Name: antibody uptake assay for tracking notch/delta endocytosis during the asymmetric division of zebrafish radial glia progenitors
Uploaded: 2023-01-20T13:15:00
Description: Watch this Scientific Journal Video about Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors at JoVE.com

The project was supported by NIH R01NS120218, the UCSF Mary Anne Koda-Kimble Seed Award for Innovation, and Chan Zuckerberg Biohub.

We have used the AB wild type line and transgenic line Tg [ef1a:Myr-Tdtomato] for the study. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco, USA (Approval Number: AN179000).
1. Preparation of zebrafish embryos
<ol>
	<li>Set up fish crossing tanks in the afternoon before 5:00 p.m., with one female wild-type fish and one male Tg [ef1a:Myr-Tdtomato] fish by using di...

<ol>
	<li>Baonza, A., Garcia-Bellido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+directly+controls+cell+proliferation+in+the+Drosophila+wing+disc.">Notch signaling directly controls cell proliferation in the Drosophila wing disc.</a> Proceedings of the National Academy of Sciences. 97 (6), 2609-2614 (2000).</li><li>Chitnis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental Dynamics. 235 (4), 886-894 (2006).</li><li>Daeden, A., Gonzalez-Gaitan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+trafficking+during+mitosis+and+notch-dependent+asymmetric+division.">Endosomal trafficking during mitosis and notch-dependent asymmetric division.</a> Progress in Molecular and Subcellular Biology. 57, 301-329 (2018).</li><li>Le Borgne, R., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling:+endocytosis+makes+delta+signal+better.">Notch signaling: endocytosis makes delta signal better.</a> Current Biology. 13 (7), 273-275 (2003).</li><li>Chapman, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+endocytosis+is+induced+by+ligand+and+is+required+for+signal+transduction.">Notch1 endocytosis is induced by ligand and is required for signal transduction.</a> Biochimica et Biophysica Acta. 1863 (1), 166-177 (2016).</li><li>Schroeter, E. H., Kisslinger, J. A., Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch-1+signalling+requires+ligand-induced+proteolytic+release+of+intracellular+domain.">Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain.</a> Nature. 393 (6683), 382-386 (1998).</li><li>Coumailleau, F., F&#252;rthauer, M., Knoblich, J. A., Gonz&#225;lez-Gait&#225;n, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+Delta+and+Notch+trafficking+in+Sara+endosomes+during+asymmetric+cell+division.">Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division.</a> Nature. 458 (7241), 1051-1055 (2009).</li><li>Derivery, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+endosome+dynamics+by+spindle+asymmetry+during+asymmetric+cell+division.">Polarized endosome dynamics by spindle asymmetry during asymmetric cell division.</a> Nature. 528 (7581), 280-285 (2015).</li><li>Matsuda, M., Chitnis, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+Notch+determines+endocytosis+of+specific+Delta+ligands+in+zebrafish+neural+tissue.">Interaction with Notch determines endocytosis of specific Delta ligands in zebrafish neural tissue.</a> Development. 136 (2), 197-206 (2009).</li><li>Kressmann, S., Campos, C., Castanon, I., F&#252;rthauer, M., Gonz&#225;lez-Gait&#225;n, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+Notch+trafficking+in+Sara+endosomes+during+asymmetric+cell+division+in+the+spinal+cord.">Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.</a> Nature Cell Biology. 17 (3), 333-339 (2015).</li><li>Dong, Z., Yang, N., Yeo, S. -. Y., Chitnis, A., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intralineage+directional+Notch+signaling+regulates+self-renewal+and+differentiation+of+asymmetrically+dividing+radial+glia.">Intralineage directional Notch signaling regulates self-renewal and differentiation of asymmetrically dividing radial glia.</a> Neuron. 74 (1), 65-78 (2012).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+endosome+dynamics+engage+cytoplasmic+Par-3+that+recruits+dynein+during+asymmetric+cell+division.">Polarized endosome dynamics engage cytoplasmic Par-3 that recruits dynein during asymmetric cell division.</a> Science Advances. 7 (24), (2021).</li><li>Zhao, X., Garcia, J., Royer, L. A., Guo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+analysis+for+cryosectioned+and+immunostained+tissue+samples+with+or+without+label+retention+expansion+microscopy+(LR-ExM)+by+JACoP.">Colocalization analysis for cryosectioned and immunostained tissue samples with or without label retention expansion microscopy (LR-ExM) by JACoP.</a> Bio-Protocol. 12 (5), 4336 (2022).</li><li>Gutzman, J. H., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+brain+ventricle+injection.">Zebrafish brain ventricle injection.</a> Journal of Visualized Experiments. (26), e1218 (2009).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calibration+of+the+injection+volume+for+microinjection+of+Xenopus+oocytes+and+embryos.">Calibration of the injection volume for microinjection of Xenopus oocytes and embryos.</a> Cold Spring Harbor Protocols. 2010 (12), (2010).</li><li>Edelstein, A., Amodaj, N., Hoover, K., Vale, R., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+control+of+microscopes+using+&#181;Manager.">Computer control of microscopes using &#181;Manager.</a> Current Protocols in Molecular Biology. , (2010).</li><li>Lukinavi&#269;ius, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+probes+for+multicolor+imaging+in+living+cells.">Fluorogenic probes for multicolor imaging in living cells.</a> Journal of the American Chemical Society. 138 (30), 9365-9368 (2016).</li></ol>

