This work was supported by the Terry Fox Program of the National Cancer Institute of Canada.

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F.S. Foster acknowledges his role as consultant to VisualSonics Inc.

Ultrasons agents de contraste ont été injectés dans des embryons de souris gestation stade avancé et des images de contraste non linéaires ont été acquis pour mesurer les paramètres de perfusion et de microbulles ciblées contraignant. Imagerie réussie des microbulles à l&#39;intérieur du système vasculaire embryonnaire était dépendant d&#39;un certain nombre de facteurs, le premier étant embryon viabilité. Tous les équipements et appareils ont été préparées à l&#39;avance afin de minimiser le temp...

échographie de rehaussement du contraste permet l&#39;utilisation d&#39;agents de contraste à microbulles de visualiser et caractériser l&#39;environnement vasculaire. Ces agents permettent l&#39;évaluation non invasive de la microcirculation, la vascularisation et la fonction cardiovasculaire. En outre, la modification de la surface de la bulle peut conduire à la liaison à microbulles ciblée biomarqueurs endotheliales, comme démontré dans des applications précliniques de l&#39;angiogenèse, l&#39;athérosclérose et l&#39;inflammation faisant 1,2 imagerie par ultrasons moléculaire des événements vasculaires possibles. Échographie de contraste amélioré peut donc être utilisée pour identifier les environnements complexes et variés qui influencent états vasculaires sains et malades 3-5. Dans le cours des dernières années, l&#39;intérêt pour l&#39;utilité de l&#39;imagerie de microbulles a étendu au modèle polyvalent d&#39;embryon de souris. Comme un modèle de développement des mammifères, l&#39;introduction de microbulles dans le système vasculaire embryonnaire améliore physiologiqueétude du système circulatoire en développement (par exemple, le flux sanguin, le débit cardiaque) et en cas de transgéniques et des modèles de souris mutantes ciblés de la maladie cardiaque 6,7, peut fournir des indications sur la façon dont les facteurs génétiques de modifier la fonction cardiovasculaire. En fait, 2D analyse quantitative et qualitative du cerveau embryonnaire vasculaire ont déjà été atteints 8. En outre, l&#39;embryon de souris présente comme un excellent modèle pour l&#39;examen de la liaison de microbulles ciblées à des marqueurs vasculaires in vivo. Bartelle et al. 9, par exemple, ont mis en place des microbulles avidine en embryon ventricules cardiaque pour évaluer la liaison ciblées dans BIO TAG-Bira embryons transgéniques et d&#39;examiner l&#39;anatomie vasculaire. La génération de modèles hétérozygotes et homozygotes souris peut également être utilisé comme un substitut pour des études de modèle de tumeur visant à définir la nature quantitative de l&#39;échographie moléculaire - une étape importante dans la traduction de cette technique à la clinique. <p class = &quot;jove_content&quot;&gt; microbulles sont le plus souvent introduit à la circulation embryonnaire par injections intra-cardiaque dans des embryons simples exposés à travers une laparotomie 8-10. Dans injections in utero, cependant, faire face à un certain nombre de défis. Il se agit notamment des conseils d&#39;injection, la nécessité de contrer le mouvement dans la mère et l&#39;embryon extériorisé, le maintien de la viabilité hémodynamique chez la mère et embryons extériorisés, portant sur ​​les effets à long terme de l&#39;anesthésie et de complications dues à un saignement 11. Par conséquent, l&#39;objectif de l&#39;enquête était de développer une technique pour injecter microbulles en vie isolé des embryons à un stade avancé 12. Cette option offre plus de liberté en matière de contrôle d&#39;injection et le positionnement, la reproductibilité du plan d&#39;imagerie sans entrave, et l&#39;analyse d&#39;image simplifiée et la quantification. Dans la présente étude, nous présentons une nouvelle procédure pour l&#39;injection de microbulles en vivant embryons murins for les fins de l&#39;étude du comportement cinétique microbulles et d&#39;étudier la liaison à des marqueurs de surface endothéliales endogènes microbulles ciblées. Non linéaire échographie de contraste spécifique est utilisé pour mesurer un certain nombre de paramètres de perfusion de base, y compris la mise en valeur de crête (PE), lavez-la fréquence et de temps pour culminer (TTP) dans isolées embryons E17.5. Nous démontrons également la validité du modèle d&#39;embryons pour évaluer la nature quantitative de l&#39;échographie moléculaire dans une perte de endogline embryonnaire de modèle de souris fonction transgénique, où endogline est une cible cliniquement pertinente en raison de sa forte expression dans les cellules endothéliales vasculaires dans les sites de l&#39;angiogenèse active 13 . L&#39;adhésion de l&#39;endogline ciblée (MB E), isotype de rat IgG 2 commande (MB C) et non ciblées (MB U) microbulles est évaluée dans endogline hétérozygote (+/- Eng) et homozygote endogline (ENG + / +) exprimant des embryons. Analyse du bindi cibléeng révèle que l&#39;échographie moléculaire est capable de différencier entre les génotypes de endogline et concernant les densités de récepteurs à des niveaux moléculaires quantifiables à ultrasons.

