Name: computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton
Uploaded: 2018-04-19T12:30:00
Description: Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

Monash Microimaging onların teknik destek için teşekkür ederiz. Bazı suşları NIH ofisi araştırma altyapı programları (P40 OD010440) tarafından finanse edilen Caenorhabditis genetik Merkezi tarafından temin edilmiştir. Bu eser bir Monash Üniversitesi Biyomedikal keşif Bursu, NHMRC proje desteği (GNT1105374), NHMRC üst düzey araştırma bursu (GNT1137645) ve veski yenilik bursu tarafından desteklenmiştir: Roger Pocock VIF 23.

1. hazırlık ve solucan yetiştiriciliği
Not: Tüm ürün bilgileri için Malzemeler tablo bakın.
<ol><li>OP50 Escherichia coli kültür: Lysogeny suyu (LB) (% 1'tryptone, % 0.5 Maya, %0,5 NaCl, pH 7,0) gecede 37 ° C'de Antibiyotikler olmadan OP50 bakteri kültürü.</li>
	<li>400 µL OP50 bakterilerin nematodunun büyüme medya (NGM) levhalar (1,5 gr NaCl, agar, pepton, 1 mL 1 M CaCl2, 1 mL etanol, 1 mL 1 M MgSO4 5 mg/mL kolesterol 1,25 g ...

