Name: live imaging of early cardiac progenitors in the mouse embryo
Uploaded: 2022-07-12T14:45:00
Description: Watch this Scientific Journal Video about Live Imaging of Early Cardiac Progenitors in the Mouse Embryo at JoVE.com

De auteurs erkennen Dr. Kenzo Ivanovitch voor eerder werk aan deze methode en de groep van Dr. Shigenori Nonaka (National Institutes of Natural Sciences, Japan) voor het leveren van de initiële expertise over embryomontage. Deze studie werd ondersteund door Grant PGC2018-096486-B-I00 van het Spaanse Ministerio de Ciencia e Innovación en Grant H2020-MSCA-ITN-2016-722427 van het EU Horizon 2020-programma aan MT en Grant 1380918 van het FEDER Andalucía 2014-2020 Operating Program aan JND. MS werd ondersteund door een La Caixa Foundation PhD fellowship (LCF/BQ/DE18/11670014) en The Company of Biologists travelling fellowship (DEVTF181145). De CNIC wordt ondersteund door het Spaanse Ministerie van Wetenschap en de ProCNIC Foundation.

Alle dierprocedures werden goedgekeurd door de ethische commissie voor dierproeven van de CNIC, door de Gemeenschap van Madrid (referentie PROEX 220/15) en voldeden aan EU-richtlijn 2010/63EU en aanbeveling 2007/526/EG betreffende de bescherming van dieren die worden gebruikt voor experimentele en andere wetenschappelijke doeleinden, afgedwongen in de Spaanse wetgeving onder Real Decreto 1201/2005.
Dit protocol omvat het gebruik van twee mannetjes uit de fluorescerende transgene muizenlijn die...

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Vroege hartvoorlopers organiseren zich in een primitieve hartbuis die begint te kloppen terwijl deze zich nog aan het vormen is. Begrijpen hoe dit proces plaatsvindt, is de sleutel tot het lokaliseren van het brede spectrum van aangeboren hartafwijkingen tot specifieke morfogenetische gebeurtenissen. Daarvoor biedt live beeldvorming de mogelijkheid om de normale en gebrekkige embryonale ontwikkeling met verhoogde temporele resolutie te bestuderen. Dit is vooral nuttig om vroege cardiale voorlopercellen te bestuderen, omd...

Het hart vormt zich vroeg tijdens de embryogenese om voedingsstoffen naar het hele embryo te pompen, terwijl het zich blijft ontwikkelen1. Bij muizenembryo's verzamelt anderhalve dag na het begin van de gastrulatie een rudimentair hartorgaan zich aan de voorste pool 2,3. In het stadium van de vroege streep (ES) dringen hartvoorlopers in de epiblast binnen via de primitieve streep naar de ontluikende mesodermale laag 4,5,6 en beginnen te migreren naar de voorste pool, waar ze differentiëren om de primitieve hartbuis te vormen. Gedurende dit proces ondergaan vroege hartvoorlopers celherschikkingen, vormtransformaties en differentiatie, naast migratie7 (figuur 1).
Vroege cardiale voorlopers hebben bijna een eeuw lang onderzoekers aangetrokken vanwege hun opmerkelijke vermogen om tegelijkertijd te differentiëren en een functioneel orgaan te bouwen. In de afgelopen twee decennia hebben klonale analyse en voorwaardelijke knock-outmodellen aangetoond dat vroege hartontwikkeling verschillende celbronnen impliceert in een zeer dynamisch proces 8,9,10. De 3D-structuur van de primitieve hartbuis en de dynamische aard van zijn morfogenese maken het echter een uitdaging om te bestuderen (figuur 1), en we zijn nog lang niet klaar met het begrijpen van de volledige complexiteitervan 11.
Om deze dynamische cellulaire processen te bestuderen, bieden live beeldvormingsmethoden nu een ongekend detail 7,12,13,14. In het muismodel zijn live benaderingen de sleutel geweest tot het ondervragen van ontwikkelingsonderwerpen die moeilijk aan te pakken zijn door statische analyse 7,13,15. Terwijl langdurige ex vivo kweek en robuuste microscoopopstellingen snel vorderen16,17, hebben maar weinig onderzoekers de expertise om levende embryo's met succes in beeld te brengen. Hoewel papieren publicaties voldoende technische details bieden om live beeldvormingsexperimenten te reproduceren, zijn sommige vaardigheden en trucs moeilijk te begrijpen zonder visuele voorbeelden of peer-to-peer-assistentie. Om dit leerproces te versnellen en het gebruik van live imaging onder laboratoria te verspreiden, hebben we een videoprotocol samengesteld (figuur 2) dat de nodige vaardigheden verzamelt om live imaging uit te voeren op gastrulerende muizenembryo's.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64273/64273fig01.jpg"> Figuur 1: Vroege differentiatie van cardiale voorlopercellen in het muizenembryo vanaf het begin van gastrulatie tot het stadium voorafgaand aan primitieve hartbuisvorming. Cardiale voorlopercellen dringen het mesoderm binnen kort na het begin van de gastrulatie en migreren naar de andere kant van het embryo. Morfologische en embryonale dag (E) fase zijn geschreven op de top van de diagrammen. Onderbroken pijlen verbeelden het migratietraject van primitieve hartbuisvoorlopers tijdens gastrulatie. Dit cijfer werd aangepast van11. Afkortingen: ES = Early Streak; MS = middelste streep; EHF = vroege hoofdplooi. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig01large.jpg" target="_blank">Klik hier om een grotere versie van deze figuur te bekijken.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64273/64273fig02.jpg"> Figuur 2: Werkstroomdiagram voor live beeldvorming van vroege hartvoorlopers. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig02large.jpg" target="_blank">Klik hier om een grotere versie van deze figuur te bekijken.</a>

