Name: spatiotemporal subcellular manipulation of the microtubule cytoskeleton in the living preimplantation mouse embryo using photostatins
Uploaded: 2021-11-30T15:00:00.000+00:00
Duration: 8 min 13 s
Description: Watch this Scientific Journal Video about Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins at JoVE.com

The authors would like to thank Dr. Oliver Thorn-Seshold and Li Gao for providing us with photostatins and advice on manuscript preparation, Monash Production for filming support, and Monash Micro Imaging for microscopy support.
This work was supported by the National Health and Medical Research Council (NHMRC) project grant APP2002507 to J.Z. and the Canadian Institute for Advanced Research (CIFAR) Azrieli Scholarship to J.Z. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

Experiments were approved by the Monash Animal Ethics Committee under animal ethics number 19143. Animals were housed in specific pathogen-free animal house conditions at the animal facility (Monash Animal Research Platform) in strict accordance with ethical guidelines.
1. Preimplantation mouse embryo collection
<ol>
	<li>Superovulate and mate mice as described previously16,18, in compliance with the institutional animal ethics guidelines. 
	NOTE: The most commonly used strains for live embryo collection are C57BL/6 or FVB/N mice. All data shown here were generated using FVB/N mice.</li>
	<li>On the morning after mating, flush zygotes from the oviduct using M2 medium as described20, or Human Tubal Fluid (HTF) medium. Using a mouth pipette apparatus as described21,22, transfer zygotes to fresh Potassium Simplex Optimised Medium (KSOM) droplets, prewarmed to 37 &#176;C and equilibrated to 5% CO2, in a 35 mm culture dish overlaid with a sufficient volume of mineral oil to ensure media coverage.</li>
	<li>Microinject zygotes as described20 with cRNA encoding for a red fluorescently-tagged microtubule plus end marker. Here, cRNA for the End Binding protein 3 (EB3)-dTomato was used at 30 ng/&#181;L concentration after preparing and purifying as described16,18 and diluting in microinjection buffer. 
	NOTE: It is recommended to prepare cRNA ahead of time and store it at -20 &#176;C until required.</li>
	<li>Culture embryos in dark at 37 &#176;C and 5% CO2 until embryos have reached the desired developmental stage for PST-1P treatment. 
	NOTE:&#160;For a comprehensive resource of the culture times required for different embryonic stages see23. For 16-cell stage embryos used here, culture to embryonic day 3 (E3) post-fertilization.</li>
</ol>
2. Drug and imaging dish preparation
NOTE: For Steps 2.1-2.10, work exclusively in the dark or red-light conditions to avoid unintended PST-1P activation. Aluminum foil or dark covers should be used for all tubes and dishes containing PSTs.
<ol>
	<li>Prepare a stock concentration of 50 mM PST-1P in ultrapure water. 
	NOTE: The molecular weight of PST-1P is 440 g/mol. The stock solution is stable at -20 &#176;C for up to 1 year. PST-1P is soluble in water or aqueous buffer but does not readily dissolve in DMSO.</li>
	<li>From step 2.1, prepare an intermediate working concentration of 800 &#181;M PST-1P in ultrapure water.</li>
	<li>Dilute PST-1P to a final concentration of 40 &#181;M in fresh KSOM. As a typical experiment requires approximately 20 &#181;L of PST-1P-treated KSOM, dilute 1 &#181;L of 800 &#181;M PST-1P in 19 &#181;L of KSOM for a final volume of 20 &#181;L to ensure sufficient medium is prepared in advance, using only red light for visibility. 