<ol>
	<li>Hubbard, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+germ+line:+a+model+for+stem+cell+biology.">Caenorhabditis elegans germ line: a model for stem cell biology.</a> Dev Dyn. 236 (12), 3343-3357 (2007).</li><li>Joshi, P. M., Riddle, M. R., Djabrayan, N. J., Rothman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+a+model+for+stem+cell+biology.">Caenorhabditis elegans as a model for stem cell biology.</a> Dev Dyn. 239 (5), 1539-1554 (2010).</li><li>Kershner, 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=Germline+stem+cells+and+their+regulation+in+the+nematode+Caenorhabditis+elegans.">Germline stem cells and their regulation in the nematode Caenorhabditis elegans.</a> Adv Exp Med Biol. 786, 29-46 (2013).</li><li>Byrd, D. T., Knobel, K., Affeldt, K., Crittenden, S. L., 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=A+DTC+niche+plexus+surrounds+the+germline+stem+cell+pool+in+Caenorhabditis+elegans.">A DTC niche plexus surrounds the germline stem cell pool in Caenorhabditis elegans.</a> PLoS One. 9 (2), 88372 (2014).</li><li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+germline+stem+cells,+entry+into+meiosis,+and+the+sperm/oocyte+decision+in+Caenorhabditis+elegans.">Controls of germline stem cells, entry into meiosis, and the sperm/oocyte decision in Caenorhabditis elegans.</a> Annu Rev Cell Dev Biol. 23, 405-433 (2007).</li><li>Hansen, D., Hubbard, E. J., 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> Dev Biol. 268 (2), 342-357 (2004).</li><li>Crittenden, S. 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=A+conserved+RNA-binding+protein+controls+germline+stem+cells+in+Caenorhabditis+elegans.">A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans.</a> Nature. 417 (6889), 660-663 (2002).</li><li>Eckmann, C. R., Crittenden, S. L., Suh, N., 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=GLD-3+and+control+of+the+mitosis/meiosis+decision+in+the+germline+of+Caenorhabditis+elegans.">GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans.</a> Genetics. 168 (1), 147-160 (2004).</li><li>Schumacher, B., 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=Translational+repression+of+C.+elegans+p53+by+GLD-1+regulates+DNA+damage-induced+apoptosis.">Translational repression of C. elegans p53 by GLD-1 regulates DNA damage-induced apoptosis.</a> Cell. 120 (3), 357-368 (2005).</li><li>McMullen, P. 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=Macro-level+modeling+of+the+response+of+C.+elegans+reproduction+to+chronic+heat+stress.">Macro-level modeling of the response of C. elegans reproduction to chronic heat stress.</a> PLoS Comput Biol. 8 (1), 1002338 (2012).</li><li>Hubbard, E. J., Korta, D. Z., Dalfo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+control+of+germline+development.">Physiological control of germline development.</a> Adv Exp Med Biol. 757, 101-131 (2013).</li><li>Wolke, U., Jezuit, E. A., Priess, 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=Actin-dependent+cytoplasmic+streaming+in+C.+elegans+oogenesis.">Actin-dependent cytoplasmic streaming in C. elegans oogenesis.</a> Development. 134 (12), 2227-2236 (2007).</li><li>Jones, A. R., Francis, R., 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=GLD-1,+a+cytoplasmic+protein+essential+for+oocyte+differentiation,+shows+stage-+and+sex-specific+expression+during+Caenorhabditis+elegans+germline+development.">GLD-1, a cytoplasmic protein essential for oocyte differentiation, shows stage- and sex-specific expression during Caenorhabditis elegans germline development.</a> Dev Biol. 180 (1), 165-183 (1996).</li><li>Hasegawa, E., Karashima, T., Sumiyoshi, E., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+CPB-3+interacts+with+DAZ-1+and+functions+in+multiple+steps+of+germline+development.">C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development.</a> Dev Biol. 295 (2), 689-699 (2006).</li><li>Austin, J., 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=glp-1+is+required+in+the+germ+line+for+regulation+of+the+decision+between+mitosis+and+meiosis+in+C.+elegans.">glp-1 is required in the germ line for regulation of the decision between mitosis and meiosis in C. elegans.</a> Cell. 51 (4), 589-599 (1987).</li><li>Berry, L. W., Westlund, B., 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=Germ-line+tumor+formation+caused+by+activation+of+glp-1,+a+Caenorhabditis+elegans+member+of+the+Notch+family+of+receptors.">Germ-line tumor formation caused by activation of glp-1, a Caenorhabditis elegans member of the Notch family of receptors.</a> Development. 124 (4), 925-936 (1997).</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 (1), 167-184 (2015).</li><li>Pasierbek, P., 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=A+Caenorhabditis+elegans+cohesion+protein+with+functions+in+meiotic+chromosome+pairing+and+disjunction.">A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction.</a> Genes and Development. 15 (11), 1349-1360 (2001).</li><li>Klass, M., Wolf, N., Hirsh, D. <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+male+reproductive+system+and+sexual+transformation+in+the+nematode+Caenorhabditis+elegans.">Development of the male reproductive system and sexual transformation in the nematode Caenorhabditis elegans.</a> Dev Biol. 52 (1), 1-18 (1976).</li><li>Korta, D. Z., Tuck, S., Hubbard, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S6K+links+cell+fate,+cell+cycle+and+nutrient+response+in+C.+elegans+germline+stem/progenitor+cells.">S6K links cell fate, cell cycle and nutrient response in C. elegans germline stem/progenitor cells.</a> Development. 139 (5), 859-870 (2012).</li><li>Laband, K., 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=Chromosome+segregation+occurs+by+microtubule+pushing+in+oocytes.">Chromosome segregation occurs by microtubule pushing in oocytes.</a> Nat Commun. 8 (1), 1499 (2017).</li></ol>

Bu iletişim kuralı doğruluğunu geliştirmek ve germline analiz için gereken süreyi azaltmak için hedeftir. Disseke germlines standart hazırlanması sonra germline çekirdeği üç boyutlu bir model Hesaplamalı işleme tarafından hazırlanmıştır. Germline çekirdeği dağıtım uzay gözlem izin verirken, üç boyutlu işleme germline belirli bölgelerinde, çekirdeği sayısını hesaplar. Önemli bir özelliği, bizim yöntemi çekirdek boyutu ve şekli parametreleri doğru tanımıdır. Bu boyama ve kullan...

