We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.

Tutti gli esperimenti sugli animali sono stati approvati dal Comitato di Cura e uso istituzionale animali (IACUC) a Albany Medical College ed eseguite secondo la guida NIH per la cura e l&#39;uso di animali da laboratorio. 1. Preparazione della soluzione NOTA: Il Rosa26Cre ERT2 15, R26R-Confetti 16, αMHC-Cre 17, e Rosa26-mTmG (mTmG) 20 linee di topo sono state acquistate sul mercato cTnT-Cre 18 è stato un dono dal Dott Jiao presso l&#39;Università di Alabama NFATc1-Cre.. era un dono di Dr. Bin Zhou all&#39;Albert Einstein college of Medicine 19. I topi sono stati sacrificati per inalazione di anidride carbonica in un locale chiuso per almeno 60 secondi seguiti da mezzi fisici di verifica morte per toracotomia bilaterale, decapitazione o dislocazione cervicale. <ol><li> Preparare Tamoxifen. Sciogliere 10 mg di tamoxifene in 10 ml di oi semi di girasolel. Una volta che si è completamente sciolto, aliquotare e conservare a -20 ° C. </li><li> Preparare soluzione salina tamponata con fosfato (PBS). Sciogliere Na 2 HPO 4 (1,41,96 mila g), KH 2 PO 4 (0,24,496 mila g), NaCl (8,0669 g) e KCl (0,20,129 mila g) in 1 L di acqua distillata e regolare il pH a 7,4. </li><li> Preparare PBST: 0.1% Tween-20 soluzione in PBS sciogliendo 100 ml di Tween-20 in 100 ml di PBS. </li><li> Preparare tampone di bloccaggio. Rendere 3% di siero albumina bovina (BSA) soluzione bloccante sciogliendo 3 g di BSA in 100 ml di PBST contenente 0,2% Tween-20. </li><li> Preparare CUBIC-1 (reattivo 1). Mescolare 25% in peso di urea, 25% in peso di N, N, N &#39;, N&#39;-tetrakis etilendiammina (2-idrossipropil) e il 15% in peso di Triton X-100 in dH 2 O. </li><li> Preparare CUBIC-2 (reattivo 2). Mescolare 50 wt% di saccarosio, 25% in peso di urea, 10% in peso di 2, 2 &#39;, 2&#39; &#39;- nitrilotrietanolo e lo 0,1% (v / v) Triton X-100 in dH 2 O. </li><li> Preparare Sca le A2: 4 M urea, 10% w / v di glicerolo e 0,1% w / v Triton X-100 in DH 2 O. Regolare il pH a 7,7. </li><li> Preparare Sca le B4: 8 M urea e 0,1% w / v Triton X-100 in dH 2 O. Regolare il pH a 8,7. </li><li> Preparare Sca le 70: 4 M urea, 70% w / v di glicerolo e 0,1% w / v Triton X-100 in dH 2 O. </li></ol> 2. Tamoxifen Induzione, isolamento degli embrioni e la fissazione <ol><li> Tamoxifene Induzione e isolamento Embryo <ol><li> Utilizzando un protocollo sondino ricurvo approvato (ACUP 13-12.007), sonda gastrica i topi di sesso femminile R26Cre ERT2 Confetti (collegato da R26Cre ERT2 topi maschi) con 10 mg / g di Tamoxifen al giorno embrionale di sviluppo E7.75 33. Al giorno embrionale 9,5 (E9.5) o E10.5, eutanasia i topi femmina con CO 2 e dislocazione cervicale. </li><li> Dopo la verifica della morte del mouse (vedi Sezione 1 Nota), aprire il mouse cavità addominale con le forbici e sezionare il corno uterino, rimuovere gli strati di tubo uterino con l&#39;aiuto di forbici e pinzette, e reaffittare gli embrioni dal 23,24 tuba uterina. <ol><li> Trasferire gli embrioni di una capsula di Petri contenente PBS (pH 7,4). Sotto il microscopio da dissezione, rimuovere gli strati non-embrionali (chorion, sacco vitellino e amnion) con l&#39;aiuto di una pinzetta. </li></ol></li><li> Utilizzando le pinzette e forbici, raccogliere i campioni femminili del mouse e della coda degli embrioni