Name: neural stem cell reactivation in cultured drosophila brain explants
Uploaded: 2022-05-18T14:00:00
Description: Watch this Scientific Journal Video about Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants at JoVE.com

We acknowledge the LSAMP Bridges to Doctorate program for funding (CNK) as well as NIH/NIGMS (R01-GM120421 and R35-GM141886). We are grateful to Dr. Conor Sipe for Figure 1. We also thank all Siegrist lab members for their continued support and mentorship. We especially thank Chhavi Sood and Gary Teeters for their careful reading of the manuscript and for providing comments.

1. Drosophila larvae collection
NOTE: Prepare the yeast plate, grape paste, and the Fly condo before starting:
<ol>
	<li>Yeast paste: In a small container, mix 5 g of active dry yeast with 10 mL of water to form a paste that has the consistency of peanut butter. Cover the yeast paste with plastic wrap and use a rubber band to firmly attach it to the container. 
	NOTE: Fresh yeast paste will expand in its container and will pop off the lid unless firmly attached...

<ol>
	<li>Suman, S., Domingues, A., Ratajczak, J., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+clinical+applications+of+stem+cells+in+regenerative+medicine.">Potential clinical applications of stem cells in regenerative medicine.</a> Advances in Experimental Medicine and Biology. 1201, 1-22 (2019).</li><li>Tabar, V., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+in+regenerative+medicine:+challenges+and+recent+progress.">Pluripotent stem cells in regenerative medicine: challenges and recent progress.</a> Nature Reviews Genetics. 15, 82-92 (2014).</li><li>Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+and+the+evolving+notion+of+cellular+identity.">Stem cells and the evolving notion of cellular identity.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 370, 20140376 (2015).</li><li>Rodrigues, M., Kosaric, N., Bonham, C. A., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing:+A+cellular+perspective.">Wound healing: A cellular perspective.</a> Physiological Reviews. 99, 665-706 (2019).</li><li>van Velthoven, C. T. J., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+quiescence:+Dynamism,+restraint,+and+cellular+idling.">Stem cell quiescence: Dynamism, restraint, and cellular idling.</a> Cell Stem Cell. 24, 213-225 (2019).</li><li>Chapman, N. M., Boothby, M. R., Chi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+coordination+of+T+cell+quiescence+and+activation.">Metabolic coordination of T cell quiescence and activation.</a> Nature Reviews Immunology. 20, 55-70 (2020).</li><li>Wosczyna, M. N., Rando, T. 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+muscle+stem+cell+support+group:+Coordinated+cellular+responses+in+muscle+regeneration.">A muscle stem cell support group: Coordinated cellular responses in muscle regeneration.</a> Developmental Cell. 46, 135-143 (2018).</li><li>Homem, C. C., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila neuroblasts: a model for stem cell biology.</a> Development. 139, 4297-4310 (2012).</li><li>Kang, K. H., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neural+stem+cell+self-renewal+and+differentiation+in+Drosophila.">Control of neural stem cell self-renewal and differentiation in Drosophila.</a> Cell and Tissue Research. 359, 33-45 (2015).</li><li>Chell, J. M., Brand, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrition-responsive+glia+control+exit+of+neural+stem+cells+from+quiescence.">Nutrition-responsive glia control exit of neural stem cells from quiescence.</a> Cell. 143, 1161-1173 (2010).</li><li>Sousa-Nunes, R., Yee, L. L., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+cells+reactivate+quiescent+neuroblasts+via+TOR+and+glial+insulin+relays+in+Drosophila.">Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila.</a> Nature. 471, 508-512 (2011).</li><li>Britton, J. S., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+control+of+the+cell+cycle+in+Drosophila:+nutrition+activates+mitotic+and+endoreplicative+cells+by+distinct+mechanisms.">Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms.</a> Development. 125, 2149-2158 (1998).</li><li>Lin, S., 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=Extremes+of+lineage+plasticity+in+the+Drosophila+brain.">Extremes of lineage plasticity in the Drosophila brain.</a> Current biology : CB. 23, 1908-1913 (2013).</li><li>Sipe, C. W., Siegrist, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eyeless+uncouples+mushroom+body+neuroblast+proliferation+from+dietary+amino+acids+in+Drosophila.">Eyeless uncouples mushroom body neuroblast proliferation from dietary amino acids in Drosophila.</a> Elife. 6, 26343 (2017).</li><li>Speder, P., Brand, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+and+local+cues+drive+neural+stem+cell+niche+remodelling+during+neurogenesis+in+Drosophila.">Systemic and local cues drive neural stem cell niche remodelling during neurogenesis in Drosophila.</a> Elife. 7, 30413 (2018).</li><li>Yuan, X., Sipe, C. W., Suzawa, M., Bland, M. L., Siegrist, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilp-2-mediated+PI3-kinase+activation+coordinates+reactivation+of+quiescent+neuroblasts+with+growth+of+their+glial+stem+cell+niche.">Dilp-2-mediated PI3-kinase activation coordinates reactivation of quiescent neuroblasts with growth of their glial stem cell niche.</a> PLoS Biology. 18, 3000721 (2020).</li><li>Colombani, J., 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=A+nutrient+sensor+mechanism+controls+Drosophila+growth.">A nutrient sensor mechanism controls Drosophila growth.</a> Cell. 114, 739-749 (2003).</li><li>Geminard, C., Rulifson, E. J., Leopold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+insulin+secretion+by+fat+cells+in+Drosophila.">Remote control of insulin secretion by fat cells in Drosophila.</a> Cell Metabolism. 10, 199-207 (2009).</li><li>Siller, K. H., Serr, M., Steward, R., Hays, T. S., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Drosophila+brain+neuroblasts+reveals+a+role+for+Lis1/dynactin+in+spindle+assembly+and+mitotic+checkpoint+control.">Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/dynactin in spindle assembly and mitotic checkpoint control.</a> Molecular Biology of the Cell. 16, 5127-5140 (2005).</li><li>Prithviraj, R., Trunova, S., Giniger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culturing+of+whole,+developing+Drosophila+brains.">Ex vivo culturing of whole, developing Drosophila brains.</a> Journal of Visualized Experiments: JoVE. (65), e4270 (2012).</li><li>Bostock, M. P., 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=An+immobilization+technique+for+long-term+time-lapse+imaging+of+explanted+drosophila+tissues.">An immobilization technique for long-term time-lapse imaging of explanted drosophila tissues.</a> Frontiers in Cell and Developmental Biology. 8, 590094 (2020).</li><li>Datta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+neuroblast+proliferation+in+explant+culture+of+the+Drosophila+larval+CNS.">Activation of neuroblast proliferation in explant culture of the Drosophila larval CNS.</a> Brain Research. 818, 77-83 (1999).</li></ol>

