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

Forfatterne anerkjenner Dr. Kenzo Ivanovitch for tidligere arbeid med denne metoden og gruppen av Dr. Shigenori Nonaka (National Institutes of Natural Sciences, Japan) for å gi den første ekspertisen på embryomontering. Denne studien ble støttet av Grant PGC2018-096486-B-I00 fra den spanske Ministerio de Ciencia e Innovación og Grant H2020-MSCA-ITN-2016-722427 fra EU Horizon 2020-programmet til MT og Grant 1380918 fra FEDER Andalucía 2014-2020 Operating Program til JND. MS ble støttet av et La Caixa Foundation PhD-stipendiat (LCF / BQ / DE18 / 11670014) og The Company of Bioologists traveling fellowship (DEVTF181145). CNIC støttes av det spanske vitenskapsdepartementet og ProCNIC Foundation.

Alle dyreprosedyrer ble godkjent av CNIC Animal Experimentation Ethics Committee, av Madrid-regionen (referanse PROEX 220/15) og i samsvar med EU-direktiv 2010/63EU og anbefaling 2007/526 / EC om beskyttelse av dyr som brukes til eksperimentelle og andre vitenskapelige formål, håndhevet i spansk lov under Real Decreto 1201/2005.
Denne protokollen inkluderer bruk av to hanner fra den fluorescerende transgene muselinjen som rapporterer NOTCH-aktivitet Tg(CBF:H2BVenus,+)18</su...

<ol>
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Tidlige hjerteprogenitorer organiserer seg i et primitivt hjerterør som begynner å slå mens det fortsatt dannes. Å forstå hvordan denne prosessen foregår er nøkkelen til å finne det brede spekteret av medfødte hjertefeil til spesifikke morfogenetiske hendelser. For det gir levende bildebehandling en mulighet til å studere normal og defekt embryonal utvikling med økt tidsmessig oppløsning. Dette er spesielt nyttig for å studere tidlige hjerteprogenitorceller da de overgår raskt gjennom flere differensierings...

Hjertet dannes tidlig under embryogenesen for å begynne å pumpe næringsstoffer til hele embryoet, mens det fortsetter å utvikleseg 1. I museembryoer, en og en halv dag etter initiering av gastrulasjon, samles et rudimentært hjerteorgan ved den fremre polen 2,3. Ved tidlig strekstadium (ES) kommer hjerteprogenitorer i epiblastingressen gjennom den primitive streken til det gryende mesodermale laget 4,5,6 og begynner å migrere til den fremre polen, hvor de differensierer for å danne det primitive hjerterøret. Gjennom denne prosessen gjennomgår tidlige hjerteprogenitorer celleomorganiseringer, formtransformasjoner og differensiering, i tillegg til migrasjon7 (figur 1).
Tidlige hjerteprogenitorer har tiltrukket forskere i nesten et århundre på grunn av deres bemerkelsesverdige evne til å differensiere og bygge et funksjonelt organ samtidig. I løpet av de siste to tiårene har klonal analyse og betingede knockout-modeller vist at tidlig hjerteutvikling impliserer forskjellige cellekilder i en svært dynamisk prosess 8,9,10. 3D-strukturen til det primitive hjerterøret og den dynamiske naturen til dets morfogenese gjør det imidlertid utfordrende å studere (figur 1), og vi er langt fra å forstå dets fulle kompleksitet11.
For å studere disse dynamiske cellulære prosessene, tilbyr levende bildebehandlingsmetoder nå en enestående detalj 7,12,13,14. I musemodellen har levende tilnærminger vært nøkkelen til å undersøke utviklingstemaer som er vanskelige å ta opp ved statisk analyse 7,13,15. Mens langsiktig ex vivo-kultur og robuste mikroskopoppsett utvikler seg raskt16,17, har få forskere ekspertisen til å lykkes med å avbilde levende embryoer. Selv om papirbaserte publikasjoner gir nok tekniske detaljer til å reprodusere live bildebehandlingseksperimenter, er noen ferdigheter og triks vanskelig å forstå uten visuelle eksempler eller peer-to-peer assistanse. For å akselerere denne læringsprosessen og spre bruken av levende bildebehandling blant laboratorier, samlet vi en videoprotokoll (figur 2) som samler de nødvendige ferdighetene til å utføre levende bildebehandling på gastrulating museembryoer.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64273/64273fig01.jpg"> Figur 1 Tidlig differensiering av hjerteprogenitorceller i museembryoet fra debut av gastrulering til stadium forut for primitiv hjerterørsdannelse. Hjerteprogenitorceller kommer inn i mesodermen kort tid etter starten av gastruleringen, og migrerer til motsatt side av embryoet. Morfologisk og embryonal dag (E) stadium er skrevet på toppen av diagrammene. Stiplede piler skildrer migrasjonsbanen til primitive hjerterørprogenitorer under gastrulasjon. Dette tallet ble tilpasset fra11. Forkortelser: ES = tidlig strek; MS = Midt strek; EHF = tidlig hodefold. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig01large.jpg" target="_blank">Klikk her for å se en større versjon av denne figuren.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64273/64273fig02.jpg"> Figur 2: Arbeidsflytdiagram for levende avbildning av tidlige hjerteprogenitorer. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig02large.jpg" target="_blank">Klikk her for å se en større versjon av denne figuren.</a>

