Name: dissection and live-imaging of the late embryonic drosophila gonad
Uploaded: 2020-10-17T14:00:00
Description: Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

Vi vil gerne takke Lindsey W. Plasschaert og Justin Sui for deres væsentlige bidrag til den tidlige udvikling af denne protokol. Forfatterne er taknemmelige for fluesamfundet for deres generøsitet med reagenser, og især til Ruth Lehmann og Benjamin Lin for deres gave af nr.5'-Lifeact-tdtomato p2a tdkatushka2 Caax nos3' linje før udgivelsen. Bestande opnået fra Bloomington Drosophila Stock Center (NIH P40OD018537) blev anvendt i denne undersøgelse. Dette arbejde blev støttet af NIH RO1 GM060804, R33AG04791503 og R35GM136270 (S.D) samt uddannelsestilskud T32GM007229 (B.W.) og F32GM125123 (LA).

1. Forberedelse dag før dissektion
<ol><li>Skærp elektrolytisk en wolframnål24 , så den resulterende diameter er ca. 0,03 mm. Juster den medfølgende spænding til ca. 14 V, og brug 3,3 M NaOH. Slibning bør ikke tage mere end 1 eller 2 minutter. FORSIGTIG: NaOH er stærkt ætsende og vil forårsage forbrændinger ved kontakt med huden. Brug handsker og beskyttelsesbriller under håndtering, og arbejd inde i en stinkhætte. BEMÆRK: Opbevar NaOH efter brug i et polypropylen Falcon-rør...

<ol>
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W., Sui, J., DiNardo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+reveals+hub+cell+assembly+and+compaction+dynamics+during+morphogenesis+of+the+Drosophila+testis+niche.">Live imaging reveals hub cell assembly and compaction dynamics during morphogenesis of the Drosophila testis niche.</a> Developmental Biology. 446, 102-118 (2019).</li><li>Rebecca Sheng, X., Matunis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+Drosophila+spermatogonial+stem+cell+niche+reveals+novel+mechanisms+regulating+germline+stem+cell+output.">Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output.</a> Development. 138, 3367-3376 (2011).</li><li>Aboim, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developpement+embryonnaire+et+post-embryonnaire+des+gonades+normales+et+agametiques+de+Drosophila+melanogaster.">Developpement embryonnaire et post-embryonnaire des gonades normales et agametiques de Drosophila melanogaster.</a> Revue Suisse De Zoologie. 52, 53 (1945).</li><li>Sano, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+actin+regulator+ENABLED+regulates+cell+shape+and+orientation+during+gonad+morphogenesis.">The Drosophila actin regulator ENABLED regulates cell shape and orientation during gonad morphogenesis.</a> PLoS One. 7, (2012).</li><li>Clark, I. B. N., Jarman, A. P., Finnegan, D. 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Under gonagenese gennemgår den embryonale gonad, og især stamcellenichen inden for den mandlige gonad15, hurtige morfologiske ændringer. Udviklingsmekanismer, der ligger til grund for disse dynamiske ændringer, forstås bedst gennem live-imaging teknikker. I embryonal fase 17 umuliggøres in vivo-billeddannelse af gonaden imidlertid ved begyndelsen af store muskelkontraktioner17. Med denne protokol leverer vi et vellykket alternativ: dissektion af gonaderne dir...

Drosophila melanogaster testiklerne har fungeret som et paradigme for vores forståelse af mange dynamiske cellulære processer. Undersøgelser af denne model har kastet lys over stamcelledelingsregulering 1,2,3, kimcelleudvikling 4,5, piRNA-biologi 6,7,8 og niche-stamcellesignalhændelser 9,10,11,12,13. Denne model er fordelagtig, fordi den er genetisk trækbar14,15 og er en af de få, hvor vi kan live-image stamceller i deres naturlige miljø 3,16,17,18. Imidlertid har levende billeddannelse af denne model været begrænset til voksenvæv og tidlige embryonale stadier, hvilket efterlader et hul i vores viden om gonadale dynamikker i det sene embryo, det nøjagtige stadium, hvor nichen først dannes og begynder at fungere.
Den sene fase embryonale gonad er en kugle, der består af somatiske nicheceller i den forreste og kimceller encysted af somatiske gonadale celler gennem mere bageste regioner19. Dette organ kan afbildes levende in vivo indtil tidligt embryonalt stadium 17 17,20,21. Yderligere billeddannelse forhindres på grund af initiering af store muskelkontraktioner. Disse sammentrækninger er så alvorlige, at de skubber gonad ud af billedrammen, og en sådan bevægelse kan ikke korrigeres med billedbehandlingssoftware. Vores laboratorium er interesseret i at afsløre mekanismerne for nichedannelse, som forekommer i denne undvigende periode for live-imaging. Derfor genererede vi en ex vivo-tilgang til levende billede af gonaden, der startede ved embryonal fase 16, hvilket letter undersøgelsen af celledynamikken i denne afgørende periode med gonadudvikling. Tidligere arbejde fra vores laboratorium viser, at denne ex vivo-billeddannelse trofast rekapitulerer in vivo gonad-udvikling17. Denne teknik er den første og eneste af sin art for Drosophila embryonad.
Her præsenterer vi dissektionsprotokollen, der kræves til ex vivo live-imaging af gonad i sene embryonale stadier. Denne protokol kan kombineres med farmakologiske behandlinger eller transgen manipulation af specifikke celleafstamninger inden for gonad. Ved hjælp af denne teknik har vi med succes afbildet trinene i stamcellenichedannelse17. Denne billeddannelsesmetode er således medvirkende til stamcellebiologi, da den vil muliggøre visualisering af de indledende faser af nichedannelse i realtid inden for sit naturlige miljø15,17. Selvom denne metode er gavnlig for stamcellebiologien, er den desuden anvendelig til visualisering af dynamiske processer, der forekommer i gonad i løbet af dette udviklingstidspunkt, herunder cellulære omlejringer22, celleadhæsion 2,12,23 og cellemigration23. Denne dissektionsprotokol vil således øge vores forståelse af mange grundlæggende cellebiologiske processer.

