%22%5BJournal%5D)">The molecular genetics of RASopathies: An update on novel disease genes and new disorders.</a> Am J Med Genet C Semin Med Genet. 190 (4), 425-439 (2022).</li>
<li>Tate, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((COSMIC%3A+the+catalogue+of+somatic+mutations+in+cancer%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">COSMIC: the catalogue of somatic mutations in cancer.</a> Nucleic Acids Res. 47 (D1), D941-D947 (2019).</li>
<li>Tartaglia, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutations+in+PTPN11%2C+encoding+the+protein+tyrosine+phosphatase+SHP-2%2C+cause+Noonan+syndrome%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.</a> Nat Genet. 29 (4), 465-468 (2001).</li>
<li>Jopling, C., van Geemen, D., den Hertog, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shp2+knockdown+and+Noonan%2FLEOPARD+mutant+Shp2-induced+gastrulation+defects%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.</a> PLoS Genet. 3 (12), e225(2007).</li>
<li>Wong, K. -L., Akiyama, R., Bessho, Y., Matsui, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ERK+activity+dynamics+during+zebrafish+embryonic+development%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">ERK activity dynamics during zebrafish embryonic development.</a> Int J Mol Sci. 20 (1), 109(2019).</li>
<li>Fasano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+the+FRET-based+Teen+sensor+to+monitor+ERK+activation+changes+preceding+morphological+defects+in+a+RASopathy+zebrafish+model+and+phenotypic+rescue+by+MEK+inhibitor%5BTitle%5D)+AND+%22Mol+Med%22%5BJournal%5D)">Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.</a> Mol Med. 30 (1), 47(2024).</li>
<li>Zimmermann, T., Rietdorf, J., Girod, A., Georget, V., Pepperkok, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectral+imaging+and+linear+un-mixing+enables+improved+FRET+efficiency+with+a+novel+GFP2-YFP+FRET+pair%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.</a> FEBS Lett. 531 (2), 245-249 (2002).</li>
<li>Sari, D. W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Time-lapse+observation+of+stepwise+regression+of+Erk+activity+in+zebrafish+presomitic+mesoderm%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Time-lapse observation of stepwise regression of Erk activity in zebrafish presomitic mesoderm.</a> Sci Rep. 8 (1), 4335(2018).</li>
<li>Wang, H., Li, J., Xue, T., Cheng, N., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+a+series+of+pCS2%2B+backbone-based+Gateway+vectors+for+overexpressing+various+tagged+proteins+in+vertebrates%5BTitle%5D)+AND+%22Acta+Biochim+Biophys+Sin+%28Shanghai%29%22%5BJournal%5D)">Construction of a series of pCS2+ backbone-based Gateway vectors for overexpressing various tagged proteins in vertebrates.</a> Acta Biochim Biophys Sin (Shanghai). 48 (12), 1128-1134 (2016).</li>
<li>Abdelrahman, D., Hasan, W., Da'as, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+quality+control+in+zebrafish+model+for+genetic+manipulations%5BTitle%5D)+AND+%22MethodsX%22%5BJournal%5D)">Microinjection quality control in zebrafish model for genetic manipulations.</a> MethodsX. 8, 101418(2021).</li>
<li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), e1113(2009).</li>
<li>Rossant, J., Nutter, L. M. J., Gertsenstein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+the+embryo%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Engineering the embryo.</a> Proc Natl Acad Sci USA. 108 (19), 7659-7660 (2011).</li>
<li>Jindal, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+severity+ranking+of+Ras+pathway+mutations+associated+with+developmental+disorders%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">In vivo severity ranking of Ras pathway mutations associated with developmental disorders.</a> Proc Natl Acad Sci USA. 114 (3), 510-515 (2017).</li>
<li>Leavesley, S. J., Rich, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overcoming+lmitations+of+FRET+measurements%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Overcoming lmitations of FRET measurements.</a> Cytometry A. 89 (4), 325-327 (2016).</li>
<li>Mascha, E., Vetter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Significance%2C+errors%2C+power%2C+and+sample+size%3A+Theblocking+and+tackling+of+statistics%5BTitle%5D)+AND+%22Anesth+Analg%22%5BJournal%5D)">Significance, errors, power, and sample size: Theblocking and tackling of statistics.</a> Anesth Analg. 126 (2), 691-698 (2018).</li>
<li>Faul, F., Erdfelder, E., Lang, A. -G., Buchner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G%2APower+3%3A+a+flexible+statistical+power+analysis+program+for+the+social%2C+behavioral%2C+and+biomedical+sciences%5BTitle%5D)+AND+%22Behav+Res+Methods%22%5BJournal%5D)">G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences.</a> Behav Res Methods. 39 (2), 175-191 (2007).</li>
<li>Cohen, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCohen%2C+J+%22Statistical+power+analysis+for+the+behavioral+sciences%22">Statistical power analysis for the behavioral sciences</a>. , Routledge. New York. (1988).</li>
<li>Anastasaki, C., Rauen, K. A., Patton, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continual+low-level+MEK+inhibition+ameliorates+cardio-facio-cutaneous+phenotypes+in+zebrafish%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Continual low-level MEK inhibition ameliorates cardio-facio-cutaneous phenotypes in zebrafish.</a> Dis Model Mech. 5 (4), 546-552 (2012).</li>
</ol>