The method described here to culture brain explants can be carried out in most lab environments. The tools required, as well as the procedure and data collection, are simple and straightforward. With this method, one can test a variety of hypotheses, including those related to the cell signaling cascades and extrinsic factors that regulate NB reactivation and proliferation. Here, using wild-type OregonR animals, we found that exogenous insulin was sufficient to reactivate NBs from quiescence independent of other animal-s...

Stem cells are of great interest because of their potential for use in regenerative medicine1,2. Many animals, especially those that are long-lived, maintain stem cells within their adult tissues. These resident stem cells function to maintain tissue homeostasis and are utilized for repair following physical injury or disease3,4. Most stem cells in adult animals are quiescent, a relatively dormant state characterized by cell cycle arrest and inactivation of growth signaling5. In response to extrinsic cues, stem cells exit from quiescence, enter the cell cycle and begin generating daughter progeny specific to their tissue type. For example, in order to mount an effective immune response, antigen-presenting cells induce quiescent naive T cells to enter cell cycle and clonally expand6. In response to skeletal muscle damage, muscle satellite stem cells enter the cell cycle and generate daughter myoblasts to replace damaged myofibrils5,7. While it is clear that quiescent stem cells respond to extrinsic signals, in many cases, the nature of the extrinsic cue remains unclear as well as the mechanism of cue-induced stem cell activation. Gaining a better understanding of how quiescent stem cells respond to extrinsic cues and enter the cell cycle will aid in the development of better stem cell therapies in the clinic and increase scientific knowledge.
For decades now, model organisms have been used to uncover the genes and cell signaling pathways that regulate stem cell proliferation during development and in adulthood. In Drosophila, neural stem cells (NSCs), known as neuroblasts (NBs), divide throughout development to generate all neurons and glia that ultimately integrate, forming the neural circuity required for brain function8,9. Like other stem cells, NBs divide asymmetrically to self-renew and, in some cases, symmetrically to expand the stem cell pool. NBs are specified during embryogenesis and most enter quiescence towards the end, coincident with declining maternal nutrient stores (Figure 1). After embryogenesis is complete, larvae hatch and begin feeding. In response to animal feeding, NBs reactivate from quiescence and resume cell divisions10,11,12,13,14,15,16. Because the Drosophila CNS is relatively simple and because NBs enter and exit quiescence at defined times, using Drosophila to investigate the regulation of quiescence, entry and exit, proves ideal.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63189/63189fig01.jpg" /> 
Figure 1: Relative proliferation of CB NBs (central brain neuroblasts, red) and MB NBs (mushroom body neuroblasts, blue) over developmental time. At the end of embryogenesis, most NBs (red line) cease proliferation and enter quiescence. Quiescence continues until freshly hatched larvae consume their first complete meal. The time points of focus for this methodology are denoted in red circles (1, quiescence and 2, reactivation). MB NBs (blue) are a subset of central brain NBs that divide continually throughout development (4 per brain hemisphere). <a href="https://www.jove.com/files/ftp_upload/63189/63189fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In response to animal feeding, PI3-kinase and TOR growth signaling pathways become active in NBs and in their glial and tracheal niche10,11,15,16. When dietary nutrients are withdrawn or when levels of PI3-kinase are reduced, NBs fail to reactivate and growth of glia and trachea are also reduced10,11,15,16. The current model posits that NB reactivation is coupled to larval growth by the fat body, which releases a systemic signal in response to animal feeding12,17,18. This signal, which remains elusive, likely promotes the expression and release of Drosophila insulin-like peptide (Dilps) in the brain, which leads to the downstream activation of PI3-kinase in NBs and their glial and tracheal niche. To better understand the nature of the systemic cue(s), we developed a method to reactivate quiescent NBs in cultured brain explants. With this method, reactivation of NBs can be assayed in the absence of whole animal systemic cues. Exogenous factors can be resupplied to the culture media and NB reactivation assayed based on the incorporation of the thymidine analog, EdU. Using this method, we determined that exogenous insulin is sufficient to reactivate quiescent NBs in brain explants. Future work will be aimed at identifying additional factors that, when added back, either positively or negatively regulate NB quiescence in brain explants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#181;L Pipette tips</td>
			<td>Denville Sci</td>
			<td>P2102</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Pipette tips</td>
			<td>Denville Sci</td>
			<td>P2103-N</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L Pipettor</td>
			<td>Gilson</td>
			<td>P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde (10 x 10 mL)</td>
			<td>Electron Microscopy Sciences</td>
			<td>2912.60.0000</td>
			<td>Used for Fixation of Larval Brains</td>
		</tr>
		<tr>
			<td>20 &#181;L Pipette</td>
			<td>Gilson</td>
			<td>P20</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipette tips</td>
			<td>Gilson</td>
			<td>P200</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L Pipette tips</td>
			<td>Denville Sci</td>