per la genotipizzazione come riportato in precedenza 22. Usando una pipetta di plastica, trasferire ogni embrione in un pozzetto di una piastra ben 48 contenente 1 ml di PBS 1x. </li><li> Sotto un microscopio a fluorescenza, identificare le GFP / RFP / CFP embrioni positivi tramite un microscopio invertito con una macchina fotografica. Sbucciare via il pericardio con una pinzetta e ruotare il cuore / embrione con l&#39;aiuto di pinzette ricurve per determinare il numero di cloni fluorescenti su entrambi i lati del cuore con i filtri GFP / RFP / CFP. Non sono necessari tagli. </li></ol></li><li> Dopo aver identificato GFP / RFP / CFP embrioni positivi, lavare rapidamente gli embrioni per due volte (2 min ciascuna) con PBS 1x in the 48 ben piastra su un agitatore a temperatura ambiente (RT). Questo rimuoverà il sangue in eccesso. </li><li> Fissare l&#39;embrione / cuore embrionale utilizzando 4% paraformaldeide (PFA) a temperatura ambiente. Il tempo di fissazione dipende dall&#39;età e del tessuto dimensioni embrionale. Per E9.5 fissare l&#39;embrione per 2 ore a temperatura ambiente. Gli embrioni fase embrionale fine richiedono un tempo più lungo di fissazione, che può anche essere prolungata fino a 24 ore per il cuore postnatale. ATTENZIONE: Paraformaldeide è tossico, evitare il contatto con la pelle, gli occhi e le mucose. </li><li> Dopo la fissazione, lavare l&#39;embrione / cuore nel 48 ben piatto due volte (10 minuti ciascuno) con 1x PBS su un agitatore a temperatura ambiente. Questo rimuove l&#39;eccesso di PFA dall&#39;embrione / cuore. Gli embrioni vengono poi ulteriormente trattati per tutto il monte di colorazione e di compensazione dei tessuti. </li></ol> 3. monte intero colorazione NOTA: Dopo PFA fissaggio l&#39;embrione / cuore è sottoposto a tutto il monte colorazione come segue. <ol><li> Permeabilize l&#39;embrione / cuore in un 48 ben piastra con 1 ml di 0,1% PBST per 2 ore su piastra agitatore a RT. </li><li> A seguito di permeabilizzazione, immergere l&#39;embrione / cuore in 1 ml di tampone di bloccaggio, per 2 ore o durante la notte su un agitatore a 4 ° C. </li><li> Immergere l&#39;embrione / cuore al endoteliali cellule-specifiche anticorpi PECAM (CD31), diluito (1: 100) in 0,3 ml di tampone di bloccaggio per 48 ore su piastra agitatore a 4 ° C. </li><li> Lavare l&#39;embrione / cuore con PBST 5 volte, 30 min ciascuno a temperatura ambiente sulla piastra shaker. Ciò elimina la non-specifico anticorpo primario legato in eccesso dal tessuto. </li><li> Immergere l&#39;embrione / cuore in Alexa Fluor 647 capra anticorpo secondario anti-topo, diluito (1: 1.000) in 1 ml di tampone di bloccaggio per 48 ore su piastra agitatore a 4 ° C. </li><li> Ripetere il passaggio 3.4 per lavare l&#39;anticorpo secondario. </li><li> Post-fix l&#39;embrione / cuore con 4% PFA per 1 ora a temperatura ambiente sulla piastra shaker e lavare due volte con PBS 5 minuti ciascuno. </li><li> Colorare l&#39;embrione / cuore con la macchia nucleare di 4 &#39;, 6-diamidino-2-phenylindole (DAPI) (diluito in PBST a concentrazione di 0,01 mg / ml) per 10 min a tavola oscillante a RT. Seguire con due lavaggi PBS su 5 minuti ciascuno. </li></ol> 4. Embrione / Clearing cuore con Sca l e o metodo CUBIC NOTA: dopo tutto il monte colorazione, l&#39;embrione / cuore è sottoposto sia