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			<td>#55 Forceps</td>
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			<td>35 mm Dish with glass coverslip bottom 14 mm Diameter</td>
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			<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>
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			<td>50 mL tubes</td>
			<td>BD Falcon</td>
			<td>352070</td>
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			<td>Distilled water</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>
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			<td>Fluorescent reporter transgenic mice (Tg(CBF:H2BVenus,+)&#160;</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>
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			<td>Z273554-1EA</td>
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			<td>pwb1</td>
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			<td>LSM 780 Upright microscope</td>
			<td>Zeiss&#160;</td>
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			<td>MaiTai Deepsee far red pulsed-laser tuned at 980 nm</td>
			<td>Spectra-Physics</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>
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			<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>
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			<td>Pipette tips</td>
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			<td>Polymethyl methacrylate&#160;</td>
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			<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>
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			<td>Set of 160 mm fines</td>
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			<td>Standard 1.0 mm glass capillaries</td>
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			<td>Bandelin</td>
			<td>BASO_17021</td>
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live imaging of early cardiac progenitors in the mouse embryo

De første trinnene i hjerteutvikling innebærer drastiske endringer i celleadferd og differensiering. Mens analyse av faste embryoer gjør det mulig å studere i detalj spesifikke utviklingsstadier i et stillbilde, fanger levende bildebehandling dynamiske morfogenetiske hendelser, for eksempel cellemigrasjon, formendringer og differensiering, ved å avbilde embryoet når det utvikler seg. Dette utfyller fast analyse og utvider forståelsen av hvordan organer utvikler seg under embryogenese. Til tross for fordelene brukes levende bildebehandling sjelden i musemodeller på grunn av tekniske utfordringer. Tidlige museembryoer er følsomme når de dyrkes ex vivo og krever effektiv håndtering. For å legge til rette for en bredere bruk av levende bildebehandling i museutviklingsforskning, presenterer dette papiret en detaljert protokoll for to-foton levende mikroskopi som muliggjør langsiktig oppkjøp i museembryoer. I tillegg til protokollen gis tips om embryohåndtering og kulturoptimalisering. Dette vil bidra til å forstå viktige hendelser i tidlig museorganogenese, og øke forståelsen av kardiovaskulær stamcellebiologi.

Vi brukte protokollen til å visualisere NOTCH-signalaktivering hos tidlige hjerteprogenitorer som var i ferd med å differensiere til endotelceller under primitiv hjerterørmorfogenese. For det krysset vi villtype C57BL/6-N-mus med Tg (CBF: H2BVenus, +) mus18 for å oppnå embryoer som rapporterte NOTCH-aktivitet gjennom gult fluorescerende protein Venus. Ved E7.5 er Venus-fluorescens tilstede gjennom hele nevralektodermen, med noen få positive kjerner ved splanknisk og ekstraembryonisk mesoderm...

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

Vi presenterer en detaljert protokoll for museembryokultur og avbildning som muliggjør 3D + tidsavbildning av hjerteprogenitorceller. Dette videoverktøysettet tar for seg de viktigste ferdighetene som kreves for vellykket live bildebehandling, ellers vanskelig å skaffe seg fra tekstpublikasjoner.