Yollar memeliler ile sinyal koruma C. elegans birden çok biyolojik süreçlerin1,2eğitim için mükemmel bir model yapar. Bizim laboratuarımızda kök hücre gelişimi, Apoptozis ve gen ekspresyonu çalışmaya C. elegans germline kullanın. Germline üç boyutlu bir yapıdır, birçok çalışma iki boyutlu üç boyutlu analiz zaman alıcı ve emek yoğun doğası gereği vardır. İki boyutlu analiz germline vivo içinde olayları yanlış muhtemeldir. C. elegans yetişkin hermafrodit iki germline kolu, her biri bir farklılaşmamış devlet3,4distal germ hücreleri korur somatik distal ucu hücre (DTC) evler vardır. Bu germ hücreleri nüfuzunu, kaçan DTC'yi uzak hareket olarak ayırt etmek başlar ve germline proksimal sonuna ulaşmak yumurta ve sperm hale gelir. Bu işlem sırasında germ hücre çekirdeği mitoz, mayoz5,6için geçiş önce tabi. Sperm üretimini geçmesi sırasında yetişkinlik yumurta üretilmektedir gelişiminin larva aşamasında 4 (L4) tarafından tamamlanır. Sperm yumurta embriyo oluşturmak için nereye gübreliyorlar spermatheca içinde depolanır.
C. elegans değişiklikler çekirdek sayısı, apoptotik olaylar, kromozom dinamikleri ve protein ifade ve/veya Yerelleştirme7 sayısı sonuçlanan germline geliştirme etkileyebilir birden çok genetik ve çevresel faktörler ,8,9,10,11. Bu olayların analizi her aşamasında nükleer morfoloji ve dağıtım göre farklılaşma tanımlaması gerekir. Doğru bir şekilde bu parametreler büyük örnek boyutu ile el ile çözümlemek için emek ve zaman alıcı. Bu sakıncaları engelleyecek ve analiz tutarlılığını sağlamak için otomatik yöntemi C. elegans germline sayma çekirdekleri, çekirdek dağıtım, protein ifade, üç boyutlu incelenmesi için geliştirdiğimiz ve hücre iskeleti yapısı. Confocal mikroskobu ile üç boyutlu render birleştirerek, boyutu ve şekli parametreleri her aşamasında germ hücre farklılaşma tanımlanması için oluşturulan. Ayrıca, bu yöntem germ hücre çekirdeği ve sperm sayım artı kromozom sayısı her yumurta içinde Puanlama sağlar.
Bir çok önemli germline germline yuvası, AIDS sitoplazmik akarsu ve koruma germline çekirdeği12istikrar sağlar sitoiskeleti yapıdır. Hesaplamalı işlemesini kullanan, biz germline sitoiskeleti üç boyutlu rekonstrüksiyon uygulandı ve farklı hücre iskeleti özellikle germline içinde teşhis etti. Burada, biz nasıl sayısal analiz confocal görüntüleme sağlar kapsamlı analiz C. elegans germline ile kombine göstermek için adım adım bir protokol tanımlamak.
C. elegans germline (şekil 1) üç boyutlu analizi için hızlı bir yöntem öneriyorum. Üç boyutlu analizi kullanılarak, otomatik germline çekirdeği (Şekil 2 ve şekil 3), üç boyutlu dağılımı hücreleri (Resim 2), ( germline sitoiskeleti yeniden inşası sayma çalışma olanağı vardır Şekil 3), proteinler (şekil 4) ve spermatheca sperm ve yumurta (şekil 5) kromozom sayısı puanlama dağıtımını. Yöntem sadece germline kolay ve doğru miktar sağlar ama fizyolojik ilgili fenotipleri tanımlar.

