Name: studying wnt signaling during patterning of conducting airways
Uploaded: 2016-10-16T13:00:00
Description: Watch this Scientific Journal Video about Studying Wnt Signaling During Patterning of Conducting Airways at JoVE.com

We acknowledge the assistance of Mike Muntifering and Matt Kofron with confocal imaging and Gail Macke with histological procedures. This work was partially supported by National Institutes of Health-NHLBI (K01HL115447 to D.S.).

Animals were housed in pathogen-free conditions. Mice were handled according to protocols approved by CCHMC Institutional Animal Care and Use Committee (Cincinnati, OH USA). Mice utilized throughout these studies were maintained in a mixed background.
1. Whole Mount X-galactosidase Staining
<ol>
	<li>Euthanize pregnant female at E11.5 to E14.5, by CO2 inhalation. Place animals in CO2 chamber, charge the chamber with CO2. Maintain animals in chamber for minimu...

<ol>
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Events underlying the morphogenesis of the respiratory tract are not completely understood, particularly the processes required for the patterning of the conducting airways. Previous studies have utilized ex vivo techniques wherein developing explants are cultured at the air-liquid interphase or embedded in matrigel21,22. These studies have shown how growth factors influence the patterning of the developing trachea and the formation of tracheal cartilage. A limitation to these studies is that the arch...

Respiratory tract development is initiated by embryonic day 9 (E9) with the appearance of Nkx2.1 positive cells in the ventral endodermal foregut1,2. Esophageal-tracheal tube separation will resolve by E11.5 when the tubes can be distinguished as distinct entities, each surrounded by mesenchymal tissue3. Wnt signaling plays a key role in the specification of the respiratory tract as deletion of Wnt2 and Wnt2b, expressed by the splanchnic mesenchyme and deletion of &#946;-catenin from the endodermal respiratory epithelium will result in lung agenesis4,5. Our previous studies determined that deletion of Wls, a cargo receptor mediating secretion of all Wnt ligands, from the endodermal respiratory tract results in lung hypoplasia, defects in pulmonary vascular development and mis-patterning of the tracheal mesenchyme6,7. These data support the importance of the epithelial-mesenchymal cross talk in cell differentiation and specification, as it has also been shown in other studies8,9.
The study of the earliest stages of lung development relies upon genetic, in vitro and ex vivo techniques that have allowed us to better understand mechanisms driving respiratory identity10-16. Whole lung explant cultures at the air liquid interphase have been widely utilized to study the effects of growth factors in early stages of pulmonary branching morphogenesis10,17,18. While this method is used as readout of morphological changes, such as branching morphogenesis, and gene expression modulation, it is limited to the study of early stages of the developmental process, as the culture itself does not support the development of vasculature17. Development of tracheal cartilage requires longer incubation times that may be not compatible with this culture technique.
To analyze the role of Wnt signaling during respiratory tract formation, we have adapted standard techniques to meet the needs of our embryonic studies. We have modified volumes, staining times, processing cycling for paraffin embedding and timing for clearing of tracheal-lung tissue. The main goal of optimizing the techniques described in the present study was to analyze the earliest stages of tracheal development in mice that take place from E11 to E14.5. Using the reporter mice line Axin2LacZ we precisely determined sites of Wnt/&#946;-catenin activity in the developing tracheal mesenchyme. We have also adapted lectin staining procedure for whole mount tracheal tissue. Thus, we were able to visualize mesenchymal condensations and predict sites where chondrogenesis will take place. Staining of whole mount and sections of embryonic tissue obtained from WlsShhCre mice, coupled with advanced microscopy techniques, allowed us to unveil the role of Wnt ligands produced by the tracheal epithelium in tracheal patterning.

