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. Desenvolvimento de Embriões Zygotic <ol><li> Material Vegetal <ol><li> Cresça o Medicago truncatula tipo selvagem Jemalong ou seu próximo isogênico, altamente re-generativas genótipo Jemalong 2HA 13 (conhecido como 2HA) em uma estufa com um fotoperíodo de 14 horas e 23 ° C / 19 ° C de temperatura dia / noite. <ol><li> Pierce a superfície do revestimento de semente (com uma agulha G 23) antes de semear a semente de modo que a água pode entrar a semente e embeber em água durante a noite. Adicionar água suficiente para cobrir integralmente a semente. </li><li> Sow 3 sementes em cada vaso diâmetro 15 cm (total de 10 potes) em mistura de envasamento (areia grossa, perlita, coco-turfa (1: 1: 1) mais de 5 g de Osmocote padrão exato: fertilizante de liberação lenta) e transferir para o estufa. Manter a plântula após a germinação saudável para que haja uma planta por vaso e cercada por uma treliça para os caules das plantas para escalar. </li></ol></li></ol></li><li> Pods Colhendo <ol><li> Colete pods do wTipo de ild Jemalong ou 2HA para obter os estágios do embrião precoce apropriados como primeiro descrito 9. NOTA: As diferentes etapas pod são mostrados na Figura 1 e as correspondentes fases de embriões são mostrados na Figura 2. </li><li> Confira os diferentes estágios pod na colheita utilizando critérios conforme tabela 1. Medir o tempo decorrido a partir da fase 6 para ajudar fases distintas 6 e 7 (Tabela 2). </li></ol></li><li> Dissecando óvulos de vagens e verificar a fase de embrião <ol><li> Isolar óvulos de vagens, usando uma pinça fina sozinho ou um microscópio de dissecação estéreo e uma pinça, se necessário. Se necessário, a fase de embrião pode ser rapidamente verificados usando um microscópio composto padrão (Figura 3B). </li><li> Prepare modificado solução de Hoyer contendo 7,5 g goma arábica, 100 g hidrato de cloral, 5 ml de glicerol, 60 ml de água desionizada destilada. Dissolve-se por agitação durante 3-5 horas ou durante a noite. </li><li> Para obter mais refinarmicroscopia d limpar os óvulos dissecados em solução 15,16 modificado de Hoyer. Com cuidado, pegue os óvulos intactas e coloque em um pequeno volume de solução de Hoyer (suficiente para cobrir o óvulo) à temperatura ambiente até claro. </li><li> Ver as amostras com contraste de interferência diferencial (DIC) óptica. Capturar as imagens iwth uma câmara digital 9 (Figura 2). </li><li> Obter os óvulos mais uniformes da região central do pod e acumular o número requerido. Um exemplo é mostrado na Figura 3A. Coletar os óvulos no gelo e armazenar a temperatura requerida (ácidos -80 ° C para nucleicos), para posterior análise. NOTA: Para detalhes sobre a análise dos cortes histológicos, microscopia eletrônica de transmissão, e hibridização in situ consulte papéis 8,9. </li></ol></li></ol> 2. Somatic Desenvolvimento de embriões in vitro <ol><li> Use um M. truncatula cultivar tchapéu é capaz de formar prontamente embriões em cultura. Para este protocolo para explantes foliares usar plantas 2HA 13,17 que são 2-4 meses de idade. </li><li> Tome folhetos da última trifólio quase totalmente expandida de uma haste de alongamento. </li><li> Mantenha os trifólios sobre papel toalha úmida em uma panela de cultura para evitar a desidratação. NOTA: Uma vez que os trifólios são colhidas eles precisam para ser utilizado com o mínimo de atraso. </li><li> Preparação do meio de cultura <ol><li> Utilizar a P4 10: 5 em meio de agar (0,8% w:: v) de 4: 1 em placas de Petri de 9 centímetros. NOTA: O P4 10: 4: 1: 5 do meio consiste na forma P4 basal 18 mais 10 uM de ácido 1-naftalenoacético (NAA), 4 uM de 6-benzilaminopurina (BAP), 1 uM de ácido abscísico (ABA) e 5 uM giberélico ácido (GA). O uso de GA + ABA acelera a formação de embriões e provoca aumentos substanciais no número de embriões 14. </li><li> Certifique-se o meio basal P4 em 1 L; que consiste em sais principais (fazer Calcium-se separadamente), sais e vitaminas menores (Tabela 3), ferro quelato (certifique-se separadamente), casaminoácidos (hidrolisado de caseína), 30 g de sacarose, 8 g de ágar. </li><li> Soluções de loja de grandes sais, de cálcio, de ácidos casamino, sais menores e vitaminas a -20 ° C em aliquotas adequadas. Armazenar ferro quelato a 4 ° C. </li><li> Perfazer o ferro quelado (200x) por dissolução de 7,44 g de Na 2 EDTA • 2H 2 O em aproximadamente 900 ml de água destilada desionizada, enquanto