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 minimum of 5 min. Perform secondary method of euthanasia by cervical dislocation.</li>
	<li>Clean abdominal area with ethanol (EtOH) 70% and perform laparotomy with a single abdominal midline incision. Utilize surgical scissors and standard pattern (straight serrate) forceps.</li>
	<li>Isolate uterine horns using forceps and fine scissors and immediately place tissue in petri dish containing chilled PBS solution. Maintain tissue on ice until proceeding to isolation of embryos.</li>
	<li>Isolate embryos from uterine horns. Dissect embryonic tracheal lung tissue using dissecting microscope. Utilize fine tip forceps and scissors to hold and puncture the uterine tissue and concepti releasing the embryos.
	<ol>
		<li>Remove remaining embryonic membranes. With assistance of needle blades cut head of embryos and lower body part below the diaphragm. Locate liver as a landmark.</li>
		<li>After isolation of the thoracic region of the embryo, proceed to remove lateral sides of the body wall, spine and heart using needle blades. Take care while removing the heart to avoid injuring tracheal tissue. Finally, carefully, separate the esophagus from the trachea by pulling the esophagus with assistance of needle blades. Maintain tissue in PBS solution on ice.</li>
	</ol>
	</li>
	<li>Place tissue in 4 ml screw cap vials using glass or transfer pipettes. Fix tracheal lung tissue in 4% paraformaldehyde (PFA) in PBS for 30 min. After fixation, rinse tissue with PBS. While rinsing, prepare X-gal staining solution (add 5 mM K3Fe(CN)6, (100 &#956;l 0.5 M stock) 5 mM K4Fe(CN)6 (100 &#956;l 0.5 M stock) 2 mM MgCl2, (20 &#956;l 1 M stock) 0.01% NaDOC (50 &#956;l 2% stock), 0.02% NP4O (100 &#956;l 2% stock), 1 mg/ml X-gal (500 &#956;l of 20 mg/ml stock) and 9.13 ml of distilled water to bring to a final volume of 10 ml).</li>
	<li>Remove any remaining PBS and add 2 ml of X-gal staining solution to the glass vial. Place vials in tray on shaker. Stain tissues 1 to 2 hr in X-gal staining solution. Incubate the tissue in X-gal solution for 4 hr for further processing and embedding.</li>
	<li>Stop the reaction by washing tissues in 3% dimethyl sulfoxide-PBS. Rinse in PBS, 3 times for 5 min each wash and store in 70% ethanol.</li>
	<li>Fill Petri dishes with a 1% agarose/PBS solution. Allow agarose to solidify. Using glass transfer pipettes, place the tissue on agarose coated plates filled with PBS solution. Photograph whole mounts using a dissecting microscope. Select appropriate filter for brightfield.</li>
	<li>To better distinguish sites of X-gal staining process samples for paraffin embedding and sectioning. Place samples in fine screen cassettes.
	<ol>
		<li>To process samples for paraffin embedding use an automated processor and set up a short cycle as follows: six changes of alcohol: 5 min first change and 3 min per each remaining change. Three changes of Xylene: 5 min first change and 3 min next two changes. The previous steps are performed at 30 &#176;C. Finally, perform three changes of paraffin at 62 &#176;C: 25 min, 8 min and 5 min.</li>
	</ol>
	</li>
	<li>Orient samples for embedding with tissue lying flat on the bottom of embedding boat. Carefully add paraffin to fill the embedding boat. Cool immediately on cold plate of embedding station until paraffin solidifies. Remove block from embedding boat. Using a microtome, generate 6 &#956;m sections. Mount sections on pretreated-slides and dry slides on slide warmer set at 42 &#176;C for 1 hr.</li>
	<li>Bake slides overnight in oven at 56 &#186;C. Place slides in racks. Deparaffinize slides with three changes of 100% Xylene, 10 min each. Use staining dishes filled with 200 ml of Xylene.</li>