alla Sca le o il metodo del tessuto compensazione cubi. La scelta del metodo dipende dalle dimensioni del tessuto ed età embrionale. Per E11.5 o embrioni età precedenti, l&#39;uso Sca l e metodo tessuto compensazione, mentre per gli embrioni di età superiore o cuori postnatali il metodo tessuto compensazione CUBIC è preferito. <ol><li> Scala del tessuto Metodo Clearing <ol><li> Incubare l&#39;embrione / cuore con 1 ml di Sca l soluzione e A2 in un flaconcino scintillazione (avvolti in carta stagnola) con occasionali agitando delicatamente (a mano) per 48 ore a 4 ° C. </li><li> Dopo l&#39;incubazione Sca l e A2, incubare l&#39;embrione / cuore con 1 ml di Sca l soluzione e B4 a 4 ° C nella stessa fiala con occasionali agitando delicatamente per anotil suo 48 hr. </li><li> Incubare l&#39;embrione / cuore per un altro 48 ore con 1 ml Sca l e 70 a 4 ° C con occasionale agitazione delicata. Monte embrione utilizzando il 90% di glicerolo (vedere paragrafo 5.1). </li></ol></li><li> CUBIC tessuto Metodo Clearing <ol><li> Per tutta la dell&#39;embrione / cuore compensazione cubo, immergere ogni embrione / cuore in 1 ml di Cubic-1 con occasionali delicata agitazione per 48 ore a 37 ° C 7. Dopo 48 ore, sostituire la soluzione con lo stesso volume di reagente CUBIC fresco 1, e incubare il campione per ulteriori 48 ore a 37 ° C, con occasionale agitazione con mano. </li><li> Lavare il CUBIC-1 trattati cuore / embrione con PBS 1X diverse volte a temperatura ambiente agitando delicatamente con la mano. Questo rimuove l&#39;eccesso di soluzione CUBIC-1. </li><li> Dopo aver lavato i cubico-1 con PBS, immergere gli embrioni / cuori in CUBIC-2 soluzione (1 g per dell&#39;embrione / cuore) per 48 ore a 37 ° C con occasionali agitando delicatamente con la mano. Questo segue dell&#39;embrione / montaggio con glicerolo cuore (vedi sezione 5.2). </li></ol></li></ol> 5. Montaggio del campione e Imaging NOTA: Gli embrioni o organi eliminato in Sca l e 70 o cubica-2 soluzione possono essere esposte direttamente con scala 70 e Cubic-2 soluzione come il mezzo di imaging, rispettivamente. Tuttavia, considerando la vulnerabilità dei campioni embrionali ai reagenti di compensazione, se i campioni non sono essere ripreso immediatamente, ma conservati a lungo termine, conservare i campioni a 90% glicerolo seguendo il protocollo di seguito. <ol><li> Direttamente immergere la Sca Le cancellata campione in 1 ml di 90% glicerolo e conservare a 4 ° C fino imaging. </li><li> Lavare il campione trattato cubica con PBS 1x due volte, 5 min ciascuna e sequenzialmente incubare il campione con 1 ml di 30%, 50%, 70% e soluzione di glicerolo 90% ciascuno per 30 min. </li><li> Per l&#39;imaging, trasferire il campione a fondo piatto di vetro Petri con una quantità minima di 90% glicerolo e illustrati cloni con un microscopio confocale equipaggiato con due funzionalità fotoni. </li><li> Immagine cuore o embrione in glicerolo con un / 0.3 NA 10X, 25X un / 0.8 NA immersione, o di una lente obiettivo correttiva 40X / 1.2 NA acqua. Immagine DAPI utilizzando 2-fotoni da un titanio coerente: zaffiro laser camaleonte. La scelta del microscopio e lente obiettivo dipenderà dallo scopo degli esperimenti, ad esempio per super-risoluzione, un microscopio a fluorescenza foglio leggero potrebbe essere utilizzato. </li></ol>

