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

Ringraziamo Monash Microimaging per il loro supporto tecnico. Alcuni ceppi sono stati forniti dal centro Caenorhabditis genetica, che è finanziato dall'ufficio di NIH di programmi di ricerca dell'infrastruttura (P40 OD010440). Quest'opera è stata sostenuta da un Monash University biomedicina Discovery Fellowship, NHMRC progetto Grant (GNT1105374), NHMRC Senior Research Fellowship (GNT1137645) e veski fellowship di innovazione: VIF 23 a Roger Pocock.

1. preparazione e allevamento della vite senza fine
Nota: Consultare la Tabella materiali per tutte le informazioni sul prodotto.
<ol><li>OP50 Escherichia coli cultura: cultura OP50 batteri in brodo di Lisogenesi (LB) (tryptone di 1%, 0,5% lievito, 0.5% NaCl, pH 7,0) durante la notte a 37 ° C senza antibiotici.</li>
	<li>400 µ l di OP50 batteri di cartilagini di accrescimento del nematode media (NGM) (1,5 g di NaCl, 8,5 g di agar, 1,25 g di peptone, 1 mL di 1 M Ca...

<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>

L'obiettivo del presente protocollo è quello di migliorare la precisione e ridurre il tempo necessario per l'analisi di germline. Dopo preparazione standard del germlines dissecata, un modello tridimensionale dei nuclei di germline è preparato dal rendering computazionale. Pur consentendo l'osservazione della distribuzione di nuclei di germline nello spazio, rendering tridimensionale calcola il numero dei nuclei alle regioni specifiche della linea germinale. L'aspetto critico del nostro metodo è accurata definizione d...

La conservazione delle vie con i mammiferi di segnalazione rende c. elegans un eccellente modello per studiare più processi biologici1,2. Nel nostro laboratorio, utilizziamo la linea germinale di c. elegans per studiare lo sviluppo delle cellule staminali, apoptosi e l'espressione genica. Mentre la linea germinale è una struttura tridimensionale, molti studi sono due dimensioni a causa della natura molto tempo e laborioso di analisi tridimensionale. È molto probabile che l'analisi bidimensionale può travisare in vivo gli eventi sulla linea germinale. L'ermafrodita adulto di c. elegans ha due braccia di germline, ognuno dei quali ospita una cellula somatica punta distale (DTC) che mantiene le cellule germinali distale in un stato indifferenziato3,4. Queste cellule germinali iniziano a differenziarsi come essi allontana il DTC, sfuggire la sua influenza e diventare ovociti e spermatozoi come raggiungono l'estremità prossimale della linea germinale. Durante questo processo, i nuclei delle cellule di germe subiscono la mitosi, prima di passare ai meiosi5,6. Produzione di spermatozoi è completata da stadio larvale 4 (L4) dello sviluppo, dopo di che gli ovociti sono prodotte durante l'età adulta. Gli spermatozoi vengono archiviati nella spermateca dove essi fertilizzare gli ovociti per generare embrioni.
Non ci sono più fattori genetici e ambientali che possono influenzare lo sviluppo di germline in c. elegans conseguente cambiamenti nel numero dei nuclei, il numero di fenomeni apoptotici, dinamiche del cromosoma e l'espressione della proteina e/o localizzazione7 ,8,9,10,11. L'analisi di questi eventi richiede l'identificazione di ogni fase di differenziazione basata sulla distribuzione e la morfologia nucleare. Per analizzare con precisione questi parametri manualmente con un campione di grandi dimensioni è laborioso e che richiede tempo. Per aggirare questi inconvenienti e per consentire la coerenza dell'analisi, abbiamo sviluppato un metodo automatizzato per l'esame tridimensionale della linea germinale per nuclei di conteggio, distribuzione di nuclei, espressione della proteina, c. elegans e del citoscheletro struttura. Combinando la microscopia confocale con rendering tridimensionale, abbiamo generato i parametri di dimensione e forma per l'identificazione di ogni fase di differenziazione delle cellule di germe. Inoltre, questo metodo consente di conteggio dei nuclei delle cellule di germe e sperma più segnando del cromosoma in ogni ovocita.
Una struttura fondamentale nella linea germinale è il citoscheletro, che fornisce stabilità al vano di germline, aids flusso citoplasmatico e protezione di germline nuclei12. Utilizzando il rendering computazionale, abbiamo eseguito la ricostruzione tridimensionale del citoscheletro germline ed abbiamo identificato caratteristiche distinte proteine citoscheletriche entro la linea germinale. Qui, descriviamo un protocollo passo-passo per illustrare come computazionale analisi combinata con confocale imaging consente analisi completa della linea germinale di c. elegans .
Proponiamo un metodo rapido per l'analisi tridimensionale di germline di c. elegans (Figura 1). Utilizzando analisi tridimensionale, è possibile studiare la distribuzione tridimensionale dei nuclei di germline (Figura 2 e Figura 3), automatizzato di conteggio delle cellule (Figura 2), ricostruzione del citoscheletro germinale ( Figura 3), distribuzione delle proteine (Figura 4) e segnando il numero di spermatozoi nella spermateca e cromosomi negli ovociti (Figura 5). Il metodo non solo consente il facile e precisa quantificazione della linea germinale, ma identifica fenotipi fisiologicamente rilevanti.