Levende avbildning av tidlige hjerteprogenitorer i museembryoet

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

Denne protokollen tillater avbildning av museembryoer når de utvikler seg. Dette gjør det mulig å studere tidlig kardiogenese i detalj. Som å ta stillbilder av falske embryoer, gir levende bildebehandling full tilgang til å studere dynamisk prosess i embryoutvikling.Ferdighetene som kreves for livet imaging er vanskelig å forstå fra tekst bare publisering. Å se en annen person gjøre det er viktig å lære. For å begynne, kutt en centimeter lange wolframtrådstykker og skjerp dem til 0,02 til 0,05 millimeter i diameter ved å senke spissen av ledningen i en mettet natriumnitratløsning.For å forberede albuede glasskapillærer, plasser midtpunktet på en standard en millimeter glasskapillær over en bunsenlighter i tre til seks sekunder, og trekk sakte fra begge ender til kapillæren blir tynn og fleksibel. Del deretter kapillæren i to og varm den kort for å produsere 90 graders bøy to centimeter fra spissen. Så et stykke polymetylmetakrylat som måler omtrent syv millimeter langt, fire millimeter bredt og to millimeter tykt.Hold metakrylatstykket ved hjelp av en benkskrustikke, og bor deretter tilpassede hull på tvers gjennom hele stykket. Roter boret sakte mens du påfører lavt, men konstant trykk. Bruk en fin fil til å jevne ut holderens kant og justere størrelsen.I tillegg lager du en mild skråning på kantene av holderen for å lette embryoposisjoneringen. Legg den ferdige holderen i en tallerken med destillert vann for å skylle den. Under stereomikroskopet kontrollerer du holderen for å se om hullene inneholder borestøv.Hvis støv er til stede, bruk wolfram nåler for å fjerne den. For å sterilisere holderen, plasser den i et konisk rør fullt av destillert vann og sonicate i 20 minutter med minimum 20 watt effekt. Trekk ut livmoren fra en avlivet embryonal dag 6,75 til 7,5 gravid mus og legg den på en tørr og ren papirserviett.Deretter kutter du livmoren, setter inn spissen av fin saks fra den mesometriale siden, og skyver bladet sammen for å avsløre decidui, og overfører dem til disseksjonsmediet. Dissekere embryoene, hold ektoplacentalkeglen intakt. Deretter skreller du av Reichters membran, og etterlater en del av den på den ekstra embryonale regionen.For å holde disseksjonsmediet varmt, bytt det ut hvert 10. minutt. Videre dissekere decidui i grupper på tre til fire mens du holder resten i et disseksjonsmedium plassert i 37 grader Celsius bad. Plasser straks de dissekerte embryoene i et kulturmedium.Velg embryoene med de ønskede fluorescensfunksjonene ved hjelp av et fluorescensstereomikroskop og plasser dem i røret som inneholder kulturmediet i cellekulturinkubatoren for å gjenopprette i to timer før avbildning. Etter at du har slått på alle mikroskopkomponenter, inkludert datamaskin, programvare og lasere, slår du på karbondioksidkontrolleren og setter den til 7 %Plasser en dråpe høyvakuum silisiumfett på en 35 millimeter tallerken med en glassdekselbunn. Plasser holderen på toppen av dråpen og trykk forsiktig for å immobilisere den.For embryomontering, overfør to til tre embryoer fra inkubatoren til parabolen med en holder. Bruk en tang og en konisk wolframnål, sett embryoene i hullene. Bruk nålen til å hekte embryoet ved ektoplacental kjegle og sett den inn i en størrelse matchende hull.Flytt forsiktig fatet som inneholder embryoene og holderen til mikroskopplaten. Senk målet til en flytende menisk dannes ved det middels objektive grensesnittet. Etter å ha plassert embryoet i fokus, monter inkubasjonskammeret og forsegle det rundt målet ved hjelp av tape.Bruk et live opptak på mikroskopkontrollprogramvaren, juster laserutgangen og forsterkningsnivåene. Dekk deretter mediet med parafinolje ved å dryppe det sakte på målet. Sett opp et tidsforløpsopptak.Sett et intervall på fem til 10 minutter med tre til fem mikron Z-mellomrom mellom stabler. La det være et tomt område på toppen av Z-Stack for å forutse embryovekst. Minimum 50 mikron, og legger til 30 mikron for hver time oppkjøpet ikke vil bli overvåket.Aktiver alternativet for automatisk lagring for å tillate tilsyn med anskaffelsen fra en datamaskin for å tillate justering av Z-Stack-størrelsen om nødvendig, for å passe interesseområdet i fokus. Levende embryohjerteutvikling for hele kroppen ved multifotonavbildning er vist. På embryonal dag 7.5 er venøs fluorescens tilstede gjennom hele nevrale ektoderm, med noen få positive kjerner ved splanknisk og ekstra embryonal mesoderm.Etter fire timer aktiverer endotelprogenitorer venøse og samles for å danne endokardial rør og underliggende aorta, som blir lukkede strukturer etter syv timer. Vær forsiktig når du fjerner Reichters membran. Det kan virkelig sitte fast i embryoet, spesielt på prevacillasjonsstadier.Under fjerning, prøv å klemme membranen ved den ekstra embryonale regionen.

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-avbildning av<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.

Protokoller for Innhenting zygotiske og somatiske embryoer for å studere regulering av tidlig embryoutvikling i 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 av Thymus- Bosetting stamfedre i Mouse Embryo hjelp<em&gt; I Vivo</em&gt; Og<em&gt; In Vitro</em&gt; Analyser

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.

Imaging subcellulære strukturer i Living Sebrafisk 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.

Observerer Mitotisk divisjon og Dynamics i en Live Sebrafisk 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.

Identifikasjon av erytromyeloidprogenitorer og deres avkom i musembryoen ved flowcytometri

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 avbildning av transgene genuttrykk i Individuell Netthinnestamfedre i kimære Sebrafisk embryoer å studere 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 ...

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 av humaniserte benmarg mikromiljøer i mus og deres visualisering av levende bildebehandling

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 Ovule dyrking for Live-celle Imaging av zygoten polarisering og Embryo mønstre 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 ...