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 morphogenesis^1,2,3, lymphatic and vascular angiogenesis^4,5,6,7, and cancer growth and metastasis^8,9,10. At the cellular level,....

Protocol

1. Preexperimental expansion and passaging of cells

1. C2C12 cell culture:
   1. Prepare 500 mL of C2C12 culture medium by combining Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1 % penicillin/streptomycin.
   2. Thaw a vial of C2C12 cells in 37 °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: C2C12 cells are murine myoblast cells that have been previously used and validated as a primary cell line for modeling cardiomyoblast progenitors.
   ....

Results

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.......

Discussion

The noncanonical Wnt/planar cell polarity (PCP) signaling pathway is a critically important cellular signaling pathway that has been implicated in multiple developmental^24,25 and disease processes^24,26. 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.......

Materials

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