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The authors declare no competing financial interests.

L&#39;isolamento embrione è un passo molto importante. embrioni E9.5 sono molto fragili e di piccole dimensioni, in modo da la cura dovrebbe essere presa per non danneggiare le strutture dell&#39;embrione / cardiaci durante l&#39;isolamento. Gli strati aggiuntivi non embrionali che avvolgono l&#39;embrione / cuore devono essere rimossi con attenzione soprattutto quando l&#39;imaging tutta dell&#39;embrione. Questo permette la penetrazione di anticorpi e di compensazione miscela in profondità all&#39;interno dei tessut...

Morfogenesi cardiaca è un evento sequenziale che richiede l&#39;organizzazione spaziotemporale di quattro diversi tipi di cellule progenitrici cardiache in settori distinti del cuore, e richiede anche più reti di regolazione genetica di orchestrare questo processo per formare il 1,2 cardiaca funzionale. Cardiac specificazione, differenziazione, patterning, e la maturazione della camera sono regolati da fattori di trascrizione cardiogeno 3. Mutazione genetica o aberrazione posttranscriptional di questi fattori nelle cellule progenitrici cardiache possono provocare sia letalità embrionale o difetti cardiaci congeniti (CHD) 4. Lo studio della morfogenesi cardiaca richiede una comprensione dei dettagli inerenti strutturali in tre dimensioni (3D) e singola etichetta lignaggio cellule progenitrici cardiache tracciamento durante lo sviluppo cardiaco promuoverà la comprensione della morfogenesi cardiaca. Un certo numero di sezione metodi basati tomografia ad alta risoluzione sono stati sviluppati in the negli ultimi decenni a immagine Struttura organo 5,6; tuttavia, questi metodi richiedono costosi, strumenti specializzati, il lavoro esteso, e la mancanza di organizzazione strutturale dettagliata alla risoluzione di singola cellula nel volumetrico finale ricostruita immagine 7,8. L&#39;imaging volumetrico 3D a livello di singola cellula fornisce un mezzo per studiare la differenziazione delle cellule progenitrici e comportamento cellulare in vivo 7. Tuttavia, la luce tessuto dispersione rimane l&#39;ostacolo principale per l&#39;imaging cellule e strutture in 3D in profondità all&#39;interno del cuore intatto. I lipidi sono una fonte importante di diffusione della luce, e la rimozione dei lipidi e / o regolazione della differenza di indice di rifrazione tra i lipidi e le aree circostanti sono potenziali approcci per aumentare la trasparenza del tessuto 8. Negli ultimi anni, un certo numero di metodi di compensazione del tessuto sono stati sviluppati, che riducono l&#39;opacità dei tessuti e diffusione della luce, come BABB (alcool benzilico e benzile benzoate miscela) e DBE (tetraidrofurano e dibenzylether); ma in questi metodi, fluorescenza quenching rimane un problema 8-10. Il solvente basa metodi idrofili, come SeeDB (fruttosio / thioglycerol) e 3DISCO (diclorometano / dibenzylether), conserva segnali fluorescenti, ma non rende l&#39;intero organo trasparente 7,8,11. In confronto, il metodo tessuto-clearing CLARITY rende l&#39;organo trasparente, ma richiede un dispositivo per elettroforesi specializzata per rimuovere i lipidi 8,12, come fa PACT (tecnica passiva chiarezza), che richiede anche idrogel incorporamento 7,13. Per informazioni dettagliate su tutti i metodi di tessuto-compensazione disponibili, fare riferimento alla Tabella 1 a Richardson e Lichtman, et al. 7. Nel 2011, Hama et al. Casualmente scoperto una miscela idrofila &#39;Sca Le&#39; (urea, glicerolo e Triton X-100 miscela) che rende il cervello di topo ed embrione traspaent mentre completamente preservando segnali fluorescenti da cloni etichettati 14. Questo permette l&#39;imaging del cervello intatto ad una profondità di diversi millimetri e ricostruzione su larga scala di popolazioni neuronali e proiezioni a una risoluzione subcellulare. Susaki et al. ulteriormente migliorata Sca Le aggiungendo amminoalcoli e sviluppato il &#39;cubica (chiare, senza ostacoli cocktail di brain imaging e analisi computazionale) Metodo tessuto compensazione, che ha aumentato la solubilizzazione fosfolipide, riduzione dei tempi di compensazione, e ha permesso per l&#39;imaging a fluorescenza multicolore 8. Nel presente studio, approfittando della Sca Le e Cubic tecniche del tessuto di compensazione e alta risoluzione sezionamento ottico 3D, i singoli cloni all&#39;interno del cuore durante cardiogenesis sono stati rintracciati utilizzando Rosa26Cre ERT2 15, R26R-Confetti 16, αMHC-Cre 17, cTnT-Cre 18, </strong&gt; NFATc1-Cre 19, e Rosa26-mTmG (mTmG) 20 linee di mouse. La combinazione del metodo tutto il monte colorazione (WMS) sviluppato in precedenza 21,22 con metodi di compensazione tessuto ulteriormente consentiti per la colorazione di altre proteine in cloni etichettati e per lo studio del loro comportamento in un contesto volumetrica 3D. La combinazione di compensazione tessuti e WMS permette una migliore comprensione dei ruoli dei diversi geni e proteine ​​durante lo sviluppo cardiaco, e l&#39;eziologia di difetti cardiaci congeniti. Questo protocollo può essere applicato per studiare altre differenziazione di cellule progenitrici, comportamento cellulare, ed eventi morfogenesi organo durante lo sviluppo.