	<li>Rehydrate slides using graded EtOH series (100%, 70%, 50% and 30%, 1 min per step) to PBS before counterstaining with Nuclear Fast Red for 10 to 30 sec. Rinse slides with tap water three times for 5 min each time to remove excess of Nuclear Fast Red.</li>
	<li>Dehydrate slides in a graded EtOH series (50%, 70% and 100% EtOH) before washing in three changes of Xylene (20 dips each change) and cover slipping with Xylene based mounting media.</li>
</ol>
2. Lectin Staining
<ol>
	<li>Dissect E13.5 and E14.5 tracheal lung tissue as described in steps 1.1 to 1.4. Using glass transfer pipettes place tissue in screw cap vials. Fix tracheal lung tissue with 2 ml of 2% PFA solution overnight at 4 &#176;C. After fixation, rinse samples in PBS three times for 10 min each wash.</li>
	<li>Prepare blocking buffer, usually a final volume of 10 ml of solution. Add 0.1 g of BSA (1%) to 8 ml of PBS. Allow BSA to dissolve. Add 0.2 ml of goat serum (2%) 0.03ml of Triton X-100 (0.3%). Bring final volume to 10 ml with PBS. Block samples for 1 hr in 0.5 ml of blocking buffer at room temperature.</li>
	<li>Remove blocking buffer using a transfer pipette and replace with lectin solution comprised of 50 &#956;g/&#956;l of lectin, 10% goat serum and PBS. Use 250 &#956;l of lectin solution per sample. Incubate overnight at 4 &#176;C. Make sure to cover the glass vial containing tissue with aluminum foil. After incubation with lectin solution, rinse samples in PBS three times for 10 min.</li>
	<li>Place explants in agarose coated petri dishes containing enough PBS to cover samples. Photograph whole mounts using a fluorescence-dissecting microscope. Select the appropriate filter to detect fluorescence. Lectin PNA is usually coupled to GFP.</li>
</ol>
3. Whole Mount Immunofluorescence Staining and Confocal Microscopy
<ol>
	<li>Isolate tracheal lung tissue, transfer to 4 ml glass screw cap vials and fix overnight in 4% PFA. E11.5 tissue can be fixed in 2% PFA. Store in methanol 100%. Samples can be stored up to several months at -20 &#176;C.</li>
	<li>Remove methanol using transfer pipette and permeabilize samples using Dent&#39;s Bleach (4 parts Methanol, 1 part DMSO and 1 part 30% H2O2) for 2 hr. Hydrate tissue in a graded series of methanol diluted in PBS as follows: 100% methanol, 75% methanol, 50% methanol, 25 % methanol, 100% PBS. Incubate 10 min at room temperature during each step of hydration.</li>
	<li>Block tissue in 0.5% blocking reagent diluted in PBS (see Materials for details) for two hours at room temperature with agitation.</li>
	<li>Dilute primary antibody in blocking solution (Sox9 1:100, Nkx2.1 1:200, &#945;SMA 1:250) and incubate samples overnight at 4 &#176;C. Wash samples with PBS five times at room temperature. Perform each wash for 1 hr.</li>
	<li>Apply secondary antibody at a 1:500 dilution in 0.5% blocking solution. Carefully select secondary antibodies to prevent binding among secondary antibodies. Incubate overnight at 4 &#176;C in dark room. Wash samples three times for 20 min at room temperature. Dehydrate samples in a graded series of methanol diluted in PBS as follows: 25% methanol, 50% methanol 75% methanol, 100% Methanol, 10 min each step. 
	NOTE: Ensure that samples remain covered with aluminum foil and minimize exposure to light. Tissue can now be stored at 4 &#176;C for several weeks until photographed.</li>
	<li>Transfer samples to custom made stage plates, remove methanol and clear with approximately 200 &#956;l of Murray&#39;s clear solution19,20 (2 parts Benzyl benzoate, 1 part Benzyl alcohol) immediately before imaging. Photograph using a confocal microscope. Process and analyze image using imaging software.</li>
</ol>
4. Cell Proliferation
<ol>
