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>
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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>
			<td></td>
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			<td>air pressure injector&#160;</td>
			<td>Narishige</td>
			<td>IM300</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse-IgG-Atto647N&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>50185</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.2H2O&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></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>
			<td></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>
		</tr>
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			<td>Dumont Medical Tweezers Style 5</td>
			<td>Thomas Scientific</td>
			<td>72877-D</td>
			<td></td>
		</tr>
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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>
			<td></td>
		</tr>
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			<td>MgSO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>M2773</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulators</td>
			<td>World Precision Instruments</td>
			<td>WPI M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Dld&#160;</td>
			<td>Abcam</td>
			<td>AB_1268496</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG blocking buffer from Zenon</td>
			<td>Thermofisher Scientific</td>
			<td>Z25008</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
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		<tr>
			<td>Stemi 2000&#160;&#160;</td>
			<td>Zeiss&#160;</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPureTM low melting point agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></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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