Name: vascular casting of adult and early postnatal mouse lungs for micro-ct imaging
Uploaded: 2020-06-20T14:00:00
Description: Watch this Scientific Journal Video about Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging at JoVE.com

This research was supported in part by the NHLBI Intramural Research Program (DIR HL-006247). We would like to thank the NIH Mouse Imaging Facility for guidance in image acquisition and analysis.

All methods described here have been approved by the Institutional Animal Care and Use Committee (ACUC) of the National Heart Lung and Blood Institute.
1. Preparation
<ol>
	<li>Inject the mouse intraperitoneally with heparin (1 unit/g mouse body weight) and allow it to ambulate for 2 min.</li>
	<li>Euthanize the animal in a CO2 chamber.</li>
	<li>Arrange the mouse in a supine position on a surgical board and secure all four limbs to the board with tape. Use magnification for fine ...

Executed properly, this method yields striking images of pulmonary arterial networks, allowing for comparison and experimentation in rodent models. Several critical steps along the way ensure success. First, investigators must heparinize the animal in the preparatory stage to prevent blood clots from forming in the pulmonary vasculature and chambers of the heart. This allows for the complete arterial transit of polymer compound. Second, when puncturing the diaphragm and removing the ribcage, take care to protect the lung...

The focus of this technique is the visualization of pulmonary arterial architecture using a polymer-based compound in mice. While extensive work has been performed on systemic vascular beds such as brain, heart, and kidney1,2,3,4,5, less information is available regarding the preparation and filling of the pulmonary arterial network. The aim of this study, therefore, is to expand upon previous work6,7,8 and provide a detailed written and visual reference that investigators can easily follow to produce high-resolution images of the pulmonary arterial tree.
While numerous methods exist for labeling and imaging lung vasculature, such as magnetic resonance imaging, echocardiography, or CT angiography9,10, many of these modalities fail to adequately fill and/or capture the small vessels, limiting the scope of what can be studied. Methods such as serial sectioning and reconstruction provide high resolution but are time/labor-intensive11,12,13. Surrounding soft tissue integrity is compromised in traditional corrosion casting10,13,14,15,16. Even animal age and size become factors when attempting to introduce a catheter or, the resolution is lacking. The polymer injection technique, on the other hand, fills arteries to the capillary level and when combined with &#181;CT, allows for unparalleled resolution5. Samples from mouse lungs as young as postnatal day 14 have been successfully casted8 and processed in a matter of hours. These can be rescanned indefinitely, or even sent for histological preparation/electron microscopy (EM) without compromising the existing soft tissue17. The main limitations to this method are the upfront cost of CT equipment/software, challenges with accurately monitoring intravascular pressure, and the inability to acquire data longitudinally in the same animal.
This paper builds on existing work to further optimize the pulmonary artery injection technique and push age/size related boundaries down to postnatal day 1 (P1) to yield striking results. It is most useful for teams that want to study arterial vascular networks. Accordingly, we provide new guidance for catheter placement/stabilization, increased control over fill rate/volume, and highlight notable pitfalls for increased casting success. Resulting casts can then be used for future characterization and morphologic analysis. Perhaps more importantly, this is the first visual demonstration, to our knowledge, that walks the user through this intricate procedure.