	<li>Inject E11.5 pregnant mice intra-peritoneally with a solution of BrdU at a concentration of 100 &#956;g BrdU/g of body weight. Sacrifice female as described in section 1 of the protocol and isolate embryos at E12.5 or E13.5. Using knife blades, excise heads and lower part of body below thoracic cavity.</li>
	<li>Place thoracic tissue in 4 ml screw cap vials and fix in 1 ml of 4% PFA overnight. Wash samples with PBS twice for 5 min and dehydrate samples through an Ethanol series diluted in distilled water up to 70% ethanol as follows: 30%, 50% and 70% ethanol, for 5 min each step.
	<ol>
		<li>Place samples in medium size screen cassettes. Process tissue for paraffin embedding using and automated processor. Set up a cycle as follows: six changes of alcohol for 8 min each, three changes of Xylene for 6 min each, three changes of paraffin, for 25 min, 9 min and 8 min.</li>
	</ol>
	</li>
	<li>Embed in paraffin orienting the tissue in desired position to generate 6 &#956;m transverse or longitudinal sections as described in section 1. Place sections on slides and allow tissue to adhere to slide on slide warmer set at 42 &#176;C for 1 hr.</li>
	<li>Bake slides, on their sides, overnight in oven at 56 &#176;C. Place slides in plastic racks. De-paraffinize slides by immersion in three changes of 100 % Xylene, 10 min per change. Use 200 ml of Xylene to completely submerge slides. Rehydrate slides using graded EtOH series (100%, 70%, 50% and 30%, 5 min per step with agitation) to PBS (5 min).</li>
	<li>Perform antigen retrieval using 10 mM citrate buffer, pH 6. To prepare 100 ml of citrate buffer mix 18 ml of 0.1 M Citric acid monohydrated and 82 ml of 0.1 M Sodium citrate.
	<ol>
		<li>Place samples in plastic coplin jars filled with antigen retrieval solution and microwave for 6.5 min at high power. Add distilled water to coplin jar to compensate for evaporated-citrate buffer and reheat at 40% power for 6 min.</li>
		<li>Fill coplin jar to top with water, and reheat again at 40% power for 6 min. Microwave times may vary based on microwave used.</li>
		<li>Allow slides to cool for 20 min at room temperature before washing slides in distilled water for 1 min, followed by PBS for 5 min. After the PBS wash, block slides for 2 hr in blocking solution containing TBS (2.425 g of Tris base and 8.765 g of NaCl diluted in 1 L of distilled water) 10% Donkey serum and 1% BSA.</li>
	</ol>
	</li>
	<li>Add primary antibodies diluted in blocking solution (TBS containing 10% Donkey serum and 1% BSA). BrdU (mitosis marker), Nkx2.1 (respiratory tract epithelium), Sox9 (cells that will give rise to cartilage) and &#945;SMA (smooth muscle cells). Incubate slides overnight, at 4 &#176;C. Use overlay method to conserve antibodies. Apply 180 &#956;l of antibody to a gasket and then attach slide as if cover slipping.</li>
	<li>Detach gasket by dipping slides into distilled water. After removal of the gasket, wash unbound primary antibody by performing six 5 min washes with TBS containing 0.1% Tween-20.
	<ol>
		<li>Incubate slides with secondary antibody diluted in blocking solution at a dilution of 1:200, for 1 hr at room temperature. Select secondary antibodies carefully to prevent undesired binding among them. Remove unbound secondary antibody by performing three five minute washes with TBS containing 0.1% Tween.</li>
		<li>Wash slides in 0.1 M Tris base twice for 5 min and then 0.05 M Tris base twice for 5 min. Slides may be left in 0.05 M Tris base while coversliping using mounting media with or without DAPI (1.5 &#956;g/ml). Store slides in slide folder at 4 &#176;C and protect from light by covering folder with aluminum foil.</li>
	</ol>
	</li>
	<li>Visualize staining and photograph using an automated fluorescence microscope. Count labeled-cells and total cells per field photographed at 20X and 40X to determine ratios of proliferating cells to total cells.</li>
</ol>