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			<td>Anti Sox9 ab.</td>
			<td>Millipore</td>
			<td>AB5535</td>
			<td>1:400 , rabbit</td>
		</tr>
		<tr>
			<td>Anti Sox9 ab.</td>
			<td>Santa Cruz</td>
			<td>Sc-20095</td>
			<td>1:50, rabbit</td>
		</tr>
		<tr>
			<td>Anti Smooth Muscle Actin ab.</td>
			<td>Sigma</td>
			<td>A5228</td>
			<td>1:2k, mouse</td>
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		<tr>
			<td>Anti NKX2.1 ab.</td>
			<td>Seven Hills</td>
			<td>n/a</td>
			<td>1:100, guinea pig</td>
		</tr>
		<tr>
			<td>Anti NKX2.1 ab.</td>
			<td>Seven Hills</td>
			<td>n/a</td>
			<td>1:400, mouse</td>
		</tr>
		<tr>
			<td>Anti Brdu ab.</td>
			<td>Abcam</td>
			<td>AB1893</td>
			<td>1:200, sheep</td>
		</tr>
		<tr>
			<td>Anti Brdu ab.</td>
			<td>Santa Cruz</td>
			<td>Sc-32323</td>
			<td>1:4k, mouse</td>
		</tr>
		<tr>
			<td>PNA Lectin</td>
			<td>Sigma</td>
			<td>L 7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td>Life technologies</td>
			<td></td>
			<td>Alexa fluor Molecular probes</td>
		</tr>
		<tr>
			<td>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>P8131</td>
			<td></td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma-Aldrich</td>
			<td>P3289</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>NaDOC</td>
			<td>Life Technologies</td>
			<td>89905</td>
			<td></td>
		</tr>
		<tr>
			<td>NP4O</td>
			<td>Life Technologies</td>
			<td>85124</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma</td>
			<td>A-3157</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisher brand super-frost plus</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA (16%)</td>
			<td>EMS</td>
			<td>15710</td>
			<td></td>
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			<td>PBS</td>
			<td>Gibco</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum</td>
			<td>Sigma</td>
			<td>11K413</td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking reagent</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Component of TSA kit #2&#160;&#160;&#160; ( T20932)</td>
		</tr>
		<tr>
			<td>BrDu</td>
			<td>Sigma</td>
			<td>B5002-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector labs</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-loc cassettes Histoscreen</td>
			<td>Fisher</td>
			<td>C-0250-GR</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy cassettes</td>
			<td>Premiere</td>
			<td>BC0109</td>
			<td>Available in different colors</td>
		</tr>
		<tr>
			<td>Nuclear fast red&#160; Kernechtrot 0.1%</td>
			<td>Sigma</td>
			<td>N3020</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma</td>
			<td>C1909-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sigma</td>
			<td>S4641-1Kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma</td>
			<td>T5941-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Pharmco-AAPER</td>
			<td>399000000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Pharmco-AAPER</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro knives</td>
			<td>FST</td>
			<td>10318-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 ceramic coated</td>
			<td>FST</td>
			<td>11252-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5CO</td>
			<td>FST</td>
			<td>11295-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont # 5</td>
			<td>FST</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo/Shandon Excelsior ES</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2135</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon i90</td>
			<td>Nikon</td>
			<td></td>
			<td>Wide field microscope</td>
		</tr>
		<tr>
			<td>NikonA1Rsi</td>
			<td>Nikon</td>
			<td></td>
			<td>Confocal microscopy. Settings:NikonA1 plus camera, scanner: Galvano, detector:DU4. Optics Plan Apo lambda 10x. Modality: Widefield fluorescence laser confocal.&#160;</td>
		</tr>
		<tr>
			<td>Leica MS 16 FA</td>
			<td>Leica</td>
			<td></td>
			<td>Fluorescence Dissecting microscope</td>
		</tr>
		<tr>
			<td>Zeiss</td>
			<td>Zeiss</td>
			<td></td>
			<td>Automated fluorescence microscope</td>
		</tr>
		<tr>
			<td>Leica Application suite</td>
			<td>Leica</td>
			<td></td>
			<td>Leica imaging software</td>
		</tr>
		<tr>
			<td>NIS</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon imaging software</td>
		</tr>
		<tr>
			<td>IMARIS</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaging processing software</td>
		</tr>
	</tbody>
</table>

studying wnt signaling during patterning of conducting airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

