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...

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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.

Research

JoVE Journal

Neuroscience

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Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

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

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