The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n&#176; 323267, from Electricit&#233; de France (EDF) and from l&#8217;Agence Nationale de la Recherche &#8211; Sant&#233;-Environnement et Sant&#233;-Travail (ANR-SEST, Neurorad).

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).
<ol>
	<li>Injections with EdU/BrdU and irradiation of E14.5 pregnant mice (Figure 2A).
	<ol>
		<li>Prepare a solution of EdU at 1 mg/ml&#160;and BrdU at 5 mg/ml&#160;in PBS. Perform an intra-peritoneal injection (IP) of 100 &#181;l 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 after, inject (IP) 200 &#181;l of BrdU solution using a 25 G&#160;needle into the pregnant mice.</li>
	</ol>
	</li>
	<li>Preparation of coronal slices of embryonic brains.
	<ol>
		<li>At 1 hr or 4 hr after irradiation, euthanize the pregnant mice by carbon dioxide.</li>
		<li>Open the abdominal cavity over three centimeters using scissors. Separate the two uterine horns from the rest of the uterus by sectioning both extremities of the horns. Transfer the uterine horns into a Petri dish containing PBS 0.6% glucose. Open the uterine horns with the forceps in order to isolate the embryos. Remove the amniotic sac of each embryo using forceps.</li>
		<li>Dissect embryonic heads with forceps and fix them by immersion in 4% paraformaldehyde (PFA, pH=7.4) O/N at 4 &#176;C.</li>
		<li>Rinse embryonic heads in PBS for at least one night at 4 &#176;C. Heads can be kept in PBS for several days.</li>
		<li>Embed heads in paraffin using a vacuum infiltration processor as indicated in Table 1. Heads embedding can also be performed under a chemical hood for ethanol and xylen baths, and then in a 65 &#176;C oven for the paraffin baths.</li>
		<li>Prepare 5-&#181;m thick coronal sections with a microtome.</li>
		<li>Mount onto polylysine-coated microscope slides.</li>
		<li>Slides can be kept at room temperature (RT) for several months.</li>
	</ol>
	</li>
</ol>
2. EdU/BrdU Staining
<ol>
	<li>Deparaffinization and antigen unmasking
	<ol>
		<li>Deparaffinize the paraffin-embedded brain sections by immersion of slides in 3 baths of toluene for 5 min.</li>
		<li>Rehydrate for 3 min in 2 baths of each solutions of decreasing ethanol concentration as following: ethanol 100%, ethanol 95%, ethanol 70%, and 5 min in H2O. Slides can be kept in deionized water for several hr.</li>
		<li>Boil the slices for 10 min in citrate solution (10 mM, pH 6), and then incubate in deionized water for 5 min.</li>
	</ol>
	</li>
	<li>EdU staining
	<ol>
		<li>Perform EdU detection according to the manufacturer&#8217;s protocol. Permeabilize cells with 0.5% triton X-100 in PBS for 10min.</li>
		<li>Prepare 1X EdU buffer additive by diluting the 10X solution in deionized water. This solution should be freshly made and used on the same day.</li>
		<li>Prepare EdU reaction cocktail, including EdU Alexa Fluor 488 azide. It is important to add the ingredients following the order listed in Table 2, otherwise, the reaction will not proceed optimally. Use the EdU reaction cocktail within 15 min of preparation.</li>
		<li>Remove the permeabilization buffer (step 2.1.1), then wash twice with PBS.</li>
		<li>Add 150 &#181;l&#160;of EdU reaction cocktail, put a coverslip, and incubate for 30 min in the dark at RT.</li>
		<li>Remove the EdU reaction cocktail, then wash once with PBS.</li>
	</ol>
	</li>
	<li>BrdU staining
	<ol>
		<li>Prepare saturation buffer with 7.5% goat serum and 7.5% fetal bovine serum in PBS. Add 150 &#181;l&#160;of saturation buffer on brain slices, put a coverslip, and incubate for 1 hr at RT to block non-specific antibody binding.</li>
		<li>Remove the coverslip. Add 150 &#181;l&#160;of mouse anti-BrdU primary antibody diluted at 1/300 in saturation buffer, put a coverslip and incubate O/N at 4 &#176;C.</li>
		<li>Remove the coverslip, then wash 3 times in PBS.</li>
		<li>Add 150 &#181;l&#160;of goat anti-mouse-Alexa594 secondary antibody diluted at 1/400 in saturation buffer, put a coverslip, and incubate for 1 hr at RT.</li>
		<li>Remove the coverslip, then wash once in PBS.</li>
		<li>Nuclear staining: add 150 &#181;l of 4&#8217;,6-diamidino-2-phenylindole (DAPI) at 1 &#181;g/ml in PBS, put a coverslip and incubate for 2 min at RT.</li>
		<li>Mount slides in mounting medium.</li>
	</ol>
	</li>
</ol>
3. Analysis
<ol>
	<li>Examine brain sections under a fluorescence microscope with a 20X objective or a confocal microscope and acquire images in three channels (488 nm, 594 nm and UV) as separates files.</li>
	<li>Stack images with Photoshop software.</li>
	<li>Analyze brain sections in a standard sector of the dorsomedial cerebral wall. This sector is 100 &#181;m in its medial-lateral dimension and is divided into 18 bins (or more) of 10 &#181;m height in its radial dimension using a grid superposed on images (Figure 1) as previously described31.
	<ol>
		<li>Align the grid such as the first bin is at the ventricular surface, with its long axis parallel to the ventricular border. Then number the labeled EdU, BrdU and/or pyknotic nuclei (corresponding to apoptotic nuclei) in each bin. Number a nucleus located on the boundary of two bins in the bin which contains its larger part, or in the lower bin, when the nucleus occupies equal areas within the two bins.</li>
	</ol>
	</li>
	<li>Statistical analysis.</li>
</ol>
Analyze at least two cortical slices per embryo. Repeat experiments on at least 3 embryos from different litters. Results should be given as mean &#177;&#160;standard error of the mean (SEM). Statistical analyses are conducted with Graphpad Prism using two-way ANOVA and Bonferroni multiple comparison posthoc tests.

<ol>
	<li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+cerebral+cortical+areas.">Specification of cerebral cortical areas.</a> Science. 241, 170-176 (1988).</li><li>Sauer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+he+neural+tube.">Mitosis in he neural tube.</a> J Comp Neurol. , 377-399 (1935).</li><li>Taverna, E., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+nuclei+IN+motion.">Neural progenitor nuclei IN motion.</a> Neuron. 67, 906-914 (2010).</li><li>Committee, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+vertebrate+central+nervous+system:+revised+terminology.">Embryonic vertebrate central nervous system: revised terminology.</a> Anat Rec. 166, 257-261 (1970).</li><li>Bystron, I., Blakemore, C., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+human+cerebral+cortex:+Boulder+Committee+revisited.">Development of the human cerebral cortex: Boulder Committee revisited.</a> Nature Reviews. Neuroscience. 9, 110-122 (2008).</li><li>Miyata, T., 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=Asymmetric+production+of+surface-dividing+and+non-surface-dividing+cortical+progenitor+cells.">Asymmetric production of surface-dividing and non-surface-dividing cortical progenitor cells.</a> Development. 131, 3133-3145 (2004).</li><li>Haubensak, W., Attardo, A., Denk, W., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+arise+in+the+basal+neuroepithelium+of+the+early+mammalian+telencephalon:+a+major+site+of+neurogenesis.">Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: a major site of neurogenesis.</a> Proc Natl Acad Sci U S A. 101, 3196-3201 (2004).</li><li>Noctor, S. C., Martinez-Cerdeno, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nat Neurosci. 7, 136-144 (2004).</li><li>Merot, Y., Retaux, S., Heng, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+projection+neuron+production+and+maturation+in+the+developing+cerebral+cortex.">Molecular mechanisms of projection neuron production and maturation in the developing cerebral cortex.</a> Seminars in Cell &amp; Developmental Biology. 20, 726-734 (2009).</li><li>Kriegstein, A. R., Noctor, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+neuronal+migration+in+the+embryonic+cortex.">Patterns of neuronal migration in the embryonic cortex.</a> Trends Neurosci. 27, 392-399 (2004).</li><li>Salomoni, P., Calegari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+of+mammalian+neural+stem+cells:+putting+a+speed+limit+on+G1.">Cell cycle control of mammalian neural stem cells: putting a speed limit on G1.</a> Trends Cell Biol. 20, 233-243 (2010).</li><li>Calegari, F., Haubensak, W., Haffner, C., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+lengthening+of+the+cell+cycle+in+the+neurogenic+subpopulation+of+neural+progenitor+cells+during+mouse+brain+development.">Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development.</a> J Neurosci. 25, 6533-6538 (2005).</li><li>Andres-Mach, M., Rola, R., Fike, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiation+effects+on+neural+precursor+cells+in+the+dentate+gyrus.">Radiation effects on neural precursor cells in the dentate gyrus.</a> Cell Tissue Res. 331, 251-262 (2008).</li><li>Semont, A., 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=Involvement+of+p53+and+Fas/CD95+in+murine+neural+progenitor+cell+response+to+ionizing+irradiation.">Involvement of p53 and Fas/CD95 in murine neural progenitor cell response to ionizing irradiation.</a> Oncogene. 23, 8497-8508 (2004).</li><li>Nowak, E., 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=Radiation-induced+H2AX+phosphorylation+and+neural+precursor+apoptosis+in+the+developing+brain+of+mice.">Radiation-induced H2AX phosphorylation and neural precursor apoptosis in the developing brain of mice.</a> Radiat Res. 165, 155-164 (2006).</li><li>Gatz, S. A., 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=Requirement+for+DNA+ligase+IV+during+embryonic+neuronal+development.">Requirement for DNA ligase IV during embryonic neuronal development.</a> J Neurosci. 31, 10088-10100 (2011).</li><li>Denekamp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+kinetics+and+radiation+biology.">Cell kinetics and radiation biology.</a> International Journal of Radiation Biology and Related Studies in Physics, Chemistry, and Medicine. 49, 357-380 (1986).