Name: modeling paracrine noncanonical wnt signaling in vitro
Uploaded: 2021-12-10T14:30:00
Description: Watch this Scientific Journal Video about Modeling Paracrine Noncanonical Wnt Signaling In Vitro at JoVE.com

This work was supported in part by NIH awards F30HL154324 to O.T. and K08HL121191 and R03HL154301 to S.R.K. The authors would like to acknowledge that the schematic in Figure 1 in this manuscript was created with biorender.com.

1. Preexperimental expansion and passaging of cells
<ol>
	<li>C2C12 cell culture:
	<ol>
		<li>Prepare 500 mL of C2C12 culture medium by combining Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 10% fetal bovine serum (FBS) and 1 % penicillin/streptomycin.</li>
		<li>Thaw a vial of C2C12 cells in 37 &#176;C water bath. While the C2C12 cells are thawing, add 5 mL of C2C12 medium to a 15 mL conical tube. Immediately transfer the thawed cells to the 15 mL tube using a P1000 pipette. 
		NOTE: C2...

<ol>
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(8), 42093 (2019).</li><li>Li, 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=Planar+cell+polarity+signaling+regulates+polarized+second+heart+field+morphogenesis+to+promote+both+arterial+and+venous+pole+septation.">Planar cell polarity signaling regulates polarized second heart field morphogenesis to promote both arterial and venous pole septation.</a> Development. 146 (20), 181719 (2019).</li><li>Lutze, G., 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=Noncanonical+WNT-signaling+controls+differentiation+of+lymphatics+and+extension+lymphangiogenesis+via+RAC+and+JNK+signaling.">Noncanonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling.</a> Scientific Reports. 9 (1), 4739 (2019).</li><li>Buttler, K., 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=Maldevelopment+of+dermal+lymphatics+in+Wnt5a-knockout-mice.">Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice.</a> Developmental Biology. 381 (2), 365-376 (2013).</li><li>Betterman, K. 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=Atypical+cadherin+FAT4+orchestrates+lymphatic+endothelial+cell+polarity+in+response+to+flow.">Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow.</a> Journal of Clinical Investigation. 130 (6), 3315-3328 (2020).</li><li>Descamps, 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=Frizzled+4+regulates+arterial+network+organization+through+noncanonical+Wnt/planar+cell+polarity+signaling.">Frizzled 4 regulates arterial network organization through noncanonical Wnt/planar cell polarity signaling.</a> Circulation Research. 110 (1), 47-58 (2012).</li><li>Weeraratna, A. T., 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+signaling+directly+affects+cell+motility+and+invasion+of+metastatic+melanoma.">Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma.</a> Cancer Cell. 1 (3), 279-288 (2002).</li><li>Henry, 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=Expression+of+the+novel+Wnt+receptor+ROR2+is+increased+in+breast+cancer+and+may+regulate+both+&#946;-catenin+dependent+and+independent+Wnt+signalling.">Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both &#946;-catenin dependent and independent Wnt signalling.</a> Journal of Cancer Research and Clinical Oncology. 141 (2), 243-254 (2014).</li><li>Anastas, J. N., 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=A+protein+complex+of+SCRIB,+NOS1AP+and+VANGL1+regulates+cell+polarity+and+migration,+and+is+associated+with+breast+cancer+progression.">A protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression.</a> Oncogene. 31 (32), 3696-3708 (2012).</li><li>Niehrs, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+world+of+WNT+receptor+signalling.">The complex world of WNT receptor signalling.</a> Nature Reviews Molecular Cell Biology. 13 (12), 767-779 (2012).</li><li>Dong, 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=Functional+redundancy+of+frizzled+3+and+frizzled+6+in+planar+cell+polarity+control+of+mouse+hair+follicles.">Functional redundancy of frizzled 3 and frizzled 6 in planar cell polarity control of mouse hair follicles.</a> Development. 145 (19), (2018).</li><li>Bernascone, I., 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=Sfrp3+modulates+stromal-epithelial+crosstalk+during+mammary+gland+development+by+regulating+Wnt+levels.">Sfrp3 modulates stromal-epithelial crosstalk during mammary gland development by regulating Wnt levels.</a> Nature Communications. 10 (1), 2481 (2019).</li><li>Hendrickx, G., 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=WNT16+requires+G&#945;+subunits+as+intracellular+partners+for+both+its+canonical+and+noncanonical+WNT+signalling+activity+in+osteoblasts.">WNT16 requires G&#945; subunits as intracellular partners for both its canonical and noncanonical WNT signalling activity in osteoblasts.</a> Calcified Tissue International. 106 (3), 294-302 (2020).