Wnt/&#946;-catenin activity
Whole mount Lac-Z staining was detected in tracheal-lung tissue of embryos isolated from reporter Axin2Lac-Z mice11. Sites of staining indicate Wnt/&#946;-catenin activity. Analysis of sections of whole mount staining determined that Wnt/&#946;-catenin activity was present in the mesenchyme of the trachea and in mesenchyme of peripheral regions of developing l...

Watch this Scientific Journal Video about Studying Wnt Signaling During Patterning of Conducting Airways at JoVE.com

Studying Wnt Signaling During Patterning of Conducting Airways

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

The overall goal of the staining procedures is to demonstrate the role of the Wnt signaling pathway in the early stages of development of the upper airways using embryonic tissue from genetically engineered mouse models. This methods can help answer key questions in the field of pulmonary biology. Such as, for example, how the supporting structures of the conducting airways are patterned.The techniques that we're going to presenting here will allow us to study cell proliferation or the process of mesenchymal condensations, which are key events in the formation of the tracheal cartilage. The main advantage of this technique is it can be used on small amounts of tissue. This includes lung explants, and tracheas isolated from E11 embryos.Generally speaking, individuals new to this procedure will struggle do to the size of the sample, as well as a limited amount of tissue available. One must feel comfortable when working with embryonic tissues, and will do so by paying attention throughout the steps and making sure that the same amount of tissue is available from step to step. After euthanizing a pregnant female mouse at E11.5 to E14.5, and performing a laparotomy according to the text protocol, use forceps and fine scissors to isolate the uterine horns, and immediately place the tissue into a Petri dish containing chilled PBS solution.Then place the dish on ice. To isolate the embryos from the uterine horns, use fine forceps to hold the uterine tissue. And with scissors, puncture the uterine tissue and conceptus, releasing the embryos.Next, remove any remaining embryonic membranes. Then, under a dissecting microscope using needle blades, cut off the head and lower body, below the diaphragm, and locate the liver as a landmark. Use needle blades to remove the lateral sides of the body wall, spine, and heart.Taking care not to injure the tracheal tissue. To dissect the embryonic tracheal lung tissue, carefully separate the esophageus from the trachea by using needle blades to pull the esophageus. Place the tissue in PBS on ice before using glass or transfer pipettes to transfer the tissue into four millimeter screw cap glass vials.Add 4%paraformaldehyde, or PFA, in PBS and incubate for 30 minutes. Then use PBS to rinse the tissue. After removing the PBS from the samples, add two millimeters of X-gal staining solution and incubate for one to two hours, or four hours for further processing and embedding.Stop the reaction by using 3%DMSO in PBS to wash the tissue three times for five minutes each. Then store the tissue in 70%ethanol. After using an automated processor to process the samples for paraffin embedding according to the text protocol, orient the samples with the tissue flat on the bottom of an embedding boat.Carefully add paraffin to fill the embedding boat, and immediately place on the cold plate of the embedding station until the paraffin solidifies. Next, remove the block from the embedding boat, and with a microtome, cut six micrometer sections. Then mount the sections on slides.And place the sections on a slide warmer at 42 degrees Celsius for one hour. Bake the slides in a 56 degree Celsius oven overnight. Then, to deparrafinize the samples, place the baked slides in racks and transfer into staining dishes filled with with 200 milliliters of 100%xylene, three times for 10 minutes each.Use a graded ethanol series for one minute each to rehydrate the slides. Then use Nuclear Fast Red to counterstain the samples for 10 to 30 seconds. With tap water, rinse the slides three times for five minutes each.To dehydrate the slides, use a graded ethanol series before washing the samples in three changes of xylene. Then use a xylene-based mounting medium and cover slips to mount the samples. To carry out lectin staining, after fixing tracheal lung tissue and preparing blocking buffer according to the text protocol, transfer samples to 0.5 milliliters of blocking buffer and incubate at room temperature for one hour.Use a transfer pipet to remove the blocking buffer and replace with 250 microliters of lectin solution per sample. Then using aluminum foil, cover the glass vials, and incubate at four degrees Celsius overnight. The following day, after using PBS to rinse the samples, place the explants in agarose-coated Petri dishes containing enough PBS to cover the samples.Then use a florescence dissecting microscope to photograph the whole mounts. To perform immunofluorescence staining on whole mounts, after fixing the samples according to the text