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