This research was supported by the Australian Research Council grant CEO348212 and the University of Newcastle. The assistance of Dr. Sam Zhang is acknowledged.

1. Zygotic Sviluppo embrionale <ol><li> Impianto Materiale <ol><li> Far crescere la Medicago truncatula wild type Jemalong o il suo vicino isogenico, altamente ri-generabile genotipo Jemalong 2HA 13 (noto come 2HA) in una serra con un fotoperiodo 14 ore e 23 ° C / 19 ° C di temperatura giorno / notte. <ol><li> Pierce la superficie del rivestimento del seme (con un ago G 23) prima di seminare il seme in modo che l&#39;acqua è permesso di entrare il seme e ammollo in acqua durante la notte. Aggiungere acqua sufficiente a coprire interamente il seme. </li><li> Seminare 3 semi in ogni 15 centimetri pentola diametro (totale di 10 vasi) in miscela di impregnazione (grossa sabbia, perlite, fibra di cocco-peat (1: 1: 1) più 5 g di Osmocote standard Exact: concime a lenta cessione) e trasferimento serra. Conservare la piantina più sano dopo la germinazione per cui vi è 1 pianta per vaso e surround un traliccio per l&#39;impianto nasce a salire. </li></ol></li></ol></li><li> Baccelli raccolta <ol><li> Raccogliere baccelli del wtipo ild Jemalong o 2HA per ottenere le opportune fasi embrione precoce come prima descritto 9. NOTA: Le diverse fasi pod sono mostrati in figura 1 e le fasi embrionali corrispondenti sono mostrati nella Figura 2. </li><li> Verificare le diverse fasi pod al momento della raccolta in base a criteri come da tabella 1. Misurare il tempo trascorso dallo stadio 6 per aiutare fasi distinte 6 e 7 (Tabella 2). </li></ol></li><li> Dissezione ovuli da baccelli e controllando la fase embrionale <ol><li> Isolare ovuli da cialde utilizzando una pinza sottile da solo o un microscopio da dissezione stereo e pinze, se necessario. Se necessario, la fase embrionale può essere rapidamente controllata usando un microscopio composto standard (Figura 3B). </li><li> Preparare modificato soluzione di Hoyer contenente 7,5 g gomma arabica, 100 g di cloralio idrato, 5 ml di glicerolo, 60 ml di acqua distillata deionizzata. Sciogliere mescolando per 3-5 ore o durante la notte. </li><li> Per ulteriori affinared microscopia cancellare gli ovuli sezionato in soluzione 15,16 modificata di Hoyer. Prendere con cautela ovuli intatti e posto in un piccolo volume di soluzione di Hoyer (sufficiente a coprire l&#39;ovulo) a temperatura ambiente fino chiaro. </li><li> Guarda i campioni con contrasto interferenziale differenziale (DIC) ottica. Catturare le immagini iwth una fotocamera digitale 9 (Figura 2). </li><li> Ottenere gli ovuli più uniformi dalla regione centrale del baccello e accumulare il numero desiderato. Un esempio è mostrato in Figura 3A. Raccogliere gli ovuli su ghiaccio e conservare a temperatura desiderata (acidi -80 ° C per nucleici) per ulteriori analisi. NOTA: Per i dettagli sulla analisi delle sezioni istologiche, microscopia elettronica a trasmissione, e ibridazione in situ consultare documenti 8,9. </li></ol></li></ol> 2. Somatic sviluppo