			<td>1158U56</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well multiwell culture plates</td>
			<td>Fisher Scientific</td>
			<td>50-197-4477</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-100A</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>4 &#176;C refrigerator</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>Provides an ideal temperature for &#62;24 h incubations in antibody solution</td>
		</tr>
		<tr>
			<td>63x Objective</td>
			<td>Lecia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Active dry yeast</td>
			<td>Most supermarkets</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>214010</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10340</td>
			<td>to label proliferating cells</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica</td>
			<td>SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 22 mm x 22 mm x 1 mm , 10 pack of 4 oz</td>
			<td>Fisher Scientific</td>
			<td>12-544-10</td>
			<td>Two Coverslips are super glued to the ends of the microscope slide. This creates a space that allows for the brains to float in antifade while being imaged.</td>
		</tr>
		<tr>
			<td>Coverslips, 22 mm x 50 mm x 1 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545E</td>
			<td>The coverslip is placed on two square coverslips on the microscope slide ensuring that the brain in the antifade does not move while imaging.</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof (100%), Decon Labs, 1 gallon bottle</td>
			<td>Fisher Scientific</td>
			<td>2701</td>
			<td>Used to wash off the larvae before the 24 hr hold in culture medium</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (10%)</td>
			<td>Sigma</td>
			<td>F4135-100ML</td>
			<td>Supplement for cell culture media.</td>
		</tr>
		<tr>
			<td>Fine forceps for dissection</td>
			<td>Fine Science Tools</td>
			<td>11295-20</td>
			<td>Forcepts used in disections. They work best when sharpened.</td>
		</tr>
		<tr>
			<td>Fly Bottles for Crossing</td>
			<td>Genessee Scientific</td>
			<td>32-130</td>
			<td>This bottle is used as a container that lets the flies lay eggs on the grape plate.</td>
		</tr>
		<tr>
			<td>Glass Dissection Dish (3 well)</td>
			<td>These are no longer available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>Sigma</td>
			<td>G6013</td>
			<td>Provides oxidative protection during cell culture.</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Sigma</td>
			<td>G9023- 10ML</td>
			<td>Blocking Agent</td>
		</tr>
		<tr>
			<td>Grape Plates</td>
			<td>Made in house</td>
			<td>Made in house</td>
			<td>Grape juice/agarose plates for collecting freshly hatched eggs</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>Imagej.net/fiji/downloads</td>
			<td>Free Download:&#160; https://fiji.sc</td>
			<td>Imaging platform that is used to count cells and Edu reactivation</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Ensures that the temperature, humidity, and light exposure is exactly the same throughout experiment.</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I0516</td>
			<td>Independant variable of the experiment</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td></td>
			<td></td>
			<td>For aliquoting culture media</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td>Provides support during cell culture</td>
		</tr>
		<tr>
			<td>Nunc 72-well Microwell Mini Trays</td>
			<td>Fisher Scientific</td>
			<td>12-565-154</td>
			<td>Immunostaining steps are performed in this tray</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>S37440</td>
			<td>Film used to seal plates in order to prevent evaporation</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Sigma</td>
			<td>P4458-100ml</td>
			<td>Antibiodics used to prevent bacterial contamination of cells during culture.</td>
		</tr>
		<tr>
			<td>Phosphate Buffer, pH7.4</td>
			<td>Made in house</td>
			<td>Made in house</td>
			<td>Solvent used to wash the brains after fixing and staining steps</td>
		</tr>
		<tr>
			<td>Pick</td>
			<td>Fine Science Tools</td>
			<td>10140-01</td>
			<td>Used to pick larvae off of the grape plate</td>
		</tr>
		<tr>
			<td>Propionic acid</td>
			<td>Fisher Scientific</td>
			<td>A-258</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>Rabbit 405</td>
			<td>Abcam</td>
			<td>ab175653</td>
			<td>Antibodies used for immunostaining</td>
		</tr>
		<tr>
			<td>Rat 555</td>
			<td>Abcam</td>
			<td>ab150166</td>
			<td>Antibodies used for immunostaining</td>
		</tr>
		<tr>
			<td>Rb Scribble</td>
			<td>A Gift from Chris Doe</td>
			<td></td>
			<td>Antibodies used for immunostaining</td>
		</tr>
		<tr>
			<td>Rt Deadpan</td>
			<td>Abcam</td>
			<td>ab195173</td>
			<td>Antibodies used for immunostaining</td>
		</tr>
		<tr>
			<td>Schneiders Culture Medium</td>
			<td>Life Tech</td>
			<td>21720024</td>
			<td>Contains nutrients that help the cells grow and proliferate</td>
		</tr>
		<tr>
			<td>SlowFade Diamond Antifade (5 x 2 mL)</td>
			<td>Life Tech</td>
			<td>S36963</td>
			<td>Reagent that provides protection against fading fluorophores</td>
		</tr>
		<tr>
			<td>Sterile Water</td>
			<td>Autoclave Milli-Q water made in house</td>
			<td></td>
			<td>Needed for Solutions</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher</td>
			<td>S2-12</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>Superfrost Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Most supermarkets</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept</td>
			<td>Genesee Scientific</td>
			<td>20-259</td>
			<td>Grape Plate Ingredients</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma</td>
			<td>T9284-100ML</td>
			<td>PBT</td>
		</tr>
		<tr>
			<td>Welch&#39;s 100% grape grape juice</td>
			<td>Most supermarkets</td>
			<td></td>
			<td>Grape Plate Ingredients</td>
		</tr>
	</tbody>
</table>