</li><li>Avgustinova, A., 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=Tumour+cell-derived+Wnt7a+recruits+and+activates+fibroblasts+to+promote+tumour+aggressiveness.">Tumour cell-derived Wnt7a recruits and activates fibroblasts to promote tumour aggressiveness.</a> Nature Communications. (7), 10305 (2016).</li><li>Tseng, J. 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=CAPE+suppresses+migration+and+invasion+of+prostate+cancer+cells+via+activation+of+noncanonical+Wnt+signaling.">CAPE suppresses migration and invasion of prostate cancer cells via activation of noncanonical Wnt signaling.</a> Oncotarget. 7 (25), 38010-38024 (2016).</li><li>Wang, Q., 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=A+novel+role+for+Wnt/Ca2++signaling+in+actin+cytoskeleton+remodeling+and+cell+motility+in+prostate+cancer.">A novel role for Wnt/Ca2+ signaling in actin cytoskeleton remodeling and cell motility in prostate cancer.</a> PLoS One. 5 (5), 10456 (2010).</li><li>Gibbs, B. 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=Prickle1+mutation+causes+planar+cell+polarity+and+directional+cell+migration+defects+associated+with+cardiac+outflow+tract+anomalies+and+other+structural+birth+defects.">Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects.</a> Biology Open. 5 (3), 323-335 (2016).</li><li>Cui, 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=a+PCP+protein+required+for+ciliogenesis,+regulates+directional+cell+migration+and+cell+polarity+by+direct+modulation+of+the+actin+cytoskeleton.">a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.</a> PLoS Biology. 11 (11), 1001720 (2013).</li><li>Gombos, R., 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=The+formin+DAAM+functions+as+molecular+effector+of+the+planar+cell+polarity+pathway+during+axonal+development+in+Drosophila.">The formin DAAM functions as molecular effector of the planar cell polarity pathway during axonal development in Drosophila.</a> The Journal of Neuroscience. 35 (28), 10154-10167 (2015).</li><li>Toubat, 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=Neural+Crest+Cell-derived+Wnt5a+Regulates+Planar+Cell+Polarity+in+Cranial+Second+Heart+Field+Progenitor+Cells.">Neural Crest Cell-derived Wnt5a Regulates Planar Cell Polarity in Cranial Second Heart Field Progenitor Cells.</a> Circulation. 142, 12540 (2020).</li><li>Li, 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=Spatial+regulation+of+cell+cohesion+by+Wnt5a+during+second+heart+field+progenitor+deployment.">Spatial regulation of cell cohesion by Wnt5a during second heart field progenitor deployment.</a> Developmental Biology. 412 (1), 18-31 (2016).</li><li>Sinha, T., 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=Loss+of+Wnt5a+disrupts+second+heart+field+cell+deployment+and+may+contribute+to+OFT+malformations+in+DiGeorge+syndrome.">Loss of Wnt5a disrupts second heart field cell deployment and may contribute to OFT malformations in DiGeorge syndrome.</a> Human Molecular Genetics. 24 (6), 1704-1716 (2015).</li><li>Humphries, A. 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T., 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=Planar+cell+polarity+in+development+and+disease.">Planar cell polarity in development and disease.</a> Nature Reviews Molecular Cell Biology. 18 (6), 375-388 (2017).</li><li>Bradshaw, 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=Dual+role+for+neural+crest+cells+during+outflow+tract+septation+in+the+neural+crest-deficient+mutant+Splotch2H.">Dual role for neural crest cells during outflow tract septation in the neural crest-deficient mutant Splotch2H.</a> Journal of Anatomy. 214 (2), 245-257 (2009).</li><li>Kodo, K., 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+Sema3c+and+the+interaction+between+cardiac+neural+crest+and+second+heart+field+during+outflow+tract+development.">Regulation of Sema3c and the interaction between cardiac neural crest and second heart field during outflow tract development.</a> Scientific Reports. 7 (1), 6771 (2017).</li><li>Waldo, K. 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=Cardiac+neural+crest+is+necessary+for+normal+addition+of+the+myocardium+to+the+arterial+pole+from+the+secondary+heart+field.">Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field.</a> Developmental Biology. 281 (1), 66-77 (2005).</li><li>Schleiffarth, J. R., 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+is+required+for+cardiac+outflow+tract+septation+in+mice.">Wnt5a is required for cardiac outflow tract septation in mice.</a> Pediatric Research. 61 (4), 386-391 (2007).</li><li>Nguyen, B. 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The noncanonical Wnt/planar cell polarity (PCP) signaling pathway is a critically important cellular signaling pathway that has been implicated in multiple developmental24,25 and disease processes24,26. During embryonic development, noncanonical Wnt signaling involves an expansive network of molecular signals from signal-sending cells that ultimately induce changes in morphology, asymmetric organ...