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			<td>#55 Forceps</td>
			<td>Dumont&#160;</td>
			<td>11295-51</td>
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			<td>35 mm Dish with glass coverslip bottom 14 mm Diameter</td>
			<td>Mattek</td>
			<td>P35G-1.5-14-C</td>
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			<td>35 mm vise table&#160;</td>
			<td>Grandado</td>
			<td>SKU 8798771617573</td>
			<td></td>
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			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
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			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco</td>
			<td>11966025</td>
			<td>&#160;with L-Glutamine, without Glucose, without Na Pyruvate</td>
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			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>10438-026</td>
			<td></td>
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			<td>Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+)&#160;</td>
			<td>JAX</td>
			<td></td>
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			<td>Fluorobrite DMEM</td>
			<td>ThermoFisher</td>
			<td>A1896701&#160;</td>
			<td>DMEM for live-cell imaging</td>
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			<td>High-vacuum silicone grease</td>
			<td>Dow Corning</td>
			<td>Z273554-1EA</td>
			<td></td>
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			<td>Holder for wires</td>
			<td>Perlen Pressen</td>
			<td>pwb1</td>
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			<td>LSM 780 Upright microscope</td>
			<td>Zeiss&#160;</td>
			<td></td>
			<td></td>
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			<td>MaiTai Deepsee far red pulsed-laser tuned at 980 nm</td>
			<td>Spectra-Physics</td>
			<td></td>
			<td></td>
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			<td>Non Descanned Detectors equipped with the filter sets 
			cyan-yellow (BP450-500/BP520-560), green-red (BP500-520/BP570-610) and yellow-red (BP520-560/BP645-710)</td>
			<td>Zeiss&#160;</td>
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			<td>Obj: 20x water dipping 1.0 NA, long working distance</td>
			<td>Zeiss&#160;</td>
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			<td>P1000 and P200 pipettes</td>
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			<td>Paraffin Oil</td>
			<td>Nidacon</td>
			<td>VNI0049</td>
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			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td>(the final concentration should be 50 &#956;g/mL penicillin and 50 &#956;g/mL streptomycin)</td>
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			<td>Petri dishes 35 mm x 10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
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			<td>Pipette tips</td>
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			<td>Polymethyl methacrylate&#160;</td>
			<td></td>
			<td></td>
			<td>Reused from old laboratory equipment</td>
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			<td>Rat Serum culture embryo, male rats SPRAGUE DAWLEY RjHan SD</td>
			<td>Janvier Labs</td>
			<td>9979</td>
			<td></td>
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			<td>Set of 160 mm fines</td>
			<td>RS PRO</td>
			<td>541-6933</td>
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			<td>Standard 1.0 mm glass capillaries</td>
			<td>Anima Lab</td>
			<td>&#160;1B100F-3</td>
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			<td>Sterile 0.22 &#956;m syringe filter</td>
			<td>Corning</td>
			<td>431218</td>
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			<td>Sterile 5 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>15809152</td>
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			<td>Tungsten needles</td>
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			<td>Ultrasonic homogeniser (sonicator)</td>
			<td>Bandelin</td>
			<td>BASO_17021</td>
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live imaging of early cardiac progenitors in the mouse embryo

