Modeling Paracrine Noncanonical Wnt Signaling In Vitro
Summary
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.
Abstract
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.
Introduction
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 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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 2-Mercaptoethanol | Sigma Aldrich | M-7522 | |
| Antifade mounting medium with DAPI | Vector Laboratories | H-1200-10 | Stored at 4 °C |
| Bovine serum albumin | Santa Cruz Biotechnology | sc-2323 | Stored at 4 °C |
| C2C12 murine myoblast cell line | ATCC | CRL-1772 | |
| Cell culture flasks, 75 cm2 | ThermoFisher Scientific | 156499 | |
| Chamber Slide System, 4-well | ThermoFisher Scientific | 154526 | |
| Dulbecco’s Modified Eagle’s Medium (DMEM), high glucose (4.5 g/L), L-glutamine (2 mM) | Corning | 10-017-CV | Stored at 4 °C |
| Falcon conical centrifuge tubes, 15 mL | Fisher Scientific | 14-959-53A | |
| Falcon permeable support for 24-well plate with 0.4 µM transparent PET membrane | Corning | 353095 | |
| Fetal bovine serum | Fisher Scientific | W3381E | Stored in 50 mL aliquots at -20 °C |
| Gelatin solution, 0.1% | ATCC | PCS-999-027 | Stored at 4 °C |
| Graduated and sterile pipette tips, 10 µL | USA Scientific | 1111-3810 | |
| Leukemia inhibitory factor (LIF), 106 unit/mL | Millipore Sigma | ESG1106 | |
| L-glutamine 200 mM (100x) | Gibco | 25030-081 | |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | 13778-075 | |
| MEM non-essential amino acids (MEM NEAA) 100x | Gibco | 11140-050 | |
| Minimum essential medium (MEM) | Corning | 10-022-CV | |
| Mitomycin C | Roche | 10107409001 | |
| Non-stick auto-glass coverslips, 24 x 55 mm | Springside Scientific | HRTCG2455 | |
| O9-1 neural crest cell line | Millipore Sigma | SCC049 | |
| Opti-MEM I, 1x | Gibco | 31985-070 | |
| Paraformaldehyde solution in PBS, 4% | Santa Cruz Biotechnology | sc-281692 | Stored at 4 °C |
| Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) | Fisher Scientific | W3470H | Stored in 10 mL aliquots at -20 °C |
| Phalloidin-iFluor 488 | Abcam | ab176753 | Stored at -20 °C, Keep out of light |
| Phosphate-buffer saline (PBS), 1x, without calcium and magnesium, pH 7.4 | Corning | 21-040-CV | Stored at 4 °C |
| Recombinant human fibroblast growth factor-basic (rhFGF-basic) | R&D Systems | 233-FB-025 | |
| Recombinant human/mouse Wnt5a protein | R&D Systems | 645-WN-010 | |
| Sodium pyruvate, 100 mM | Gibco | 11360-070 | |
| Square Petri dish with grid | Thomas Scientific | 1219C98 | |
| STO murine fibroblast feeder cells | ATCC | CRL-1503 | |
| Triton X-100 solution | Sigma Aldrich | X100-100ML | |
| Trypsin-EDTA, 0.25% | Fisher Scientific | W3513C | Stored at 4 °C |
| Zeiss Apotome.2 fluoresence microscope | Carl Zeiss AG | ||
| Zeiss inverted Axio Vert.A1 light microscope | Carl Zeiss AG | ||
| Zen lite 2012 microscopy software | Carl Zeiss AG | imaging software |
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