Noncanonical Wnt signaling is an evolutionarily conserved pathway that regulates cellular filament organization and directional migration. This pathway has been implicated in multiple biological processes, including embryonic tissue morphogenesis1,2,3, lymphatic and vascular angiogenesis4,5,6,7, and cancer growth and metastasis8,9,10. At the cellular level, noncanonical Wnt signaling is carried out through coordinated paracrine interactions between signal-sending and signal-receiving cells. These interactions frequently occur between cells of different lineages or types and involve a diverse molecular network that includes up to 19 ligands and multiple receptors, co-receptors, and downstream signal transduction effectors11. Further complicating this signaling process, previous studies have shown that ligand-receptor combinations can vary in a context- and tissue-dependent manner12,13, and that the same source ligands that drive noncanonical Wnt signaling in signal-receiving cells can be produced by multiple signal-sending cell types14,15. Given the cellular and molecular complexity associated with noncanonical Wnt signaling, the ability to study individual and clinically relevant mechanisms in vivo has been limited.
Attempts have been made to study noncanonical Wnt signaling using cell culture&#160;techniques in vitro. For example, wound-healing assays performed in cellular monolayers have been used to functionally assess cellular directional migration4,16,17,18,19. Immunostaining techniques have been used to perform spatial analyses of surface protein expression to evaluate noncanonical Wnt-induced changes in cellular morphology7,10, architecture, and asymmetric polarization18,19,20. Although these approaches have provided important tools for characterizing Wnt-related phenotypes in signal-receiving cells, the lack of signal-sending components in these protocols limits their ability to accurately model paracrine signaling mechanisms observed in vivo. As a result, there remains a critical need to develop in vitro systems that allow robust and reproducible evaluation of paracrine signaling interactions between signal-sending and receiving cells of the noncanonical Wnt pathway, particularly those of different cell types.
To this end, the primary objective of this study was to establish a protocol to model paracrine noncanonical Wnt signaling interactions in vitro. We developed a non-contact coculture system that recapitulates signal-sending and signal-receiving components of these interactions and allows the use of standard molecular, genetic, or pharmacologic approaches to independently study specific ligand-receptor mechanisms in the noncanonical Wnt pathway. Mechanisms of NCC-mediated Wnt signaling were examined in myoblasts using established murine cell lines. As proof of principle, this model was used to corroborate findings of prior in vivo studies in mice that implicate the Wnt5a-ROR2 axis as a relevant noncanonical Wnt signaling pathway between NCCs21 and SHF cardiomyoblast progenitors3,22,23.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M-7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Antifade mounting medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200-10</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-2323</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>C2C12 murine myoblast cell line</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flasks, 75 cm2</td>
			<td>ThermoFisher Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Chamber Slide System, 4-well</td>
			<td>ThermoFisher Scientific</td>
			<td>154526</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM), high glucose (4.5 g/L), L-glutamine (2 mM)</td>
			<td>Corning</td>
			<td>10-017-CV</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Falcon conical centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon permeable support for 24-well plate with 0.4 &#181;M transparent PET membrane</td>
			<td>Corning</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>W3381E</td>
			<td>Stored in 50 mL aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Gelatin solution, 0.1%</td>
			<td>ATCC</td>
			<td>PCS-999-027</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Graduated and sterile pipette tips, 10 &#181;L</td>
			<td>USA Scientific</td>
			<td>1111-3810</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukemia inhibitory factor (LIF), 106 unit/mL</td>
			<td>Millipore Sigma</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAiMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>13778-075</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (MEM NEAA) 100x</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Corning</td>
			<td>10-022-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Roche</td>
			<td>10107409001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-stick auto-glass coverslips, 24 x 55 mm</td>
			<td>Springside Scientific</td>
			<td>HRTCG2455</td>
			<td></td>
		</tr>
		<tr>
			<td>O9-1 neural crest cell line</td>
			<td>Millipore Sigma</td>
			<td>SCC049</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I, 1x</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution in PBS, 4%</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 &#956;g/mL streptomycin)</td>
			<td>Fisher Scientific</td>
			<td>W3470H</td>
			<td>Stored in 10 mL aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Phalloidin-iFluor 488</td>
			<td>Abcam</td>
			<td>ab176753</td>
			<td>Stored at -20 &#176;C, Keep out of light</td>
		</tr>
		<tr>
			<td>Phosphate-buffer saline (PBS), 1x, without calcium and magnesium, pH 7.4</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant human fibroblast growth factor-basic (rhFGF-basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human/mouse Wnt5a protein</td>
			<td>R&#38;D Systems</td>
			<td>645-WN-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate, 100 mM</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Square Petri dish with grid</td>
			<td>Thomas Scientific</td>
			<td>1219C98</td>
			<td></td>
		</tr>
		<tr>
			<td>STO murine fibroblast feeder cells</td>
			<td>ATCC</td>
			<td>CRL-1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Fisher Scientific</td>
			<td>W3513C</td>
			<td>Stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Zeiss Apotome.2 fluoresence microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss inverted Axio Vert.A1 light microscope</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen lite 2012 microscopy software</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td>imaging software</td>
		</tr>
	</tbody>
</table>

