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 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.
Laboratoire de Radiopathologie, CEA DSV iRCM SCSR; INSERM, U967; Université Paris Diderot, Sorbonne Paris Cité; Université Paris Sud, UMR 967.
Olivier
O
Etienne
Laboratoire de Radiopathologie, CEA DSV iRCM SCSR; INSERM, U967; Université Paris Diderot, Sorbonne Paris Cité; Université Paris Sud, UMR 967.
Amandine
A
Bery
Laboratoire de Radiopathologie, CEA DSV iRCM SCSR; INSERM, U967; Université Paris Diderot, Sorbonne Paris Cité; Université Paris Sud, UMR 967.
Telma
T
Roque
Laboratoire de Radiopathologie, CEA DSV iRCM SCSR; INSERM, U967; Université Paris Diderot, Sorbonne Paris Cité; Université Paris Sud, UMR 967.
Chantal
C
Desmaze
Laboratoire de Radiopathologie, CEA DSV iRCM SCSR; INSERM, U967; Université Paris Diderot, Sorbonne Paris Cité; Université Paris Sud, UMR 967; boussin@cea.fr.
François D
FD
Boussin
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology* / Q000528:radiation effects; D001973:Bromodeoxyuridine / Q000008:administration & dosage / Q000737:chemistry*; D002453:Cell Cycle / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D003857:Deoxyuridine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry; D005260:Female; D059585:G1 Phase Cell Cycle Checkpoints / Q000235:genetics / Q000528:radiation effects; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D058953:Neural Stem Cells / Q000166:cytology / Q000502:physiology*; D011247:Pregnancy; D059807:S Phase Cell Cycle Checkpoints / Q000235:genetics / Q000528:radiation effects; D013234:Stem Cells / Q000166:cytology / Q000502:physiology* / Q000528:radiation effects; D014916:Whole-Body Irradiation
D001973:Bromodeoxyuridine; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine