Name: cell cycle analysis in the c. elegans germline with the thymidine analog edu
Uploaded: 2018-10-22T14:00:00
Description: Watch this Scientific Journal Video about Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU at JoVE.com

We are grateful to the E. coli stock center for MG1693; Wormbase; the Caenorhabditis Genetics Center which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40OD010440) for strains; Zach Pincus for statistical advice; Aiping Feng for reagents; Luke Schneider, Andrea Scharf, Sandeep Kumar, and John Brenner for training, advice, support, and helpful discussion; and the Kornfeld and Schedl labs for feedback on this manuscript. This work was supported in part by National Institutes of Health [R01 AG02656106A1 to KK, R01 GM100756 to TS] and a National Science Foundation predoctoral fellowship [DGE-1143954 and DGE-1745038 to ZK]. Neither the National Institutes of Health nor the National Science Foundation had any role in the design of the study, collection, analysis, and interpretation of data, nor in writing the manuscript.

1. Preparation of EdU-labeled Bacteria
<ol>
	<li>Grow a starter culture of MG1693. Escherichia coli (E. coli) MG1693 carries a mutation in thyA.
	<ol>
		<li>Streak out E. coli MG1693 from a frozen glycerol stock onto a 120 mm lysogeny broth (LB) agar Petri dish. Culture at 37 &#176;C overnight.</li>
		<li>Inoculate from two individual E. coli MG1693 colonies into two duplicate 4 mL tubes of liquid LB. Culture at 37 &#176;C for ~16 h. 
		NOTE: MG1693 grows fine i...

<ol>
	<li>Fox, P. M., Vought, V. E., Hanazawa, M., Lee, M. -. H., Maine, E. M., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+E+and+CDK-2+regulate+proliferative+cell+fate+and+cell+cycle+progression+in+the+C.+elegans+germline.">Cyclin E and CDK-2 regulate proliferative cell fate and cell cycle progression in the C. elegans germline.</a> Development. 138 (11), 2223-2234 (2011).</li><li>Crittenden, S. L., Leonhard, K. A., Byrd, D. T., Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+analyses+of+the+mitotic+region+in+the+Caenorhabditis+elegans+adult+germ+line.">Cellular analyses of the mitotic region in the Caenorhabditis elegans adult germ line.</a> Molecular biology of the cell. 17 (7), 3051-3061 (2006).</li><li>Seidel, H. S., Kimble, 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-cycle+quiescence+maintains+Caenorhabditis+elegans+germline+stem+cells+independent+of+GLP-1/Notch.">Cell-cycle quiescence maintains Caenorhabditis elegans germline stem cells independent of GLP-1/Notch.</a> eLife. 4, (2015).</li><li>Fox, P. M., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Germline+Stem+Cell+Differentiation+Following+Loss+of+GLP-1+Notch+Activity+in+Caenorhabditis+elegans.">Analysis of Germline Stem Cell Differentiation Following Loss of GLP-1 Notch Activity in Caenorhabditis elegans.</a> Genetics. 201 (9), 167-184 (2015).</li><li>Kocsisova, Z., Kornfeld, K., Schedl, T. <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+accumulation+of+the+proliferating+cell+nuclear+antigen+PCN-1+transitions+from+continuous+in+the+adult+germline+to+intermittent+in+the+early+embryo+of+C.+elegans.">Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.</a> BMC Developmental Biology. 18 (1), (2018).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences. 105 (7), 2415-2420 (2008).</li><li>vanden Heuvel, S., Kipreos, E. T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+Cell+Cycle+Analysis.">elegans Cell Cycle Analysis.</a> Methods in Cell Biology. , 265-294 (2012).</li><li>ThermoFisher. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Click-iT EdU Imaging Kits. , (2011).</li><li>Pazdernik, N., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Germ Cell Development in C. elegans. , 1-16 (2013).</li><li>Hirsh, D., Oppenheim, D., Klass, M. <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+reproductive+system+of+Caenorhabditis+elegans.">Development of the reproductive system of Caenorhabditis elegans.</a> Developmental Biology. 49 (1), 200-219 (1976).</li><li>Brenner, 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+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Hansen, D., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+proliferation+versus+meiotic+fate+decision+in+Caenorhabditis+elegans.">Stem cell proliferation versus meiotic fate decision in Caenorhabditis elegans.</a> Advances in Experimental Medicine and Biology. 757, 71-99 (2013).</li><li>Jaramillo-Lambert, A., Ellefson, M., Villeneuve, A. M., Engebrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+timing+of+S+phases,+X+chromosome+replication,+and+meiotic+prophase+in+the+C.+elegans+germ+line.">Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.</a> Developmental Biology. 308 (1), 206-221 (2007).</li><li>Agarwal, I., Farnow, C., 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=HOP-1+presenilin+deficiency+causes+a+late-onset+notch+signaling+phenotype+that+affects+adult+germline+function+in+Caenorhabditis+elegans.">HOP-1 presenilin deficiency causes a late-onset notch signaling phenotype that affects adult germline function in Caenorhabditis elegans.</a> Genetics. 208 (2), 745-762 (2018).</li><li>Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R., Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+programmed+cell+death+in+the+Caenorhabditis+elegans+hermaphrodite+germline.">Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline.</a> Development. 126 (5), 1011-1022 (1999).</li><li>Hendzel, M. J., Wei, Y., 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=Mitosis-specific+phosphorylation+of+histone+H3+initiates+primarily+within+pericentromeric+heterochromatin+during+G2+and+spreads+in+an+ordered+fashion+coincident+with+mitotic+chromosome+condensation.">Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.</a> Chromosoma. 106 (6), 348-360 (1997).</li><li>Zetka, M. C., Kawasaki, I., Strome, S., M&#252; Ller, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapsis+and+chiasma+formation+in+Caenorhabditis+elegans+require+HIM-3,+a+meiotic+chromosome+core+component+that+functions+in+chromosome+segregation.">Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation.</a> Genes &amp; development. 13 (17), 2258-2270 (1999).</li><li>Hansen, D., Hubbard, E. J. A., Schedl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-pathway+control+of+the+proliferation+versus+meiotic+development+decision+in+the+Caenorhabditis+elegans+germline.">Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline.</a> Developmental Biology. 268 (2), 342-357 (2004).</li><li>Crawley, O., Barroso, C., 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=Cohesin-interacting+protein+WAPL-1+regulates+meiotic+chromosome+structure+and+cohesion+by+antagonizing+specific+cohesin+complexes.">Cohesin-interacting protein WAPL-1 regulates meiotic chromosome structure and cohesion by antagonizing specific cohesin complexes.</a> eLife. 5 (2), 1-26 (2016).</li><li>Zhao, H., Halicka, H. 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=DNA+damage+signaling,+impairment+of+cell+cycle+progression,+and+apoptosis+triggered+by+5-ethynyl-2&#8242;-deoxyuridine+incorporated+into+DNA.">DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2&#8242;-deoxyuridine incorporated into DNA.</a> Cytometry Part A. 83 (11), 979-988 (2013).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , 1-11 (2006).</li><li>Gervaise, A. L., Arur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+Temporal+Analysis+of+Active+ERK.">Spatial and Temporal Analysis of Active ERK.</a> in the C. elegans Germline. Journal of Visualized Experiments. 117 (117), e54901-e54901 (2016).</li><li>Vos, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> De Cell Counter Plugin. , (2015).</li><li>Schindelin, J., Arganda-Carreras, I., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Rasband, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (2016).</li><li>Michaelson, D., Korta, D. Z., Capua, Y., Hubbard, E. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+signaling+promotes+germline+proliferation+in+C.+elegans.">Insulin signaling promotes germline proliferation in C. elegans.</a> Development. 137 (4), 671-680 (2010).</li><li>Qin, Z., Jane, E., Hubbard, A., Hubbard, E. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-autonomous+DAF-16/FOXO+activity+antagonizes+age-related+loss+of+C.+elegans+germline+stem/progenitor+cells.">Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells.</a> Nature communications. 6 (5), 7107 (2015).</li><li>Luo, S., Kleemann, G. A., Ashraf, J. M., Shaw, W. M., Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFB+and+Insulin+Signaling+Regulate+Reproductive+Aging+via+Oocyte+and+Germline+Quality+Maintenance.">TGFB and Insulin Signaling Regulate Reproductive Aging via Oocyte and Germline Quality Maintenance.</a> Cell. 143 (2), 299-312 (2010).</li><li>Narbonne, P., Maddox, P. S., Labbe, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-18/PTEN+locally+antagonizes+insulin+signalling+to+couple+germline+stem+cell+proliferation+to+oocyte+needs+in+C.+elegans.">daf-18/PTEN locally antagonizes insulin signalling to couple germline stem cell proliferation to oocyte needs in C. elegans.</a> Development. , 4230-4241 (2015).</li><li>Cinquin, A., Chiang, M., 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=Intermittent+Stem+Cell+Cycling+Balances+Self-Renewal+and.">Intermittent Stem Cell Cycling Balances Self-Renewal and.</a> Senescence of the C. elegans Germ Line. PLoS Genetics. 12 (4), 1005985 (2016).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Invitrogen EdU (5-ethynyl-2’-deoxyuridine). , 1-7 (2010).</li><li>Tuttle, A. H., Rankin, M. M., 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> Journal of Visualized Experiments. 46 (46), e2166-e2166 (2010).</li></ol>

Preparation of EdU-labeled bacteria (step 1) is critical for this protocol, and the first point for troubleshooting. Wild-type young adult hermaphrodites label very reliably in a 4 h EdU-pulse, making this a useful control for every new batch of EdU-labeled bacteria. Additionally, intact EdU-labeled bacteria that enter the intestine (in older animals or certain pharynx/grinder defective mutants) will label with click chemistry and appear as bright oblong puncta in the gut. An alternative technique for labeling hermaphrod...

In animal development, hundreds, thousands, millions, billions, or even trillions of cell divisions are required to form the adult organism. The cell cycle, the set of cellular events composed of G1 (gap), S (synthesis), G2 (gap), and M (mitosis) define the series of events that are executed each cell division. The cell cycle is dynamic and best appreciated in real time, which can be technically difficult. The techniques presented in this protocol allow one to make the measurements of the phases and timing of the cell cycle from still images.
Labeling with nucleoside analogs such as 5-ethynyl-2&#39;-deoxyuridine (EdU) or 5-bromo-2&#39;-deoxyuridine (BrdU) is the gold standard to identify S-phase in the studies of cell cycle dynamics in the Caenorhabditis elegans (C. elegans) adult hermaphrodite germline1,2,3,4,5. Both EdU and BrdU can be used in nearly any genetic background, as they do not rely on any genetic construct. Visualizing BrdU requires harsh chemical treatment to expose the antigen for anti-BrdU antibody staining, which is often incompatible with the assessment of other cellular markers visualized by co-staining with additional antibodies. By contrast, visualizing EdU occurs by click chemistry under mild conditions and thus is compatible with antibody co-staining6,7.
The specificity of the label is clear, since nuclei only incorporate the thymidine (5-ethynyl-2&#39;- deoxyuridine) analogs into DNA during S-phase. Visualization takes place in fixed tissue. The EdU label is invisible by itself until an azide-containing dye or fluorophore reacts covalently with the alkyne in EdU by copper-catalyzed click chemistry8. EdU labeling can provide immediate information on which nuclei are in S-phase, using a short pulse of labeling. EdU can also provide dynamic information, using pulse-chase or continuous labeling; for example, in a pulse-chase experiment, the label is diluted at each cell division or propagated as nondividing cells progress through development.
The C. elegans hermaphrodite germline is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle. The adult germline is a polarized assembly-line with stem cells found at the distal end followed by entry and progression through meiotic prophase, coordinated with the stages of gametogenesis more proximally (Figure 1). At the proximal end, oocytes mature, are ovulated and fertilized and begin embryogenesis in the uterus9,10,11. The ~20 cell-diameter long region near the distal tip cell, which includes the mitotically cycling germline stem, progenitor cells and meiotic S-phase cells but not the cells in meiotic prophase, is called the progenitor zone2,4,9,12. The cell membranes provide incomplete separation between the nuclei in the distal germline, but the progenitor zone cells undergo mitotic cell cycling largely independently. The median mitotic cell cycle duration of germline progenitor zone cells in young adult hermaphrodites is ~6.5 h; G1 phase is brief or absent, and quiescence is not observed1,2,13. Germline stem cell differentiation occurs through essentially direct differentiation and thus lacks transit-amplifying divisions4. During differentiation in the pachytene stage, approximately 4 out of 5 nuclei will not form oocytes but instead undergo apoptosis, acting as nurse cells by donating their cytoplasmic contents to the developing oocyte12,14,15.
In addition to labeling cells in S-phase with nucleoside analogs, one can identify the cells in mitosis and meiosis using antibody staining. Nuclei in mitosis are immunoreactive to anti-phospho-histone H3 (Ser10) antibody (called pH3)7,16. Nuclei in meiosis are immunoreactive to anti-HIM-3 antibody (a meiotic chromosome axis protein)17. Nuclei in the progenitor zone can be identified by the absence of HIM-3, the presence of nucleoplasmic REC-818, or the presence of WAPL-119. WAPL-1 intensity is highest in the somatic gonad, high in the progenitor zone, and low during early meiotic prophases19. Several cell cycle measurements are possible with a few variations in the protocol: I) identify nuclei in S-phase and measure S-phase index; II) Identify nuclei in M-phase and measure the M-phase index; III) determine whether nuclei were in mitotic or meiotic S-phase; IV) measure the duration of G2; V) measure the duartion&#160;of G2+M+G1 phases; VI) measure the rate of meiotic entry; VII) estimate the rate of meiotic progression.
One can make multiple cell cycle measurements from only a few types of wet-lab experiments. The protocol below describes a 30 min pulse labeling by feeding C. elegans adult hermaphrodites with EdU labeled bacteria and co-labeling M-phase cells by staining with anti-pH3 antibody and progenitor zone cells by staining with anti-WAPL-1 antibody. Only changes in the duration&#160;of EdU feed (Step 2.5), type of antibodies employed (Step 5), and analyses (Step 8.3) are required for the additional measurements.

<table border="0" cellpadding="0" cellspacing="0" style="width:1019px;" width="1020">
	<tbody>
		<tr height="42">
			<td >E. coli MG1693</td>
			<td style="width:255px;">Coli Genetic Stock Center</td>
			<td style="width:255px;">6411</td>
			<td style="width:255px;">grows fine in standard unsupplemented LB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">E. coli OP50</td>
			<td style="width:255px;">Caenorhabditis Genetics Center</td>
			<td style="width:255px;">OP50</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:255px;">C10337</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">5-Ethynyl-2&#8242;-deoxyuridine</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">900584-50MG</td>
			<td style="width:255px;">or use EdU provided in kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Glucose</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">D9434-500G</td>
			<td style="width:255px;">D-(+)-Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thiamine (Vitamin B1)</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">T4625-5G</td>
			<td style="width:255px;">Reagent Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Thymidine</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">T1895-1G</td>
			<td style="width:255px;">BioReagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Magnesium sulfate heptahydrate</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">M1880-1KG</td>
			<td style="width:255px;">MgSO4, Reagent Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Sodium Phosphate, dibasic, anhydrous</td>
			<td style="width:255px;">Fisher</td>
			<td style="width:255px;">BP332-500G</td>
			<td style="width:255px;">Na2HPO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Potassium Phosphate, monobasic</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">P5379-500G</td>
			<td style="width:255px;">KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ammonium Chloride</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">A4514-500G</td>
			<td style="width:255px;">NH4Cl, Reagent Plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Bacteriological Agar</td>
			<td style="width:255px;">US Biological</td>
			<td style="width:255px;">C13071058</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Calcium Chloride dihydrate</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">C3881-500G</td>
			<td style="width:255px;">CaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">LB Broth (Miller)</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">L3522-1KG</td>
			<td style="width:255px;">Used at 25g/L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Levamisole</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">L9756-5G</td>
			<td style="width:255px;">0.241g/10ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phosphate buffered saline</td>
			<td style="width:255px;">Calbiochem Omnipur</td>
			<td style="width:255px;">6506</td>
			<td style="width:255px;">homemade PBS works just as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween-20</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:255px;">P1379-500ML</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">16% Paraformaldehyde, EM-grade ampules</td>
			<td style="width:255px;">Electron Microscopy Sciences</td>
			<td style="width:255px;">15710</td>
			<td style="width:255px;">10ml ampules</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">100% methanol</td>
			<td style="width:255px;">Thermo Fisher Scientific</td>
			<td style="width:255px;">A454-1L</td>
			<td style="width:255px;">Gold-label methanol is critical for proper morphology with certain antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Goat Serum</td>
			<td style="width:255px;">Gibco</td>
			<td style="width:255px;">16210-072</td>
			<td style="width:255px;">Lot 1671330</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">rabbit-anti-WAPL-1</td>
			<td style="width:255px;">Novus biologicals</td>
			<td style="width:255px;">49300002</td>
			<td style="width:255px;">Lot G3048-179A02, used at 1:2000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">mouse-anti-pH3 clone 3H10</td>
			<td style="width:255px;">Millipore</td>
			<td style="width:255px;">05-806</td>
			<td style="width:255px;">Lot#2680533, used at 1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">goat-anti-rabbit IgG-conjugated Alexa Fluor 594</td>
			<td style="width:255px;">Invitrogen</td>
			<td style="width:255px;">A11012</td>
			<td style="width:255px;">Lot 1256147, used at 1:400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">goat-anti-mouse IgG-conjugated Alexa Fluor 647</td>
			<td style="width:255px;">Invitrogen</td>
			<td style="width:255px;">A21236</td>
			<td style="width:255px;">Lot 1511347, used at 1:400</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:255px;">Vectashield antifade mounting medium containing 4&#39;,6-Diamidino-2-Phenylindole Dihydrochloride (DAPI)</td>
			<td style="width:255px;">Vector Laboratories</td>
			<td style="width:255px;">H-1200</td>
			<td style="width:255px;">mounting medium without DAPI can be used instead, following a separate DAPI incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">nail polish</td>
			<td style="width:255px;">Wet n Wild</td>
			<td style="width:255px;">DTC450B</td>
			<td style="width:255px;">any clear nail polish should work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">S-medium</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">M9 buffer</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">M9 agar</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">same recipe as M9 buffer, but add 1.7% agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nematode Growth Medium</td>
			<td style="width:255px;">various</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">see wormbook.org for protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">dissecting watch glass</td>
			<td style="width:255px;">Carolina Biological</td>
			<td style="width:255px;">42300</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm laboratory film</td>
			<td style="width:255px;">Pechiney Plastic Packaging</td>
			<td style="width:255px;">PM-996</td>
			<td style="width:255px;">4 inch wide laboratory film</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">petri dishes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;">60 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Long glass Pasteur pipettes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">1ml centrifuge tubes</td>
			<td style="width:255px;">MidSci Avant</td>
			<td style="width:255px;">2926</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tips</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Serological pipettes</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">500 mL Erlenmyer flask</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Aluminum foil</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">25G 5/8&#8221; needles</td>
			<td style="width:255px;">BD PrecisionGlide&#160;</td>
			<td style="width:255px;">305122</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">5ml glass centrifuge tube</td>
			<td style="width:255px;">Pyrex&#160;</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Borosilicate glass tubes 1ml</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glass slides</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">no 1 coverslips 22 x 40 mm</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;">no 1.5 may work, also</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">37 &#176;C Shaker incubator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tabletop Centrifuge</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clinical Centrifuge</td>
			<td style="width:255px;">IEC</td>
			<td style="width:255px;">428</td>
			<td style="width:255px;">with 6 swinging bucket rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Mini Centrifuge</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">20 &#176;C incubator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:255px;">4 &#176;C refrigerator</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">-20 &#176;C freezer</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:255px;">Observer Z1 microscope</td>
			<td style="width:255px;">Zeiss</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Plan Apo 63X 1.4 oil-immersion objective lens</td>
			<td style="width:255px;">Zeiss</td>
			<td style="width:255px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:255px;">Ultraview Vox spinning disc confocal system</td>
			<td style="width:255px;">PerkinElmer&#160;</td>
			<td style="width:255px;"></td>
			<td style="width:255px;">Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy&#160;</td>
		</tr>
	</tbody>
</table>

cell cycle analysis in the c. elegans germline with the thymidine analog edu

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Since DNA synthesis is required to incorporate EdU, one can conclude that EdU-labeled nuclei underwent S-phase during the EdU-labeling time window. One may interpret the nuclei that label in a 30 min feeding with EdU labeled bacteria as nuclei in S-phase at the time of dissection. Nuclei that label in a longer continuous EdU feeding experiment may have labeled early in the time window and since left S-phase, or may have labeled in the late part of the EdU time window. EdU signal co-locali...

Watch this Scientific Journal Video about Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU at JoVE.com

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

This method can help answer key questions about the cell cycle and germ line stem cell population dynamics in C.elegans. The main advantages of this technique are that it requires no transgenes, it is compatible with immunofluorescent staining, and it can be modified to study cells under many different conditions. I was very excited when I first saw this method being used in the Scheda lab.Though this method can provide insight into the C.elegans cell cycle, it can also be applied to broader questions about aging, nutrition, or altered genotypes. Generally labs new to this method may struggle with the initial idiodisprepation or C.elegans hematoxylin staining. Begin by using sterile technique to add 200 microliters of 10 millimolar EdU in 100 milliliters of M9 buffer and the appropriate supplements to four milliliters of freshly grown overnight MG 1693 E.coli culture.Making idiodiscious requires a delicate balance between EdU and thymidine supplementation. As the amount of thymidine must be sufficient for the E coli to grow well, while the amount of EdU must be sufficient for the robust shield against leveling. After no more than 24 hours at 37 degrees Celsius and 200 RPM, use sterile technique to split the culture between two to four sterile 50 milliliter conical tubes for centrifugation.Resuspend the pellatin four milliliters of fresh M9 buffer and use the same pipette to apply about 8 drops of EdU labeled E Coli MG1693 solution to the center of individual room temperature 60 milliliter M9 A guard petri dishes. To feed the EdU labeled bacteria to the nematodes, use fresh PBS to wash the C.Elegans from a nematode growth medium dish into a 1.5 milliliter tube and allow the animals to settle briefly by gravity. Wash the animals one to two times with one milliliter of PBS and use a glass pasteur pipette to transfer the nematodes onto the center of the EdU lawn in a tiny drop of PBS.Wait a few minutes for the liquid to be absorbed and incubate the culture for at least 30 minutes at 20 degrees Celsius according to the experimental protocol. At the end of the incubation, use two milliliters of PBS to flush the worms from the EdU culture plate into a glass dissecting dish. After dissection and fixation of the nematode gonads as previously described, rinse a small one milliliter borosilicate glass tube and a long glass pasteur pipette with PBS supplemented with 0.1%tween 20 and use the pipette to transfer the fixed gonads to the tube in a small amount of PBS tween.Collect the gonads by centrifugation and use a long, drawn out glass pasture pipette to remove as much supernatent as possible without disturbing the gonad pellet. For EdU detection, add 100 microLiters of click EdU cocktail to the gonads and cover the tube with laboratory film for a 30 to 60 minute incubation at room temperature. At the end of the incubation, wash the gonads one time with 100 microLiters of reaction rinse buffer, and four times with one milliliter of PBS tween.After the last wash, add 25 microLiters of anti-fade mounting medium supplemented with DAPI to the sample. While the medium is settling over the gonads, place a large 2.5%agarose pad onto a standard glass microscope slide. Then flatten with a second slide.Next, use a long glass pasteur pipette and keep all of the liquid and gonads in the narrow tip of the pipette to minimize sample loss when transferring the gonads to the pad. Using an eyelash glued to a toothpick, distribute the gonads over the agarose pad and remove any dust particles. Then slowly lower a rectangular glass cover slip onto the gonads, taking care to avoid air bubbles, and removing any excess solution with a lab tissue.Counting cells in three dimensions reliably takes some practice. Using Fiji's cell counter plug-in and a script like marks the cells to remove any duplicates helps make the counts reproducible. After allowing the cover slip to settle overnight, use the cell counter plug-in and Fiji to manually count each nucleus, labeling each individual nucleus according to the presence or absence of phosphohistone three EdU or pregenetor zone cell labeling.EdU signal co-localizes with DAPI signal. In some nuclei, the EdU signal covers all of the chromosomes, while in other nuclei, the EdU signal localizes to one to two bright punkta, likely on the X-chromosome, which replicates late in the S-phase. EdU signal from a successful 30 minute labeling localizes to approximately half of the nuclei in the pregenetor zone.While the technique works consistently in wild type young adult animals, a significant fraction of mated 5 day old hermaphrodites fails to label in a 30 minute EdU pulse. The duration of G2 can be estimated by analyzing the percentage of nuclei in the M-phase that are EdU positive over the time course, allowing calculation of the median and maximum duration of G2.The duration of G2 and G1 can be estimated from the percentage of all of the pregenetor zone nuclei that are EdU positive, allowing calculation of the maximum duration of the combined phases. While attempting this procedure, it's important to remember to use the same batch of EdU dishes to minimize inter-experimental variability.Following this procedure, other methods like staining with additional antibodies can be performed or answer additional questions about the cell cycle, cell fate, or signaling pathways. Don't forget that working with nucleicide analogs and parafermeldahyde can be hazardous, and that precautions, such as wearing nitrol gloves, should always be taken while performing this procedure.

cell-cycle-analysis-c-elegans-germline-with-thymidine-analog

Research

JoVE Journal

Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a&#160;model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism&#39;s genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analy

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...