embrione in vitro <ol><li> Utilizzare un M. truncatula cultivar tcappello è in grado di formare facilmente embrioni in coltura. Per questo protocollo per espianti fogliari utilizzare le piante 2HA 13,17 che sono 2-4 mesi di età. </li><li> Prendere volantini dall&#39;ultima foglia trifogliato quasi completamente ampliata di uno stelo allungamento. </li><li> Mantenere le foglie trifogliolate su carta umida spugna in una pentola coltura per evitare la disidratazione. NOTA: Una volta che le foglie trifogliolate vengono raccolti hanno bisogno di essere utilizzato con un ritardo minimo. </li><li> Preparazione del terreno di coltura <ol><li> Utilizzare il P4 10: 4: 1: 5 medie agar (0,8% w: v) in 9 piatti cm Petri. NOTA: Il P4 10: 4: 1: 5 media consiste mezzo P4 basale 18 più 10 mM di acido 1-naftalenacetico (NAA), 4 mM 6-benzilaminopurina (BAP), 1 mM di acido abscissico (ABA) e 5 mM gibberellico Acido (GA). L&#39;uso di GA + ABA accelera la formazione dell&#39;embrione e provoca un aumento sostanziale nel numero di embrioni 14. </li><li> Portare il supporto basale P4 in 1 L; costituito da importanti sali (fare Calcium separatamente), sali e vitamine minori (Tabella 3), di ferro chelato (make up a parte), Casamino acidi (caseina idrolizzato), 30 g di saccarosio, 8 g di agar. </li><li> Conservare le soluzioni stock di grandi sali, calcio, acidi, sali Casamino minori e vitamine a -20 ° C in aliquote adatti. Conservare ferro chelato a 4 ° C. </li><li> Portare il ferro chelato (200x) sciogliendo 7,44 g di Na 2 EDTA • 2H 2 O in circa 900 ml di acqua distillata deionizzata agitando, quindi portare la soluzione a 98-99 ° C durante l&#39;aggiunta di 1.853 g di FeSO 4 • 7H 2 O. Infine portare a 1 L quando gli ingredienti sono sciolti. Conservare in ambra bottiglie a 4 ° C colorato. </li><li> Portare il mezzo basale P4 con le soluzioni madre come in Tabella 4. Gli ormoni nel mezzo sono 10 pM NAA, 4 mM BAP, 1 pM ABA, 5 mM GA (vedere Tabella 4 e Materiali specifici Tabella). Aggiungere la NAAe BAP prima autoclave e aggiungere ABA e GA dopo autoclavaggio e raffreddamento a circa 55 ° C, per mezzo del filtro di sterilizzazione con un filtro di 0,22 micron attaccato ad una siringa. Mescolare bene agitando delicatamente. </li><li> Regolare i media a pH 5,8 con 1 M KOH prima della sterilizzazione in autoclave. Autoclave a 121 ° C per 20 min. </li><li> Dopo autoclave aggiungere il filtro ABA sterilizzato e GA, e versare circa 25 ml di media in sterili 9 centimetri piastre di Petri in una cappa a flusso laminare o un armadietto a rischio biologico. Fresco e lasciare che l&#39;agar impostato con il coperchio. Poi mettere il coperchio e assicurarsi che non vi è alcuna condensa sul coperchio. Etichetta come richiesto e conservare a 4 ° C (in una scatola di cartone per evitare che il coperchio di condensa). </li></ol></li><li> Sterilizzazione di Leaf Tissue <ol><li> Lavorare in una cappa a flusso laminare-UV sterilizzati o armadio rischio biologico. </li><li> Inserire lascia in palla maglie di una precedenza autoclavato tè