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&#34;The authors have nothing to disclose.&#34;

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 architecture of the tissue is not properly maintained and therefore, they may not quite recapitulate the in vivo process of chondrogenesis.
The approach that we undertook to study the role of Wnt signaling in the process of conducting airways patterning included in vivo studies coupled with whole mount staining techniques, immunofluorescence and imaging. X-gal staining of whole mount tissue followed by sectioning and counter-staining, presents advantages over the staining of paraffin sections. Whole mount staining allows for direct detection of sites where Wnt/&#946;-catenin activity is operative. Furthermore, sectioning of the stained explants determined precise sites in the mesenchyme wherein the activity was located as development progressed. The latter is a better and accurate readout than detection of expression of &#946;-galactosidase enzyme in paraffin sections. While enzymatic activity could be detected in sections, this procedure will require cryosections that will not always be easy to generate and may not preserve the architecture of small samples. A limitation in the protocol described in this paper, is that processing of samples for paraffin embedding after whole mount staining will cause a loss of the signal. Therefore, samples need to remain longer in X-gal solution and a shorter cycle for paraffin embedding should be used to obtain good signal on sections.
Processing of small tissue for paraffin embedding will result in reduction of the size of the tissue. This is an undesired result while working with reduced amount of tissue. Therefore, we developed a short processing cycle that simultaneously allows for adequate preservation of the structure without causing reduction in the size of the tissue.
On the other hand, whole mount immunofluorescence procedure, modified after the technique described by Ahnfelt-Ronne23, allowed for tridimensional visualization of developing tracheal and pulmonary tissue, with a defined expression pattern of proteins encoded by key transcription factors such as Nkx2.1 and Sox9. When desired, the whole mount staining can be presented as confocal-aided z stacks of images, depicting different depths of the tracheal lung tissue. While imaging small explants, clearing of the tissue should be performed in a stage plate. We utilized thick metallic custom made stage plates with optical bottom where samples are cleared and imaged. This prevents the loss of material while transferring small tissue after clearing step. While whole mount immunofluorescence is a suitable technique for staining of small tissue, we acknowledge limitations to the technique. In particular, we observed dissimilar performances of antibodies that recognize antigen better in sections as opposed to whole mount. This issue may preclude the performance of the technique if no alternative antibodies are available. It is also possible that the use of methanol and clearing with Murray&#39;s clear may have a negative impact in the signal observed. In the future, we will test other non-organic clearing solutions and adjust the protocol properly for this purpose24. A final consideration is that whole mount will allow the study of a few proteins expressed in a tissue at one given time.
Previous reports have shown the usefulness of PNA lectin staining to detect mesenchymal condensations in sections of tracheal tissue21,25. In the present study, we report a methodology by which the staining was performed in whole mount, which facilitates a better visualization of the regions of the trachea wherein the cartilage will be formed. While performing lectin staining care should be placed in using the appropriate concentration of lectin to not over-stain, as commercially available PNA-lectin is coupled to GFP. Auto-fluorescence of the tissue may obscure the results making difficult to distinguish bands corresponding to mesenchymal condensations.
Finally, in vivo labeling of tissue with BrdU followed by sectioning and immunofluorescence staining is a feasible technique to determine cell proliferation that presents advantages over typical and static staining such as PPH3 or PCNA26,27. The latter technique presents limitations, as staining will vary depending on the cell cycle stage. This problem is eliminated by our described technique. Combining in vivo labeling followed by embedding, sectioning and immunofluorescence allowed us to determine that epithelial Wnt ligands balance the differential proliferation of Sox9 and &#945;-SMA expressing cells that will give rise to tracheal cartilage and muscle7.
In summary, a multiple-technique approach to studying the patterning of the developing respiratory tract allows for a deep analysis of the role of Wnt signaling in conducting airway morphogenesis. These studies determined the precise sites of Wnt/&#946;-catenin in the tracheal tissue, as well as inductive and instructive signaling provided by the tracheal epithelium that is required for specification, differential proliferation of tracheal mesenchyme cell lineages, as well as cartilage formation. Our future directions include the optimization of the whole mount staining for recently identified Wls target genes, as well as performing whole mount immunofluorescence in combination with lectin staining.