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.,&#160;MEK inhibitors) being proposed to treat a subset of RASopathie...

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&#39; 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&#160;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 (&#955;) from CFP to YFP followed by spectral &#34;unmixing&#34; 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&#160;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.

<table><tbody><tr><td>&lt;strong&gt;Plasticwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1.7 L Breeding Tank - Beach style Design</td><td>Tecniplast</td><td>1.7L SLOPED</td><td>Breeding tank</td></tr><tr><td>Capillaries GC100F-10</td><td>Harvard apparatus</td><td>30-0019</td><td>One-cell stage embryo microinjection</td></tr><tr><td>Cell and Tissue Culture Plates - 12 well</td><td>BIOFIL</td><td>TCP011012</td><td>Embryo collection and treatment</td></tr><tr><td>Cell and Tissue Culture Plates - 6 well</td><td>BIOFIL</td><td>TCP011006</td><td>Embryo collection and treatment</td></tr><tr><td>Cell Culture Dish</td><td>SPL Life Sciences</td><td>20100</td><td>Embryo collection&amp;nbsp;</td></tr><tr><td>Nunc Glass Dishes 12mm</td><td>Thermo Fisher</td><td>150680</td><td>Embryo FRET spectral imaging</td></tr><tr><td>Pipette Pasteur</td><td>Corning</td><td>357524</td><td>Embryo transfer</td></tr><tr><td>Protein Lobind Tubes 2ml</td><td>Eppendorf</td><td>30108450</td><td>IHC assay</td></tr><tr><td>&lt;strong&gt;Reagents and others&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Caviar 500-800 &amp;micro;m</td><td>Rettenmaier Italia</td><td>BE2269 (500-800)</td><td>Dry fish food</td></tr><tr><td>Great Salt Lake Blue Artemia Cysts</td><td>Sanders</td><td>00004727</td><td>Live fish food</td></tr><tr><td>Instant Ocean salt</td><td>Tecniplast</td><td>XPSIO25R</td><td>Dehydrated sea salt for live food preparation</td></tr><tr><td>&lt;em&gt;Tg(EF1a:ERK Biosensor-nes) (Teen)&lt;/em&gt;</td><td>Contacts for ordering*:&lt;br/&gt; 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 &lt;em&gt;*upon MTA signature.&amp;nbsp;&lt;/em&gt;</td><td>-</td><td>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).</td></tr><tr><td>6x loading dye</td><td>Cell Signaling</td><td>B7024S</td><td>Gel Elecrophoresis</td></tr><tr><td>100 bp DNA ladder</td><td>NEB</td><td>N3231S</td><td>Gel Elecrophoresis</td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>1,01,236</td><td>Gel Elecrophoresis</td></tr><tr><td>Agarose, low gelling temperature</td><td>Sigma-Aldrich</td><td>A9414-10G</td><td>Embryo mounting for FRET spectral imaging and IHC assay</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A8022</td><td>IHC assay</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>223506</td><td>E3 medium component</td></tr><tr><td>Calcium nitrate</td><td>Sigma-Aldrich</td><td>237124</td><td>Danieau stock solution component</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418-100ML</td><td>IHC assay</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>TBE buffer component for gel preparation</td></tr><tr><td>Ethanol 99%+</td><td>Fisher Scientific</td><td>10048291</td><td>In vitro RNA purification</td></tr><tr><td>Formaldeide 16%</td><td>Thermo Fisher</td><td>28908</td><td>Embryo fixation</td></tr><tr><td>Formamide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F9037</td><td>Gel Elecrophoresis</td></tr><tr><td>Gel Loading Buffer II (Denaturing PAGE)</td><td>Thermo Fisher</td><td>AM8546G</td><td>In vitro RNA transcription</td></tr><tr><td>Glacial Acetic Acid</td><td>Sigma-Aldrich</td><td>695092</td><td>TBE buffer component for gel preparation</td></tr><tr><td>Glycerol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G6279-1L</td><td>IHC assay</td></tr><tr><td>Goat anti-mouse Alexa Fluor 488</td><td>Thermo Fisher</td><td>A11001</td><td>IHC assay</td></tr><tr><td>Goat anti-rabbit Alexa Fluor 633</td><td>Thermo Fisher</td><td>A21070</td><td>IHC assay</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>Danieau stock solution component</td></tr><tr><td>KpnI - HF (Enzyme + rCutSmart Buffer)</td><td>NEB</td><td>R3142</td><td>Plasmid linearization&amp;nbsp;</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>230391</td><td>E3 medium component/Danieau stock solution component</td></tr><tr><td>Millennium RNA Markers</td><td>Thermo