</li><li>Hartwell, L. H., Weinert, 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=Checkpoints:+controls+that+ensure+the+order+of+cell+cycle+events.">Checkpoints: controls that ensure the order of cell cycle events.</a> Science. 246, 629-634 (1989).</li><li>Weinert, T. A., Hartwell, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RAD9+of+Saccharomyces+cerevisiae+and+evidence+that+its+function+acts+posttranslationally+in+cell+cycle+arrest+after+DNA+damage.">Characterization of RAD9 of Saccharomyces cerevisiae and evidence that its function acts posttranslationally in cell cycle arrest after DNA damage.</a> Mol Cell Biol. 10, 6554-6564 (1990).</li><li>Tolmach, L. J., Jones, R. W., Busse, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+action+of+caffeine+on+X-irradiated+HeLa+cells.+I.+Delayed+inhibition+of+DNA+synthesis.">The action of caffeine on X-irradiated HeLa cells. I. Delayed inhibition of DNA synthesis.</a> Radiat Res. 71, 653-665 (1977).</li><li>Painter, R. B., Young, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitivity+in+ataxia-telangiectasia:+a+new+explanation.">Radiosensitivity in ataxia-telangiectasia: a new explanation.</a> Proc Natl Acad Sci U S A. 77, 7315-7317 (1980).</li><li>Etienne, O., Roque, T., Haton, C., Boussin, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+radiation-sensitivity+of+neural+stem+and+progenitor+cell+populations+within+the+developing+mouse+brain.">Variation of radiation-sensitivity of neural stem and progenitor cell populations within the developing mouse brain.</a> Int J Radiat Biol. 88, 694-702 (2012).</li><li>Roque, T., 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=Lack+of+a+p21waf1/cip+-dependent+G1/S+checkpoint+in+neural+stem+and+progenitor+cells+after+DNA+damage+in+vivo.">Lack of a p21waf1/cip -dependent G1/S checkpoint in neural stem and progenitor cells after DNA damage in vivo.</a> Stem Cells. 30, 537-547 (2012).</li><li>Rousseau, L., 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=In+vivo+importance+of+homologous+recombination+DNA+repair+for+mouse+neural+stem+and+progenitor+cells.">In vivo importance of homologous recombination DNA repair for mouse neural stem and progenitor cells.</a> PLoS One. 7, 12 (2012).</li><li>Bradford, J. A., Clarke, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-pulse+labeling+using+5-ethynyl-2'-deoxyuridine+(EdU)+and+5-bromo-2'-deoxyuridine+(BrdU)+in+flow+cytometry.">Dual-pulse labeling using 5-ethynyl-2'-deoxyuridine (EdU) and 5-bromo-2'-deoxyuridine (BrdU) in flow cytometry.</a> Curr Protoc Cytom. , (2011).</li><li>Deckbar, D., 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=The+limitations+of+the+G1-S+checkpoint.">The limitations of the G1-S checkpoint.</a> Cancer Res. 70, 4412-4421 (2010).</li><li>Manual, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click-iT+EdU+Imaging+Kit.">Click-iT EdU Imaging Kit.</a> , (2011).</li><li>Weidtkamp-Peters, S., Rahn, H. P., Cardoso, M. C., Hemmerich, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+centromeric+heterochromatin+in+mouse+fibroblasts+takes+place+in+early,+middle,+and+late+S+phase.">Replication of centromeric heterochromatin in mouse fibroblasts takes place in early, middle, and late S phase.</a> Histochem Cell Biol. 125, 91-102 (2006).</li><li>Wu, R., Terry, A. V., Singh, P. B., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subnuclear+localization+and+replication+timing+of+histone+H3+lysine+9+methylation+states.">Differential subnuclear localization and replication timing of histone H3 lysine 9 methylation states.</a> Mol Biol Cell. 16, 2872-2881 (2005).</li><li>Wu, R., Singh, P. B., Gilbert, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+global+and+fine-tuning+replication+timing+determinants+for+mouse+pericentric+heterochromatin.">Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin.</a> J Cell Biol. 174, 185-194 (2006).</li><li>Takahashi, T., Nowakowski, R. S., Caviness Jr, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle+of+the+pseudostratified+ventricular+epithelium+of+the+embryonic+murine+cerebral+wall.">The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.</a> J Neurosci. 15, 6046-6057 (1995).</li><li>Tuttle, A. H., 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=Immunofluorescent+detection+of+two+thymidine+analogues+(CldU+and+IdU)+in+primary+tissue.">Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.</a> J Vis Exp. , (2010).</li></ol>