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><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>K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>P8131</td><td></td></tr><tr><td>K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3289</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</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>NP&lt;sub&gt;4&lt;/sub&gt;O</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&amp;nbsp;&amp;nbsp;&amp;nbsp; ( 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&amp;nbsp; 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.&amp;nbsp;</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 lungs. In WlsShhCre embryos (wherein secretion of Wnt ligands from the epithelium of respiratory tract was abrogated) Lac-Z staining was almost absent (Figure 1).
Mesenchymal condensations in tracheal mesenchyme
A critical step in chondrogenesis is the process by which mesenchymal cells fated to give rise to cartilage condense. These tight aggregations of cells are known as mesenchymal condensations. To test whether the lack of cartilage observed in WlsShhCre mice was due to an absence of mesenchymal condensations, tracheal lung tissues from gestational ages E12.5 to E14.5 were stained with PNA lectin. At E13.5 and E14.5, fluorescence was detected as periodic bands in the tracheal mesenchyme at sites where cartilage will be formed. Lack of detectable fluorescence in the tracheal tissue of WlsShhCre embryos indicates that endodermal Wnt signaling to the tracheal mesenchyme is necessary for mesenchymal condensations (Figure 2).
Respiratory tract cell specification
Whole mount immunofluorescence of tracheal lung tissues determined the expression pattern of Sox9, Nkx2.1 and &#945;SMA at E11.5. In the tracheal tissue, Sox9 expression is limited to the mesenchyme of the trachea as a continuous stripe (arrow in Figure 3A and B) while in the developing lung Sox9 is observed at the periphery of the respiratory epithelium (arrow heads in Figure 3A, B and C). Nkx2.1 presents a distinct expression pattern restricted to the epithelium of the trachea of the lung. Preventing secretion of Wnt ligands from epithelium causes diminished expression of Sox9 in tracheal mesenchyme but does not affect Sox9 expression in peripheral lung epithelium. Expression of &#945;SMA is not readily detected in tracheal mesenchyme, however staining was detected at the level of the larynx (yellow arrow, Figure 3) and stomach (Figure 3).
Cell proliferation in developing trachea
Proliferation in epithelium and mesenchyme of the trachea was detected after in vivo labeling of tissue with BrdU. Sections of tracheal tissue were stained with antibodies recognizing BrdU, Sox9 and &#945;SMA. The proliferation profile in the tracheal mesenchyme indicates that the levels of Sox9 stained cells undergoing proliferation is higher than the level of &#945;SMA stained cells undergoing proliferation. This proliferation pattern seems to be reverted in WlsShhCre tracheas (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/53910/53910fig1.jpg" /> 
Figure 1: Wnt/&#946;-catenin activity is operative in tracheal mesenchyme. E13.5 tracheal lung tissue, isolated from Axin2-LacZ and WlsShhCre:Axin2-LacZ mice, were stained with X-gal (A and B). Sections of tracheal lung tissue were counterstained with fast red (A&#39;, B&#39;). Note the lack of X-gal staining in WlsShhCre;Axin2-Lac-Z mice demonstrating absence of Wnt/&#946;-catenin activity (B&#39;). E14.5 tissue from Axin2 LacZ mice stained with X-gal depicts sites of future cartilaginous rings (C); however, Axin2 activity was restricted to the periphery (arrow head) of the mesenchymal condensations (arrow) (D). Scale bar A, B and C = 500 &#956;m; B&#39;, D&#39; and D = 100 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53910/53910fig2.jpg" /> 
Figure 2: Mesenchymal condensation in developing tracheal tissue. E13.5 tracheal-lung explants were stained with PNA lectin. Control tracheal tissue depicting the regions were mesenchymal cells condensed is shown (arrows A, C). No mesenchymal condensations were detected in WlsShhCre mice (B and arrow in D). C and D depict lower magnification of tracheal-lung tissue shown in A and B. Scale bar A and B = 1 mm; C and D = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53910/53910fig3.jpg" /> 
Figure 3: Expression pattern in prechondrogenic whole mount trachea. Nkx2.1 and Sox9 staining was detected in E11.5 explants. Panel A depicts an image of a control tissue, while panel B shows an optical section of a 20 &#956;m control tissue. Note the lack of mesenchymal expression of Sox9 in WlsShhCre tissue (arrow in C), but maintenance of peripheral expression of Sox9 (arrowhead in C). &#945;SMA staining was detected at low levels in trachealmesenchyme of E11.5 tissue but observed in the laryngeal region (yellow arrowhead in D and E), stomach (S) and remaining cardiac tissue (CT) (E). Nkx2.1 is expressed in epithelium of developing trachea and lungs (D, E). Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53910/53910fig4.jpg" /> 
Figure 4: Cell proliferation pattern in prechondrogenic mesenchyme. Sections of E13.5 embryos were stained with anti BrdU, Sox9 and &#945;SMA antibody. Panels C and D are higher magnifications of A and B respectively. Dotted lines represent limits of the tracheal epithelium. Note Sox9 stained proliferative cell (arrowhead C) and &#945;SMA stained proliferative cell (arrowhead in D). T = trachea, E = esophagus. Scale bar A and B = 50 &#956;m, C and D = 20 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53910/53910fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.3K Views.  Cincinnati Children’s Hospital Medical Center.  Het gebruik van reporter muizen gekoppeld aan ganse berg en rubriek vlekken, microscopie en in vivo assays vergemakkelijkt de analyse van de onderliggende mechanismen van het normale patroon van de luchtwegen. Hier beschrijven we hoe deze technieken hebben bijgedragen aan de analyse van Wnt signalering gedurende tracheale ontwikkeling. 

