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.

<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. 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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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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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><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 &amp;deg;C</td></tr><tr><td>Bovine serum albumin</td><td>Santa Cruz Biotechnology</td><td>sc-2323</td><td>Stored at 4 &amp;deg;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 cm&lt;sup&gt;2&lt;/sup&gt;</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&amp;rsquo;s Modified Eagle&amp;rsquo;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 &amp;deg;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 &amp;micro;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 &amp;deg;C</td></tr><tr><td>Gelatin solution, 0.1%</td><td>ATCC</td><td>PCS-999-027</td><td>Stored at 4 &amp;deg;C</td></tr><tr><td>Graduated and sterile pipette tips, 10 &amp;micro;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 &amp;deg;C</td></tr><tr><td>Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 &amp;mu;g/mL streptomycin)</td><td>Fisher Scientific</td><td>W3470H</td><td>Stored in 10 mL aliquots at -20 &amp;deg;C</td></tr><tr><td>Phalloidin-iFluor 488</td><td>Abcam</td><td>ab176753</td><td>Stored at -20 &amp;deg;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 &amp;deg;C</td></tr><tr><td>Recombinant human fibroblast growth factor-basic (rhFGF-basic)</td><td>R&amp;amp;D Systems</td><td>233-FB-025</td><td></td></tr><tr><td>Recombinant human/mouse Wnt5a protein</td><td>R&amp;amp;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 &amp;deg;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...

Watch this Scientific Journal Video about Modeling Paracrine Noncanonical Wnt Signaling In Vitro at JoVE.com

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 ...

modeling-paracrine-noncanonical-wnt-signaling-in-vitro

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Developmental Biology

1.5K Views.  Keck School of Medicine of USC. Dit protocol schetst een zeer reproduceerbare methode om paracriene niet-canonieke Wnt-signalering functioneel en moleculair te evalueren in elke celpopulatie van belang. Functionele en moleculaire beoordelingen worden uitgevoerd in dezelfde celpopulatie en kunnen worden toegepast om elk onderdeel van de Wnt / PCP-route te bestuderen. Om te beginnen resuspenseer je de eerder gepelletiseerde C2C12-cel in 10 milliliter C2C12-medium.Om de cellen te verdunnen in de verhouding van 1: 20, vo...

Video: Modeling Paracrine Noncanonical Wnt Signaling In Vitro

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 ...

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.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

The 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 ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

Neuroscience

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

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

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

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

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Biology

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

Wound Assay to Evaluate the Migratory Capacity of Murine Myoblasts in Co-Culture

Source: Toubat, O. et al., <a href="https://app.jove.com/v/63247">Modeling Paracrine Noncanonical Wnt Signaling In Vitro.</a> J. Vis. Exp. (2021).
This video describes a non-contact co-culture wound healing assay to evaluate the migratory capacity of murine myoblasts. This helps to understand signal-sending and signal-receiving components of the paracrine non-canonical Wnt signaling interactions in vitro.

Source: Toubat, O. et al., Modeling Paracrine Noncanonical Wnt Signaling In Vitro. J. Vis. Exp. (2021).
This video describes a non-contact co-culture ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Canonical Wnt Signaling Pathway

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

Modeling Paracrine Noncanonical Wnt Signaling In Vitro

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