We have developed an antibody uptake assay for labeling and imaging endosomal Notch/Delta trafficking in zebrafish radial glia progenitors with high efficiency. Compared to previous methods used for tracking labeled anti-DeltaD antibody in Drosophila SOP cells7,8, our method used microinjection instead of incubation of samples in the conjugated antibody. Fluorescently conjugated anti-Dld antibodies were microinjected into the hindbrain ventricle; this technique d...

Notch signaling controls cell fate decision and patterning during development in metazoans1, and recent studies have shown that Notch signaling in stem cell division mainly depends on endocytic trafficking2,3. Endocytosed Notch/Delta can activate Notch signaling in the nucleus and enhance the transcription of Notch target genes4,5,6. Directional Notch/Delta endosomal trafficking was first observed in Drosophila sensory organ precursor (SOP) cells during its asymmetric cell division (ACD), resulting in a higher Notch signaling activity in pIIa than in pIIb7,8. Antibody uptake assays with anti-Delta and anti-Notch antibodies have been applied to monitor the endocytic process in mitotic SOP cells. Notch/DeltaD endosomes move along with a kinesin motor protein to the central spindle during cytokinesis, and are asymmetrically translocated into the pIIa cell due to the antiparallel array of the asymmetric central spindle at the last moment of cell division3,8. These studies have shed light on the molecular mechanisms regulating asymmetric division in Drosophila SOP cells, but it is unclear whether similar endocytic processes occur in vertebrate radial glia progenitors (RGPs).
Moreover, the molecular mechanisms that regulate asymmetric Notch/DeltaD signaling during vertebrate RGP division are not well understood. In zebrafish, it has been reported that the interaction of Notch and Delta facilitates the endocytosis of the DeltaD ligand9. It is unknown whether DeltaD endocytosis can impact the cell fate choice of daughter cells in the developing vertebrate brain. Recent studies show injecting fluorescently conjugated anti-DeltaD antibodies into the neural tube could label Sara endosomes specifically in neuroepithelial cells, and anti-DeltaD containing Sara endosomes preferentially segregate into proliferating daughter cells10. It has been suggested that Notch signaling from the endosomes could regulate daughter cell fate. Previous results have shown that most zebrafish RGP cells in the developing forebrain undergo ACD, and the daughter cell fate determination is dependent on intralineage Notch/DeltaD signaling11. In order to elucidate the nature of the intralineage Notch/DeltaD signaling in zebrafish RGPs, we have developed the anti-DeltaD antibody uptake assay in the zebrafish developing brain. Using this protocol, we have successfully performed live labeling and imaging of DeltaD endocytic trafficking in mitotic RGPs.
The fluorescently labeled anti-DeltaD-antibody is efficiently internalized into the RGPs along the forebrain ventricle. It has greatly facilitated the discovery of directional trafficking of DeltaD endosomes in the asymmetrically dividing RGPs12,13. Compared to previous antibody uptake protocols developed for Drosophila notum cultures and the zebrafish spinal cord10, this protocol has achieved long-lasting and highly efficient anti-DeltaD labeling in the brain ventricle cell layer, specifically with less than 10 nL of microinjected antibody mixture. The hindbrain ventricle injection is very convenient for applying the antibody uptake assay in the developing brain, as the hindbrain ventricle is well expanded in zebrafish embryos and filled up with cerebrospinal fluid at early development stage14. By injecting the antibody mixture into the hindbrain ventricle without injuring any crucial developing tissues, the protocol has minimized possible damage to the imaging zone in the forebrain as much as possible. The reduced dosage of the injected primary antibody has also avoided potential side-effects of interfering with endogenous Delta-Notch signaling in vivo. This protocol can be easily combined with other pharmacological or genetic perturbations, utilized at different developmental stages, and possibly adapted to the adult brain as well as human pluripotent stem cell-derived 2D/3D brain organoids. Taken together, the protocol has made it possible to understand how and when Notch signaling asymmetry is established during ACD. The main challenge for successful implementation of this protocol is to achieve precise delivery of appropriate concentrations of the antibody based on specific experimental conditions.