Video: Studying Wnt Signaling During Patterning of Conducting Airways

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 ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

Biology

Flow Cytometry and Confocal Imaging Analysis of Low Wnt Expression in Axin2-mTurquoise2 Reporter Thymocytes

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity.
This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or &#946;-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.

Signaling levels are known to regulate cell fate, indicating that regulation of Wnt signaling constitutes an interesting therapeutic target. Here, we describe flow cytometry and confocal microscopy analysis methods for a robust murine canonical Wnt signaling reporter model that measures distinct Wnt signaling levels.

Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent stem cells (hiPSCs) can be differentiated stepwise into 3D multicellular lung organoids, made of both epithelial and mesenchymal cell populations. We recapitulate embryonic developmental cues by temporally introducing a variety of growth factors and small molecules to efficiently generate definitive endoderm, anterior foregut endoderm, and subsequently lung progenitor cells. These cells are then embedded in growth factor reduced (GFR)-basement membrane matrix medium, allowing them to spontaneously develop into 3D lung organoids in response to external growth factors. These whole lung organoids (WLO) undergo early lung developmental stages including branching morphogenesis and maturation after exposure to dexamethasone, cyclic AMP and isobutylxanthine. WLOs possess airway epithelial cells expressing the markers KRT5 (basal), SCGB3A2 (club) and MUC5AC (goblet) as well as alveolar epithelial cells expressing HOPX (alveolar type I) and SP-C (alveolar type II). Mesenchymal cells are also present, including smooth muscle actin (SMA), and platelet-derived growth factor receptor A (PDGFR&#945;). iPSC derived WLOs can be maintained in 3D culture conditions for many months and can be sorted for surface markers to purify a specific cell population. iPSC derived WLOs can also be utilized to study human lung development, including signaling between the lung epithelium and mesenchyme, to model genetic mutations on human lung cell function and development, and to determine the cytotoxicity of infective agents.

