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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M., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+promotes+respiratory+progenitor+identity+in+mouse+foregut.">beta-Catenin promotes respiratory progenitor identity in mouse foregut.</a> Proc Natl Acad Sci U S A. 106, 16287-16292 (2009).</li><li>Cornett, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wntless+is+required+for+peripheral+lung+differentiation+and+pulmonary+vascular+development.">Wntless is required for peripheral lung differentiation and pulmonary vascular development.</a> Dev Biol. 379, 38-52 (2013).</li><li>Snowball, J., Ambalavanan, M., Whitsett, J., Sinner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Endodermal+Wnt+signaling+is+required+for+tracheal+cartilage+formation&amp;#34;.">34;Endodermal Wnt signaling is required for tracheal cartilage formation&amp;#34;.</a> Dev Biol. , (2015).</li><li>Shannon, J. 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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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<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>
		</tr>
		<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>
		</tr>
		<tr>
			<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.

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Research

JoVE Journal

Developmental Biology

7.3K Views.  Cincinnati Children’s Hospital Medical Center. 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...