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			<td>35mm glass bottom culture dish&#160;</td>
			<td>MatTek corporation</td>
			<td>P35GC-1.5-10-C</td>
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			<td>air pressure injector&#160;</td>
			<td>Narishige</td>
			<td>IM300</td>
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			<td>Anti-Mouse-IgG-Atto647N&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>50185</td>
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			<td>CaCl2.2H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
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			<td>Capillaries, 1.2 mm OD, 0.9 mm ID, with filament</td>
			<td>World Precision Instruments</td>
			<td>1B120F-6</td>
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			<td>CSU-W1 Spinning Disk/High Speed Widefield</td>
			<td>Nikin</td>
			<td>N/A</td>
			<td>Nikon&#160;Ti inverted fluorescence microscope with CSU-W1 large field of view confocal.&#160;</td>
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			<td>Dumont Medical Tweezers Style 5</td>
			<td>Thomas Scientific</td>
			<td>72877-D</td>
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			<td>Flaming-Brown P897 puller</td>
			<td>Sutter Instruments</td>
			<td>N/A</td>
			<td>https://www.sutter.com/manuals/P-97-INT_OpMan.pdf</td>
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			<td>KCl</td>
			<td>Millipore</td>
			<td>529552</td>
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			<td>MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2773</td>
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			<td>micromanipulators</td>
			<td>World Precision Instruments</td>
			<td>WPI M3301R</td>
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			<td>Mouse anti-Dld&#160;</td>
			<td>Abcam</td>
			<td>AB_1268496</td>
			<td></td>
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			<td>Mouse IgG blocking buffer from Zenon</td>
			<td>Thermofisher Scientific</td>
			<td>Z25008</td>
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			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
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			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
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			<td>Stemi 2000&#160;&#160;</td>
			<td>Zeiss&#160;</td>
			<td>N/A</td>
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			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
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			<td>UltraPureTM low melting point agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16520050</td>
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antibody uptake assay for tracking notch/delta endocytosis during the asymmetric division of zebrafish radial glia progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

In Figure 2A, the embryos injected with Atto647N, without binding with the primary antibody, showed background fluorescence in the brain ventricle. Very few engulfed fluorescent particles can be observed in the cells. The anti-Dld-Atto647N injected zebrafish embryos showed large amounts of internalized fluorescent particles in most cells of the developing forebrain (Figure 2A, right panel). After zooming in to focus on mitotic RGPs, as shown in <strong class="xf...

Watch this Scientific Journal Video about Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors at JoVE.com