The article describes step wise directed differentiation of induced pluripotent stem cells to three-dimensional whole lung organoids containing both proximal and distal epithelial lung cells along with mesenchyme.

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This process requires complex and coordinated paracrine interactions between signal-sending and signal-receiving cells. Given that these interactions can occur between various types of cells from different lineages, in vivo evaluation of cell-specific defects can be challenging. The present study describes a highly reproducible method to evaluate paracrine noncanonical Wnt signaling in vitro. This protocol was designed with the ability to (1) conduct functional and molecular assessments of noncanonical Wnt signaling between any two cell types of interest; (2) dissect the role of signal-sending versus signal-receiving molecules in the noncanonical Wnt signaling pathway; and (3) perform phenotypic rescue experiments with standard molecular or pharmacologic approaches.
This protocol was used to evaluate neural crest cell (NCC)-mediated noncanonical Wnt signaling in myoblasts. The presence of NCCs is associated with an increased number of phalloidin-positive cytoplasmic filopodia and lamellipodia in myoblasts and improved myoblast migration in a wound-healing assay. The Wnt5a-ROR2 axis was identified as a crucial noncanonical Wnt signaling pathway between NCC and second heart field (SHF) cardiomyoblast progenitors. In conclusion, this is a highly tractable protocol to study paracrine noncanonical Wnt signaling mechanisms in vitro.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

The present study outlines a highly reproducible and tractable method to study paracrine noncanonical Wnt signaling events in vitro. This protocol was applied to evaluate the impact of paracrine Wnt5a signaling in murine neural crest cells and myoblasts.

Noncanonical Wnt signaling regulates intracellular actin filament organization and polarized migration of progenitor cells during embryogenesis. This ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Bioengineering

A Comprehensive Review of Avian Embryonic Skin Explant Culture Protocols

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Author Spotlight: Insight into the Current Experimental Avian Skin Explant Methodologies

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

Medicine

Canonical Wnt Signaling Pathway

The gene encoding the main signaling molecules of the Wnt signaling pathways (the Wnt proteins) was discovered almost four decades ago by N&#252;sslein-Volhard and Wieschaus. They identified and originally named the gene &#34;wingless&#34; (wg) after a phenotype discovered during their landmark genetic screen in Drosophila for body pattern defects. At around the same time, another researcher named Harold Varmus found that a murine tumor virus activates the mammalian wg homolog, Int-1, which results in tumor formation in mice. Consequently, this gene family was named &#34;Wnt&#34; - a portmanteau of the wingless and integrated gene names.
The Wnt Signaling Mechanism
The Wnt proteins, secreted from the signaling cell, activate Frizzled receptors on the target cell via paracrine or autocrine signaling. The signaling pathway can subsequently proceed via three different pathways. In a nutshell, unlike the canonical Wnt signaling pathway that is &#946;-catenin dependent, the non-canonical pathways are &#946;-catenin independent and use different secondary messengers. Nevertheless, according to recent discoveries, these pathways are not autonomous and have considerable overlap and crosstalk between them.
Wnt Signaling Functions and Associated Diseases
The Wnt protein is evolutionarily conserved in all animals. It plays a central role in mediating intercellular communication during embryonic development, as well as adult tissue homeostasis. Given the crucial role of Wnt signaling in developmental patterns such as cell differentiation, polarization, and migration, the association of the pathway with cancer progression is predictable. Aberrant Wnt signaling has long been implicated as the primary cause of colorectal cancers.
Due to the vital role of Wnt signaling in the development of the nervous system and its prominence at the synapse, there is recent evidence that supports its role in the aging brain - and even Alzheimer&#39;s Disease. Additionally, dysregulation of the Wnt pathway is linked to bone and cardiovascular diseases.

Studying Wnt Signaling During Patterning of Conducting Airways

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