protocol, use a transfer pipette to remove the methanol, then add dense bleach and incubate the tissue for two hours to permeabilize the samples. Using a graded series of methanol, diluted in PBS, hydrate the tissue by incubating it at room temperature for 10 minutes at each step.After blocking the tissue according to the text protocol, add primary antibodies and blocking solution, and incubate the samples at four degrees Celsius overnight. Once the samples have been washed in PBS, add a one to 500 dilution of secondary antibodies in PBS, and incubate in the dark at four degrees Celsius overnight. The following day, after washing the samples three times for 20 minutes each in PBS, use a graded methanol series in PBS to dehydrate the samples for 10 minutes at each step.Transfer the samples to custom-made stage plates. Remove the methanol. Then use 200 microliters of Murray's clear solution to clear the samples.Image immediately using a confocal microscope. After injecting E11.5 pregnant mice with BrdU, and isolating the embryos at E12.5 or E13.5 according to text protocol, use knife blades to excise the embryonic heads and lower parts of the bodies, below the thoracic cavities. Place the thoracic tissue in four milliliter screw cap vials, and add one milliliter of 4%PFA.After embedding, sectioning, and rehydrating samples according to text protocol, place the slides in Coplin jars and fill with 10 millimolar citrate buffer, pH six. Microwave the samples at high power for six and a half minutes before using distilled water to replace the evaporated citrate buffer and heating again. After cooling, washing, and blocking the slides according to the text protocol, apply 180 microliters of primary antibodies, diluted in blocking solution, to a gasket and attach the slide as if applying a cover slip.The following day, dip the slides in distilled water to detach the gasket. Then use TBS, with 0.1%Tween 20, to wash the slides six times for five minutes each. After incubating the slides with secondary antibodies, use 0.1 molar Tris Base to wash the slides twice for five minutes each, before washing twice with 05 molar Tris Base.Keep slides in 05 molar Tris Base while adding mounting medium, with or without DAPI, and add a cover slip. Finally, use an automated florescence microscope to image the samples, and analyze according to the text protocol. In this figure of whole-mount tracheal lung tissue, isolated from Reporter and two lacZ mice, lacZ staining was detected in the mesenchyme of the trachea, and the peripheral regions of developing lungs, indicating Wnt beta catenin activity.In this experiment, E12.5 to E14.5 tracheal lung tissues were stained with PNA lectin. In control animals, florescence was detected as periodic bands in mesenchyme where cartilage forms. However, the lack of staining in Wntless sonic hedgehog Cree embryos, indicates that Wnt signaling is necessary for mesenchymal condensations.In whole mount tracheal tissue of E11.5 embryos, SOX9 expression is limited to mesenchyme as a continues stripe, while in the developing lung, SOX9 is observed at the periphery of the respiratory epithelium. As shown here, NKX2.1 is restricted to the epithelium of the trachea. Prevention of secretion of Wnt ligands from epithelium diminishes expression of SOX9 in mesenchyme, but does not effect SOX9 expression in peripheral lung epithelium.Alpha smooth muscle actin staining is detected at low levels in tracheal tissue, but it is clearly observed in larynx and stomach. Finally, as indicated in this figure, in vivo labeling of tracheal tissue with BrdU, SOX9, and alpha smooth muscle actin, demonstrates that SOX9 stained cells undergo proliferation at a higher level than alpha smooth muscle actin stained cells. The proliferation pattern is reverted in Wntless sonic hedgehog Cree tracheal tissue.Once mastered, most of these techniques can be done in two days if performed correctly. While attempting this procedure, it is important to remember the source of the primary antibody when choosing the secondary antibody. This is important for all immunostainings, but it is especially important when you have more than one primary antibody.Following the X-galactosidase staining, additional techniques such as in situ hybridization can be performed to acquire additional information, as well as answer additional questions, such as whether or not a particular gene of interest will be expressed during Wnt signaling. The techniques presented here would be useful for researchers that are interest in a silent patterning mechanisms mediated by Wnt signaling, or other key signaling powers to development. After watching this video, you should have a good understanding of how to study differentiation process during the development of the respiratory tract.And also have a better understanding of the roll of Wnt signaling in the patterning of the conducting airways. Don't forget when working with paraformaldehyde and benzyl benzoate to take the proper precautions, like earing gloves, preparing solutions under the hood, and disposing of waste solutions in accordance to your local laws and regulations.