Fisher</td><td>AM7150</td><td>Gel Elecrophoresis</td></tr><tr><td>Monarch Genomic DNA purification Kit</td><td>NEB</td><td>T3010L</td><td>Plasmid linearization&amp;nbsp;</td></tr><tr><td>Mouse monoclonal p44/42 MAPK</td><td>Cell Signaling</td><td>4696S</td><td>IHC assay</td></tr><tr><td>mMACHINE SP6 Transcription Kit&amp;nbsp;</td><td>Thermo Fisher</td><td>AM1340</td><td>In vitro RNA transcription</td></tr><tr><td>Normal Goat serum (NGS)</td><td>Sigma-Aldrich</td><td>G9023</td><td>IHC assay</td></tr><tr><td>Nuclease-free water Ambion</td><td>Thermo Fisher</td><td>AM9937</td><td>In vitro RNA transcription</td></tr><tr><td>PD0325901</td><td>Sigma-Aldrich</td><td>PZ0162</td><td>MEK inhibitor</td></tr><tr><td>Phenol Red solution</td><td>Sigma-Aldrich</td><td>P0290</td><td>Microinjection mix component&amp;nbsp;</td></tr><tr><td>Poly A Tailing Kit</td><td>Thermo Fisher</td><td>AM1350</td><td>In vitro RNA transcription</td></tr><tr><td>Potassium chloride bioxtra&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P9333</td><td>E3 medium component/Danieau stock solution component/PBS stock solution component</td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>P0662</td><td>PBS stock solution component</td></tr><tr><td>Proteinase K</td><td>Sigma-Aldrich</td><td>P2308</td><td>IHC assay</td></tr><tr><td>Rabbit polyclonal phospho-p44/42 MAPK&amp;nbsp;</td><td>Cell Signaling</td><td>4695S</td><td>IHC assay</td></tr><tr><td>SYBR safe DNA gel staining</td><td>Thermo Fisher</td><td>S33102</td><td>Gel Elecrophoresis</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>31434-M</td><td>E3 medium component/Danieau stock solution component/PBS stock solution component</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma-Aldrich</td><td>71643</td><td>PBS stock solution component</td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td>TBE buffer component for gel preparation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>PBSTr buffer component</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alliance Mini HD9&amp;nbsp;</td><td>Uvitec</td><td>-</td><td>Imaging system</td></tr><tr><td>Centrifuge 5430 R</td><td>Eppendorf</td><td>5428000205</td><td>Microcentrifuge</td></tr><tr><td>Eppendorf ThermoMixer C</td><td>Eppendorf</td><td>-</td><td>Embryo mounting&amp;nbsp;</td></tr><tr><td>FemtoJet 4x</td><td>Eppendorf</td><td>-</td><td>Microinjection system</td></tr><tr><td>Infinite M Plex&amp;nbsp;</td><td>Tecan</td><td>-</td><td>Multimode plate reader</td></tr><tr><td>Leica M205FA</td><td>Leica Microsystems</td><td>-</td><td>Fluorescence stereo microscope</td></tr><tr><td>Leica TCS-SP8X equipped with incubator (OkoLab)</td><td>Leica Microsystems</td><td>-</td><td>Confocal microscope</td></tr><tr><td>Mini-sub Cell GT Horiziontal Electrophoresis System&amp;nbsp;</td><td>Bio-Rad</td><td>1704406</td><td>Gel Elecrophoresis</td></tr><tr><td>PC-100 Vertical puller</td><td>Narishige</td><td>-</td><td>Needle puller</td></tr><tr><td>PowerPac Universal Power Supply</td><td>Bio-Rad</td><td>1645070</td><td>Gel Elecrophoresis</td></tr><tr><td>Stellaris 5&amp;nbsp;</td><td>Leica Microsystems</td><td>-</td><td>Confocal microscope</td></tr><tr><td>Vortex MiniStar silverline</td><td>VWR</td><td>-</td><td>Plasmid preparation</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biorender</td><td>Biorender</td><td>CC-BY 4.0 license</td><td>Cartoon elaboration for Figures</td></tr><tr><td>Excel</td><td>Microsoft Office Professional Plus 2019</td><td>-</td><td>Data analyses</td></tr><tr><td>Fiji software</td><td>ImageJ</td><td>15.3t</td><td>Imaging rendering and quantitative analyses (FRET signals measurements, ERK fluorescence intensity in IHC assay, embryo axes lenght)</td></tr><tr><td>GraphPad Prism&amp;nbsp;</td><td>GraphPad Software LLC</td><td>v. 9</td><td>Statistical data analyses</td></tr><tr><td>iControl spectrophotometer software</td><td>Tecan</td><td>v. 2.0</td><td>RNA quantification</td></tr><tr><td>Illustrator</td><td>Adobe</td><td>26.0.3 (64-bit)</td><td>Figure assembling</td></tr><tr><td>LASX software</td><td>Leica Microsystems</td><td>v. 4.5 (Stellaris 5), v. 3.0 (M205FA), v. 3.5 (TCS-SP8X)</td><td>Imaging acquisition for spectral FRET experiments and embryo imaging for axes lenght measurements</td></tr><tr><td>Q9 Mini 18.02-SN software</td><td>Uvitec</td><td>-</td><td>Gel image acquisition</td></tr></tbody></table>

