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. Author manuscript; available in PMC: 2009 Nov 25.
Published in final edited form as: Stem Cells. 2009 May;27(5):1130–1141. doi: 10.1002/stem.36

Figure 2.

Figure 2

Cell autonomous Wnt signaling in AHPs. (A–D): OTP-GFP assay detects AHP secreted Wnt. HEK293 cells stably expressing LEF/TCF-GFP Wnt reporter (OTG293) were generated as described in Experimental Procedures Section. Either naïve fibroblasts as a negative control (A: Control), AHPs (B: AHP), or fibroblasts engineered to overexpress Wnt3a as positive controls, (C: Wnt-3a) were plated onto established monolayers of OTG reporter cells and the overall green fluorescent protein (GFP) fluorescence measured at 7 days in vitro (DIV). (Insets) Fluorescent micrographs of LEF/TCF-GFP Wnt reporter cells imaged using equal camera exposures (4 seconds). As summarized (D), Wnt3a production by L9-Wnt3a cells induces a substantial increase in GFP fluorescence among OTG293 cells, whereas native L9 cells do not produce any significant increase in GFP expression, demonstrating the sensitivity and specificity of this assay. As summarized, AHPs cocultured on OTG293 cells induce a significant, but submaximal increase in GFP expression, demonstrating that AHPs secrete active Wnt. (E): Basal AHP Wnt activity. AHPs were lentivirally transduced with either the OTP-GFP Wnt reporter (open bar) or the OFF-GFP negative control (gray bar) that contains nonfunctional LEF binding sites (similar to FOPflash above) and the fluorescence measured after 7 DIV. These AHPs were cultured in either control medium (base) or in the presence of the Wnt antagonist Fz8CRD (1 μg/ml), Wnt3a (300 ng/ml), or a combination of the two. At baseline, AHPs exhibit significant canonical activity, whereas application of saturating recombinant Wnt3a (for 7 days) induced a larger, significant increase in reporter activity. In contrast, application of Fz8CRD effectively blocked both endogenous (i.e., baseline) and exogenous (i.e., bath applied) Wnt activity. Together, these data indicate the presence of baseline canonical Wnt activity and validate the specificity of the antagonist. Mean ± s.e.m.; (*OTP vs. OFF activity, #Drug vs. Baseline OTP activity, p < .05; ANOVA with Tukey post hoc test). Abbreviations: AHPs, adult hippocampal progenitors.