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.

Effects of NCCs on migratory capacity of murine myoblasts 
This assay was first applied to evaluate the impact of NCCs on the migratory capacity of myoblasts. Figure 1 outlines the schematic model of the assay. To test this impact, scratch assays were performed with myoblasts that were grown in isolation (without NCC inserts) compared to those grown in the presence of inserts. As a positive control, 500 ng/mL of recombinant Wnt5a (rWnt5a) was added to chamber wells with...

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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.

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

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

This protocol outlines a highly reproducible method to functionally and molecularly evaluate paracrine non-canonical Wnt signaling in any cell population of interest. Functional and molecular assessments are performed in the same cell population and can be applied to study any component of the Wnt/PCP pathway. To begin, resuspend the previously pelleted C2C12 cell in 10 milliliters of C2C12 medium.To dilute the cells at the ratio of 1:20, add 0.5 milliliters of cell suspension in a 15 milliliter tube containing 9.5 milliliters of fresh C2C12 medium and gently mix with a serological pipette using a P1000 pipette, add one milliliter of the diluted cell suspension to each well of a new four chambered well and place it in the incubator. For plating O9-1 cell inserts, resuspend the O9-1 cell pellet in 10 milliliters of O9-1 growth medium. And similar to C2C12 cell dilution, dilute the O9-1 cells at the ratio of 1:20.Next, place a single permeable insert inside each well of a new four chambered well filled with one milliliter of O9-1 growth medium. Add 300 microliters of the diluted O9-1 cell suspension to each insert and ensure that insert is submerged completely. Then incubate the well at 37 degrees Celsius.To perform siRNA knockdown in the O9-1 cells, dilute the siRNA and transfection reagent in the reduced serum medium according to the manufacturer's recommendations at the desired concentrations. Gently mix the diluted siRNA and transfection reagent and incubate the mixture at room temperature for seven minutes. After 18 to 24 hours of cell plating, add the siRNA lipid complexes to the O9-1 cell inserts and incubate for approximately 34 to 48 hours.When the cells reach 70 to 80%confluency, remove the medium from the C2C12 chamber well and wash the cells once with PBS. After removing the PBS, begin scratching the cells by passing a sterile P10 pipette tip firmly in a single direction to span the entire length or width of the cell monolayer. Once the cells are scratched, quickly add one milliliter of PBS.Next, turn on the computer and the microscope. Place the chamber slide on the stage and rotate the objective dial to 5X magnification. Open the imaging software.Click the camera tab and then click the Live button to visualize the cells on the AxioCam IC tab. Ensure that the light filter is pulled all the way out to allow light to pass to the camera and computer screen, manually move or rotate the chamber slide to position the wound area in the center of the live image on the AxioCam IC tab. To take images, click snap to open a new tab next to the AxioCam IC tab that contains the image.To save this still image, click on the file, select save as, select desktop on the bar on the left to save the file to the desktop, enter the file name in the file name box and save the figure in czi file format. To save the picture as a tiff, click file, select save as, enter the file name in the file name box. Click the save as type button and select tiff from the dropdown menu.Manually reposition the chamber slide to take two to three more images at other points of the wound in the same well. After imaging, remove the PBS