an efficient protocol to assess erk activity modulation in early zebrafish noonan syndrome models via live fret microscopy and immunofluorescence

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

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 F&#246;rster 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.

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 whole...

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. ...

We generate zebrafish model of complex developmental diseases, linking patient sequencing to functional genomics, to support variant interpretation and also preclinical drug testing. Despite improved FRET sensors, robust in vivo assay detecting subtle pathogenic changes of Ras-MAPK signaling with sufficient spatiotemporal sensitivity remain lacking. To begin, place the custom microinjection plate containing molded 2%agarose in E3 medium on a working platform.Arrange and align fertilized teen zebrafish eggs in the plate to appropriate lanes to restrict egg movement during microinjection. Back load the needle with two microliters of injection material using a micro loader pipette. Adjust the pressure and time settings to calibrate each injection based on the needle and embryo quality on the microinjection device.Inject the solution into the one-cell stage of teen zebrafish embryos. Raise microinjected teen embryos under controlled husbandry conditions. Clean out any eggs that appear cloudy or degenerated within three hours of deposition.At approximately four hours post-fertilization, using a standard fluorescence stereo microscope, set to a wavelength range of 465-to-500 nanometers. Screen the embryos for fluorescence reporter expression. Select teen positive fish for FRET imaging and reserve negative siblings for immunohistochemical assessments.Place embryo pools of equal numbers into different wells of a six-well plate. To start treatment, immerse the embryos in three milliliters of E3 medium containing vehicle control as DMSO or diluted MEK at the desired concentration. To begin, melt the 1.5%to-2%low-melting agarose aliquot in a ThermoMixer at 50 degrees Celsius.Once dissolved, reduce the temperature to approximately 30 degrees Celsius. Position a single injected teen positive embryo at the center of a 35-millimeter glass bottom dish for live imaging. Remove excess E3 medium, and add a drop of low-melting agarose to immobilize the embryo.Allow the agarose to polymerize at room temperature. Turn on the incubator controller at least one hour before acquisition and set the temperature to 28 degrees Celsius to maintain embryo health. Once the incubator stabilizes, place the imaging dish with the embryo on the sample holder and use a 10x dry objective to visualize the sample.Switch on the argon ion laser and adjust the laser power to 50%In Hardware Settings, choose eight-bit depth resolution to acquire images. Next, in the Acquisition panel, select the spectral detection xy, gamma, lambda, z scanning mode, and set the format image to 512 by 512 pixels, scanning speed of 400 hertz, and optical zoom of 0.75. Activate the 458-nanometer laser line of the argon ion laser, and set its intensity value to 8.5%Then select a hybrid detector and set its sensitivity to a gain value of 500.Open the dropdown menu in the detector bar to select the cyan fluorescence protein or ECFP emission curve. To display the YPET emission curve, activate a second detector. Then select the yellow fluorescence protein YFP emission curve.To start live acquisitions, position the detection cursor in the range of the most intense YFP signal to visualize the sample. Set the start and end positions of the sample thickness in the Z-Stack LASX window. Set the gamma scan range properties to begin at 460 nanometers and end at 570 nanometers of the detection range.Define the detection bandwidth as five nanometers and scan step size as five nanometers. Start z-stack acquisition. Insert and save two adapted reference emission spectra for CFP and YFP in the dye