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			<td height="20" style="height:20px;width:419px;">Reagents</td>
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			<td style="width:207px;"></td>
			<td></td>
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			<td height="19" style="height:19px;">Antibodies (biotinylated, eBioscience)</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td>Antibody choice depends on the experiment</td>
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		<tr height="25">
			<td height="25" style="height:25px;">&#160;&#160;&#160;&#160;&#160; rat isotype IgG2 control</td>
			<td style="width:215px;">eBioscience</td>
			<td style="width:207px;">13-4321-85</td>
			<td>This antibody/microbubble combination is often required as experimental control&#160;</td>
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		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160;&#160; biotin anti-mouse CD309</td>
			<td style="width:215px;">eBioscience</td>
			<td style="width:207px;">13-5821-85</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Biotinylated rat MJ 7/18 antibody to mouse endoglin</td>
			<td style="width:215px;">In house hybridoma</td>
			<td style="width:207px;"></td>
			<td>Outside antibodies may also be appropriate: we&#160; have used eBioscience (13-1051-85 ) in the past</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Distilled water</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Embryo media</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; 500 mL Dulbecco&#8217;s Modified Eagle&#8217;s Medium with high glucose</td>
			<td>Sigma</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; 50 mL Fetal Bovine Serum</td>
			<td>ATCC</td>
			<td style="width:207px;">30-2020</td>
			<td>lot # 7592456</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; Hepes&#160;</td>
			<td style="width:215px;">Gibco</td>
			<td style="width:207px;">15630</td>
			<td>5mL, 1M</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">&#160;&#160;&#160;&#160; Penicillin-Streptomycin&#160;</td>
			<td style="width:215px;">Gibco</td>
			<td style="width:207px;">15140-122</td>
			<td>5 mL, 10,000 units Pen., 10,000 ug Strep</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ethanol, 70%</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ice</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Paraformaldehyde</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">76240</td>
			<td>4%</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Phosphate Buffered Saline [1x]&#160;</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">D8537</td>
			<td>1x, w/o calcium chloride &#38; magnesium chloride</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pregnant mouse, CD-1</td>
			<td style="width:215px;">Charles River Laboratories Inc.&#160;</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">0.9% sodium chloride (saline)</td>
			<td style="width:215px;">Hospira</td>
			<td style="width:207px;">0409-7984-11</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound contrast agent, target ready and untargeted</td>
			<td style="width:215px;">MicroMarker; VisualSonics Inc.</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound gel (Aquasonic 100, colourless)</td>
			<td style="width:215px;">CSP Medical</td>
			<td style="width:207px;">133-1009</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell culture plates (4) :&#160; 100x20 mm</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">08-772-22</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell culture plates (12) : 60x15 mm</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:207px;">D8054</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:419px;">Centrifuge</td>
			<td>Sorvall Legend RT centrifuge&#160;</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Conical tubes, 50 mL BD Falcon</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">21008-938</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Diluent</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:207px;">Isoton II Diluent, 8448011</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Dissection scissors (Wagner)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">Wagner 14068-12</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps (2), Dumont SS (0.10x0.06 mm)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">11200-33</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Forceps, splinter</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">25601-134</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass beaker, 2 L (Griffin Beaker)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89000-216</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass capillaries, 1x90 mm GD-1 with filament</td>
			<td style="width:215px;">Narishige</td>
			<td style="width:207px;">GD-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glass needle puller</td>
			<td style="width:215px;">Narishige</td>
			<td style="width:207px;">PN-30</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gloves</td>
			<td style="width:215px;">Ansell</td>
			<td style="width:207px;">4002</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Gross anatomy probe</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">10088-15</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Hot plate</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89090-994</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ice bucket</td>
			<td style="width:215px;">Cole Parmer</td>
			<td style="width:207px;">RK 06274-01</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Imaging Platform</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">Integrated Rail System</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Light source, fiber-optic</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">12-562-36</td>
			<td>Ideally has adjustable arms</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Luers (12), polypropylene barbed female &#188;-28 UNF thread</td>
			<td style="width:215px;">Cole Parmer</td>
			<td style="width:207px;">45500-30</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Micro-ultrasound system, high-frequency</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">Vevo2100</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Needles, 21 gauge&#160; (1&#8221;)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">305165</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Particle size analyzer</td>
			<td style="width:215px;">Beckman Coulter</td>
			<td style="width:207px;">Multisizer 3 Coulter Counter</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Perforated spoon (Moria)</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">MC 17 10373-17</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pins (6), black anodized minutien 0.15 mm</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">26002-15</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;">Pipettors [2-20 uL, 20-200uL, 100-1000uL]</td>
			<td style="width:215px;">Eppendorf</td>
			<td style="width:207px;">Research Plus&#160; adjustable 3120000038;&#160;&#160;&#160;&#160;&#160;&#160; 3120000054;&#160;&#160;&#160;&#160;&#160;&#160; 3120000062</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Pipettor tips [2-200uL, 50-1000uL]</td>
			<td style="width:215px;">Eppendorf</td>
			<td style="width:207px;">epT.I.P.S.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 22491334;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 022491351</td>
			<td style="width:639px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Scissors</td>
			<td style="width:215px;"></td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td style="width:215px;">Dow Corning</td>
			<td style="width:207px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tubing, Tygon laboratory 1/32x3/32&#8221;</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">63010-007</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Wooden applicator stick (swab, cotton head)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">CA89031-270</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Surgical microscope 5-8x magnification</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:207px;">Steromaster</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringes, 1 mL Normject</td>
			<td style="width:215px;">Fisher</td>
			<td style="width:207px;">14-817-25</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringes (10), 30 mL</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">CA64000-041</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe infusion pump&#160;</td>
			<td style="width:215px;">Bio-lynx&#160;</td>
			<td style="width:207px;">NE-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermometer, -20-110oC</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">89095-598</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Timer</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">33501-418</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tubes, Eppendorf</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">20170-577</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Tube racks (3)</td>
			<td style="width:215px;">VWR</td>
			<td style="width:207px;">82024-462</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Ultrasound transducer, 20 MHz</td>
			<td style="width:215px;">VisualSonics Inc.</td>
			<td style="width:207px;">MS250</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Vannas-Tubingen, angled up</td>
			<td style="width:215px;">Fine Science Tools</td>
			<td style="width:207px;">15005-08</td>
			<td></td>
		</tr>
	</tbody>
</table>

