Name: live imaging of mouse secondary palate fusion
Uploaded: 2017-07-27T14:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Vi takker M. Douglas Benson for indledende samtaler vedrørende sekundær ganebilleddannelse. Vi anerkender også David Castaneda-Castellanos (Leica) og Chris Rieken (Zeiss) for deres hjælp til at justere billeddannelsesforhold i konfokal mikroskopi. Vi sætter pris på Lynsey Hamilton (Bitplane) for nyttige forslag til kvantitativ billedanalyse ved hjælp af Imaris software. Dette arbejde blev finansieret af NIH / NIDCR R01 DE025887.

Alle dyreforsøg blev udført i overensstemmelse med protokollerne fra University of California i San Francisco Institutional Animal Care and Use Committee. 1. Forberedelse af Live Imaging Media <ol><li> Fremstilling af flydende dyrkningsmedier <ol><li> Tilsæt 20% føtalt bovint serum (FBS), 2 mM L-glutamin, 100 U / ml penicillin, 100 μg / ml streptomycin, 200 μg / ml L-ascorbinsyre, 15 mM HEPES til Dulbecco&#39;s Modified Eagle Medium (DMEM) / F12 medier. BEMÆR...

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Z., Ding, J. <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+cell+migration,+transdifferentiation+and+apoptosis+during+mouse+secondary+palate+fusion.">Analysis of cell migration, transdifferentiation and apoptosis during mouse secondary palate fusion.</a> Development. 133, 3341-3347 (2006).</li><li>Xu, X., 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=Cell+autonomous+requirement+for+Tgfbr2+in+the+disappearance+of+medial+edge+epithelium+during+palatal+fusion.">Cell autonomous requirement for Tgfbr2 in the disappearance of medial edge epithelium during palatal fusion.</a> Dev. Biol. 297, 238-248 (2006).</li><li>Carette, M. J., Ferguson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+medial+edge+epithelial+cells+during+palatal+fusion+in+vitro:+an+analysis+by+Dil+labelling+and+confocal+microscopy.">The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by Dil labelling and confocal microscopy.</a> Development. 114, 379-388 (1992).</li><li>Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li><li>Dassule, H., Lewis, P., Bei, M., Mass, R., McMahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+growth+and+morphogenesis+of+the+tooth.">Sonic hedgehog regulates growth and morphogenesis of the tooth.</a> Development. 127, 4775-4785 (2000).</li><li>Eisenhoffer, G., 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=Crowding+induces+live+cell+extrusion+to+maintain+homeostatic+cell+numbers+in+epithelia.">Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia.</a> Nature. 484, 501-546 (2012).</li><li>Riedl, J., 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Riedl, J., 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=Lifeact+mice+for+studying+F-actin+dynamics.">Lifeact mice for studying F-actin dynamics.</a> Nat Methods. 7, 168-169 (2010).</li><li>Can, 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=Attenuation+correction+in+confocal+laser+microscopes:+a+novel+two-view+approach.">Attenuation correction in confocal laser microscopes: a novel two-view approach.</a> J Microsc. 211, (2003).</li><li>Veenman, C., Reinders, M., Backer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+motion+correspondence+for+densely+moving+points.">Resolving motion correspondence for densely moving points.</a> IEEE Trans Pattern Anal Mach Intell. 23, 54-72 (2001).</li><li>Udan, R., Piazza, V., Hsu, C., Hadjantonakis, A., Dickinson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+cell+dynamics+in+mouse+embryos+using+light-sheet+microscopy.">Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.</a> Development. 141, 4406-4414 (2014).</li><li>Stoller, J., 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=Cre+reporter+mouse+expressing+a+nuclear+localized+fusion+of+GFP+and+beta-galactosidase+reveals+new+derivatives+of+Pax3-expressing+precursors.">Cre reporter mouse expressing a nuclear localized fusion of GFP and beta-galactosidase reveals new derivatives of Pax3-expressing precursors.</a> Genesis. 46, 200-204 (2008).</li></ol>

Levende billeddannelse af vævsmorfogenese med 3D-organteksplanteringskultur kan give detaljeret information om cellulære processer, der ikke kan vises i konventionel farvningsanalyse af faste vævssektioner. Ved at bruge in vitro eksplantatkultur af musembryonisk sekundær gane observerede vi adskillige interessante cellulære adfærd, der førte os til at foreslå en ny mekanisme af ganefusion, der involverer epithelkonvergens og celleekstrudering. En fælles udfordring i unders?...