database available in the Configuration window to exclude spectral bleed-through.Select the resulting spectral image file from the imaging session and open the process window. Next, in the dye separation tool, select Spectral Dye Separation, and configure the settings for the dye separation. In the dropdown lists on the left side of the dialogue, select the new CFP emission spectrum in the first position and the new YFP emissions spectrum from the spectrum database.In the gamma scan of the images, identify the image with the greatest signal intensity. Then move along the z-scan to select the optical section that highlights the area of interest on the margin zone. Activate the ROI selection mode on the margin zone of the animal pole to define the area with the best spectrum.Click on ROI crosshair in the display window and adjust the size of the reference ROI to 40 voxels in the measurements area. Open the newly generated file containing the two separated channels. Produce a two-dimensional projection image from the three-dimensional image series using the maximum intensity projection.In the Process window, select crop to separate channels into two separate files, the channel one and channel two channels. In the Process window, select Combine Images, then select the channel two file and insert it in the first option. Then select the channel one file and insert it in the second option.Set a rescale with factor five and choose the ratio operation. Click on Apply to generate the new file containing the ratio metric image. Save the file for data analysis.Import the spectral imaging and dye separation image files into the open-source Fiji software for analysis. Configure parameters for readout measurements in Fiji using Analyze and Set Measurements. Select area, integrated density and mean gray value as the parameters of interest.Using the polygon selection tool from the toolbar, select the region of interest corresponding to the margin of the gastrula. Sequentially, click Analyze, Tools and ROI Manager to save the ROI xy specifications. To perform measurements in a selected ROI, click on Analyze and Measure.Organize FRET by CFP ratio values for each ROI in a worksheet with experimental groups in columns and raw values in rows. For a single biological replicate, create a column table with one grouping variable, where each group is defined by a column. At the end of gastrulation, fix the embryos through immersion in 4%paraldehyde prepared in PBS for 20 minutes at room temperature.After fixation, wash the embryos several times with PBS. Using a pastor pipette, orient the embryos laterally in a single well of a 12-well plate containing fresh PBS. Using a stereo microscope with a 2.7x magnification objective with 0.63x scanning objective and 8.6 zoom factor, capture the images in a brightfield mode to evaluate the presence of an oval shape.Import the captured embryo image into Fiji software. To measure the embryo's axes'length, select the straight tool from the toolbar and click on Analyze, followed by Measure. After performing selected measurements, add them to the ROI manager list and save the ROI file.Export the data into a worksheet. Calculate the axes'ratio by dividing the length of the major axis by the length of the minor axis. Calculate the mean, the standard deviation of the mean, or the standard error of the mean of different replicates.Our live FRET pipeline in zebrafish enables early sensitive detection of pathogenic ERK signaling activation, supporting variant interpretation, and MEK inhibitor efficiency testing.

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Learn live FRET microscopy to assess ERK activity modulation in zebrafish Noonan syndrome models. This protocol enables sensitive detection of pathogenic Ras-MAPK signaling changes during embryogenesis.

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence (Video) | JoVE Journal

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

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