studying-wnt-signaling-during-patterning-of-conducting-airways

Research

JoVE Journal

Developmental Biology

Method of Studying Palatal Fusion using Static Organ Culture

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that adheres to form the midline epithelial seam (MES). The theories suggest that MES cells follow an epithelial to mesenchymal transition (EMT), apoptosis and migration, making a fused palate 1. Complete disintegration of the MES is the final essential phase of palatal confluence with surrounding mesenchymal cells. We provide a method for palate organ culture. The developed in vitro protocol allows the study of the biological and molecular processes during fusion. The applications of this technique are numerous, including evaluating responses to exogenous chemical agents, effects of regulatory and growth factors and specific proteins. Palatal organ culture has a number of advantages including manipulation at different stages of development that is not possible using in vivo studies.

Studies of palate development are motivated by the incidence of cleft palate, a birth defect that imposes a tremendous health burden and can leave lasting disfigurement. We demonstrate here a technique to culture palatal shelves that can be used to study different signaling pathways involved in palatal development and fusion.

Cleft lip and palate are among the most common of all birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium that ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

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 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 Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

A Microfluidics Approach for the Functional Investigation of Signaling Oscillations Governing Somitogenesis

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and gradients are thought to control the timing and spacing of segment formation, respectively. While the involved signaling pathways have been studied extensively over the last decades, direct evidence for the function of signaling oscillations in controlling somitogenesis has been lacking. To enable the functional investigation of signaling dynamics, microfluidics is a previously established tool for the subtle modulation of these dynamics. With this microfluidics-based entrainment approach endogenous signaling oscillations are synchronized by pulses of pathway modulators. This enables modulation of, for instance, the oscillation period or the phase-relationship between two oscillating pathways. Furthermore, spatial gradients of pathway modulators can be established along the tissue to study how specific changes in the signaling gradients affect somitogenesis.
The present protocol is meant to help establish microfluidic approaches for the first-time users of microfluidics. The basic principles and equipment needed to set up a microfluidic system are described, and a chip design is provided, with which a mold for chip generation can conveniently be prepared using a 3D printer. Finally, how to culture primary mouse tissue on a microfluidic chip and how to entrain signaling oscillations to external pulses of pathway modulators are discussed.
This microfluidic system can also be adapted to harbor other in vivo and in vitro model systems such as gastruloids and organoids for functional investigation of signaling dynamics and morphogen gradients in other contexts.

A protocol for the culture and manipulation of mouse embryonic tissue on a microfluidic chip is provided. By applying pulses of pathway modulators, this system can be used to externally control signaling oscillations for the functional investigation of mouse somitogenesis.

Periodic segmentation of the presomitic mesoderm of a developing mouse embryo is controlled by a network of signaling pathways. Signaling oscillations and ...