Vævsfusion er et vigtigt skridt i udviklingen af ​​flere organer. Større menneskelige fødselsdefekter som spaltet læbe og gane, spina bifida og misdannelser i hjertet kan skyldes defekter i vævsfusion 1 . Mus sekundær gane fusion er blevet grundigt studeret for at identificere de cellulære og molekylære mekanismer, der styrer vævsfusion i udvikling 2 , 3 , 4 . I musen begynder sekundær ganeudvikling på omkring E11.5 med udvidelsen af ​​en sekundær palatalhylde fra hver af de bilaterale maksillære processer. Den oprindelige vækst af palatalhyllerne foregår lodret langs tungen, indtil ca. E14.0, hvorefter palatalhyllerne bliver hævet vandret over tungen. Medialt rettet vækst resulterer i fysisk kontakt mellem de to palatale hylder, der tilslutter sig epithelia, der danner midterlinjen epitheliAl søm (MES) ved E14.5. Den mellemliggende MES skal fjernes fra mellem de sekundære palatalhyller for at muliggøre mesenkymkonfluens og udvikling af en intakt, fuldstændigt smeltet sekundær gane ved E15.5 3 . Hvordan er et fælles epithelial MES-cellelag dannet mellem to separate palatale hylder og derefter fjernet for at opnå mesenkym konfluens, har været et centralt spørgsmål i ganeudvikling. Baseret på histologiske og elektronmikroskopi (EM) undersøgelser, eksplanterende kulturstudier og funktionelle musegenetikforsøg har flere grundlæggende celleadfærd været impliceret i denne proces. Filopodi-lignende fremskrivninger fra medialkantepitelet MEE af hver palatalhylde letter initial kontakt 5 , 6 efterfulgt af interkalering af disse epithelceller til en fælles enkelt MES 6 , 7 . Fjernelse af den resulterende delte MESEr blevet foreslået at gå videre med tre ikke-eksklusive mekanismer. Tidlige undersøgelser, der anvendte histologisk observation og ex vivo linjesporing med vitale farvestoffer, viste, at MES kunne fjernes ved epithelial til mesenchymal overgang (EMT) af MES-celler 8 , 9 , men for nylig har genetisk afstamning af epithelceller skabt usikkerhed omkring Det langsigtede bidrag fra epithelceller til palatal hylde mesenchymme 10 , 11 , 12 . Signifikant antal apoptotiske celler og en reduktion i deres antal i nogle mutanter, der ikke gennemgår ordentlig palatfusion, har ført til ideen om, at apoptose kan være en vigtig drivkraft for MES opløsning 2 , 3 . Endelig baseret på undersøgelser, der involverer epitelmærkning og statisk observation ved progressive tidspunkter, MES-cellerBlev foreslået at migrere i oronasale og anteroposterior dimensioner 11 , 13 , men sådanne dynamiske celleadfærd var oprindeligt ubekræftet på grund af manglende evne til at observere dem i levende palatalvæv. For nylig var vi i stand til direkte at observere disse adfærd ved at udvikle en ny levende billeddannelsesmetode, der kombinerer musegenetiske metoder til fluorescerende mærkning med konfokal levende billeddannelse af eksplanterede palatalhyller. For at visualisere dynamisk cellulær adfærd i palatepitelceller under ganefusion genererede vi en epithelspecifik reportermus ved at krydse ROSA26-mTmG floxmus med Keratin14-cre mus 14 , 15 . Konfokal levende billeddannelse af ganen eksplanterende kultur af de resulterende embryoner bekræftede nogle tidligere foreslåede cellulære adfærd og identificerede nye begivenheder i fusionsprocessen 6 </sup&gt;. Epithelcelle-membran fremspring forud for begyndelsen af ​​celle-cellekontakt efterfulgt af epithelisk konvergens ved celleinterkalering og oronasal celleforskydning. Navnlig opdagede vi også, at celleekstrudering, en proces rapporteret at spille vigtige roller i epithelial homeostase, var en vigtig mekanisme, der kørte mus sekundær ganefusion 6 , 16 . Denne billeddannelsesmetode kan bruges sammen med andre reporterlinjer; Vi benyttede Lifeact-mRFPruby- transgene mus 17 , 18 til at undersøge actin cytoskeletaldynamik under fusionsprocessen. Andre journalister kan også bruges til at observere andre specifikke aspekter af ganefusion, og denne metode kan tilpasses enten til laserskanning af konfokal mikroskopi eller spin-disk-konfokalmikroskopi afhængigt af billedbehov og tilgængelighed af mikroskop. Levende billeddannelse bliver i stigende grad en keystone-tilgang i udviklingsbiologi. PartiCularly, kraniofacial morfogenese er kompleks og menneskelige fødselsdefekter, der påvirker ansigtet er almindelige. Denne konfokale levende billeddannelsesmetode vil bidrage til at muliggøre en bedre forståelse af underliggende grundlæggende udviklingsmekanismer såvel som oprindelse af humane kraniofaciale abnormiteter.