	NOTE: Both the concentration and the activation status of a PST-1P dilution can be checked by taking a UV-Vis absorbance spectrum using a spectrophotometer which should be done during assay establishment. The absorbance at 380 nm (A380) of a fully-trans 40 &#181;M dilution in a 1 cm cuvette should be approximately 0.8. Both cis- and trans-forms have the same absorbance at 455 nm (A455). When the ratio of the A380 to the A455 is approximately 9:1, the dilution is inactive (fully trans). When the ratio A380:A455 is 1:2, the dilution is fully activated (fully cis). Intermediate ratios reflect intermediate states of activation.</li>
	<li>To prepare the chamber slide for live imaging, pipette 10 &#181;L of PST-1P-treated KSOM into the center of one well to form a hemispherical droplet (Figure 1A).</li>
	<li>Gently add sufficient mineral oil to cover the droplet, ensuring that it does not disperse the media. This will ensure that the droplet does not evaporate.</li>
	<li>Pre-warm and CO2-equilibrate the chamber slide dish in an incubator at 37 &#176;C and 5% CO2 for a minimum of 3 h or at most, overnight.</li>
	<li>At the end of the equilibration period in step 2.6, prepare a 35 mm culture dish with a 10 &#181;L droplet of PST-1P-treated, prewarmed, and equilibrated KSOM as a wash step. Do not overlay with oil.</li>
	<li>Transfer embryos by mouth pipetting into the PST-1P-treated KSOM droplet from step 2.7. 
	NOTE: Steps 2.7 and 2.8 are recommended for mouth pipetting of embryos but are optional.</li>
	<li>Immediately transfer embryos by mouth pipetting into the center of the PST-1P-treated KSOM droplet in the imaging chamber slide prepared in steps 2.4-2.6 (Figure 1A).</li>
	<li>Incubate embryos at 37 &#176;C and 5% CO2 in PST-1P-treated KSOM in the imaging chamber slide for at least 1 h before imaging. If possible, mount the chamber slide on the microscope inside an environmental chamber at 37 &#176;C and 5% CO2, and in complete darkness to ensure all PST-1Ps are in the inactive trans-configuration and that embryos can sink to the bottom of the dish.</li>
</ol>
3. Live imaging and PST-1P photoactivation
NOTE: Steps 3.1-3.13 are performed on a laser scanning confocal microscope fitted with avalanche photodiode detectors (APDs) and a dark environmental chamber. These instructions refer specifically to the imaging setup using the acquisition software described in the Table of Materials; however, they can also be applied to other confocal microscopy systems.
<ol>
	<li>Prepare a 63x/1.2 NA water oil immersion objective with the prescribed immersion medium.</li>
	<li>Using a red light torch to guide positioning, advance the objective to contact the immersion medium. At this stage, avoid using white or brightfield light to find the embryos as this could precociously activate PST-1P.</li>
	<li>Using the eyepiece and while under red light illumination, locate the edge of the droplet of medium and position the objective directly over this location. This can assist the user to establish orientation and find the focal plane.</li>
	<li>Next, through the eyepiece or on software-enabled live-mode scanning, use a red wavelength filter or 561 nm laser to locate the embryos within the droplet.</li>
	<li>Use stage controllers and live-scanning mode to set the start- and end-points for acquiring a z-stack of the whole embryo.</li>
	<li>Adjust the laser power settings (typically, with highly sensitive detectors such as the APDs, a 561 nm laser power of less than 5% is sufficient), digital offset (typically at -0.900) to optimize the appearance of EB3-dTomato comets and minimize background noise, pinhole at 2 &#181;m, pixel resolution of 512 x 512, and pixel dwell time of 3.15 &#181;s.</li>
	<li>Acquire a z-stack of the whole embryo with 1 &#181;m section intervals to assess areas of microtubule growth in the whole organism (Figure 1B).</li>