primavera infusore. </li><li> Immergere tè infusore contenente le foglie nel 70% (v: v) di etanolo in un culpentola ture per 30 sec. NOTA: per tutti i passaggi di questa procedura usa da 250 ml con tappo a vite pentole cultura policarbonato che vengono sterilizzati in autoclave. </li><li> Scolare e poi trasferire e immergere il tessuto fogliare in 1: 8 (v: v) candeggina diluita (0,5% cloro) per 10 min. Agitare delicatamente di tanto in tanto. </li><li> Scolare la candeggina e trasferire l&#39;infusore tè in una pentola di coltura contenente acqua distillata deionizzata sterile. Agitare delicatamente e drenare l&#39;acqua in eccesso. Ripetere ancora una volta con una cultura pentola fresco di acqua distillata deionizzata sterile. </li><li> Togliere le foglie dalla infusore tè con pinza sterile a una cultura pentola fresco di acqua distillata deionizzata sterile. Avvitare il tappo sterile e lavare capovolgendo e vorticoso. Lascia foglie galleggianti in acqua sterile fino al momento di preparare espianti. </li></ol></li><li> Preparazione e placcatura espianti <ol><li> Usando tecniche sterili, tagliare singoli depliant sterilizzati dal bordo con un bisturi, e tagliare il rettangolo rimanenti in due o three espianti rettangolari (8-10 x 3-5 mm) con la metà vena del centro di ogni espianto (Figura 5A). NOTA: La dimensione del espianto è molto importante nell&#39;avvio del prima formazione del callo. Effettuare il taglio sui coperchi sterili da contenitori di cibo take-away. </li><li> Piastra 6 espianti di agar-solidificato terreno di coltura in 9 cm di diametro di Petri (Figura 5B). Piatto il espianti lato abaxial giù per mantenere consueto orientamento foglia. </li><li> Sigillare le piastre di Petri con Parafilm (Figura 5C) allungate intorno al piatto. Il tessuto è ora pronto per l&#39;incubazione. </li></ol></li><li> Tissue Incubazione <ol><li> Incubare le piastre al buio a 28 ° C in un ambiente a temperatura controllata o armadio crescita durante il periodo di coltura. NOTA: Le espianti avvieranno dedifferenziazione, subire la divisione cellulare, la proliferazione (formazione del callo) e embriogenesi (differenziazione). </li><li> Sottocultura il fornire i chiarimenti di differenziazionenti dopo 3 settimane a mezzo fresco. A questo subcultura trasferire l&#39;intera espianto usando una spatola inox sterili e pinze, in una cappa a flusso laminare o un armadietto rischio biologico. Come avviene formazione del callo e il callo supera la dimensione del più grande in figura 5C, trasferire il 50% del callo specifico quando subcoltura terreno fresco. </li><li> Osservare i primi embrioni in circa 4 settimane. Mentre vi è un grado di sincronia, raccogliere diverse fasi di embrione in un periodo di incubazione di 4-8 settimane (Figura 6A, B). Raccogliere sotto un microscopio e di processo dissezione in funzione degli obiettivi sperimentali. </li><li> Sottocultura ogni 3 settimane e rimuovere periodicamente embrioni in modo che gli espianti continueranno a produrre embrioni per 3 mesi. </li></ol></li></ol>