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	<tbody>
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			<td height="20" style="height:20px;width:348px;">Reagents</td>
			<td style="width:360px;"></td>
			<td style="width:225px;"></td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM/F12</td>
			<td>Life technology</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Life technology</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-glutamine</td>
			<td>Life technology</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pennicillin/Streptomycin</td>
			<td>Life technology</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Low melting agarose</td>
			<td>BioExpress</td>
			<td>E-3111-125</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">35mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petrolieum Jelly (Vaseline)</td>
			<td>Sigma</td>
			<td>16415-1Kg</td>
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		</tr>
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			<td height="20" style="height:20px;">Mice</td>
			<td></td>
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			<td height="19" style="height:19px;">Keratin14-cre</td>
			<td></td>
			<td>MGI: J:65294</td>
			<td>Allele = Tg(KRT14-cre)1Amc</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ROSA26mTmG</td>
			<td></td>
			<td>MGI: J:124702</td>
			<td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Lifeact-mRFPruby</td>
			<td></td>
			<td>MGI: J:164274</td>
			<td>Allele = Tg(CAG-mRuby)#Rows</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">White Light SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of mouse secondary palate fusion

Sammensmeltningen af ​​de sekundære palatalhylder til dannelse af den intakte sekundære gane er en nøgleproces i udvikling af pattedyr, og dets forstyrrelse kan føre til spaltet sekundær gane, en fælles medfødt anomali hos mennesker. Sekundær ganefusion er blevet grundigt undersøgt, hvilket fører til flere foreslåede cellulære mekanismer, som kan mediere denne proces. Disse undersøgelser er dog hovedsageligt udført på faste embryonale væv ved progressive tidspunkter under udvikling eller i faste eksplanterede kulturer analyseret ved statiske tidspunkter. Statisk analyse er begrænset til analyse af dynamiske morfogenetiske processer, sådan en ganefusion, og hvilke typer af dynamiske cellulære adfærd medier palatal fusion er ufuldstændigt forstået. Her beskriver vi en protokol til levende billeddannelse af ex vivo sekundær ganefusion i musembryoer. For at undersøge cellulære opførsel af ganefusion blev epithel-specifik Keratin14- cre brugt til at mærke ganeepitelceller i ROSA26-mTmG flox reporter embryoner. For at visualisere filamentøs actin blev Lifeact-mRFPruby- reportermus anvendt. Levende billeddannelse af sekundær ganefusion blev udført ved dissekering af nyligt adhærerede sekundære palatalhyller af embryonale (E) 14.5-fosters embryoner og dyrkning i agaroseholdige medier på en glasbunds skål for at muliggøre billeddannelse med et inverteret konfokalmikroskop. Ved hjælp af denne metode har vi opdaget en række nye cellulære adfærd under sekundær ganefusion. En forståelse for, hvor adskillige celleadfærd er koordineret i rum og tid, bidrager meget til vores forståelse af denne dynamiske morfogenetiske proces. Denne protokol kan anvendes på mutante muselinier eller kulturer behandlet med farmakologiske inhibitorer for yderligere at fremme forståelse for, hvordan sekundær ganefusion styres.

