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

Los autores reconocen al Dr. Kenzo Ivanovitch por su trabajo previo sobre este método y al grupo del Dr. Shigenori Nonaka (Institutos Nacionales de Ciencias Naturales, Japón) por proporcionar la experiencia inicial en el montaje de embriones. Este estudio ha contado con el apoyo de la subvención PGC2018-096486-B-I00 del Ministerio de Ciencia e Innovación y la subvención H2020-MSCA-ITN-2016-722427 del programa Horizonte 2020 de la UE a MT y la subvención 1380918 del Programa Operativo FEDER Andalucía 2014-2020 a JND. MS ha contado con el apoyo de una beca de doctorado de la Fundación La Caixa (LCF/BQ/DE18/11670014) y una beca itinerante de la Compañía de Biólogos (DEVTF181145). El CNIC cuenta con el apoyo del Ministerio de Ciencia y de la Fundación ProCNIC.

Todos los procedimientos con animales fueron aprobados por el Comité Ético de Experimentación Animal del CNIC, por la Comunidad de Madrid (Referencia PROEX 220/15) y conformados con la Directiva 2010/63UE de la UE y la Recomendación 2007/526/CE relativa a la protección de los animales utilizados para experimentación y otros fines científicos, aplicados en la legislación española bajo el Real Decreto 1201/2005.
Este protocolo incluye el uso de dos machos de la línea de ratón transgé...

<ol>
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Los primeros progenitores del corazón se organizan en un tubo cardíaco primitivo que comienza a latir mientras aún se está formando. Comprender cómo se lleva a cabo este proceso es clave para identificar el amplio espectro de defectos cardíacos congénitos a eventos morfogenéticos específicos. Para eso, las imágenes en vivo ofrecen la oportunidad de estudiar el desarrollo embrionario normal y defectuoso con una mayor resolución temporal. Esto es especialmente útil para estudiar las células progenitoras cardí...

El corazón se forma temprano durante la embriogénesis para comenzar a bombear nutrientes a todo el embrión, mientras continúa desarrollándose1. En embriones de ratón, un día y medio después del inicio de la gastrulación, un órgano cardíaco rudimentario se ensambla en el polo anterior 2,3. En la etapa de Raya Temprana (ES), los progenitores cardíacos en el epiblasto ingresan a través de la raya primitiva a la capa mesodérmica naciente 4,5,6 y comienzan a migrar al polo anterior, donde se diferencian para formar el tubo cardíaco primitivo. A lo largo de este proceso, los progenitores cardíacos tempranos experimentan reordenamientos celulares, transformaciones de forma y diferenciación, además de la migración7 (Figura 1).
Los primeros progenitores cardíacos han atraído a los investigadores durante casi un siglo debido a su notable capacidad para diferenciar y construir un órgano funcional simultáneamente. En las últimas dos décadas, el análisis clonal y los modelos de knockout condicional han demostrado que el desarrollo temprano del corazón implica distintas fuentes celulares en un proceso altamente dinámico 8,9,10. Sin embargo, la estructura 3D del tubo cardíaco primitivo y la naturaleza dinámica de su morfogénesis hacen que sea difícil de estudiar (Figura 1), y estamos lejos de comprender toda su complejidad11.
Para estudiar estos procesos celulares dinámicos, los métodos de imagen en vivo ofrecen ahora un detalle sin precedentes 7,12,13,14. En el modelo de ratón, los enfoques en vivo han sido clave para interrogar temas de desarrollo que son difíciles de abordar mediante análisis estático 7,13,15. Si bien el cultivo ex vivo a largo plazo y las configuraciones robustas de microscopios avanzan rápidamente16,17, pocos investigadores tienen la experiencia para obtener imágenes exitosas de embriones vivos. Aunque las publicaciones en papel proporcionan suficientes detalles técnicos para reproducir experimentos de imágenes en vivo, algunas habilidades y trucos son difíciles de comprender sin ejemplos visuales o asistencia entre pares. Para acelerar este proceso de aprendizaje y difundir el uso de imágenes en vivo entre los laboratorios, ensamblamos un protocolo de video (Figura 2) que reúne las habilidades necesarias para realizar imágenes en vivo en embriones de ratón gastrulados.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64273/64273fig01.jpg"> Figura 1: Diferenciación temprana de las células progenitoras cardíacas en el embrión de ratón desde el inicio de la gastrulación hasta la etapa anterior a la formación primitiva del tubo cardíaco. Las células progenitoras cardíacas ingresan al mesodermo poco después del inicio de la gastrulación, migrando al lado opuesto del embrión. La etapa morfológica y embrionaria del día (E) se escribe en la parte superior de los diagramas. Las flechas discontinuas representan la trayectoria de migración de los progenitores primitivos del tubo cardíaco durante la gastrulación. Esta figura fue adaptada de11. Abreviaturas: ES = Early Streak; MS = Racha media; EHF = Pliegue temprano de la cabeza. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig01large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64273/64273fig02.jpg"> Figura 2: Diagrama de flujo de trabajo para imágenes en vivo de progenitores cardíacos tempranos. <a href="https://www.jove.com/files/ftp_upload/64273/64273fig02large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