se agitava, em seguida, levar a solução a 98-99 ° C, enquanto a adição de 1,853 g de FeSO4 • 7H 2 O. Finalmente completar a 1 L quando os ingredientes são dissolvidos. Loja em âmbar garrafas a 4 ° C colorido. </li><li> Adicione-se o meio basal P4 com as soluções de reserva como na Tabela 4. As hormonas no meio são 10 uM de NAA, 4 uM BAP, 1 uM de ABA, 5 uM GA (ver Tabela 4 e Tabela materiais específicos). Adicione o NAAe BAP antes da autoclavagem e adicionar ABA e GA após autoclavagem e arrefecimento a cerca de 55 ° C, utilizando-se esterilização por filtração com um filtro de 0,22 um ligado a uma seringa. Misture bem por agitação suave. </li><li> Ajuste a mídia para pH 5,8 com 1 M KOH antes da autoclavagem. Autoclave a 121 ° C durante 20 min. </li><li> Após autoclavagem adicionar o filtro ABA esterilizado e GA, e despeje cerca de 25 ml de mídia na estéreis nove centímetros placas de Petri numa câmara de fluxo laminar ou gabinete de risco biológico. Legal e deixar o agar conjunto com a tampa fora. Em seguida, coloque sobre a tampa e certifique-se que não há condensado na tampa. Etiqueta tal como exigido e armazenar a 4 ° C (em uma caixa de papelão para impedir tampa condensado). </li></ol></li><li> Esterilização de tecido foliar <ol><li> Trabalhar em uma capela de fluxo laminar, esterilizada por UV ou gabinete de risco biológico. </li><li> Coloque as folhas em malha a bola de um infusor de chá mola previamente autoclavada. </li><li> Imergir infusor de chá que contém as folhas em 70% (v: v) de etanol em um culpot tura durante 30 segundos. NOTA: Para todas as etapas deste procedimento utilizar 250 ml tampa de rosca potes de cultura de policarbonato que são autoclavados. </li><li> Drenar e, em seguida, transferir e imergir o tecido de folha em 1: 8 (v: v) de lixívia diluída (0,5% de cloro) durante 10 min. Homogeneizar suavemente ao longo do tempo. </li><li> Escorra a água sanitária e transferir o infusor de chá em uma panela de cultura contendo água destilada deionizada estéril. Agite suavemente e drenar o excesso de água. Repita mais uma vez com um pote de cultura fresco de água destilada deionizada estéril. </li><li> Retire as folhas do infusor de chá com uma pinça estéril para um pote de cultura fresco de água destilada deionizada estéril. Parafuso na tampa estéril e enxaguar invertendo e rodando. Deixe as folhas que flutuam na água estéril até que esteja pronto para preparar explantes. </li></ol></li><li> Preparação e chapeamento explantes <ol><li> Usando técnicas estéreis, guarnição folhetos esterilizados individuais a partir da borda com um bisturi, e cortar o retângulo restante em dois ou tree explantes rectangulares (8-10 x 3-5 mm) com o meio da veia no centro de cada explante (Figura 5A). NOTA: O tamanho do explante é bastante importante no início do primeiro dos calos. Realizar o corte em tampas estéreis a partir de recipientes de comida take-away. </li><li> Placa 6 explantes no meio de cultura solidificado com agar de 9 cm de diâmetro em placas de Petri (Figura 5B). Placa explantes lado do abaxial para baixo para manter a orientação da folha de costume. </li><li> Selar as placas de Petri com Parafilm (Figura 5C) se estenderam ao redor do prato. O tecido está agora pronto para a incubação. </li></ol></li><li> A incubação de tecidos <ol><li> Incubam-se as placas no escuro a 28 ° C numa sala de temperatura controlada ou câmara de crescimento durante todo o período de cultura. NOTA: Os explantes iniciará desdiferenciação, sofrem divisão celular, proliferação (formação de calos) e embriogênese (diferenciação). </li><li> Subcultura do EXPOSIÇÃO diferenciandonts após 3 semanas em meio fresco para. Neste subcultura transferir todo o explante usando uma espátula de aço inoxidável estéril e uma pinça, em uma câmara de fluxo laminar ou um armário de risco biológico. Como ocorre calos e o calos excede o tamanho do maior na Figura 5C, transferir 50% do calo indivíduo quando subcultura para meio fresco. </li><li> Observar os primeiros embriões em cerca de 4 semanas. Embora haja um certo grau de sincronia, recolher os diferentes estágios de embriões, durante um período de incubação de 4-8 semanas (Figura 6A, B). Recolha sob um microscópio de dissecação e processo de acordo com objetivos experimentais. </li><li> Subcultura a cada 3 semanas e remover embriões periodicamente de modo que os explantes continuará a produzir embriões durante 3 meses. </li></ol></li></ol>

