Activation of Wnt/ß-catenin signaling in ESC promotes rostral forebrain differentiation in vitro
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Wnt/ß-catenin signaling is crucial for maintenance of pluripotent state of embryonic stem cell (ESC). However, it is unclear how Wnt/ß-catenin signaling affects the differentiation ability of ESC, especially with regard to rostral forebrain cells. Here, using Rax, rostral forebrain marker, and Wnt/ß-catenin reporter lines, we report ratio of Rax+ and Wnt responding tissue (Wnt+) patterns, which were affected by seeding number of ESC in three-dimensional culture system. Surprisingly, we found ß-catenin level and localization are heterogeneous in ESC colony by immunostaining and time-laps imaging of ß-catenin-mEGFP signals. Moreover, activation of Wnt signaling in ESC promoted expression level and nuclear localization of ß-catenin, and mRNA levels of Wnt antagonists, axin2 and dkk1, leading to upregulating Wnt/ß-catenin reporter in ESC state and Rax expression at differentiation culture day 7. Together, our results suggest that activation of Wnt signaling in ESC promotes the differentiation efficacy of rostral forebrain cells. Wnt-priming culture method may provide a useful tool for applications in the areas of basic science and molecular therapeutics for regenerative medicine.
KeywordsWnt/ß-catenin signaling Wnt antagonist Rostral forebrain Rax Embryonic stem cells Regenerative medicine
Forebrain is derived from neural plate, called neural ectoderm (Ruiz i Altaba 1998), which is subdivided into telencephalon and diencephalon (Markakis 2002). Diencephalon contains the hypothalamus and eyes in vertebrate (Sinn and Wittbrodt 2013). These developmental events involving forebrain patterning are largely conserved among species (Wilson and Houart 2004).
In rostral forebrain cells, Rax, transcriptional factor is expressed during early development (Furukawa et al. 1997; Ohuchi et al. 1999). In fact, disruption of Rax gene led to severe defects involving forebrain and eye development (Mathers and Jamrich 2000; Zhang et al. 2000; Andreazzoli et al. 2003; Nakagawa 2004). Additionally, misexpression of Rax gene led to duplication of rostral neural tube and eye (Mathers et al. 1997). Moreover, genetic mutations of the human RAX were found in a patient with anophthalmia and microphthalmia, which were ocular birth defects and a cause of congenital blindness (Voronina et al. 2004; Abouzeid et al. 2012). Thus, Rax gene plays crucial roles in forebrain and eye development.
Although its roles are well know as described above, Rax gene has another role as a specific marker for forebrain development. In this regard, for regenerative medicine, monitoring of Rax gene expression is a powerful tool for estimation of forebrain differentiation in vitro.
Previously, using embryonic stem cell (ESC) culture system, Wataya et al. showed efficient induction of hypothalamic tissues expressing Rax gene (Wataya et al. 2008), and Eiraku et al. showed optic cup morphogenesis by monitoring Rax expression in live condition (Eiraku et al. 2011). These reports demonstrated clearly that the use of Rax gene monitoring facilitated the analysis of forebrain development, suggesting that innovation of generation and manipulation of such tissues make it possible for regenerative tools to become closer to reality. However, in both methods, Rax expression patterns slightly vary among tissues in vitro (high and low expressions among tissues). Lacking of efficient and stable culture methods may hamper the reliability of future regenerative approaches.
In another aspect of ESC culture system, ESC state is maintained by Wnt signaling activity (Nusse 2008; Nusse et al. 2008; ten Berge et al. 2011). Namely, activation of the Wnt signaling maintains ESCs in a pluripotent state. In addition, ESCs that harbor Wnt activating mutations in the negative regulator APC or in positive regulator ß-catenin indeed have a profound reduction in their ability to differentiate, even following LIF (a ESC maintenance factor) withdrawal (Kielman et al. 2002). Wnt signaling also regulates the lineage differentiation potential of ESCs (Atlasi et al. 2013; Price et al. 2013). 2i conditioned medium (ESC medium containing Wnt agonist, CHIR99021 (CHIR) and Fgf antagonist, PD0325901 (Ying et al. 2008; Li and Ding 2010; Plusa and Hadjantonakis 2014)) improves ESC differentiation rate of neuronal lineages, expressing pan-neural marker, sox1 (Marks et al. 2012). Therefore, Wnt signaling in ESC maintenance is important for a potential lineage commitment. However, it is unclear how Wnt activation in ESC state affects rostral forebrain differentiation in vitro.
Here, we observed Rax gene expression with Wnt/ß-catenin signaling to explore the efficient method, which potentially promotes rostral forebrain differentiation from mouse ESCs.
