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 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 ...

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

Research

JoVE Journal

Developmental Biology

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

Video: Studying Wnt Signaling During Patterning of Conducting Airways

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 ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

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 ...