Fig 2: Heterogenous multicellular WNT signaling models.A, B RT-qPCR and GAG/DNA ratios of pellets treated with various WNTs during pellet culture. Different letters are significantly different from each other (p < 0.05). Mean ± SEM. n = 3–4 pellets per group. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. C WNT treatment increased infiltration of off-target cells (pink arrowheads and white dashed lines) into the pellets, decreased COL2A1 staining, but increased COL1A1 (yellow arrowheads) and COL10A1 staining in the pellets. The pellets with C59 treatment exhibited homogenous COL2A1 staining and decreased COL1A1 and COL10A1 staining. Scale bar = 0.2 mm. The experiment was performed twice with similar results. D Heatmap showing distinct expression levels of various WNTs in various cellular subpopulations in d14 TGF-ß3-treated pellets. A total of 2148 cells from the TGF-ß3-treated d14 pellets was analyzed and used to generate the heatmap. E Percentage of the cells expressing WNT3A and its putative receptors in d14 TGF-ß3-treated pellets. F, G Heterogenous multicellular signaling models in d14 TGF-ß3-treated pellets.

Fig 3: Notch and Wnt signaling affect the expression of methyltransferases in DPSCs. (A,B): Q-PCR showing relative differences in DNA-methyltransferases DNMT1, DNMT3A and DNMT3B expression in DPSCs between control (DMSO, MBIO) and DAPT, BIO, WNT-3A, adipoinduction and osteoinduction conditions. Data are normalized to reference ß-ACTIN and GAPDH levels and represented as the mean + SEM (n = 3). (C,D): Q-PCR analysis showing relative differences in transcript expression for the Nicotinamide-N-methyltransferase NMMT in DPSCs subjected to different treatments. Data are normalized to reference ß-ACTIN and GAPDH levels and represented as the mean+SEM (n = 3) *: p < 0.05; **: p < 0.01; ***: p < 0.001. Dunn’s Test, Kruskal- Wallis H Test. Asterisks (*) report significance with respect to controls PBS/DMSO, and Hash symbol (#) represents significance with respect to control MBIO. #: p < 0.05; ##: p < 0.01.

Fig 4: IGF2BP3 is required for mediating Wnt/ß-catenin signaling in vitro.(A–J) Knockdown of IGF2BP3 inhibited Wnt/ß-catenin signaling. HKC-8 cells were incubated with Wnt3a (100 ng/mL) in the presence of control siRNA or IGF2BP3-specific siRNA for 2 days, as indicated. Representative Western blot (A) and quantitative data for IGF2BP3 (B), ß-catenin (C), active ß-catenin (D), Snail1 (E), fibronectin (F), a-SMA (G), KIM-1 (H), p53 (I), and p16INK4A (J) are presented. *P < 0.05, **P < 0.01 versus controls; †P < 0.05, ††P < 0.01 versus the Wnt3a plus control siRNA group (n = 3, ANOVA with Student-Newman-Keuls test). (K) Immunofluorescence staining shows fibronectin expression after various treatments. HKC-8 cells were incubated with Wnt3a (100 ng/mL) in the presence of control siRNA or BP3 siRNA for 2 days as indicated. Arrows indicate positive staining. Scale bar, 50 µm. (L–R) Representative Western blots (L) and quantitative data show the expression of IGF2BP3 (M), active ß-catenin (N), MMP-7 (O), fibronectin (P), p-H3 (Q), and p16INK4A (R). HKC-8 cells were pretreated with ICG-001 (10 µM) for 1 hour, then incubated with Wnt3a (100 ng/mL) for 2 days. *P < 0.05 versus controls; †P < 0.05, ††P < 0.01 versus the Wnt3a group (n = 3, ANOVA with Student-Newman-Keuls test). (S) Schematic presentation of reciprocal feed-forward activation loop between IGF2BP3 and ß-catenin.

Fig 5: Stimulation of Wnt/ß-Catenin Signaling by Either Wnt-3a or a GSK3ß Inhibitor Promotes CM Cell Fate at the Cost of NR Identity at the Periphery of Mouse Eyecups(A–D) CM marker Cdon was expanded in E14.5 DKO retinas (D) compared to that in Six3F/F retinas (A), Six3F/F;a-Cre retinas (B), and Six3F/F;a-Cre; Six6+/- retinas (C).(E–H) Compared to vehicle controls (E), Wnt-3a (G) or GSK3ß inhibitor CHIR99021 (H), but not Wnt-16b (F), expanded the size of CM (Cdon-positive Tubb3-negative) at the cost of NR identity at the periphery of mouse eyecups.(I–L) A working model. At E10.5–11.5, both Six3 and Six6 expression displayed a gradient along peripheral-central axis (I). In most-peripheral regions, low levels of Six3 and Six6 permitted Wnt/ ß-catenin signaling, leading to CM specification (blue in (K), an illustration of Otx1 expression in E15.5 control retina in Figure 4U). Moving centrally in the neuroretina, high levels of Six3 and Six6 protected multipotent NR progenitors directly or indirectly through (1) suppressing Wnt/ß-catenin signaling and (2) maintaining the expression of retinogenic factors. When Six3 was deleted in E10.5 neuroretina using a-Cre in combination with Six6 deletion (J), the suppression of Wnt/ß-catenin signaling was removed and the expression of retinogenic factors was disrupted. Retinal cells in far-peripheral regions gained CM identity at the expense of NR progenitors under the stimulation of Wnt/ß-catenin signaling, whereas retinal cells in mid-peripheral regions maintained NR identity but lost multipotency (L, an illustration of Otx1 and Neurod1 expression in E15.5 DKO retinas in Figures 4P and 4X).Scale bars, 100 µm (A), 50 µm (E). n = 3 retinas.