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

Los primeros pasos del desarrollo del corazón implican cambios drásticos en el comportamiento y la diferenciación celular. Mientras que el análisis de embriones fijos permite estudiar en detalle etapas específicas del desarrollo en una instantánea fija, las imágenes en vivo capturan eventos morfogenéticos dinámicos, como la migración celular, los cambios de forma y la diferenciación, al obtener imágenes del embrión a medida que se desarrolla. Esto complementa el análisis fijo y amplía la comprensión de cómo se desarrollan los órganos durante la embriogénesis. A pesar de sus ventajas, las imágenes en vivo rara vez se utilizan en modelos de ratón debido a sus desafíos técnicos. Los embriones tempranos de ratón son sensibles cuando se cultivan ex vivo y requieren un manejo eficiente. Para facilitar un uso más amplio de imágenes en vivo en la investigación del desarrollo de ratones, este documento presenta un protocolo detallado para la microscopía viva de dos fotones que permite la adquisición a largo plazo en embriones de ratón. Además del protocolo, se proporcionan consejos sobre el manejo de embriones y la optimización del cultivo. Esto ayudará a comprender los eventos clave en la organogénesis temprana del ratón, mejorando la comprensión de la biología del progenitor cardiovascular.

Utilizamos el protocolo para visualizar la activación de la señalización NOTCH en progenitores cardíacos tempranos a punto de diferenciarse a las células endoteliales durante la morfogénesis primitiva del tubo cardíaco. Para eso, cruzamos ratones C57BL / 6-N de tipo salvaje con ratones Tg (CBF: H2BVenus, +)18 para obtener embriones que informan actividad NOTCH a través de la proteína fluorescente amarilla Venus. En E7.5, la fluorescencia de Venus está presente en todo el ectodermo neural...

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

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

Presentamos un protocolo detallado para el cultivo de embriones de ratón y la obtención de imágenes que permite obtener imágenes 3D + tiempo de células progenitoras cardíacas. Este kit de herramientas de video aborda las habilidades clave requeridas para obtener imágenes en vivo exitosas que de otro modo serían difíciles de adquirir de publicaciones de solo texto.