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The authors declare that they have no competing interests.

Os protocolos descritos são relativamente simples e permitir a investigação de embriogênese legume com toda a célula contemporânea e técnicas moleculares. Reconhecemos que há vantagens e desvantagens de ambos in vivo e in vitro abordagens. Ambos permitem mais foco na embriogênese inicial em relação à cultura de sementes imaturas 19. No caso de estudos in vivo que é descrito é predominantemente o isolamento do óvulo da vagem que é adequado para mui...

Legumes são a terceira maior família de plantas superiores com cerca de 20.000 espécies e da (ou Fabaceae) família Leguminosae são segundo para os cereais produzidos na área colhida e produção total de 1. A soja é a terceira maior safra cultivada. Leguminosas fornecer cerca de um terço da proteína da dieta e um terço de óleo vegetal para consumo humano 2. Legumes com a sua capacidade de fixação de N 2 também contribuir para sistemas agrícolas sustentáveis. Medicago truncatula, como soja, lojas de proteína e óleo nos cotilédones de suas sementes e é um modelo legume genética e genómica com consideráveis ​​recursos genéticos e genômicos 3,4. Enquanto M. truncatula permitiu avanços na compreensão da simbiose rizóbio-leguminosa 4 tem sido cada vez mais utilizados para estudar biologia sementes de leguminosas 5-7 e embriogênese 8,9. Arabidopsis embriogénese tem sido extensivamente estudada 10,11 mas isa não leguminosa e os detalhes da embriogénese não são idênticos aos Medicago 8,10. Embriogênese Zygotic em M. truncatula tem características interessantes, com um hipófise multicelular distintivo, um suspensor endoployploid e célula de transferência basal 8. Embriogênese somática (SE) é comumente usado para regeneração de plantas 12. No modelo de leguminosa M. truncatula, a linha de semente Jemalong 2HA (2HA) foi desenvolvido a partir do pai Jemalong a ter altas taxas de embriogênese somática 13. O número de embriões produzidos recentemente tem sido substancialmente aumentada pela adição tanto de ácido giberélico (GA) e ácido abscísico (ABA) para a forma longa estabelecida 14. Neste caso GA e agir sinergicamente ABA, o que é incomum, dado que GA e ABA normalmente agem antagonicamente 14. Os embriões produzidos a partir de calo desenvolver na superfície que permite que a fase de embriogénese a ser prontamente determinada Visually e facilmente colhidas. Tendo em perto de linhas isogénicas que são embriogénicos (2HA) e não embriogénico (Jemalong) facilita a investigação de embriogénese somática e tendo tanto in vivo como em sistemas in vitro proporciona diferentes possibilidades experimentais. A compreensão dos mecanismos celulares e moleculares do desenvolvimento do embrião é essencial para a compreensão de sementes e o desenvolvimento da planta. Em leguminosas, como em outros dicotiledóneas, são os cotilédones do embrião que armazenar os produtos que são utilizados para a nutrição humana. Embriogênese inicial envolve a divisão celular rápida e padronização embrião correta. Em cerca de 8 dias após a fertilização, a M. truncatula embrião atinge estágios iniciais de cotilédones. A caracterização morfológica não é exatamente indicado por dias após a fertilização em condições de casa de vegetação. Assim, uma abordagem padronizada eficiente para indicar a fase de embriões em desenvolvimento é valioso no estudo da genética regulção da embriogênese zigótica cedo. Neste artigo, nós fornecemos dois protocolos padronizados para coletar embriões em desenvolvimento para estudos biológicos da embriogênese no modelo legume M. truncatula. O primeiro deles é coletar embriões zigóticos, associando a embriogênese e pod morfologia enquanto o segundo é embriogênese somática via explantes foliares cultura para fornecer um grande número de embriões facilmente acessados.