The authors have no conflict of interest.

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 1st 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 ...

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)4,5. IPC migrate to an overlying proliferating layer called the subventricular zone (SVZ), where after one last symmetric division they generate two immature projection neurons6-8. Contrary to RGC, IPC do not undergo INM (reviewed in 9). Newly generated neurons migrate radially along the radial fibers through the intermediate zone (IZ) to reach their final destination in the cortical plate (CP)10,8. The perfect timing of all these events is essential for a correct cortical development. For instance the switch from proliferation to differentiation of neural progenitor populations is controlled by the G1 phase duration11,12. The lengthening of the G1 phase correlates thus with cell differentiation.
Genotoxic stress, such as ionizing radiation, severely impair brain development (reviewed in 13). We and others have shown that NSPC are highly prone to radiation-induced apoptosis14,15,16. Ionizing radiations induce DNA double strand breaks that are the most severe damage to proliferating cells. One essential component of DNA Damage Response (DDR) in cycling cells is the activation of cell cycle checkpoints at the G1/S or G2/M transitions or during S phase (intra-S checkpoint)17-21. They block the cell cycle progression to provide time for DNA damage repair or elimination of too damaged cells. Consequently, cell death as well as delayed cell cycle progression may alter brain development in response to ionizing radiation exposure22-24. It was thus interesting to develop a method to assess the activation of cell cycle checkpoints in NSPC in the irradiated mouse embryonic brain.
The progression of the cell cycle is routinely followed using the incorporation of a thymidine analog, 5-Bromo-2&#8217;-deoxyUridine (BrdU). BrdU is incorporated during the S phase of the cell cycle, when DNA is replicating. The use of an antibody against BrdU allows thereafter the detection of cells that were in S phase during the pulse of BrdU.
A novel thymidine analog, 5-Ethynyl-2&#8217;-deoxyUridine (EdU) is detected by a fluorescent azide. The different ways to detect EdU and BrdU do not cross-react25, enabling the simultaneous detection of both thymidine analogs, which is useful for the study of cell cycle progression. Usually cells are first pulsed with EdU and then pulsed with BrdU, where the time between both incorporations lasts couple of hours25,26. Addition of BrdU in culture media containing EdU results in preferentially incorporation of BrdU into the DNA with the exclusion of EdU, while simultaneous addition of EdU25 with equimolar or half equimolar BrdU to the media results in only BrdU incorporation27. This simplifies the dual labeling protocol by eliminating the wash steps that are normally required to remove the first label from the culture media prior to addition of the second label. This also is of a particular interest for in vivo study, where the washing steps are not possible, to determine the precise timing of S phase entry or exit of cell population.
Recently, a method of dual-pulse labeling in embryonic mouse brain using EdU and BrdU23,24,22 has been developed to analyze cell cycle progression and INM of NSPC after in utero irradiation. Moreover it has been demonstrated that, during S phase, mouse cells replicate first the euchromatin regions and then the pericentric heterochromatin28,29,30. Interestingly, pericentric heterochromatin of different chromosomes clustered in interphasic nuclei to form heterochromatic foci also known as chromocenters and easily detectable by DAPI staining as bright foci. Therefore, the differential EdU and BrdU stainings of euchromatin and chromocenters helped us to analyze more precisely S phase progression of NSPC.
This method allowed the demonstration of the apparent lack of G1/S checkpoint in NSCP22-24, which is quite surprising since this checkpoint is supposed to be critical for genome stability.&#160; Several experimental designs based on various combinations of EdU and BrdU pulses have been used to analyze cell cycle progression in the ventral and dorsal telencephalon. Here, we give an example of protocols allowing the study of the acute DNA damage of NSPC within the first 4 hr&#160;following in utero irradiation of E14.5 mouse embryos.

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

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&#160;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.

In the experiment described in Figure 2, EdU was administered 1.5 hr&#160;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 on...

Watch this Scientific Journal Video about Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress at JoVE.com

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

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

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.

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

assessing-cell-cycle-progression-neural-stem-progenitor-cells-mouse

Research

Education

JoVE Journal

JoVE Core

Biology

Neuroscience

Cellular Processes

Cell Cycle and Division

SCIENTISTS IN ACTION

15.2K Views.  CEA DSV iRCM SCSR. 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.

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

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Cancer Research

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...