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Notch signaling controls sulfate decision and patterning during development in metazoans. We have developed an antibody uptake assay for labeling the notch ligand delta-D and imaging its endocytic trafficking in radial glia progenitors in the developing zebra fish forebrain. The zebrafish hidden brain ventricular injection enables selective Anti-Dld-Atto-647N uptake by myototic radial glia progenitors lining the brain ventricles in development with high efficiency and the long lasting effects.This protocol can be easily combined with other pharmacological or genetic perturbations utilized at different developmental stages and possibly adapted to the adult brain as well as human pluripotent stem cell derived 2D or 3D brain organoids. After generating the fertilized embryos and incubating them for 18 to 20 hours, remove them from the incubator. Observe them under an epifluorescence microscope using white light first at a 20 times magnification.Discard dead embryos that appear cloudy or ruptured under the microscope using a glass pipette. Then turn the fluorescent lamp on and choose the RFP filter setting of the microscope. Select the embryos with strong red fluorescence and transfer them to a new dish with egg water.Now dechorionate the embryos manually with two fine forceps under white light. Hold the chorion with one pair of forceps and make a tear in the chorion with the other forceps. Open the tear carefully using the forceps and make it large enough for the embryo to pass through by gently pushing the embryo with the tips of the other forceps.Transfer the dechorionated embryos to a new Petri dish with fresh embryonic medium for a quick rinse before microinjection. After pulling fine injection needles on a puller, open the tip of the needle with forceps under a stereo dissection microscope. Make the diameter of the tip around 10 micrometers and the taper angle around 30 degrees.Now mix 0.5 microliters of the anti-Dld antibody with two microliters of the anti mouse L G Atto-647N antibody by pipetting five to 10 times to conjugate them and incubate at room temperature for at least 30 minutes. At the end of the incubation, add 2.5 microliters of blocking buffer and 0.5 microliters of 0.5%phenol red to the antibody mixture and mixed by pipetting to block any unconjugated antibodies remaining in the mixture. Next, prepare 1%low melting point agarose in the embryo medium.Heat the agarose-contained mixture at 70 degrees Celsius till the mixture turns transparent. After aliquoting the agarose solution, keep them in a heat block at 40 degrees Celsius. Rinse the embryo in the tube containing 1%low melting point agarose for three seconds.Place the embryo on an inverted plastic Petri dish lid with individual agarose drops to mount each embryo separately. Lay the embryos flat laterally in the agarose and keep this position until the agarose has solidified at room temperature. Cover all the mounted embryos in the agarose with egg medium.Put the embedded embryos under the stereo microscope and cover the agarose with egg water. Set the air pressure injector with micro manipulators, placing them close to the microscopes. Use the steel gas cylinder containing gaseous nitrogen under high pressure as the air resource.Once the embryos have been mounted in the agarose, open the gas valve. While using the front fill module of the micro injector, frontload the prepared glass needle with two microliters of antibody mixture on the micro manipulator. Now tune the input pressure to 80 to 90 pounds per square inch and the injection pressure to 20 pounds per square inch.Calibrate the injection volume using a micrometer under the microscope. Set the tune time duration according to the size of the needle opening from 10 to 120 milliseconds. Tap the paddle to deliver each injection pulse and tune the injection volume of each delivery to four to five nanoliters.Then poke the tip of the micro injection needle through the dorsal roof plate of the hind brain, posterior to the rondimere zero-one hinge point and inject about 10 nanoliters of antibody mixture without hitting the brain tissue. Observed the flowing of red fluids in the brain ventricle. After injection, remove the needle tip from the embryo swiftly by rotating the knob of the micro manipulator.For a successful injection, the red dye of the injected mixture remains in the brain ventricle stably without leaking into the surrounded agarose. Move the mounting plate under the microscope to locate another mounted embryo at a suitable position for repeats. After injecting six to eight embryos, peel the agarose with a microsurgical knife to release the embryos from the embedded agarose.Transfer the micro injected embryos to a fresh dish with 30 milliliters of embryo medium and place them at room temperature for the next steps. After 30 minutes, transfer the selected embryos to 10 milliliters of embryo medium. To mount the embryos, prepare 0.8%low melting point agarose containing the same concentration of tricane in the tube.Use a glass pipette to immerse the injected embryos in the warm agarose for three seconds. Then immediately remove the embryos from the agarose with the same glass pipette and place the embryos in the center of 35 millimeter glass bottom culture dishes with a drop of agarose from the tube. Orient the embryos gently with a fiber probe or loading tip to keep the dorsal side of the embryonic brain as close to the glass bottom as possible.Turn the embryo position gently to extend the embryo without curling as the agarose solidifies gradually. Afterward, check the embryo position by flipping the glass bottom dish over. Ensure that the whole dorsal forebrain with the correctly mounted embryos can be seen under the microscope.Add two to three milliliters of 28.5 degrees Celsius preheated embryonic medium containing tricane to cover the embryo. Place the dish properly on the temperature controlled stage of the confocal microscope and adjust the temperature of the imaging chamber to 28.5 degrees Celsius. The embryo is now ready for imaging.The Atto-647N injected embryos showed background fluorescence in the brain ventricle. The anti-Dld-Atto-647N injected zebrafish embryos showed large amounts of internalized fluorescent particles in most cells of the developing forebrain. Time lapse imaging showed the movement of intracellular Anti-Dld-Atto-647N endosomes during cell division.Most mitotic radial glia progenitors showed anterior or posterior asymmetric segregation of anti-Dld-Atto-647N endosomes into two daughter cells. The asymmetry stabilized toward the end of anaphase, resulting in asymmetric inheritance of notch signaling endosomes by the daughter cells afterward. Make sure the needle has been put into the hidden brand ventricle at the right place and check if there is any leakage after microinjection.In addition to labeling notch delta signaling, the protocol is applicable for labeling other membrane protein or extracellular proteins using similar strategies, if a good antibody to the extracellular domain is available.

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Developmental Biology

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Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

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The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

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Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...