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			<td height="21" style="height:21px;width:231px;">1cc syringe</td>
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			<td>For sample Storage and scanning</td>
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			<td height="20" style="height:20px;width:231px;">Heparin (1000USP Units/ml)</td>
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			<td style="width:117px;">NDC 0409-2720-01</td>
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			<td height="21" style="height:21px;width:231px;">induction chamber</td>
			<td style="width:135px;">n/a</td>
			<td style="width:117px;">n/a</td>
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			<td>fiber optic cleaning wipe</td>
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			<td height="21" style="height:21px;width:231px;">Micro-CT system</td>
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			<td></td>
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			<td height="21" style="height:21px;width:231px;">Microfil (Polymer Compound)</td>
			<td style="width:135px;">Flowech Inc.</td>
			<td style="width:117px;">Kit B - MV-122</td>
			<td>8 oz. of MV compound; 8 oz. of diluent; MV-Curing Agent</td>
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			<td height="21" style="height:21px;width:231px;">Micromanipulator</td>
			<td style="width:135px;">Stoelting</td>
			<td style="width:117px;">56131</td>
			<td></td>
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			<td style="width:135px;">Bemis company, Inc.</td>
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			<td height="21" style="height:21px;width:231px;">PE-10 tubing</td>
			<td style="width:135px;">Instech</td>
			<td style="width:117px;">BTPE-10</td>
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			<td>Height 24in.</td>
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			<td height="21" style="height:21px;width:231px;">Sodium Nitroprusside</td>
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			<td style="width:135px;">N/A</td>
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			<td>16 x 16 in. area, 1/16 in thick</td>
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			<td height="21" style="height:21px;width:231px;">Surgical Board</td>
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			<td>This is a converted slide holder</td>
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			<td style="width:135px;">Zeiss</td>
			<td style="width:117px;">n/a</td>
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	</tbody>
</table>

vascular casting of adult and early postnatal mouse lungs for micro-ct imaging

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

A successful cast will exhibit uniform filling of the entire pulmonary arterial network. We demonstrate this in C57Bl/6J mice ranging in age: Postnatal day P90 (Figure 4A), P30 (Figure 4B), P7 (Figure 4C), and P1 (Figure 4D). By controlling the rate of flow and visually monitoring the fill in real-time, reliable endpoints of the most distal vasculature were achieved (<st...

Watch this Scientific Journal Video about Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging at JoVE.com