from each well and add one milliliter of C2C12 medium. Assemble the well insert co-culture system by manually placing the inserts containing the O9-1 cells in each well of the chamber well.Gently push the inserts down into the well, such that the bottom of the insert sits just above the underlying C2C12 cells. Return the well insert constructs to the incubator and allow the cells to migrate for a total of nine to 12 hours. To determine the optimal migration time, check the cells at six hours following wound creation, then every two to three hours after that.End the experiment when the cells in control conditions completely cover the wound. Begin migration assay termination and well insert system deconstruction by removing the O9-1 cell inserts after nine to 12 hours of the migration period. Carefully aspirate the C2C12 medium.Add 0.5 milliliters of PBS to the chamber wells and take final images of cells following migration. Carefully aspirate all PBS mixed with medium and remove the plastic chambers from the chamber wells using kit instructions. Leaving the underlying slide containing the C2C12 cells.For immunostaining, immediately incubate the slide with 4%paraform aldehyde for 10 minutes at room temperature. Then remove paraformaldehyde and wash the slide with 0.1%Triton X-100 containing PBST for 15 minutes at room temperature. After 15 minutes, pour out PBST and wash the slide twice with PBS for 10 minutes each.Then create a hydrophobic boundary with a hydrophobic pen around the slide to prevent solutions from spilling from the slide preventing disruption of the adherence cells. Next, add approximately 0.5 milliliters of 1%BSA blocking solution to the slide within the hydrophobic boundary and incubate the slide for one hour at room temperature in a humidified slide chamber. At the end of the incubation, remove the blocking solution and add the phalloidin antibody to this slide within the hydrophobic boundary, and incubate at four degrees Celsius overnight.The next day, place the slide in a slide holder protected from light exposure. Wash the cells thrice with PBS for 10 minutes each at room temperature. Add DAPI-containing mounting medium, mount the slides with glass cover slips and capture the cells using a standard fluorescence microscope.In the present study, the addition of recombinant Wnt5a to co-culture wells accelerated myoblast migration with complete recovery of some areas by nine hours. The migratory myoblasts in all three conditions exhibited normal migratory cellular morphology, including well-formed and protruding filopodia and lamellipodia and asymmetric polarization of actin cytoskeletal projections. The paracrine effect of NCC-derived Wnt5a on myoblast migration was studied.Treatment with 50 nanomolar siRNA against Wnt5a reduced Wnt5a gene expression by 64%compared to negative control. The migration of C2C12 mile blasts with significantly reduced following Wnt5a knockdown in NCCs and the addition of exogenous recombinant Wnt5a rescued this migratory deficit and morphological defect in these myoblasts. In order to better understand the signal receiving cell mechanisms in the paracrine model, the ROR2 receptor was knocked down and gene expression was reduced by 54%Knockdown of ROR2 in myoblasts reduces their migratory capacity despite the presence of NCCs.Exogenous recombinant Wnt5a failed to rescue myoblast migration after ROR2 knockdown suggesting that ROR2 depletion disrupts the ability of myoblasts to receive Wnt5a signals. Culture cells to adequate density scratch with P10 tip only once assemble and unassembled co-culture system with care without disrupting underlying cell. This technique was used as a proof of concept to study a specific molecular signaling axis within the complex Wnt/PCP pathway with relevance for neural crest cell and second heart field biology.

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