De eerste stappen van de ontwikkeling van het hart impliceren drastische veranderingen in celgedrag en differentiatie. Terwijl analyse van vaste embryo's het mogelijk maakt om specifieke ontwikkelingsstadia in een stilstaande momentopname in detail te bestuderen, legt live beeldvorming dynamische morfogenetische gebeurtenissen vast, zoals celmigratie, vormveranderingen en differentiatie, door het embryo in beeld te brengen terwijl het zich ontwikkelt. Dit is een aanvulling op vaste analyse en vergroot het begrip van hoe organen zich ontwikkelen tijdens embryogenese. Ondanks de voordelen wordt live imaging zelden gebruikt in muismodellen vanwege de technische uitdagingen. Vroege muizenembryo's zijn gevoelig wanneer ze ex vivo worden gekweekt en vereisen een efficiënte behandeling. Om een breder gebruik van live imaging in onderzoek naar de ontwikkeling van muizen mogelijk te maken, presenteert dit artikel een gedetailleerd protocol voor twee-foton live microscopie dat langdurige acquisitie in muizenembryo's mogelijk maakt. Naast het protocol worden er tips gegeven over embryobehandeling en kweekoptimalisatie. Dit zal helpen bij het begrijpen van belangrijke gebeurtenissen in de vroege organogenese van muizen, waardoor het begrip van de cardiovasculaire voorloperbiologie wordt verbeterd.

We gebruikten het protocol om NOTCH-signaleringsactivatie te visualiseren in vroege cardiale voorlopers die op het punt stonden te differentiëren naar endotheelcellen tijdens primitieve morfogenese van de hartbuis. Daarvoor kruisten we wild-type C57BL / 6-N-muizen met Tg (CBF: H2BVenus, +) muizen18 om embryo's te verkrijgen die NOTCH-activiteit rapporteerden via geel fluorescerend eiwit Venus. Bij E7.5 is venusfluorescentie aanwezig in het neurale ectoderm, met enkele positieve kernen bij het spl...

Watch this Scientific Journal Video about Live Imaging of Early Cardiac Progenitors in the Mouse Embryo at JoVE.com

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

We presenteren een gedetailleerd protocol voor het kweken en beeldvorming van muizenembryo's dat 3D + tijdbeeldvorming van cardiale voorlopercellen mogelijk maakt. Deze video-toolkit behandelt de belangrijkste vaardigheden die nodig zijn voor succesvolle live imaging die anders moeilijk te verkrijgen zijn uit tekstpublicaties.