<ol>
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The authors declare that they have no competing interests.

I protocolli descritti sono relativamente semplice e permettono indagini dell&#39;embriogenesi legume con tutta la cellula contemporanea e tecniche molecolari. Si riconosce che ci sono vantaggi e svantaggi di entrambi in vivo e in vitro approcci. Entrambi permettono maggiore attenzione per embriogenesi precoce rispetto alla cultura dei semi immaturi 19. Nel caso di studi in vivo quanto descritto è prevalentemente l&#39;isolamento dell&#39;ovulo dal bacc...

I legumi sono la terza più grande famiglia di piante superiori con circa 20.000 specie e la (o Fabaceae) famiglia delle leguminose sono secondi a cereali in superficie coltivata e produzione totale 1. La soia è la terza più grande coltivato. Legumi granella forniscono circa un terzo delle proteine ​​alimentari e un terzo di olio vegetale per il consumo umano 2. Legumi con la loro capacità di fissaggio N 2 contribuiscono a sistemi agricoli sostenibili. Medicago truncatula, come la soia, i negozi di proteine ​​e olio nelle cotiledoni di suoi semi ed è un modello di legume genetica e genomica, con notevoli risorse genetiche e genomiche 3,4. Mentre M. truncatula ha consentito progressi nella comprensione del legume-rhizobium simbiosi 4 è stato sempre impiegato per studiare sementi di leguminose biologia 5-7 e embriogenesi 8,9. Arabidopsis embriogenesi è stato ampiamente studiato 10,11 ma isa non legume ei dettagli dell&#39;embriogenesi non sono identici a Medicago 8,10. Zigotica in M. truncatula ha caratteristiche interessanti, con un ipofisi multicellulare distintivo, un suspensor endoployploid e cellule basali trasferimento 8. Embriogenesi somatica (SE) è comunemente utilizzato per la rigenerazione piante 12. Nel modello legume M. truncatula, la linea seme Jemalong 2HA (2HA) è stato sviluppato dal genitore Jemalong di avere alti tassi di embriogenesi somatica 13. Il numero di embrioni prodotti è stata recentemente sostanzialmente aumentato aggiungendo sia l&#39;acido gibberellico (GA) e l&#39;acido abscissico (ABA) al mezzo di lunga data 14. In questo caso, GA e ABA agiscono in sinergia, che è inusuale visto che GA e ABA di solito agiscono antagonisticamente 14. Gli embrioni ottenuti da callo sviluppano sulla superficie che permette la fase dell&#39;embriogenesi per essere facilmente determinata Visually e prontamente raccolto. Avendo in prossimità di linee isogeniche che sono embryogenic (2HA) e non-embriogenetica (Jemalong) facilita l&#39;indagine di embriogenesi somatica e avendo sia in vivo che in vitro sistemi fornisce diverse possibilità sperimentali. La comprensione dei meccanismi cellulari e molecolari di sviluppo embrionale è essenziale per le sementi di comprensione e lo sviluppo delle piante. In legumi, come in altre dicotiledoni, sono i cotiledoni dell&#39;embrione che immagazzinare i prodotti che vengono utilizzati per l&#39;alimentazione umana. Embriogenesi precoce coinvolge rapida divisione cellulare, e corretta patterning dell&#39;embrione. In circa 8 giorni dopo la fecondazione, la M. truncatula embrione raggiunge stadi cotyledon iniziali. La caratterizzazione morfologica non è esattamente indicato da giorni dopo la fecondazione in condizioni serra. Così, un approccio standardizzato efficace per indicare la fase di embrioni in via di sviluppo è prezioso per lo studio della genetica regulzione del primo zigotica. In questo lavoro, mettiamo a disposizione due protocolli standardizzati per raccogliere embrioni in via di sviluppo per gli studi biologici della embriogenesi nel modello legume M. truncatula. Il primo è quello di raccogliere gli embrioni zigotici associando embriogenesi e pod morfologia mentre il secondo è embriogenesi somatica via espianti fogliari coltura di fornire un gran numero di embrioni facilmente accessibili.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >P4 medium</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Use Sigma-Aldrich Chemicals or other analytical grade supplier</td>
		</tr>
		<tr >
			<td >Major salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Minor salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Vitamins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Agar</td>
			<td>Bacto Laboratories</td>
			<td>214010</td>
			<td>Bacto agar</td>
		</tr>
		<tr >
			<td >Plant hormones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1-Naphthaleneacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>N0640</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >Abscisic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A1049</td>
			<td>Dissolve in small amount of 1 M NaOH</td>
		</tr>
		<tr >
			<td >6-Benzylaminopurine</td>
			<td>Sigma-Aldrich</td>
			<td>B3274</td>
			<td>Dissolve in MQ water with heating and few drops 1N HCl</td>
		</tr>
		<tr >
			<td >Gibberellic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>G7645</td>
			<td>Dissolve in small amount of ethanol</td>
		</tr>
		<tr >
			<td >Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo dissecting microscope</td>
			<td>Leica</td>
			<td>MZFLIII</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Light microscope</td>
			<td>Zeiss</td>
			<td>Axiophot</td>
			<td>Or similar, with suitable optics</td>
		</tr>
		<tr >
			<td >Digital camera</td>
			<td>Zeiss</td>
			<td>AxioCam HRc</td>
			<td>Or similar</td>
		</tr>
		<tr >
			<td >Sterilising leaves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >250 mL screw cap polycarbonate container with polypropylene lid</td>
			<td>SARSTEDT</td>
			<td>75.9922.519</td>
			<td>Autoclavable</td>
		</tr>
	</tbody>
</table>

protocols for obtaining zygotic and somatic embryos for studying the regulation of early embryo development in the model legume medicago truncatula

