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

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

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

<ol>
	<li>Maeda, Y., Dave, V., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+lung+morphogenesis.">Transcriptional control of lung morphogenesis.</a> Physiol Rev. 87, 219-244 (2007).</li><li>Morrisey, E. E., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+for+the+first+breath:+genetic+and+cellular+mechanisms+in+lung+development.">Preparing for the first breath: genetic and cellular mechanisms in lung development.</a> Dev Cell. 18, 8-23 (2010).</li><li>Fausett, S. R., Klingensmith, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalization+of+the+foregut+tube:+developmental+origins+of+the+trachea+and+esophagus.">Compartmentalization of the foregut tube: developmental origins of the trachea and esophagus.</a> Wiley Interdiscip Rev Dev Biol. 1, 184-202 (2012).</li><li>Goss, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt2/2b+and+beta-catenin+signaling+are+necessary+and+sufficient+to+specify+lung+progenitors+in+the+foregut.">Wnt2/2b and beta-catenin signaling are necessary and sufficient to specify lung progenitors in the foregut.</a> Dev Cell. 17, 290-298 (2009).</li><li>Harris-Johnson, K. S., Domyan, E. T., Vezina, C. M., Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+promotes+respiratory+progenitor+identity+in+mouse+foregut.">beta-Catenin promotes respiratory progenitor identity in mouse foregut.</a> Proc Natl Acad Sci U S A. 106, 16287-16292 (2009).</li><li>Cornett, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wntless+is+required+for+peripheral+lung+differentiation+and+pulmonary+vascular+development.">Wntless is required for peripheral lung differentiation and pulmonary vascular development.</a> Dev Biol. 379, 38-52 (2013).</li><li>Snowball, J., Ambalavanan, M., Whitsett, J., Sinner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Endodermal+Wnt+signaling+is+required+for+tracheal+cartilage+formation&amp;#34;.">34;Endodermal Wnt signaling is required for tracheal cartilage formation&amp;#34;.</a> Dev Biol. , (2015).</li><li>Shannon, J. M., Hyatt, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+interactions+in+the+developing+lung.">Epithelial-mesenchymal interactions in the developing lung.</a> Annu Rev Physiol. 66, 625-645 (2004).</li><li>Shannon, J. M., Nielsen, L. D., Gebb, S. A., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchyme+specifies+epithelial+differentiation+in+reciprocal+recombinants+of+embryonic+lung+and+trachea.">Mesenchyme specifies epithelial differentiation in reciprocal recombinants of embryonic lung and trachea.</a> Dev Dyn. 212, 482-494 (1998).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt5a+regulates+Shh+and+Fgf10+signaling+during+lung+development.">Wnt5a regulates Shh and Fgf10 signaling during lung development.</a> Dev Biol. 287, 86-97 (2005).</li><li>Loscertales, M., Mikels, A. J., Hu, J. K., Donahoe, P. K., Roberts, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+pulmonary+Wnt5a+directs+airway+and+vascular+tubulogenesis.">Chick pulmonary Wnt5a directs airway and vascular tubulogenesis.</a> Development. 135, 1365-1376 (2008).</li><li>Yin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+FGF-WNT+gene+regulatory+network+controls+lung+mesenchyme+development.">An FGF-WNT gene regulatory network controls lung mesenchyme development.</a> Dev Biol. 319, 426-436 (2008).</li><li>Shu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+acts+upstream+of+N-myc,+BMP4,+and+FGF+signaling+to+regulate+proximal-distal+patterning+in+the+lung.">Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung.</a> Dev Biol. 283, 226-239 (2005).</li><li>Bretholz, A., Morrisey, R., Hoffman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+OpdA+in+rat+models+of+organic+phosphorus+(OP)+poisoning.">The use of OpdA in rat models of organic phosphorus (OP) poisoning.</a> Toxicology. 257, (2009).</li><li>Goss, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt2+signaling+is+necessary+and+sufficient+to+activate+the+airway+smooth+muscle+program+in+the+lung+by+regulating+myocardin/Mrtf-B+and+Fgf10+expression.">Wnt2 signaling is necessary and sufficient to activate the airway smooth muscle program in the lung by regulating myocardin/Mrtf-B and Fgf10 expression.</a> Dev Biol. 356, 541-552 (2011).