Materials and Methods
ESCs maintenance, differentiation, and Wnt-priming method. Mouse ESCs; E14Tg2a (Hooper et al. 1987), Rax::GFP (Wataya et al. 2008), and Rax::GFP//TOP::DsRed (Andrabi et al. 2015) murine ESCs were maintained, and SFEBq culture were performed as described thoroughly by Wataya et al. (2008). Briefly, SFEBq (serum-free floating culture of embryoid body-like aggregates with quick reaggregation) culture is a method that starts with quick reaggregation of dissociated mouse ESCs in each well of a low cell-adhesive coating 96-well plate (Nunclon Sphera 96U Bottom Plate 174925; Thermo Scientific, Waltham, MA). For differentiation medium, we used gfCDM (a modified growth factor free chemically defined medium (Wiles and Johansson 1999; Bouhon et al. 2005)), which is free of knockout serum replacement and other growth factors including insulin. Composition of gfCDM was as follows: Iscove’s modified Dulbecco’s medium (Gibco, Carlsbad, CA) and Ham’s F-12 Nutrient mixture (Gibco) are mixed in a one-to-one, supplemented with 1x chemically defined lipid concentrate (Gibco), monothioglycerol (450 μM), purified BSA (5 mg/ml), and human apo-transferrin (15 μg/ml). In Wnt-priming method, we maintained ESCs with 10 μM CHIR99021 (Stemgent, Cambridge, MA) for 2 or 3 d and then performed SFEBq differentiation in gfCDM. This method has one advantage, because it is not necessary to add additional chemical reagents during forebrain differentiation, meaning not necessary to remove chemical reagents completely (when collecting forebrain tissues for regenerative medicine). Differentiation status was quickly checked by BZ-9000 microscope (KEYENCE, Osaka, Japan), which allows us to monitor fluorescent and transillumination images.
FACS analysis. Fluorescence-activated cell sorting (FACS) analysis was performed as previously described (Kamiya et al. 2011). For cell preparation, cells were dissociated to single cells by TrypLE™ Express (Gibco, 12605–010) treatment and filtration through Cell Strainer (BD Biosciences, East Rutherford, NJ). Cells in tube were kept on ice until analysis. For population analysis, FACSDiva (BD Biosciences) was used. For data analysis, data were analyzed with FlowJo software. All processes were performed, based on manufacturer’s instruction.
Immunohistochemistry, confocal microscope, and image analysis. Immunostaining and cryosectioning were performed as previously described (Wataya et al. 2008; Ohgushi et al. 2015). Briefly, for ESC staining, the ESCs were seeded onto a gelatin-coated 8-well chamber slide (Biocoat Collagen TypeI Cellware 8well Culture Slide 354630; CORNING, Corning, NY), and fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min and then permeabilized with 0.3% Triton-X100/PBS solution. After incubation of ESCs in 2% skim milk/PBS blocking solution, we used specific antibodies in blocking solution as follows: ß-catenin (mouse, 1:500, BD transduction 610153: rabbit, 1:500, C-2206; Sigma-aldrich): GFP (rat, 1/500, 04404–84; Nacalai, San Diego, CA): Nanog (rabbit, 1/1000, RCAB0001P; ReproCell, Yokohama, Japan): Oct3/4 (mouse, 1/200, BD 611202). After washing primary antibodies in 0.05% Tween/PBS, the staining was visualized using appropriate secondary antibodies conjugated with the fluorescent probes, Alexa Fluor-488 (1:1000, Molecular Probes, Eugene, OR) or Cy3/Cy5 (1:200, Jackson ImmunoResearch, West Grove, PA). We used DAPI (#11034-56, Nacalai) for counter staining. F-actin was visualized with AlexaFluor-conjugated phalloidin (546 and 647, 1/500, A22283 and A22287, respectively; Invitrogen, Carlsbad, CA). For cryosectioned sample staining, day-7 tissues were fixed with 4% PFA at room temperature for 30 min and washed in PBS. Then fixed tissues were in 15% sucrose/PBS at 4°C for overnight for cryoprotection, followed by cryosection. Immunostaining of cryosectioned sample slides is basically the same as above. Imaging analysis was performed using LSM 710 or 780 confocal laser-scanning microscope (Zeiss, Oberkochen, Germany). The Z-scanning images in video 1 were reconstituted from the serial slices of confocal images using imageJ software.
Rosa26 locus targeting vector construction, introduction, and genotyping of ß-catenin-mEGFP knock-in ESCs. For Rosa26 locus knock-in, we obtained Rosa26 mT/mG (Muzumdar et al. 2007), a gift from Liqun Luo (plasmid # 17787; Addgene, Cambridge, MA) and performed chimeric-PCR to amplify ß-catenin-mEGFP-SV40pA from mouse ESC cDNA and pDONR221-mEGFP (monomeric EGFP). Subsequently, by restriction enzymes, we subcloned ß-catenin-mEGFP-SV40pA PCR products into downstream of pCAG (consisting of the cytomegalovirus enhancer fused to the chicken beta-actin promoter) in Rosa26 mT/mG as a backbone vector, which has 5′ (1.1 kbp) and 3′ (4.3 kbp) homology arms with pPGK::neor-bGHpA (mT/mG cDNA was removed at this time). Then, we linearized Rosa26 ß-catenin-mEGFP targeting vector by a restriction enzyme and purified it by phenol-chloroform extraction to eliminate enzyme proteins. Using electroporation grade of linearized vector (7.5 μg), we introduced it into 1.0×106 E14Tg2a line via A24 program of Amaxa Nucleofector (Lonza, Basel, Switzerland) based on the manufacturer’s instruction (day 0). Next at day 2, we add 200 μg/ml G418 sulfate (10131–035, Gibco) to select pooled homologous recombinant ESCs. Finally, at day 10, we extracted genome of pooled-drug-resistant ESCs and performed genotyping of ß-catenin-mEGFP knock-in allele by primers (rosa3, 5′-CCACTGACCGCACGGGGATTC-3′; rosa4, 5′-TCAATGGGCGGGGGTCGTT-3′). We used pooled knock-in ESCs for Figs. 3 and 4.