Embriogenesi precoce a partire da una singola cellula zigote attraversa rapida divisione cellulare e la morfogenesi, ed è morfologicamente caratterizzato da fasi di pre-globulare, globulari, cuore, siluri e cotyledon. Questo progressivo sviluppo è sotto la stretta regolazione di una rete molecolare complessa. Raccolta sufficienti embrioni in uno stadio di sviluppo è essenziale per studiare la regolazione cellulare e molecolare del primo embriogenesi. Questo non è semplice da inizio embriogenesi subisce una rapida morfogenesi in breve tempo ad esempio, 8 giorni per i Medicago truncatula per raggiungere la fase cotyledon precoce. Qui, affrontiamo il problema da due approcci. Il primo stabilisce un legame tra lo sviluppo embrionale e pod morfologia nell&#39;aiutare indica la fase dell&#39;embrione zigotico. Questo è particolarmente basa sul numero di spirali pod e sviluppo delle spine. Un modo alternativo per integrare in vivo sTUDI è via espianti fogliari coltura per la produzione di embrioni somatici. Il supporto include una combinazione di ormoni insolito - un&#39;auxina (acido 1-naftalenacetico), un citochinine (6-benzilaminopurina), acido abscissico e acido gibberellico. Le diverse fasi possono essere individuate a crescere senza il callo senza dissezione.

Per zigotici embriogenesi differenti strutture pod corrispondenti alle diverse fasi embrionali sono mostrate in Figura 1A - F mentre le diverse fasi embrionali sono mostrati in Figura 2A - F. Selezionando baccelli nella stessa fase, campioni di ovuli che sono piuttosto uniforme può essere ottenuta (Figura 3A). Usando RT-qPCR embrione geni specifici possono essere facilmente individuati e gli studi del corso di tempo valutato 9.</s...

Watch this Scientific Journal Video about Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula at JoVE.co

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.

Protocolli per ottenere Zygotic e Somatic embrioni per lo studio della regolazione dello sviluppo embrionale precoce nel Modello leguminose<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 ...

protocols-for-obtaining-zygotic-somatic-embryos-for-studying

Research

JoVE Journal

Developmental Biology

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

13.8K Views.  University of Newcastle, Callaghan, New South Wales, Australia. 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. 

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Ricerca

Biologia dello sviluppo

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 strutture subcellulari nell&#39;embrione Living Zebrafish

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

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Trascrittoma Profiling di<em&gt; In-Vivo</em&gt; Embrioni bovini pre-impianto prodotto con piattaforma microarray a due colori

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Visualizzazione dell'attività elettrica cellulare in Zebrafish embrioni in anticipo e tumori

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Trasferimento embrionale minimamente invasivo e vetrificazione embrionale nella fase embrionale ottimale nel modello Rabbit

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Preparazione di piccole librerie di RNA per il sequenziamento da embrioni di topo precoce

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Un protocollo per l'immunoistochimica e la distribuzione dell'RNA in situ all'interno dell'embrione di drosofila precoce

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Un dispositivo di trapianto semplice ed efficace per embrioni di zebrafish

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Isolamento delle cellule staminali mesenchimali del tessuto adiposo di ratto per la differenziazione in cellule produttrici di insulina

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face Preparazione del cuscino endocardico per l'analisi della morfogenesi planare negli embrioni di topo

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...