	<li>Use the 3D z-stack image from step 3.7 to identify regions of interest (ROIs) for EB3-dTomato tracking experiments. Increase the zoom to 3x and draw a rectangular ROI around the specific subcellular area of interest.</li>
	<li>Acquire a time-lapse movie of a single z-plane using the typical values of imaging parameters: 561 nm laser power at 5%, digital offset of -0.900, pixel resolution of 512 x 512, pinhole at 3 &#181;m, pixel dwell time of 3.15 &#181;s, zoom of 3x, time interval of 500 ms. 
	NOTE: 120 time frames will provide a tracking movie of 1 min and should be sufficient for data analysis. The acquisition can continue for longer, provided that bleaching of the fluorophore is minimal (Figure 1C).</li>
	<li>To activate PST-1P, switch to a 405 nm laser and acquire another time-lapse movie with the 405 nm laser set to 10% power, pixel resolution of 512 x 512, pinhole opened maximally, pixel dwell time of 3.15 &#181;s, zoom of 3x, time interval of 500 ms, and a total of 20 frames (Figure 1D).</li>
	<li>Switch back to 561 nm laser and repeat acquisition as in step 3.9 to confirm the loss of EB3-dTomato comets after activation of PST-1P (Figure 1E). Ensure that this acquisition takes place as soon as possible following activation. 
	NOTE: Step 3.10 can be performed repetitively for longer inhibition of EB3-dTomato comets. However, embryos must be monitored carefully to avoid any harm caused by UV light.</li>
	<li>To reverse PST-1P back to its inactive trans-state, engage a 514 nm laser at 10% power. Acquire a time-lapse movie with the 514 nm laser set to 10% power, pixel resolution of 512 x 512, pinhole opened maximally, pixel dwell time of 3.15 &#181;s, zoom of 3x, time interval of 500 ms, and a total of 20 time frames&#160;(Figure 1F).</li>
	<li>Repeat step 3.11 to visualize recovery of EB3-dTomato comets (Figure 1G).</li>
</ol>
4. Image data analysis
<ol>
	<li>For analyzing and quantifying the inhibition of microtubule polymerization by PSTs, use software programs available to researchers to suit their specific needs. Those recommended for use will possess a tracking tool, that can manually or automatically track the movement, direction, and speed of EB3-dTomato comets16,18.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63290/63290fig01.jpg" /> 
Figure 1: Schematic representation of PST-1P photoactivation and deactivation in the live 3D preimplantation mouse embryo. All experiments are performed in complete darkness (black background) or only by red light illumination. (A)&#160;Live preimplantation mouse embryos expressing EB3-dTomato are cultured to 16-cell stage and then transferred to a droplet of KSOM containing 40 &#181;M PST-1P in an imaging chamber slide. (B)&#160;A 3D image of the whole embryo allows the assessment of microtubule growth by visualizing the distribution of EB3-dTomato comets. (C)&#160;To start the experiment, EB3-dTomato comets are tracked in a subcellular region using time-lapse imaging. (D)&#160;Subsequent PST-1P photoactivation in the same subcellular region using a 405 nm laser results in the loss of EB3-dTomato comets (E).&#160;Intensified PST-1P activation can be implemented, if necessary, by sequential 405 nm light illumination. (F-G)&#160;To reverse PST-1P back to its inactive state and restore EB3-dTomato comets, a 514 nm laser is applied to the same subcellular region. If required, multiple rounds of photoactivation and deactivation can be performed. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/63290/63290fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no competing or financial interest.