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	<tbody>
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			<td height="21" style="height:21px;width:335px;">C. elegans strains: wild type (N2, Bristol), rnp-8(tm2435) I/hT2[bli-4(e937) let-?(q782) qIs48] (I;III), cpb-3(bt17) I, glp-1 (e2141) III&#160;</td>
			<td style="width:264px;">Caenorhabditis Genetics Center (CGC)</td>
			<td style="width:168px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OP50 Escherichia coli bacteria</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nematode Growth Media (NGM) plates</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">polyclonal rabbit anti-REC-8&#160;</td>
			<td>SDIX</td>
			<td>29470002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488 conjugated antibody raised in goat</td>
			<td>Thermofisher Scientific</td>
			<td>A-21236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytoskeletal dye phalloidin&#160;</td>
			<td>Thermofisher Scientific</td>
			<td style="width:168px;">A-12380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetramisol&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P5899</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M HEPES buffer, pH 7.4&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:168px;">G0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X PBS pH 7.4&#160;</td>
			<td>Thermofisher Scientific</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20&#160;</td>
			<td>Sigma Aldrich</td>
			<td>P1389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37% Paraformaldehyde solution</td>
			<td>Merck Millipore</td>
			<td>1040031000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluoroshield fixing reagent&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F6182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol&#160;</td>
			<td>Millipore&#160;</td>
			<td>1009832511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20&#176;C &#38; 25&#176;CIncubator&#160;</td>
			<td>Any brand</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope</td>
			<td style="width:264px;">Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope&#160;&#160;</td>
			<td style="width:264px;">Any brand (Leica, Zeiss)</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Computer equipped with Imaris suit 8.4.1 or later version, full licence to use the software and Matlab software.</td>
			<td style="width:264px;">Bitplane</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Phospho buffered saline, pH 7.4</td>
			<td style="width:264px;">Homemade</td>
			<td style="width:168px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon microscope slides&#160;</td>
			<td style="width:264px;">Tekdon&#160;</td>
			<td style="width:168px;">&#160;941-322-8288</td>
			<td></td>
		</tr>
	</tbody>
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computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton

Caenorhabditis elegans (C. elegans) germline kök hücre geliştirme, Apoptozis ve kromozom dinamikleri dahil olmak üzere çeşitli biyolojik olarak önemli işlemleri incelemek için kullanılır. Germline mükemmel bir model olsa da, genellikle iki boyutlu zaman ve emek üç boyutlu analiz için gerekli nedeniyle analizidir. Bu tür çalışmalarda önemli dökümanları numarası/konumunu çekirdeği ve protein dağıtım germline içinde vardır. Burada, otomatik sayı ve germline her bölgede çekirdeği konumunu belirlemek için confocal mikroskobu ve Hesaplamalı yaklaşımları kullanarak germline analizini gerçekleştirmek için bir yöntem mevcut. Bizim yöntem da farklı genetik geçmişleri protein ifade üç boyutlu incelenmesi sağlar germline protein dağıtım analiz eder. Ayrıca, bizim çalışma belirli kayma gelişimsel gereksinimleri karşılamak germline farklı bölgelerinde hücre iskeleti mimaride varyasyonlar gösterir. Son olarak, bizim yöntemi otomatik her germline spermatheca Sperm sayımı etkinleştirir. Birlikte ele alındığında, bizim yöntemi C. elegans germline hızlı ve tekrarlanabilir fenotipik analizini etkinleştirir.

Şekil 1 üç boyutlu germline analiz için gereken süreyi gösterir. 20 ° C'de inkübe L4 hünsa germlines yalıtmak için disseke ve DAPI, phalloidin ve germline proteinler karşı antikor lekeli. Germlines kullanarak confocal mikroskobu görüntüsü. Boyama ve confocal mikroskobu gerektirir yaklaşık 24 h sayısal analiz için tam germline sayısını ve konumunu çekirdeği saymak, protein dağıtım tanımlamak, puan ve hücre iskeleti yapısını an...

Watch this Scientific Journal Video about Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton at JoVE.com

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

Çekirdeklerin dağıtım, proteinler ve sitoiskeleti çalışmaya Caenorhabditis elegans Germline sayısal analizi

Biz üç boyutlu Caenorhabditis elegans germline. inşası için otomatik yöntemi mevcut Bizim yöntem sayısını ve her çekirdek germline ve analizleri germline protein dağıtım ve hücre iskeleti yapısı içindeki konumunu belirler.