<table>
	<tbody>
		<tr>
			<td>Alfa Aesar&#160;Tungsten wire</td>
			<td>Fisher Scientific</td>
			<td>AA10408G6</td>
			<td>0.25mm (0.01 in.) dia., 99.95% (metals basis)</td>
		</tr>
		<tr>
			<td>D. melanogaster: His2Av::mRFP1</td>
			<td>Bloomington Drosophila Stock Center (BDSC)</td>
			<td>FBtp0056035</td>
			<td>Schuh, Lehner, &#38; Heidmann, Current Biology, 2007</td>
		</tr>
		<tr>
			<td>D. melanogaster: nos-lifeact::tdtomato</td>
			<td>Gift from Ruth Lehmann Lab</td>
			<td></td>
			<td>Lin, Luo, &#38; Lehmann, Nature Communications, 2020: nos5&#39;- Lifeact-tdtomato p2a tdkatushka2 Caax nos3&#39;</td>
		</tr>
		<tr>
			<td>D. melanogaster: P-Dsix4-eGFP::Moesin</td>
			<td></td>
			<td>FBtp0083398</td>
			<td>Sano et al., PLoS One, 2012</td>
		</tr>
		<tr>
			<td>Diamond-tipped knife</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td>665</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging dish</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>MetaMorph Microscopy Automation and Image Analysis Software v7.8.4.0</td>
		</tr>
		<tr>
			<td>Insulin, bovine</td>
			<td>Sigma</td>
			<td>l0516</td>
			<td>Store aliquots at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fisher Scientific</td>
			<td>08-955</td>
			<td></td>
		</tr>
		<tr>
			<td>Nytex basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td></td>
			<td></td>
			<td>2 mM MgCl2, 2 mM CaCl2, 130 mM NaCl, 5mM KCl, 36 mM Sucrose, 5mM Hepe&#8217;s Buffer; adjusted with NaOH until pH of 7.3 is achieved</td>
		</tr>
		<tr>
			<td>Schneider&#39;s Insect Media</td>
			<td>GIBCO</td>
			<td>21720-024</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and live-imaging of the late embryonic drosophila gonad

Drosophila melanogaster mandlig embryonal gonad er en fordelagtig model til at studere forskellige aspekter af udviklingsbiologi, herunder, men ikke begrænset til, kimcelleudvikling, piRNA-biologi og nichedannelse. Her præsenterer vi en dissektionsteknik til at live-image gonad ex vivo i en periode, hvor in vivo live-imaging er yderst ineffektiv. Denne protokol skitserer, hvordan man overfører embryoner til en billedbehandlingsskål, vælger passende iscenesatte mandlige embryoner og dissekerer gonaden fra dets omgivende væv, mens man stadig opretholder sin strukturelle integritet. Efter dissektion kan gonoder afbildes ved hjælp af et konfokalmikroskop for at visualisere dynamiske cellulære processer. Dissektionsproceduren kræver præcis timing og fingerfærdighed, men vi giver indsigt i, hvordan man forebygger almindelige fejl, og hvordan man overvinder disse udfordringer. Så vidt vi ved, er dette den første dissektionsprotokol for Drosophila embryonad, og vil tillade levende billeddannelse i et ellers utilgængeligt tidsvindue. Denne teknik kan kombineres med farmakologiske eller celletypespecifikke transgene manipulationer for at studere eventuelle dynamiske processer, der forekommer i eller mellem cellerne i deres naturlige gonadale miljø.

Vi illustrerer tilberedningen af billedbehandlingsskålen i figur 1, som beskrevet i "Forberedelse af dissektionsdagen". Disse metoder bør i sidste ende resultere i, at velhydrerede embryoner klæbes til en dækstrimmel, som midlertidigt fastgøres til bunden af fadet og nedsænkes i Ringers opløsning (figur 1F). En diamantspidskniv gør det muligt for en rent at skære et dæksel på 22 x 22 mm i tre til fire mindre strimler (figur 1A</st...

Watch this Scientific Journal Video about Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad at JoVE.com

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Her leverer vi en dissektionsprotokol, der kræves for at levende afbilde den sene embryonale Drosophila mandlige gonad. Denne protokol vil gøre det muligt at observere dynamiske cellulære processer under normale forhold eller efter transgen eller farmakologisk manipulation.

Dissektion og levende billeddannelse af den sene embryonale Drosophila Gonad

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Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

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The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

Levende-billeddannelse af<em&gt; Drosophila</em&gt; Puppe Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Dissection and Culture of Mouse Embryonic Kidney

Dissektion og kultur af mus embryonisk nyre

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Long-term Live Imaging of Drosophila Eye Disc

Long-term Live Imaging of Drosophila Eye Disc

Langsigtet Live-Imaging af<em&gt; Drosophila</em&gt; Disc Eye

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Bioengineering af humaniserede knoglemarv mikro miljøer i mus og deres visualisering ved Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Dissection and Staining of Drosophila Pupal Ovaries

Dissection and Staining of Drosophila Pupal Ovaries

Dissektion og farvning af Drosophila puppe æggestokke

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Dissektion af Drosophila puppe nethinden for immunhistokemi, vestlige analyse og RNA isolering

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Levende billeddannelse og analyse af muskelsammentrækninger i Drosophila embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...