contrast imaging in mouse embryos using high-frequency ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

L&#39;injection d&#39;agents de contraste ultrasonore dans ex utero des embryons de souris dépend de l&#39;isolement réussi de vie, les embryons au stade gestationnel tardif de l&#39;utérus et le maintien de la viabilité au cours de l&#39;injection et l&#39;imagerie par ultrasons associée. Une fois que l&#39;embryon a été extériorisé et positionné, comme le montre la figure 1, l&#39;injection soigneuse de l&#39;agent de contraste dans le système vasculaire embryonnaire est possible. ...

Watch this Scientific Journal Video about Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound at JoVE.com

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ici, nous présentons un protocole d&#39;injecter des agents de contraste ultrasonores de microbulles dans la vie, isolés en fin de gestation embryons murins de la scène. Cette méthode permet l&#39;étude des paramètres de perfusion et de marqueurs moléculaires vasculaires au sein de l&#39;embryon en utilisant à haute fréquence échographie de contraste amélioré.

Imagerie de contraste dans les embryons de souris utilisant à haute fréquence à ultrasons

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

contrast-imaging-in-mouse-embryos-using-high-frequency-ultrasound

Research

JoVE Journal

Developmental Biology

15.2K vues.  University of Toronto.  Ici, nous présentons un protocole d'injecter des agents de contraste ultrasonores de microbulles dans la vie, isolés en fin de gestation embryons murins de la scène. Cette méthode permet l'étude des paramètres de perfusion et de marqueurs moléculaires vasculaires au sein de l'embryon en utilisant à haute fréquence échographie de contraste amélioré. 

Vidéo: Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

en cours d&#39;analyse<em&gt; In Vivo</em&gt; Migration cellulaire en utilisant Transplantations cellulaires et imagerie time-lapse en Zebrafish Embryons

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Recherche

Biologie du développement

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.

Imagerie en direct de la souris Fusion de palais secondaire

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 ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

Une nouvelle utilisation d’échographie en trois dimensions de haute fréquence pour la caractérisation de grossesse précoce chez la souris