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="934">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<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>
</table>

computational analysis of the caenorhabditis elegans germline to study the distribution of nuclei, proteins, and the cytoskeleton

La linea germinale Caenorhabditis elegans (c. elegans) è usata per studiare i diversi processi biologicamente importanti tra cui dinamiche di sviluppo, apoptosi e cromosoma della cellula formativa. Mentre la linea germinale è un eccellente modello, l'analisi è spesso due dimensioni a causa del tempo e la manodopera necessaria per l'analisi tridimensionale. Principali letture in tali studi sono il numero/posizione dei nuclei e della distribuzione della proteina all'interno della linea germinale. Qui, presentiamo un metodo per eseguire l'analisi automatica della linea germinale usando microscopia confocale e approcci computazionali per determinare il numero e la posizione dei nuclei in ogni regione della linea germinale. Il nostro metodo analizza anche distribuzione di proteina di germline che consente l'esame tridimensionale dell'espressione proteica in differenti ambiti di provenienza genetici. Ulteriormente, il nostro studio mostra variazioni nell'architettura del citoscheletro in regioni distinte della linea germinale che può ospitare fino a specifiche esigenze di sviluppo spaziale. Infine, il nostro metodo consente un conteggio automatizzato dello sperma nella spermateca di ogni linea germinale. Presi insieme, il nostro metodo permette l'analisi fenotipica rapida e riproducibile della linea germinale di c. elegans .

Figura 1 indica il tempo necessario per l'analisi tridimensionale di germline. L4 ermafroditi incubate a 20 ° C sono stati sezionati per isolare germlines e macchiati con DAPI, falloidina e anticorpi contro le proteine di germline. Germlines sono imaging usando la microscopia confocal. Microscopia confocale e colorazione richiede circa 24 h. analisi computazionale per la linea germinale completa richiede 10-15 min a contare il numero e la posizione dei nucle...

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

Analisi computazionale della linea germinale Caenorhabditis elegans di studiare la distribuzione dei Nuclei, le proteine ed il citoscheletro

Presentiamo un metodo automatico per la ricostruzione tridimensionale della linea germinale Caenorhabditis elegans . Il nostro metodo determina il numero e la posizione di ogni nucleo all'interno del germline e la distribuzione di analisi germinale della proteina e la struttura del citoscheletro.

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

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Valutazione completa della linea germinale Chemical Tossicità Utilizzando il nematode<em&gt; Caenorhabditis elegans</em

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

Ricerca

Biologia dello sviluppo

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Utilizzando Analisi confocale di<em&gt; Xenopus laevis</em&gt; Per Indagare modulatori di Wnt e Shh morphogen gradienti

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

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

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

Spaziale e temporale Analisi di attiva ERK nel<em&gt; C. elegans</em&gt; Linea germinale

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

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Valvola di isolamento e caratterizzazione del ratto primario cellule interstiziali: un nuovo modello per studiare la calcificazione della valvola aortica

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Immobilizzazione del Caenorhabditis elegans per analizzare il trasporto intracellulare nei neuroni

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

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

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

Applicazione di RNAi ed espressione di fattore di trascrizione indotta da calore-shock per riprogrammare le cellule germinali ai neuroni in c. elegans

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

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

Valutazione Lysosomal alcalinizzazione l' intestino di Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

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

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

Analisi del ciclo cellulare nella linea germinale di c. elegans con la timidina analogico EdU

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

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Studiando lo Stress ossidativo causato dagli streptococchi di gruppo Mitis in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Microscopia isotropica leggera e analisi di lignaggio cellulare automatizzate al catalogo Caenorhabditis elegans embriogenesi con risoluzione subcellulare

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...