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

Summary

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.

Abstract

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 Axin2^em1Fstl 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 β-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.

Introduction

Canonical Wnt signaling is a conserved signaling pathway implicated in healthy tissue homeostasis as well as in disease. Precise regulation of Wnt signaling levels has been shown to be important in embryonic development, but is also of great importance in adult tissues. Canonical Wnt signaling has been found to play an important role in tissue regeneration of several organs such as the gut, the skin, and the hematopoietic system. Hence, when Wnt signaling is deregulated, severe pathologies arise. Colorectal, liver, and skin cancer, neurological disease, as well as certain hematological malignancies are exemplary pathologies wherein deregulated Wnt signaling is the cau....

Protocol

NOTE: All mouse procedures were performed with the approval of the Leiden University Medical Centre (LUMC) Ethical Committee on Animal Experiments. Male and female, 6-12-week-old, wild-type (wt), which have no insertion of the Axin2-mTurquoise2 reporter construct, heterozygous (Tg/0) with one insertion of the Axin2-murquoise2 reporter construct and thus, one disrupted Axin2 gene, and homozygous (Tg/Tg) with the insertion of the Axin2-mTurquoise2 reporter construct in both alleles and thus, two .......

Discussion

Several canonical Wnt reporters are available with differing reporter sensitivity and actual reporter proteins. Reporter models using synthetically introduced multimerized TCF/LEF binding sites are available with fluorescent reporter proteins; however, such repeats of transgenes can be lost during breeding or long in vivo experiments and can be sensitive to non-Wnt signals from surrounding genomic sequences that influence reporter expression. Therefore, the most used reporter remains the older variant Axin2-LacZ.......

Materials

Name	Company	Catalog Number	Comments
BD FACScantoII flow cytometer	BD Biosciences	not aplicable	Serial number V96300710. The flow cytometer setup in this protocol contains a 405 nm laser line with 505 longpass filter and 530/30 nm bandpass filter, and 470/20 nm bandpass filter; a 488 nm laser with 735 nm longpass filter and 780/60 nm bandpass filter, 670 nm longpass filter and 655 nm longpass filter, 610 nm longpass filter, 550 nm longpass filter and 575/26 nm bandpass filter, 505 nm longpass filter and 530/30 nm bandpass filter, and 488/10 nm bandpass filter; and a 633 nm laser line with 735 nm longpass filter and 780/60 nm bandpass filter, 685 nm longpass filter, and 660/20 nm bandpass filter.
BSA	Sigma	A9647
Corning 70 μm cell strainer	Falcon/Corning	352350
Cytospin 4 Type A78300101	Thermo Scientific	not aplicable
DMSO	Sigma Aldrich	D5879-1L
DNAse I	Sigma	A9647
Falcon 50 mL Conical Centrifuge tubes	Greiner bio-one	227261
Falcon round-bottom Polystyrene Test tubes with cell strainer snap cap	Fisher Scientific	352235
Fetal Calf Serum (FCS)	Greiner Bio-One B.V.	not aplicable	Depends on origin
Fiji software	ImageJ	not aplicable	Version 1.53
Filter card white (for cytospin)	VWR	SHAN5991022
FlowJo 10 software	Treestar	not aplicable	Version 10.5.3
Frost slides	Klinipath
Gibco IMDM medium	Fisher Scientific	12440053
HCX PL APLO 40x 1.4 OIL lens	Leica microsystems	not aplicable
Hydrophobic pen: Omm Edge pen	Vector	not aplicable
Leica TCS SP5 DMI6000	Leica microsystems	not aplicable	The microscope setup in this protocol consisted of an HCX PL APO 40x/1.2 oil-immersion objective with 8-bit resolution, 1024 pixels x 1024 pixels, 400 Hz speed, pinhole 68 µm, and zoom factor of 1.5 at room temperature. This system contains a 405 diode laser, argon laser, DPSS 561 laser, HeNe 594 laser and HeNe 633 laser with 4 hybrid detectors (HyDs) and 5 photomultiplier tubes (PMTs).
Methanol	VWR	1060091000
NaN3/sodium azide	Hospital farmacy	not aplicable
Normal mouse serum	Own mice	not aplicable
PBS	Lonza	BE17-517Q
ProLong Diamond Antifade Mountant	Fisher Scientific	P36965
Purified mouse anti-β-catenin (CTNNB1)	BD Biosciences	610154
TO-PRO-3 Iodide	Thermofisher	T3605
Transparent nailpolish	at any drugstore	not aplicable
Tween-20	Sigma Aldrich	P1379-500ml
Zenon Alexa Fluor 568 Mouse IgG1 labeling kit	Thermofisher	Z25006