<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

No início da embriogênese a partir de uma única célula zigoto passa por rápida divisão celular e morfogênese, e é caracterizada morfologicamente por estágios de pré-globular, globulares, coração, torpedo e cotilédones. Esta evolução progressiva está sob a regulação apertada de uma rede molecular complexa. Colhendo embriões suficientes em um estágio similar de desenvolvimento é essencial para a investigação da regulação celular e molecular da embriogênese precoce. Isso não é simples, uma vez embriogênese sofre morfogênese rápido em um curto espaço de tempo por exemplo, oito dias para Medicago truncatula para chegar à fase de cotilédones cedo. Aqui, nós resolver a questão através de duas abordagens. O primeiro estabelece uma ligação entre o desenvolvimento do embrião e morfologia pod em ajudar indicar a etapa do embrião zigótico. Isto é particularmente com base no número de espirais e desenvolvimento das espinhas pod. Uma forma alternativa para complementar o s in vivotudies é através de cultura de explantes de folhas para produzir embriões somáticos. O meio inclui uma combinação de hormônio incomum - uma auxina (ácido 1-naftalenoacético), uma citocinina (6-benzilaminopurina), ácido abscísico e giberelina. As diferentes fases pode ser discernido que cresce fora do calo sem dissecção.

Para diferentes estruturas de embriogénese pod zigóticos correspondentes às diferentes fases de embriões são mostrados na Figura 1A - F enquanto os diferentes estágios embrionários são mostrados na Figura 2A - F. Ao seleccionar vagens na mesma fase, as amostras de óvulos que são bastante uniforme pode ser obtida (Figura 3A). Usando RT-qPCR embrião genes específicos podem ser facilmente detectada e os estudos do curso de temp...

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.

Protocolos para obtenção de embriões somáticos e Zygotic para estudar a regulação do desenvolvimento embrionário precoce no Modelo de leguminosas<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 ...

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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 visualizações.  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. 

Vídeo: 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.

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

Biologia do Desenvolvimento

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.

Imagiologia estruturas celulares no embrião vivo 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.

Transcriptoma Profiling de<em&gt; In-Vivo</em&gt; embriões produzidos pré-implantação dos bovinos por meio de duas cores Plataforma Microarray

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.

Visualização da atividade elétrica celular no Zebrafish embriões adiantados e tumores

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.

Transferência de embriões minimamente invasivos e vitrificação embrionária na fase de embrião ideal em modelo de coelho

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.

Preparação de Pequenas Bibliotecas de RNA para Sequenciamento de Embriões de Camundongos Primitivos

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.

Um Protocolo para Imunohistoquímica e Distribuição In-situ de RNA dentro do Embrião Drosophila 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.

Um dispositivo de transplante simples e eficaz para embriões de 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 de células-tronco mesenquimais de tecido adiposo de rato para diferenciação em células produtoras de 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 Preparação de Almofadas Endocárdicas para Análise de Morfogênese Planar em Embriões de Camundongos

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