Live beeldvorming van vroege cardiale voorlopers in het muizenembryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Dit protocol maakt het mogelijk om muizenembryo's in beeld te brengen terwijl ze zich ontwikkelen. Dit maakt het mogelijk om vroege cardiogenese in detail te bestuderen. Net als het maken van stilstaande beelden van nepembryo's, geeft live beeldvorming volledige toegang tot het dynamische proces in de ontwikkeling van embryo's.De vaardigheden die nodig zijn voor het beeldvorming van het leven zijn moeilijk te begrijpen uit publicatie alleen tekst. Kijken naar een andere persoon die het doet, is essentieel om te leren. Knip om te beginnen een centimeter lange wolfraamdraadstukken en slijp ze tot 0,02 tot 0,05 millimeter in diameter door de punt van de draad onder te dompelen in een verzadigde natriumnitraatoplossing.Om glazen haarvaten met ellebogen te bereiden, plaatst u het middelpunt van een standaard glazen capillair van één millimeter gedurende drie tot zes seconden over een bunsen-aansteker en trekt u langzaam aan beide uiteinden totdat het capillair dun en flexibel wordt. Breek vervolgens het capillair doormidden en verwarm het kort om een bocht van 90 graden te maken op twee centimeter van de punt. Zaag een stuk polymethylmethacrylaat van ongeveer zeven millimeter lang, vier millimeter breed en twee millimeter dik.Houd het methacrylaatstuk vast met behulp van een bankschroef en boor vervolgens aangepaste gaten dwars door het hele stuk. Draai de boor langzaam terwijl u een lage maar constante druk uitoefent. Gebruik een fijne vijl om de rand van de houder glad te strijken en de grootte aan te passen.Zorg bovendien voor een milde helling aan de randen van de houder om de positionering van het embryo te vergemakkelijken. Plaats de afgewerkte houder in een schaal met gedestilleerd water om deze af te spoelen. Controleer onder de stereomicroscoop de houder om te zien of de gaten boorstof bevatten.Als er stof aanwezig is, gebruik dan wolfraamnaalden om het te verwijderen. Om de houder te steriliseren, plaatst u deze in een conische buis vol gedestilleerd water en soniceert u gedurende 20 minuten met een vermogen van minimaal 20 watt. Haal de baarmoeder uit een geëuthanaseerde embryonale dag 6,75 tot 7,5 zwangere muis en plaats deze op een droog en schoon papieren doekje.Knip vervolgens de baarmoeder, steek de punt van de fijne schaar van de mesometrische kant in en schuif het mes langs om de decedui bloot te leggen en breng ze over naar het dissectiemedium. Ontleed de embryo's en houd de ectoplacentale kegel intact. Pel vervolgens het membraan van de Reichter af en laat een deel ervan achter op het extra embryonale gebied.Om de dissectie medium warm te houden, vervang je deze elke 10 minuten. Ontleed bovendien decidui in groepen van drie tot vier terwijl je de rest in een dissectiemedium in een bad van 37 graden Celsius houdt. Plaats de ontleedde embryo's onmiddellijk in een kweekmedium.Selecteer de embryo's met de gewenste fluorescentiekenmerken met behulp van een fluorescentiestereoscoop en plaats ze in de buis met het kweekmedium in de celkweekincubator om gedurende twee uur vóór de beeldvorming te herstellen. Nadat u alle microscoopcomponenten hebt ingeschakeld, inclusief computer, software en lasers, schakelt u de kooldioxideregelaar in en stelt u deze in op 7%Plaats een druppel hoogvacuüm siliciumvet op een schaal van 35 millimeter met een glazen afdeklaagbodem. Plaats de houder bovenop de druppel en druk zachtjes om deze te immobiliseren.Voor het monteren van embryo's, breng twee tot drie embryo's over van de couveuse naar de schaal met een houder. Zet met behulp van een tang en een taps toelopende wolfraamnaald de embryo's in de gaten. Gebruik de naald om het embryo aan de ectoplacentale kegel te haken en in een gat te steken dat overeenkomt met de grootte.Beweeg de schaal met de embryo's en de houder voorzichtig naar de microscoopplaat. Verlaag het objectief totdat een vloeibare meniscus wordt gevormd op het medium objectieve grensvlak. Nadat u het embryo in focus hebt geplaatst, monteert u de incubatiekamer en verzegelt u deze met tape rond het objectief.Met behulp van een live opname op de microscoopbesturingssoftware kunt u de laseruitvoer en versterkingsniveaus aanpassen. Bedek het medium vervolgens met paraffineolie door het langzaam op het doel te druppelen. Stel een timelapse-opname in.Stel een interval van vijf tot 10 minuten in met drie tot vijf micron Z ruimte tussen stapels. Laat een lege ruimte bovenop de Z-Stack om te anticiperen op de groei van het embryo. Een minimum van 50 micron, het toevoegen van 30 micron voor elk uur dat de acquisitie wordt uitgevoerd, wordt niet gecontroleerd.Schakel de optie voor automatisch opslaan in om toezicht op de acquisitie vanaf een computer mogelijk te maken, zodat de Z-Stack-grootte indien nodig kan worden aangepast aan het interessegebied. Live whole body embryo heart development door multiphoton imaging wordt getoond. Op embryonale dag 7,5 is veneuze fluorescentie aanwezig in het hele neurale ectoderm, met een paar positieve kernen bij het splanchnische en extra embryonale mesoderm.Na vier uur activeren endotheelvoorlopers veneuze en assembleren zich om een endocardiale buis en onderliggende aorta's te vormen, die na zeven uur ingesloten structuren worden. Wees voorzichtig bij het verwijderen van het membraan van de Reichter. Het kan echt aan het embryo worden geplakt, vooral in prevacillatiestadia.Probeer tijdens het verwijderen het membraan te knijpen door het extra embryonale gebied.

live-imaging-of-early-cardiac-progenitors-in-the-mouse-embryo

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-beeldvorming van de<em&gt; Drosophila</em&gt; Pupal Eye

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

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protocollen voor het verkrijgen Zygotic en somatische embryo&#39;s voor het bestuderen van de verordening van de vroege embryo ontwikkeling in de Model Legume<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Karakterisering van Thymic Settling voorouders in de Mouse Embryo behulp<em&gt; In Vivo</em&gt; En<em&gt; In Vitro</em&gt; Testen

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Beeldvorming Subcellulaire Structuren in het Living zebravis embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Observeren mitotische deling en Dynamiek in een live zebravis embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Identificatie van Erythromyeloid Progenitors En Hun Nakomelingen In De Muis Embryo Door Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In vivo beeldvorming van transgene Gene Expression Individuele retinale voorouders in chimère zebravis embryo&#39;s to Cell Nonautonomous Invloeden Studie

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Bioengineering van Humanized Botmarg Microenvironments in Mouse en hun visualisatie door 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 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; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

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

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

In Vitro Zaadknop teelt voor Live-cel Imaging van Zygote polarisatie en Embryo patronen in 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 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 cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

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 ...