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imaging cleared embryonic and postnatal hearts at single-cell resolution

Singolo tracing clonale e analisi a livello intero cuore può determinare il comportamento delle cellule progenitrici cardiache e differenziazione durante lo sviluppo cardiaco, e consentire lo studio delle basi cellulari e molecolari della morfogenesi cardiaca normale e anormale. Recenti tecnologie emergenti di retrospettive singole analisi clonali rendono lo studio della morfogenesi cardiaca a risoluzione singola cella fattibile. Tuttavia, l&#39;opacità dei tessuti e la dispersione della luce del cuore come la profondità delle immagini è aumentata Hinder di imaging intero-cuore con una risoluzione di singola cellula. Per superare questi ostacoli, un sistema di compensazione intero embrione che può rendere il cuore altamente trasparente sia per l&#39;illuminazione e la rilevazione devono essere sviluppati. Fortunatamente, negli ultimi anni, sono stati segnalati molti metodologie per sistemi di compensazione intero organismo, come la chiarezza, la Sca le, SeeDB, ClearT, 3DISCO, cubi, DBE, BABB e PACT. Questo laboratorio è interessato ai meccanismi cellulari e molecolari della cartaIAC morfogenesi. Recentemente, abbiamo stabilito singola linea cellulare tracciare tramite il ROSA26-Cre ERT2; sistema ROSA26-Confetti alle cellule di etichette scarsamente durante lo sviluppo cardiaco. Abbiamo adattato diverse metodologie embrio-clearing interi compresi Sca le e cubi (chiare, senza ostacoli cocktail di imaging cerebrale e analisi computazionale) per cancellare l&#39;embrione in combinazione con tutto il monte colorazione di immagine singoli cloni all&#39;interno del cuore. Il cuore è stato ripreso con successo ad una risoluzione di singola cellula. Abbiamo trovato che le Sca può cancellare il cuore embrionale, ma non può efficacemente eliminare cuore postnatale, mentre CUBIC può cancellare cuore postnatale, ma danneggia il cuore embrionale sciogliendo il tessuto. I metodi qui descritti permetteranno lo studio della funzione genica in una sola risoluzione clone durante morfogenesi cardiaca, che, a sua volta, può rivelare basi cellulari e molecolari dei difetti cardiaci congeniti.

Imaging del cuore embrionale eliminato Formazione del cuore dei vertebrati è un processo morphogenic spatiotemporally regolata e dipende dall&#39;organizzazione e la differenziazione delle cellule progenitrici da quattro diverse fonti 1. Le cellule del primo campo cuore della mezzaluna cardiaca si piega verso la linea mediana ventrale per formare un tubo lineari cuore. Le cellule dal secondo campo cuore, inizialmen...

Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Imaging Azzerato embrionale e postnatale Cuori a risoluzione di una singola cella

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

imaging-cleared-embryonic-postnatal-hearts-at-single-cell

Research

JoVE Journal

Developmental Biology

8.1K Views.  Albany Medical College. We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately. 

Video: Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Calcio Imaging in<em&gt; Drosophila</em&gt; All&#39;attivazione Egg

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Ricerca

Biologia dello sviluppo

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

Riprogrammazione fibroblasti embrionali di topo con fattori di trascrizione per indurre un programma Hemogenic

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Analisi dei vasi coronarici in sgomberati cuori embrionali

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

Modulazione reversibile mediata dalla luce del percorso proteico di kinasi attivato da Mitogen durante la differenziazione cellulare e<em&gt; Xenopus</em&gt; Sviluppo embrionale

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

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

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

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

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

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

Catturare la risposta alle lesioni cardiache delle popolazioni cellulari mirate tramite l'imaging tridimensionale del cuore autorizzato

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Microdissezione laser di cattura della cartilagine embrionale del topo e dell'osso per l'analisi dell'espressione genica

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

Differenziazione neurale efficiente utilizzando la coltura a cella singola di cellule staminali embrionali umane

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Analisi del sequenziamento dell'mRNA a cella singola multiplexed delle cellule embrionali del topo

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

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

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

Casting vascolare di polmoni di mouse postnatali adulti e precoci per l'imaging Micro-CT

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