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

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

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

This protocol gives precise, step-by-step guidance for casting the pulmonary arterial vasculature in both adult and early postnatal mice. This technique enables users to fully cast the entire vascular network for visualization by a variety of methods, including micro-CT, while maintaining soft tissue integrity. This technique is particularly useful for looking at pulmonary-vascular morphology and architecture, but can easily be adapted for the targeting of other vascular beds.Like many surgeries and dissections, there's no substitute for actually visualizing a procedure, and viewers may benefit from seeing some of the more challenging maneuvers being performed. Once the xiphoid process is exposed, gently grasp and elevate the rib cage. Carefully make an incision in the now-exposed, semi-transparent diaphragm.The lungs will visibly collapse, and retract away from the diaphragm. After exposing the lung and trachea, thread 15 centimeters of PE10 tubing onto the hub of a 30-gauge needle. And attach this unit to a one-milliliter syringe, containing 10 to the 4 molar of sodium nitroprusside, in PBS.Advance the plunger to prime the tubing until all of the air has been purged. As an effective alternative to the text, penetrate the apex of the heart on one side, and pass the tips of curved forceps through the muscle, and out the other side of the tissue. Grasp one end of a 10-centimeter length of 7-0 silk, and pull approximately two centimeters through the tissue.After tying off the suture, use the remaining eight centimeters of suture to pull the heart caudally. And tape the end of the suture to the surgical board. Hook the tips of curved forceps under both the ascending aorta and pulmonary artery trunk.And pull a three-centimeter length of 7-0 silk back through the opening to create a single-throw loose suture. Use scissors to make a one-to-two-millimeter incision towards the apex of the heart, penetrating the thin-walled right ventricle. And introduce the primed tubing into the right ventricle, gently advancing the catheter into the semi-transparent, thin-walled pulmonary artery trunk.Visually verify that the catheter has not advanced into either the left or right pulmonary branches, and does not abut the pulmonary artery branch point. Tape the distal portion of the tubing to the surgical board, and gently tighten the loose suture around both of the great vessels. Cut the eight-centimeter piece of suture to return the heart to a natural resting position.And clip the left oracle of the heart to allow the perfusate to exit the vasculature. Then, use a syringe pump, to infuse the sodium nitroprusside at a 0.5 ml/min flow rate to both flush the blood, and to maximally dilate the vasculature, until the perfusate runs clear. To construct the lung inflation unit, cut the flexible plastic 24-gauge intravenous catheter from a tub and connect to the needle of a butterfly infusion set.Attach this unit to the stopcock, and attach the stopcock to an open 50-milliliter syringe. After loading the syringe with formalin and priming the catheter, with the stopcock closed, position the syringe in a ring stand, to the point at which the meniscus is 20 centimeters above the trachea. Place two loose sutures, two to four millimeters apart, inferior to the cricoid cartilage, and use scissors to make a small incision in the cricothyroid ligament, superior to the sutures.Insert the catheter into the opening, and advance the tip beyond the two loose sutures. Tighten the sutures around the trachea, and open the stopcock to allow the formalin to enter the lungs by gravity. Wait for five minutes for the lungs to fully inflate.If the lungs adhere to the rib cage during inflation, use blunt-tipped forceps to grasp the outside of the rib cage, and move the ribs in all directions to assist in freeing the lobes without making direct contact with the lungs. After five minutes, retract the catheter behind the first suture and ligate. Then retract the catheter behind the second suture and ligate.The lungs should now be inflated in a closed, pressurized state. To cast the vasculature, load one milliliter of an 8-1-1 solution of polymer diluent curing agent into a one-milliliter syringe. And carefully insert the plunger.With the syringe inverted, depress the plunger to remove the air, and to allow the formation of a meniscus at the tip of the syringe. Remove the sodium nitroprusside syringe from the hub of the needle, and drip addition PBS into the hub, to create a meniscus. Join the polymer-compound syringe to the hub, and start the infusion at 0.02 millimeters per minute.Monitor the compound as it moves freely through the tubing, noting the syringe volume as it enters the pulmonary artery trunk. Continue the profusion until all lobes are completely filled, down to the smallest vessel. Stop the pump, and once again, note the syringe volume.Cover the lungs with a fiber-optic cleaning wipe, and liberally apply PBS. Allow the specimen to sit undisturbed for 30 to 40 minutes at room temperature. Once the polymer compound is cured and hardened, sever the limbs and lower half of the mouse.And place the head and thorax into a 50-milliliter conical filled 10%buffered formalin, overnight. The next morning, grasp the trachea to gently separate the heart and lung unit from the remaining rib cage and thorax. Place the harvested tissue block in a formalin-filled scintillation vial.A successful cast will result in a uniform filling of the entire pulmonary arterial network. If there is damage to the lung or lung airways, small leaks will prevent the lungs from holding pressure. Patchy or incomplete filling can arise from an airlock as a result of air being introduced into the vascular system via the catheter, blocking downstream flow of the compound.Underfilling occurs when too little compound is introduced into the vasculature. Alternatively, overfilling, or introducing too much polymer compound too rapidly, can cause arterial rupture, or, more commonly, venous transit. Advancing the catheter too far down the pulmonary trunk can cause the tip to wedge into one pulmonary artery branch, creating an imbalance in flow, and causing one side to fill faster than the other.Careful performance of the technique as demonstrated, using the appropriate direct monitoring of distal vasculature end points, and standard infusion rates can result in optimal filling of pulmonary vasculature. Furthermore, adapting this technique to the systemic vasculature can yield equally favorable results in other vascular beds. After casting, samples can be processed for micro-computed tomography scanning.For post-processing, a commercial software package can be used to generate a 3D volume rendering of the pulmonary-vascular tree. When attempting this technique for the first time, be especially careful around the fragile lungs, and the pulmonary artery. Most importantly, avoid introducing air into the vascular circuit.Following a successful cast, a sample can be scanned by micro-CT, and questions involving vascular morphometry and architecture can be explored.

vascular-casting-adult-early-postnatal-mouse-lungs-for-micro-ct

Research

JoVE Journal

Developmental Biology

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

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

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

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

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.

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.

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 ...

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.

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

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.

Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.