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

The overall goal of this method is to automate the analysis of a Caenorhabditis elegans germline to study its nuclei, protein, and cytoskeletal distribution. This method can help answer the key questions in the C.Elegans field about the number of nuclei and sperm, nuclear distribution, and cytoskeletal structure of the germline. The main advantages of this technique are it reduces the analysis time, increases the sample size, and eliminate the human error associated with the manual analysis.To image the samples, place slides with stained germlines into the slide holder above the 63X objective of a confocal microscope, and locate a germline. Focus on the top of the germline, and mark it with the confocal microscope software. When the total germline thickness has been established, define the thickness of each slice up to 0.5 micrometers, and mark the complete germline.Then, start the acquisition, scanning each slice eight times and averaging the values to improve the image quality. When all of the images have been obtained, import the images to an appropriate image analysis software program, and use the surface function tool to select the mitotic region. An image of the distal end of the germline will appear.Cancel the automatic surface creation wizard, and manually draw a region of interest around the mitotic region at the first and last slices of the image from the three-dimensional stack. Click Create Surface, followed by Mask Channel, to mask all of the channels except the DAPI channel. Using the spot function tool, define the size parameters of the mitotic region with an XY diameter of two micrometers and with the Z diameter undefined.Click subtract background to subtract the background. To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files.For the transition zone, create surface as explained before. Then, click Mask Channel to mask all the channels except the DAPI channel. Using spot function, define an XY diameter of two micrometers and Z diameter of 1.5 micrometers, and click subtract background to subtract the background.To define the minimum threshold, increase the minimum 3D rendering threshold on a DAPI-stained wild-type germline until the first spot appears outside of the germline, and generate a 3D model for each region. After saving the images, click table to view the number of nuclei, as generated automatically by the software, and export the images as TIFF files. For scoring the sperm number, select the spermatheca as the region of interest, as demonstrated for the nuclei regions, and use the spot function tool to define the XY diameter between 0.75 and one micrometers with an undefined Z diameter to detect each sperm.Select the Background Subtract box, and use the background correction values calculated by the software to subtract the background. Then, adjust the 3D rendering threshold to detect all of the DAPI-stained sperm in the spermatheca. For chromosome counting in the oocytes, select the oocytes and define the XY diameter as less than 0.75 micrometers, and define the threshold before developing the 3D models.Then, save the images for export as TIFF files. For cytoskeletal germline reconstruction, identify the mitotic region of interest in the germline and create a surface mask as previously demonstrated. Click Mask Channels to mask all of the channels except the phalloidin channel, and use the surface function tool to define a surface detail of 0.25 micrometers.After subtracting the background, adjust the 3D rendering threshold to save the developed 3D image for export as a TIFF file. The nuclei distribution is dependent on the stage of differentiation. For example, at the mitotic region, the inner actin appears as a solid mass upon which the nuclei are organized.In a wild-type germline, the mitotic region contains approximately 250 nuclei and is tightly packed with nuclei compared to the rest of the germline. As the nuclei move away from the distal end, however, they appear closer to the circumference of the germline, with the change in distribution starting at the transition zone and finishing as the nuclei enter the third stage of prophase in meiosis. At the transition zone, the germline inner actin assumes a more cylindrical structure, becoming a hollow cylinder as it reaches the pachytene.The second layer of actin covers the inner layer, giving shape to the germline. This cylinder within a cylinder actin structure disappears toward the oocyte region, at which point the actin appears as thick fibers covering the oocytes. In the spermatheca, the actin also forms into thick bundles of fiber, although these fibers appear to be packed more closely together than in the oocyte region.Three-dimensional rendering of the proximal end of the germline, as demonstrated, reveals an average of 151 sperm in each spermatheca. Once mastered, this analysis can be completed in 10 minutes per germline. While attempting this procedure, it is important to remember to define the analysis parameters according to the magnification of the objective used to visualize the germline in your experiment.After watching this video, you should have a good understanding of how to perform an automatic analysis of a germline and to study cytoskeletal structure and to score the number of the nuclei within the germline.

Research

JoVE Journal

Developmental Biology

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

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