Time-laps imaging and analysis. Live-imaging was performed using an incubator-combined confocal optic system (Olympus, Tokyo, Japan) as previously described (Eiraku et al. 2011) using a thin plastic-bottom dish (μ-Dish, 35 mm, low; ibidi, 80136), supplying penicillin/streptomycin and then filmed using a LCV110 equipped with 488-nm excitation lasers. We edited acquired images using MetaMorph software (Molecular Devices, Sunnyvale, CA) and Image J (free software).
Statistics. Statistical analyses were performed with Prism (GraphPad Software, Inc., La Jolla, CA). Data sets were first checked for standard error of the mean. The appropriate test for comparison was performed as follows; t tests (two samples) and Dunnett’s test (control and others) were used to generate P values (*P < 0.05; **P < 0.01; ***P < 0.001).
Results and Discussion
In this study, we developed a new method for efficient differentiation of Rax+ rostral forebrain by activating Wnt signaling through ß-catenin stabilization in mouse ESCs.
To date, the level of Wnt signaling activity in individual stem cells correlates with differences in lineage-specific differentiation propensity (Blauwkamp et al. 2012). Here, we endeavored to extend the idea involving Wnt signaling/ß-catenin in ESC state and its roles for differentiation, especially in rostral neural lineage.
Next, we tested weather seeding number of cells in SFEBq affect the expression patterns of Rax::GFP+ and 7Tcf::Cherry+. Compared with 3000 cells experimental condition, we found that decreasing seeding cell number significantly increased Wnt+ ratio but decrease Rax+ ratio as seen in 1000 and 500 cells (Fig. 2b ). Furthermore, via FACS analysis, Rax::GFP+ cells were significantly decreased in 1000 and 500 cells condition (Fig. 2c, d ), indicating that population of Rax+ rostral forebrain cells are correlated inversely with Wnt signals and proportionally with seeding cell numbers.
These results indicate that activation of Wnt signal directly upregulates ß-catenin in nucleus expression and Wnt antagonists, which then have a potential role for inhibiting Wnt signaling (Kawano and Kypta 2003).
Although, Wnt inhibition in ESC differentiation promote rostral neural fate (Haegele et al. 2003; Nasu et al. 2012), these results raised a possibility that Wnt antagonists in Wnt-priming method minimized Wnt signaling during rostral forebrain tissue development in vitro. However, future work requires how Wnt antagonists act during in vitro rostral forebrain differentiation in this culture system.
Research of the relationship between Wnt/ß-catenin and rostral forebrain lineage using our results will provide useful insights into mechanisms of Wnt/ß-catenin signaling in ESC culture system for the future improvement of regenerative medicine.
We are grateful to M. Eiraku, H. Inomata and M. Ohgushi for invaluable comments and to members of the laboratory for discussion. We also thank H. Kiyonari for critical information about ESC culture, T. Abe for technical advises of Rosa26 knock-in strategy, and M. Kawada for technical advices of SFEBq and vector construction; L. Luo for Rosa26 mTmG. This work was supported by grants-in-aid from Ministry of Education, Culture, Sports, Science, and Technology in Japan (MEXT) (to Y.S.), and the Network Program for Realization of Regenerative Medicine from the Japan Science and Technology Agency (JST) (to Y.S.).
N.T. designed the research, N.T. and E.S. performed the experiments, N.T. analyzed the data, N.T. prepared the figures and wrote the paper, and Y.S. supervised the project.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
Z-scanning of ß-catenin expression along the apical-basal of ESC colony, related to Fig. 3 b. Left, ß-catenin (green); right, ß-catenin and DAPI (magenta) were observed by confocal microscope. The movie was taken at 1 frame per 0.5 μm step and played at 15 fps. (MP4 4442 kb)
Dynamics of ß-catenin-mEGFP expression in ESC colony, related to Fig. 3 f. Left, ß-catenin-mEGFP; right, DIC were observed by time-lapse imaging. The movie was taken at 1 frame per 15 min and played at 15 fps. (MP4 7882 kb)
- Atlasi Y, Noori R, Gaspar C, Franken P, Sacchetti A, Rafati H, Mahmoudi T, Decraene C, Calin GA, Merrill BJ et al (2013) Wnt signaling regulates the lineage differentiation potential of mouse embryonic stem cells through Tcf3 down-regulation. Plos Genet 9Google Scholar
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