neural stem cell reactivation in cultured drosophila brain explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes are controlled by the complex interplay between NSC intrinsic genetic programs with NSC extrinsic factors, local and systemic. In the genetic model organism, Drosophila melanogaster, NSCs, known as neuroblasts (NBs), switch from quiescence to proliferation during the embryonic to larval transition. During this time, larvae emerge from their eggshells and begin crawling, seeking out dietary nutrients. In response to animal feeding, the fat body, an endocrine organ with lipid storage capacity, produces a signal, which is released systemically into the circulating hemolymph. In response to the fat body-derived signal (FBDS), Drosophila insulin-like peptides (Dilps) are produced and released from brain neurosecretory neurons and glia, leading to downstream activation of PI3-kinase growth signaling in NBs and their glial and tracheal niche. Although this is the current model for how NBs switch from quiescence to proliferation, the nature of the FBDS extrinsic cue remains elusive. To better understand how NB extrinsic systemic cues regulate exit from quiescence, a method was developed to culture early larval brains in vitro before animal feeding. With this method, exogenous factors can be supplied to the culture media and NB exit from quiescence assayed. We found that exogenous insulin is sufficient to reactivate NBs from quiescence in whole-brain explants. Because this method is well-suited for large-scale screens, we aim to identify additional extrinsic cues that regulate NB quiescence versus proliferation decisions. Because the genes and pathways that regulate NSC proliferation decisions are evolutionarily conserved, results from this assay could provide insight into improving regenerative therapies in the clinic.