The microtubule network is integral to the fundamental inner workings of a cell. Consequently, this presents challenges in manipulating microtubule dynamics in living organisms, as any perturbation to the network tends to have widespread consequences for all aspects of cellular function. The emergence of photoswitchable microtubule-targeting compounds presents a way to precisely manipulate the cytoskeleton at a subcellular level, with superior control for the induction and reversal of microtubule growth inhibition<sup cl...

The microtubule architecture varies widely across different cell types to support diverse functions1,2. Its dynamic nature of growth and shrinkage allows rapid adaptation to extra- and intracellular cues and to respond to the ever-changing needs of a cell. Hence, it can be considered as the &#34;morphological fingerprint&#34; playing a key role in cellular identity.
Pharmacological targeting of the microtubule cytoskeleton using small molecule inhibitors has led to a plethora of fundamental discoveries in developmental biology, stem cell biology, cancer biology, and neurobiology3,4,5,6,7. This approach, while indispensable, presents various limitations such as toxicity and off-target effects. For example, one of the most widely used microtubule-targeting agents, nocodazole, is a powerful microtubule-depolymerizing drug8. However, small-molecule inhibitors such as nocodazole are active from the time of application and, given the essential nature of the microtubule cytoskeleton to many critical cellular functions, global depolymerization of microtubules can produce off-target effects, which may be unsuitable for many applications. Additionally, nocodazole treatment is irreversible unless samples are washed free of the drug, preventing&#160;continuous live imaging and, thus, precise tracking of individual microtubule filaments.
The development of light-activated compounds began with the creation of photouncaged molecules and has heralded a new era in targeting and monitoring the effects of microtubule growth inhibition in a precise and spatiotemporally-controlled manner. One family of reversibly photoswitchable drugs, photostatins (PSTs), were developed by replacing the stilbene component of combretastatin A-4 with azobenzene9. PSTs are inactive until illumination with UV light, whereby the inactive trans-configuration converts to the active cis-configuration by reversible isomerization. Cis-PSTs inhibit microtubule polymerization by binding to the colchicine binding site of &#946;-tubulin, blocking its interface with &#946;-tubulin and preventing dimerization required for microtubule growth10. Among a cohort of PSTs, PST-1P has emerged as a lead compound as it has the highest potency, is fully water-soluble, and shows a rapid onset of bioactivity after illumination.
The most effective trans- to cis-isomerization of PSTs occurs at wavelengths between 360-420 nm, which enables dual options for PST activation. A 405 nm laser line on a typical confocal microscope can be administered for optimal spatial targeting of microtubule growth inhibition. The ability to pinpoint the location and timing of PST activation through 405 nm laser illumination facilitates precise temporal and spatial control, allowing disruption of microtubule dynamics on a subcellular level, within sub-second response times9. Alternatively, an affordable LED UV light allows whole organism illumination to induce organism-wide disruption of the microtubule architecture. This may be a cost-effective alternative for researchers for whom the precisely-timed onset of inhibition, rather than spatial targeting, is the goal. Another feature of PSTs is their on-demand inactivation by applying green light of a wavelength in the 510-540 nm range9. This enables tracing of microtubule filaments before, during, and after PST-mediated growth inhibition.
PSTs, while still a relatively recent design, have been used in numerous in vitro applications across diverse research fields11, including investigating new mechanisms of cell migration in amoeboids12, in neurons isolated from the brain of the newborn mouse13, and wing epithelium development in Drosophila melanogaster14. Other light-reactive drugs have proven to be valuable tools in targeted disruption of cellular function. For instance, an analog of blebbistatin,&#160;azidoblebbistatin, was used for enhanced myosin inhibition under illumination15,16. This highlights the potential for new discoveries owing to the ability for spatiotemporally controlled inhibition of cellular function.
Live 3D organisms present superb yet more delicate systems to manipulate microtubule dynamics on a whole-animal, single-cell, or subcellular level under physiological conditions. In particular, the preimplantation mouse embryo offers exceptional insight into the inner workings of the cell as well as intercellular relationships within an organism17. Temporally and spatially targeted consecutive cycles of activation and deactivation of PSTs contributed to the characterization of the interphase bridge, a post-cytokinetic structure between cells, as a non-centrosomal microtubule-organizing center in the preimplantation mouse embryo16. A similar experimental setup demonstrated the involvement of growing microtubules in the sealing of the mouse embryo to allow blastocyst formation18. Furthermore, PSTs were also used in whole zebrafish embryos to investigate neuronal cell migration by inhibiting microtubule growth in a subset of cells in the hindbrain19.
This protocol describes the experimental setup and use of PST-1P in the preimplantation mouse embryo. The instructions presented here can also guide the application of PSTs for a wide array of objectives such as studying chromosome segregation and cell division, trafficking of intracellular cargo, and cell morphogenesis and migration. Furthermore, such studies will assist the implementation of PSTs&#160;in organoid systems, blastoids, and other embryo models such as Caenorhabditis elegans and Xenopus laevis, as well as potentially expand the use of PSTs for in vitro fertilization technologies.

