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

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 causative factor or contributor¹. Therefore, several inhibitors for different Wnt targets are currently being tested in clinical trials as Wnt-associated cancer therapeutics².

Additionally, interesting advances are taking place in Wnt therapeutic potential for neurological recovery, age-related neurological disorders, and congenital autism spectrum disorders^3,4,5. Wnt signals have been explored for ex vivo expansion of stem cells for subsequent transplantation⁶. However, therapeutic targeting of canonical Wnt signaling is a difficult endeavor due to its importance in many basic cell functions and cross-talk with other pathways^7,8,9, resulting in the need to precisely measure the effects of these Wnt therapeutic agents in an easy-to-interpret model. Canonical Wnt signaling is driven by short-range, soluble Wnt ligands, which are secreted by neighboring cells or as autocrine excretion as reported in various Wnt-responsive stem cell types.

The Wnt Frizzled receptor and lipoprotein receptor-related protein (LRP) co-receptors are responsive to these ligands, which triggers an intracellular signaling cascade. When Wnt signaling is off, a destruction complex composed of Axis Inhibitor (Axin), tumor suppressor gene product, Adenomatous Polyposis Coli (APC), Casein Kinase1 (CK1α), and Glycogen Synthase Kinase (GSK-3β), prevents the accumulation of β-catenin (CTNNB1) by proteasomal degradation. Upon Wnt ligand-receptor binding, the destruction complex is inactivated, leading to accumulation and stabilization of β-catenin in the cytoplasm. The active β-catenin can migrate to the nucleus where it binds to the Transcription Factor/Lymphoid Enhancer-binding Factor (TCF/LEF) transcription factors to initiate the transcription of Wnt target genes. Axin2 is considered a target gene as it is a direct target of the Wnt pathway¹⁰. Additionally, Axin2 serves as a negative regulator as well as a reporter gene for active canonical Wnt signaling^11,12.

Several canonical Wnt signaling reporters have been described in literature and have been of great use in understanding the role of Wnt signaling in embryonic development. Most of these reporters make use of synthetically inserted TCF/LEF binding sites, which do not use an endogenous target gene^{13,14,15,16,17,18,19}. Additionally, Axin2 knock-in strategies have been used that respect the natural location of the gene^{11,20,21,22,23}, of which Axin2-LacZ is generally accepted as the most robust canonical Wnt reporter¹¹. However, the reporter protein LacZ, albeit easy-to-use in most tissues, requires a β-galactosidase substrate, which is recognized to be harsh for live cells²⁴. Especially for stem cells and thymocytes, the harsh LacZ detection conditions increase cellular death (own unreported data) when handling cell suspensions.

Although the signal amplification caused by the LacZ staining is convenient to detect low signals, it makes the quantification less direct and thus arguably less reliable. Therefore, a murine reporter model was designed to mimic the Axin2-LacZ genetic strategy, but with an mTurquoise2 reporter protein²¹, to provide a readout that is more direct and closer to the physiological expression levels. The mTurquoise2 fluorescent protein is an excellent substitute for LacZ due to its high brightness (quantum yield (QY)= 0.93), flexibility in combination with other fluorescent proteins for extensive cell surface characterization, and its lack of needing an exogenous substrate. Furthermore, its close genetic relationship to green fluorescent protein (GFP) offers the possibility to use most GFP-recognizing fluorescent antibodies for stronger signal detection, if necessary, in extremely Wnt-sensitive cells²⁵.

The Axin2-mTurquoise2 model is not only a canonical Wnt reporter, but also offers the possibility to study Axin2 heterozygote and homozygote (Axin2 knock-out) phenotypes. The targeted insertion of mTurquoise2 at the start site of Axin2 results in a disrupted Axin2 protein²¹. As Axin2, also known as Conductin, is part of the Wnt destruction complex, and the destruction complex tightly regulates β-catenin mediated transcription, its partial or complete absence could be of interest to study diverse pathologies. For instance, in colorectal cancer, Axin2 levels are relatively high due to Wnt hyperactivation¹¹; however, its role in other pathologies is still largely unknown. Even though Axin2 is considered to play a limited role in the degradation of β-catenin, its role in Wnt regulation can be enhanced by the addition of a small peptide, which blocks Wnt-mediated colorectal cancer growth²⁶.

Altogether, careful Wnt regulation via Wnt therapeutic targets can open up opportunities to change the onset or development of severe pathologies and should be further investigated in models with reporter capacity. In this report, we explain our best-practice analysis method of the Axin2-mTurquoise2 murine model for flow cytometry and confocal imaging. In the context of Wnt dosage levels, very low canonical Wnt signaling levels are difficult to detect, for which advanced detection and analysis abilities provide an advantage to fully derive the benefits of this model. Thymocytes are used as a model system due to their fragile cell viability, low canonical Wnt signaling expression, and condensed cytoplasm area to represent the detection sensitivity of the Axin2-mTurquoise2 model. Additionally, a histological total β-catenin-staining procedure for thymocyte cell suspensions is explained to measure cytoplasmic β-catenin levels and verify nuclear active canonical Wnt signaling in combination with the reporter.