Freshly hatched OregonR wild-type brains were dissected and cultured for 24 h in supplemented Schneider&#39;s media (SSM) with insulin. Tissues were fixed and stained according to the protocol. Primary antibodies generated against Deadpan (Dpn) to detect NBs and Scribble to label cell membranes were used. The thymidine analog 5-Ethynyl-2&#8242;-deoxyuridine (Edu) was added to detect S-phase entry and NB reactivation. We found large sized Edu positive and Dpn positive NBs after 24 h in culture (Figure...

Watch this Scientific Journal Video about Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants at JoVE.com

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit.

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants

Neural stem cells (NSCs) have the ability to proliferate, differentiate, undergo apoptosis, and even enter and exit quiescence. Many of these processes ...

We developed a method to reactivate quiescent neural stem cells in cultured drosophila brain explants. This method can supply exogenous factors to the culture media and can assay neuroblast reactivation. Better stem cell therapies could be developed by a better understanding of how quiescent stem cells respond to extrinsic cues and enter the cell cycle.Demonstrating the procedure will be Susan Doyle, a research scientist from my laboratory. To begin carefully pick approximately 20 to 25 freshly hatched larvae from the grape agar plate under a dissecting microscope. Fill a Petri dish with about two milliliters of PBS and submerge the tool's tip containing larvae in PBS for two minutes.After two minutes, tip the dish at an angle to pool the liquid at the bottom, and using a small paint brush, brush the larvae out of the liquid up the bottom of the Petri dish. Collect all larvae on the paint brush and rinse the larvae briefly in 70%ethanol before transferring them back to the Petri dish containing PBS. Spray the work area, dissection tools, forceps, and two glass watch dishes with 70%ethanol and let them dry on the bench.Make the supplemented Schneider's culture medium, or SSM, and place it on ice. Pipette one milliliter of the medium into each glass watch dish. Using a micropipette with a sterile tip, transfer the freshly hatched larvae from the plate of PBS to the SSM in the first glass watch dish.Dissect the brains out of the larvae placed in the second glass watch dish with SSM using forceps under a dissecting microscope and adjusting the magnification as needed. Use one forceps to grab the mouth hooks, and with the other, gently grab the body halfway down and pull in the opposite direction to split the larva into two pieces. After dissecting 15 to 20 brains, add one milliliter of SSM into one well of a sterile 24-well culture tray.Using a micropipette and a sterile tip, transfer the freshly dissected brains into the SSM, followed by incubation of the media with brains for 24 hours at 25 degrees Celsius. Pipette 10 microliters of a 10-millimolar stock of 5-ethynyl-2'deoxyuridine, or EDU, with 990 microliters of SSM into a sterile microcentrifuge tube, mix, then pipette one milliliter of EDU SSM into one well of the sterile 12-well culture tray. Transfer the brains using a micropipette with a sterile tip from the well containing SSM to the new well containing the EDU SSM solution and incubate for one hour at 25 degrees Celsius.Next, transfer the EDU-labeled brains to another well in the same culture tray containing one milliliter of fixative and allow the brains to be fixed for 20 minutes. After fixation, quickly transfer the brains to a 72-well minitray wells using a micropipette. Rinse the brains three times in 10 microliters of PBT and repeat three washes for 10 minutes each, ensuring the brains are always covered with some liquid.Pipette 10 microliters of blocking solution onto the brains. Cover the tray and seal it with a strip of parafilm around the edge. The figure shows large-sized EDU-positive and deadpan-positive neuroblast cells after 24 hours of culturing in SSM with insulin and staining.After 24 hours in supplemented Schneider's media without insulin, the freshly hatched Oregon R wild type brains had no large-sized EDU-positive and deadpan-positive neuroblast cells, other than the four-mushroom-body neuroblast cells and one ventrolateral neuroblast cell. During confocal imaging, some brain hemispheres with damage, like small to large sized holes in the explanted brain tissue, were occasionally observed. Any brains with holes were not used for analysis.Dissect tissues carefully and pipette gently to avoid any tissue damage. Also try to be as sterile as possible and not contaminate your cultures. Because the culture media can be easily manipulated by adding different factors, This technique can be used to address future hypotheses regarding extrinsic signaling and neuroblast quiescence entry and exit.

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Research

JoVE Journal

Neuroscience

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...