<table><tbody><tr><td>Aspirator tube</td><td>Sigma-Aldrich</td><td>A5177</td><td>For mouth aspiration apparatus</td></tr><tr><td>Chamber slides - LabTek</td><td>Thermo Fisher Scientific</td><td>NUN155411</td><td></td></tr><tr><td>cRNA encoding for EB3-dTomato</td><td>N/A</td><td>N/A</td><td>Prepared according to manufacturers instructions using mMessage &lt;em&gt;in vitro &lt;/em&gt;Transcription kit</td></tr><tr><td>Culture dishes - 35mm</td><td>Thermo Fisher Scientific</td><td>150560</td><td></td></tr><tr><td>Human chorionic growth hormone</td><td>Sigma-Aldrich</td><td>C8554</td><td></td></tr><tr><td>Human Tubal Fluid (HTF) medium</td><td>Cosmo-Bio</td><td>CSR-R-B071</td><td></td></tr><tr><td>Imaris Image Analysis Software</td><td>Bitplane</td><td></td><td></td></tr><tr><td>Immersion Oil W 2010</td><td>Carl Zeiss</td><td>444969-0000-000</td><td>For use with microscope immersion objective</td></tr><tr><td>LED torch - Red light</td><td>Celestron</td><td>93588</td><td></td></tr><tr><td>M2 medium</td><td>Sigma-Aldrich</td><td>M7167</td><td></td></tr><tr><td>Mice - wild-type FVB/N, males and females</td><td>N/A</td><td>N/A</td><td>Females 8-9 weeks old. Males 2-6 months old.</td></tr><tr><td>Microcapillary Pipettes - Kimble</td><td>Sigma-Aldrich</td><td>Z543306</td><td>For mouth aspiration apparatus</td></tr><tr><td>Microinjection buffer</td><td>N/A</td><td>N/A</td><td>5 mM Tris, 5 mM NaCl, 0.1 mM EDTA, pH 7.4</td></tr><tr><td>Mineral oil</td><td>Origio</td><td>ART-4008-5P</td><td></td></tr><tr><td>mMessage &lt;em&gt;In vitro &lt;/em&gt;Transcription kit</td><td>Thermo Fisher Scientific</td><td>AM1340</td><td></td></tr><tr><td>NanoDrop Spectrophotometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>Potassium Simplex Optimised Medium (KSOM) medium</td><td>Cosmo-Bio</td><td>CSR-R-B074</td><td></td></tr><tr><td>Pregnant mare serum gonadotrophin</td><td>Prospec Bio</td><td>HOR-272</td><td></td></tr><tr><td>PST-1P</td><td>N/A</td><td>N/A</td><td>Borowiak, M. &lt;em&gt;et al.&lt;/em&gt;, Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. &lt;em&gt;Cell.&lt;/em&gt; 162 (2), 403-411, doi:10.1016/j.cell.2015.06.049, (2015).</td></tr><tr><td>RNA purification kit</td><td>Sangon</td><td>B511361-0100</td><td></td></tr><tr><td>Ultrapure water</td><td>Sigma-Aldrich</td><td>W1503</td><td></td></tr><tr><td>ZEN Black Software</td><td>Carl Zeiss</td><td></td><td></td></tr></tbody></table>

spatiotemporal subcellular manipulation of the microtubule cytoskeleton in the living preimplantation mouse embryo using photostatins

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of &#946;-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

In line with the protocol, preimplantation mouse embryos were microinjected with cRNA for EB3, tagged with red fluorescent dTomato (EB3-dTomato). This enables the visualization of growing microtubules as EB3 binds to polymerizing microtubule plus ends24.
The experiments were performed 3 days post-fertilisation (E3) when the mouse embryo is comprised of 16 cells. Any other preimplantation developmental stage can be used, depending on the scientific question to be investi...

Watch this Scientific Journal Video about Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins at JoVE.com

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. ...

spatiotemporal-subcellular-manipulation-microtubule-cytoskeleton

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

2.3K Views.  Monash University. Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.

Video: Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Neuroscience

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Biology

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...

Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

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Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

Mouse In Vivo Placental Targeted CRISPR Manipulation

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis