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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Drosophila larvae collection

NOTE: Prepare the yeast plate, grape paste, and the Fly condo before starting:

  1. 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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
10 µL Pipette tipsDenville SciP2102
1000 µL Pipette tipsDenville SciP2103-N
1000 µL PipettorGilsonP1000
16% paraformaldehyde (10 x 10 mL)Electron Microscopy Sciences2912.60.0000Used for Fixation of Larval Brains
20 µL PipetteGilsonP20
200 µL Pipette tipsGilsonP200
200 µL Pipette tipsDenville Sci1158U56
24-well multiwell culture platesFisher Scientific50-197-4477
35 mm Petri dishesFisher Scientific08-757-100AGrape Plate Ingredients
4 °C refrigeratorFisher ScientificProvides an ideal temperature for >24 h incubations in antibody solution
63x ObjectiveLecia
Active dry yeastMost supermarkets
AgaroseFisher Scientific214010Grape Plate Ingredients
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dyeThermo Fisher ScientificC10340to label proliferating cells
Confocal MicroscopeLeicaSP8
Coverslips 22 mm x 22 mm x 1 mm , 10 pack of 4 ozFisher Scientific12-544-10Two 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 mmFisher Scientific12-545EThe coverslip is placed on two square coverslips on the microscope slide ensuring that the brain in the antifade does not move while imaging.
Dissecting microscopeZeissStemi 2000
Ethanol 200 proof (100%), Decon Labs, 1 gallon bottleFisher Scientific2701Used to wash off the larvae before the 24 hr hold in culture medium
Fetal Bovine Serum (10%)SigmaF4135-100MLSupplement for cell culture media.
Fine forceps for dissectionFine Science Tools11295-20Forcepts used in disections. They work best when sharpened.
Fly Bottles for CrossingGenessee Scientific32-130This 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
GlutathioneSigmaG6013Provides oxidative protection during cell culture.
Goat SerumSigmaG9023- 10MLBlocking Agent
Grape PlatesMade in houseMade in houseGrape juice/agarose plates for collecting freshly hatched eggs
Image JImagej.net/fiji/downloadsFree Download:  https://fiji.scImaging platform that is used to count cells and Edu reactivation
IncubatorThermo Fisher ScientificEnsures that the temperature, humidity, and light exposure is exactly the same throughout experiment.
InsulinSigmaI0516Independant variable of the experiment
Laminar flow hoodFor aliquoting culture media
L-GlutamineSigmaG7513Provides support during cell culture
Nunc 72-well Microwell Mini TraysFisher Scientific12-565-154Immunostaining steps are performed in this tray
ParafilmFisher ScientificS37440Film used to seal plates in order to prevent evaporation
Pen-StrepSigmaP4458-100mlAntibiodics used to prevent bacterial contamination of cells during culture.
Phosphate Buffer, pH7.4Made in houseMade in houseSolvent used to wash the brains after fixing and staining steps
PickFine Science Tools10140-01Used to pick larvae off of the grape plate
Propionic acidFisher ScientificA-258Grape Plate Ingredients
Rabbit 405Abcamab175653Antibodies used for immunostaining
Rat 555Abcamab150166Antibodies used for immunostaining
Rb ScribbleA Gift from Chris DoeAntibodies used for immunostaining
Rt DeadpanAbcamab195173Antibodies used for immunostaining
Schneiders Culture MediumLife Tech21720024Contains nutrients that help the cells grow and proliferate
SlowFade Diamond Antifade (5 x 2 mL)Life TechS36963Reagent that provides protection against fading fluorophores
Sterile WaterAutoclave Milli-Q water made in houseNeeded for Solutions
SucroseFisherS2-12Grape Plate Ingredients
Superfrost Microscope SlidesFisher Scientific12-544-7
SuperglueMost supermarkets
TegoseptGenesee Scientific20-259Grape Plate Ingredients
Triton-X 100SigmaT9284-100MLPBT
Welch's 100% grape grape juiceMost supermarketsGrape Plate Ingredients

References

  1. Suman, S., Domingues, A., Ratajczak, J., Ratajczak, M. Z. Potential clinical applications of stem cells in regenerative medicine. Advances in Experimental Medicine and Biology. 1201, 1-22 (2019).
  2. Tabar, V., Studer, L.

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