Levende billeddannelse af ganeeksplanterende kultur afslørede flere cellulære processer medierende ganefusion 6 . Indledende kontakter mellem to epithelceller fremstilles af membranfremspring ( Figur 2 B-2E ). Når to epithelag mødes, danner de en multicellelagret MES efterfulgt af interkalering for at gøre et integreret enkeltcellelag MES gennem epithelkonvergens ( Figur 2 A-...

Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Live Imaging of Mouse Secondary Palate Fusion

Her præsenterer vi en protokol til levende billeddannelse af musekonfliktfusion med konfokal mikroskopi. Denne protokol kan anvendes i kombination med en række fluorescerende reportermuslinjer, og med pathway-hæmmere til mekanistisk indsigt. Denne protokol kan tilpasses til levende billeddannelse i andre udviklingssystemer.

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 ...

The overall goal of this confocal live imaging method is to directly observe the cellular processes, the drive's secondary palate fusion during cranial facial development. Begin by separating the head from the body of the embryo under a dissecting microscope in a petri dish containing fresh, room temperature PBS. Grasp the head with one pair of fine number five forceps and use a second pair of forceps to remove the upper brain, cutting away any extra tissue outside of the palatal shelves with a second pair of forceps.Remove the mandible carefully without damaging the palatal shelves. When the mandible has been detached, carefully remove the tongue and confirm the presence of a robust reporter signal in the midline epithelia seam. It's important not to damage it, palatal explained during dissection to keep the midline epithelia seam intact.Next, remove the hind brain, brain stem and both maxilli with the adhered palatal shelves. Cut the anterior upper jaw tissue, keeping both the primary and secondary palates intact. Remove the nasal septum on the nasal side of the explant to expose the midline epithelia seam and confirm that the epithelia layer between the tissue is intact.Now, add live imaging medium to a 35 millimeter glass bottom dish and place the dissected palatal shelves, oral side down in the dish as close to the glass bottom as possible. Then, place dry ice pellets on a conductive surface near the culture dish to expedite the solidification of the agarose within the imaging medium. When the agarose is semi-solid, mount the culture dish in an inverted confocal microscope equipped with a 37 degree celsius incubation chamber and use a syringe to apply petroleum jelly between the culture dish and the culture cover to prevent evaporation of the imaging medium.Select a low magnification objective to locate the region of interest. Then, select a high magnification objective and scan multiple z stacks with five micron steps for 16 to 24 hours, using the appropriate laser excitation. Live imaging of palate explant culture reveals multiple cellular processes that mediate palate fusion, with the initial contacts between the two epithelia cells being made by membrane protrusions.When the two epithelia layers meet, they form a multi cell layered midline epithelia seam, followed by intercalation to generate an integrated single cell layer, midline epithelia seam through epithelia convergence. The epithelia cells in deeper z levels are also progressively displaced to the oral side of the midline epithelia seam, contributing to the epithelia convergence process. Posterior directed cell migration is observed in middle palate midline epithelia seam.With epithelia cell extrusion events observed during palate fusion, suggesting that the epithelia cells in the midline epithelia seam can be removed by this active process. This integrated epithelia seam will ultimately break down to achieve confluency. Further, filamentis actin becomes enriched at the border between the epithelia and mazenkemo areas, forming a cable like structure along the anterior posterior axis.Software can be used to track specific cellular behaviors of interest, during palate fusion with the best method for cell tracking depending on the reporter signal used in the experiment.

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Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Live Confocal Imaging of Developing Arabidopsis Flowers

Lev Konfokal Imaging af Udvikling Arabidopsis Blomster

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

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 ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule dyrkning for Live-celle Imaging af Zygote polarisering og Embryo mønstre i Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Levende celle billeddannelse af kromosom Segregation under mitosen

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

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 ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Vaskulær støbning af voksne og tidlige postnatal mus lunger til Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Dissektion og levende billeddannelse af den sene embryonale Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...