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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.
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.
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,....
1. Preexperimental expansion and passaging of cells
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.......
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.......
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.
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.
....Name | Company | Catalog Number | Comments |
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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