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound (30/45MHZ) System

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular mechanisms of CHDs is&#160;crucial for inventing new preventive and therapeutic strategies. Mutant mouse models are powerful tools to discover new&#160;mechanisms&#160;and environmental stress modifiers that drive cardiac development and their potential&#160;alteration in CHDs. However, efforts to establish the causality of these putative contributors have been limited to histological&#160;and molecular studies&#160;in non-survival animal experiments, in which&#160;monitoring the key physiological and hemodynamic parameters is&#160;often absent. Live imaging technology has become an essential tool to establish the&#160;etiology of CHDs. In particular,&#160;ultrasound imaging can be used prenatally without surgically exposing the fetuses,&#160;allowing maintaining&#160;their baseline physiology while monitoring the impact of environmental stress&#160;on the hemodynamic and structural aspects of cardiac chamber development. Herein, we use the High-Frequency Ultrasound (30/45)&#160;system to examine the cardiovascular system in fetal mice at E18.5 in utero at the baseline and in response to prenatal hypoxia exposure. We demonstrate the feasibility of the system to measure cardiac chamber size, morphology, ventricular function, fetal heart rate, and umbilical artery flow indices, and their alterations in fetal mice exposed to systemic chronic hypoxia in utero in real time.

Fetal Mouse Cardiovascular Imaging Using a High-frequency Ultrasound 30/45MHZ System

High-frequency ultrasound imaging of the fetal mouse has improved imaging resolution and can provide precise non-invasive characterization of cardiac development and structural defects. The protocol outlined herein is designed to perform real-time fetal mice echocardiography in vivo.

Souris foetale Cardiovascular Imaging à l’aide d’un système à ultrasons de haute fréquence (30 / 45MHZ)

Congenital heart defects (CHDs) are the most common cause of childhood morbidity and early mortality. Prenatal detection&#160;of the underlying molecular ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualiser le nœud et la notochorde plaque en Gastrulating des embryons de souris à l’aide de la microscopie électronique à balayage et toute monture Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and painless, and the application is easy, fast, and can be done anywhere, even with mobile devices. During pregnancy, ultrasound imaging is standardly used to closely monitor fetal development. The technique is important to assess intrauterine growth restriction (IUGR), a pregnancy complication with short- and long-term health consequences for both the mother and fetus. Understanding the process of IUGR is indispensable for developing effective therapeutic strategies. 
The ultrasound system used in this manuscript is an ultrasound device produced for the analysis of small animals and can be used in various research fields, including pregnancy research. Here we describe the usage of the system for in vivo analysis of fetuses from natural killer (NK) cell/mast cell (MC)-deficient mothers that give birth to growth-restricted pups. The protocol includes preparation of the system, handling of the mice before and during measurements, and the usage of the B-mode, color doppler mode, and pulse-wave doppler mode. Fetal size, placental size, and blood supply to the fetus were analyzed. We found reduced implantation sizes and smaller placentas in NK/MC-deficient mice from mid-gestation onwards. In addition, MC/NK-deficiency was associated with absent and reversed end diastolic flow in the fetal Arteria umbilicalis(UmA) and an elevated resistance index. The methods described in the protocol can easily be used for related and non-related research topics.

High Frequency Ultrasound for the Analysis of Fetal and Placental Development In Vivo

Here we describe the technique of high frequency ultrasound for in vivo analysis of fetuses in mice. This method allows the follow-up of fetuses and the analysis of placental parameters as well as maternal and fetal blood flow throughout pregnancy.

Échographie de haute fréquence pour l’analyse du développement foetale et placentaire In Vivo

Ultrasound imaging is a widespread method used to detect organ anomalies and tumors in human and animal tissues. The method is non-invasive, harmless, and ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Analyse des malformations cardiaques congénitales chez les embryons de souris à l’aide de méthodes histologiques qualitatives et quantitatives

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

Échocardiographie à ultrasons à haute fréquence pour évaluer la fonction cardiaque des poissons zèbres

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Imagerie Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos Imaging Intra

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Is My Mouse Pregnant? High-Frequency Ultrasound Assessment

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant mice to determine evolving processes at various timepoints. Moreover, optimal and efficient breeding of model mice requires an assessment of timed pregnancies. Most commonly, mice are mated overnight, and the presence of a vaginal plug is determined; however, the positive predictive value of this technique is suboptimal, and one needs to wait to know if the mouse is truly pregnant. High-resolution ultrasound biomicroscopy is an effective and efficient tool for imaging: 1) Whether a mouse is pregnant; 2) What gestational stage the mouse has reached; and 3) Whether there are intrauterine losses. In addition to the embryos and fetuses, the investigator must also recognize common artifacts in the abdominal cavity so as not to mistake these for a gravid uterus. This article provides a protocol for imaging along with illustrative examples.

High-resolution ultrasound can help streamline experiments requiring timed-pregnant mice by determining the state of pregnancy, gestational age, and pregnancy losses. Presented here is a protocol to illustrate methods to assess mouse pregnancies as well as potential pitfalls (image artifacts) that may mimic pregnancy.

Ma souris est-elle enceinte? Évaluation par ultrasons à haute fréquence

The mouse is the mammalian animal model of choice for many human diseases and biological processes. Developmental biology often requires staged-pregnant ...