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

Summary

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.

Abstract

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 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.

Introduction

During embryonic brain development, projection neurons of the cerebral cortex are generated in the ventricular zone, a pseudostratified epithelium composed of different types of neural stem and progenitor cells (NSPC) that lines the lateral ventricles. Among NSPC, the radial glial cells (RGC), which serve as neural stem cells, undergo interkinetic nuclear migration (INM): they perform mitosis at the surface of the ventricle and S-phase at the basal limit of the ventricular zone (VZ)^1,2,3. They can divide either symmetrically to generate two RGC or asymmetrically to generate one RGC and one neuron or an intermediate progenitor cell (IPC)⁴

Protocol

1. Animal Procedures

This protocol has been designed in compliance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and has been approved by our institutional committee on animal welfare (CETEA-CEA DSV IdF).

1. Injections with EdU/BrdU and irradiation of E14.5 pregnant mice (Figure 2A).
   1. Prepare a solution of EdU at 1 mg/ml and BrdU at 5 mg/ml in PBS. Perform an intra-peritoneal injection (IP) of 100 µl of EdU solution using a 25 G needle into pregnant mice. 1.5 hr later, irradiate the mice (total body irradiation) at 0 Gy or 2 Gy (0.6 Gy/min). Immediately ....

Results

In the experiment described in Figure 2, EdU was administered 1.5 hr before irradiation and BrdU just after irradiation. Four types of cells were then distinguished in the cortical slices prepared at 1 or 4 hr post-irradiation, according to the incorporation of either EdU or BrdU, both or none (Figures 2A and 2B). Importantly, neither EdU nor BrdU incorporation changed the level of radiation-induced apoptosis (data not shown). Moreover, the staining methods allow only th.......

Discussion

The experimental design described here based on incorporation of EdU 1.5 hr before irradiation and incorporation of BrdU immediately after irradiation allowed the demonstration that NSPCs are able to activate S and G2/M checkpoints but not the G1/S checkpoint during the 1^st hr after a genotoxic stress in the fetal mouse brain. We performed other experiments in which EdU has been injected at different times after irradiation and BrdU, just 1 hr before mice sacrifices, allowing us to specifically appreciate the .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EdU	Life Technologies	A10044	1 mg/ml
BrdU	Life Technologies	B5002	5 mg/ml
IBL 637	CIS BIO International
Tissu Tek VIP	Leica
Microtome	Leica	RM 2125 RT
Triton X-100	Sigma-Aldrich	93443
Click-iT EdU Alexa 488 imaging kit	Life Technologies	C10083
Anti-BrdU	GE Healthcare	RPN202	1/300
Goat anti mouse-Alex594	Life Technologies	A11001	1/400
Fluoromount	SouthernBiotech	0100-01
Polysine slide	Thermo scientific	J2800AMNZ
Paraformaldehyde	Sigma-Aldrich	P6148
PBS	Life Technologies	20012-068
DAPI	Sigma-Aldrich	D9542
Microscope BX51	Olympus
Confocal microscope SPE	Leica
Prism software	Graphpad		Version 5.0c
Photoshop software	Adobe