</li><li>Mucenski, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+is+required+for+specification+of+proximal/distal+cell+fate+during+lung+morphogenesis.">beta-Catenin is required for specification of proximal/distal cell fate during lung morphogenesis.</a> J Biol Chem. 278, 40231-40238 (2003).</li><li>Hyatt, B. A., Shangguan, X., Shannon, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-10+induces+SP-C+and+Bmp4+and+regulates+proximal-distal+patterning+in+embryonic+tracheal+epithelium.">FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium.</a> Am J Physiol Lung Cell Mol Physiol. 287, L1116-L1126 (2004).</li><li>Del Moral, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-A+signaling+through+Flk-1+is+a+critical+facilitator+of+early+embryonic+lung+epithelial+to+endothelial+crosstalk+and+branching+morphogenesis.">VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis.</a> Dev Biol. 290, 177-188 (2006).</li><li>Ott, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+in+large+insect+brains:+zinc-formaldehyde+fixation+improves+synapsin+immunostaining+and+preservation+of+morphology+in+whole-mounts.">Confocal microscopy in large insect brains: zinc-formaldehyde fixation improves synapsin immunostaining and preservation of morphology in whole-mounts.</a> J Neurosci Methods. 172, 220-230 (2008).</li><li>Jahrling, N., Becker, K., Dodt, H. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-reconstruction+of+blood+vessels+by+ultramicroscopy.">3D-reconstruction of blood vessels by ultramicroscopy.</a> Organogenesis. 5, 145-148 (2009).</li><li>Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Sox9+by+Sonic+Hedgehog+(Shh)+is+essential+for+patterning+and+formation+of+tracheal+cartilage.">Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage.</a> Dev Dyn. 239, 514-526 (2010).</li><li>Elluru, R. G., Thompson, F., Reece, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+18+gives+growth+and+directional+cues+to+airway+cartilage.">Fibroblast growth factor 18 gives growth and directional cues to airway cartilage.</a> Laryngoscope. 119, 1153-1165 (2009).</li><li>Ahnfelt-Ronne, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+three-dimensional+reconstruction+of+protein+expression+patterns+in+intact+mouse+and+chicken+embryos+and+organs.">An improved method for three-dimensional reconstruction of protein expression patterns in intact mouse and chicken embryos and organs.</a> J Histochem Cytochem. 55, 925-930 (2007).</li><li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing.">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158, 945-958 (2014).</li><li>Gillotte, D. M., Fox, P. L., Mjaatvedt, C. H., Hoffman, S., Capehart, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+method+for+analysis+of+chondrogenesis+in+limb+mesenchyme+from+individual+transgenic+(hdf)+embryos.">An in vitro method for analysis of chondrogenesis in limb mesenchyme from individual transgenic (hdf) embryos.</a> Methods Cell Sci. 25, 97-104 (2003).</li><li>Cohen, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+regulates+smooth+muscle+precursor+development+in+the+mouse+lung+via+a+tenascin+C/PDGFR+pathway.">Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway.</a> J Clin Invest. 119, 2538-2549 (2009).</li><li>Boucherat, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+functional+redundancy+between+Hoxa5+and+Hoxb5+paralog+genes+during+lung+morphogenesis.">Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis.</a> Am J Physiol Lung Cell Mol Physiol. 304, L817-L830 (2013).</li></ol>

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 ...

Research

JoVE Journal

Developmental Biology

Method of Studying Palatal Fusion using Static Organ Culture

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

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

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 ...

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

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 ...

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 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; ...

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 ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

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 ...

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 ...

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 ...

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 ...