Imágenes en vivo de progenitores cardíacos tempranos en el embrión de ratón

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

Este protocolo permite obtener imágenes de embriones de ratón a medida que se desarrollan. Esto permite estudiar la cardiogénesis temprana en detalle. Al igual que tomar imágenes fijas de embriones falsos, las imágenes en vivo brindan acceso completo al proceso dinámico de estudio en el desarrollo embrionario.Las habilidades requeridas para las imágenes de la vida son difíciles de comprender a partir de la publicación de solo texto. Ver a otra persona hacerlo es esencial para aprender. Para comenzar, corte trozos de alambre de tungsteno de un centímetro de largo y afilarlos a 0.02 a 0.05 milímetros de diámetro sumergiendo la punta del alambre en una solución saturada de nitrato de sodio.Para preparar capilares de vidrio con codo, coloque el punto medio de un capilar de vidrio estándar de un milímetro sobre un encendedor de bunsen durante tres a seis segundos, tirando lentamente de ambos extremos hasta que el capilar se vuelva delgado y flexible. Luego, rompe el capilar por la mitad y caliéntalo brevemente para producir una curva de 90 grados a dos centímetros de la punta. Vio una pieza de polimetacrilato de metilo que mide aproximadamente siete milímetros de largo, cuatro milímetros de ancho y dos milímetros de espesor.Sostenga la pieza de metacrilato usando un vicio de banco, luego perfore agujeros personalizados transversalmente a través de toda la pieza. Gire el taladro lentamente mientras aplica una presión baja pero constante. Utilice una lima fina para suavizar el borde del soporte y ajustar su tamaño.Además, cree una pendiente suave en los bordes del soporte para facilitar el posicionamiento del embrión. Coloque el soporte terminado en un plato con agua destilada para enjuagarlo. Bajo el microscopio estéreo, revise el soporte para ver si los orificios contienen polvo de perforación.Si hay polvo, use agujas de tungsteno para eliminarlo. Para esterilizar el soporte, colóquelo en un tubo cónico lleno de agua destilada y sonicar durante 20 minutos con un mínimo de 20 vatios de potencia de salida. Extraiga el útero de una ratona embrionaria eutanasiada del día 6.75 a 7.5 y colóquelo en una toallita de papel seca y limpia.Luego, corte el útero, insertando la punta de las tijeras finas desde el lado mesometrial y deslizando la cuchilla para exponer los decidui, y transfiéralos al medio de disección. Diseccionar los embriones, manteniendo intacto el cono ectoplacentario. Luego, retire la membrana del Reichter, dejando parte de ella en la región embrionaria adicional.Para mantener la disección medio caliente, reemplácela cada 10 minutos. Además, disecciona los decidui en grupos de tres a cuatro mientras mantienes el resto en un medio de disección colocado en un baño de 37 grados centígrados. Colocar inmediatamente los embriones disecados en un medio de cultivo.Seleccione los embriones con las características de fluorescencia deseadas utilizando un microscopio estereoscópico de fluorescencia y colóquelos en el tubo que contiene el medio de cultivo en la incubadora de cultivo celular para recuperarlos durante dos horas antes de la obtención de imágenes. Después de encender todos los componentes del microscopio, incluyendo la computadora, el software y los láseres, encienda el controlador de dióxido de carbono y configúrelo al 7%Coloque una gota de grasa de silicio de alto vacío en un plato de 35 milímetros con un fondo deslizante de cubierta de vidrio. Coloque el soporte encima de la gota y presione suavemente para inmovilizarlo.Para el montaje de embriones, transfiera de dos a tres embriones de la incubadora al plato con un soporte. Usando un par de pinzas y una aguja de tungsteno cónica, coloque los embriones en los agujeros. Use la aguja para enganchar el embrión por el cono ectoplacentario e insértelo en un orificio de tamaño equivalente.Mueva con cuidado el plato que contiene los embriones y el soporte a la placa del microscopio. Baje el objetivo hasta que se forme un menisco líquido en la interfaz del objetivo medio. Después de colocar el embrión enfocado, monte la cámara de incubación y séllela alrededor del objetivo con cinta adhesiva.Usando una captura en vivo en el software de control del microscopio, ajuste la salida del láser y los niveles de ganancia. Luego, cubra el medio con aceite de parafina goteándolo lentamente sobre el objetivo. Configurar una grabación de lapso de tiempo.Establezca un intervalo de cinco a 10 minutos con un espacio Z de tres a cinco micras entre pilas. Deje un espacio en blanco encima de la Z-Stack para anticipar el crecimiento del embrión. Un mínimo de 50 micras, añadiendo 30 micras por cada hora que la adquisición no será supervisada.Habilite la opción de guardado automático para permitir la supervisión de la adquisición desde una computadora para permitir el ajuste del tamaño Z-Stack si es necesario, para que se ajuste al área de interés dentro del foco. Se muestra el desarrollo del corazón de un embrión de cuerpo entero vivo mediante imágenes multifotónicas. En el día embrionario 7.5, la fluorescencia venosa está presente en todo el ectodermo neural, con algunos núcleos positivos en el mesodermo esplácnico y extra embrionario.Después de cuatro horas, los progenitores endoteliales se activan venosos y se ensamblan para formar el tubo endocárdico y las aortas subyacentes, que se convierten en estructuras cerradas después de siete horas. Tenga cuidado al quitar la membrana del Reichter. Puede estar realmente pegado al embrión, especialmente en las etapas de prevacilación.Durante la extracción, trate de pellizcar la membrana por la región embrionaria adicional.

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

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

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

Live-imaging of the Drosophila Pupal Eye

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

Live-proyección de imagen de la<em&gt; Drosophila</em&gt; Pupal Ojo

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

Investigación

Biología del Desarrollo

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

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

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

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

Protocolos para la obtención de embriones somáticos y cigóticos para el estudio de la regulación del desarrollo embrionario temprano en el 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 ...

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

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

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

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

Caracterización de tímico Instalarse Progenitores en el embrión de ratón Uso<em&gt; En Vivo</em&gt; Y<em&gt; In Vitro</em&gt; Ensayos

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

Imaging Subcellular Structures in the Living Zebrafish Embryo

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

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

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

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

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

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

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

La observación de la división mitótica y dinámica en un embrión de pez cebra en vivo

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

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

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

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

Identificación de progenitores eritromioideos y su progenie en el embrión de ratón por citometría de flujo

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

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

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

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

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

En vivo de imágenes de transgénicos de la Expresión Génica en el Estimado de retina progenitores en embriones de pez cebra quiméricos para estudiar celulares Influencias no autónomos

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

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

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

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

Bioingeniería de los microambientes humanizados de la médula ósea en el ratón y su visualización por imágenes en directo

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

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

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

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

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

In Vitro Cultivo de óvulos para la proyección de imagen de cigoto polarización de Live-celular y patrones del embrión en Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

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

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

Imágenes en vivo de la fusión secundaria del paladar del ratón

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...