Fig. 6.

Comparison of overexpressed and exogenously applied WNT7A in the induction of local calcium signalling and spine spacing. (A) Quantification of calcium transient amplitude identified significant increases compared with control if neurons overexpressed WNT7A (O.E. WNT7A) or if the soluble protein was exogenously applied to the culture (recWNT7A). These increases were significantly attenuated when cells were co-transfected with memNotum. (B) A significant calcium amplitude peak prominence increase was observed in cells overexpressing WNT7A (O.E. WNT7A). (C) Calcium transient width was significantly altered in cultures expressing memNotum or after exogenous application of WNT7A (recWNT7A), compared to control or O.E. WNT7A groups. (D) Quantification of the dendritic calcium transient intensity identified a significant increase when comparing the overexpression of WNT7A to the exogenous application of recombinant WNT7A. (E) Representative images from iPSC-derived cortical neuron cultures either transfected with mem-mCherry and WNT7A or with mem-mCherry plus exogenous application of recombinant (rec.) WNT7A, followed by analysis of protrusion spacing along dendrites. (F) Quantification of the distance between dendritic protrusions identified a significant reduction in spacing when exogenously applying recombinant WNT7A, compared to overexpression of the protein. For statistical analysis of the calcium transient data, we performed direct comparisons within between groups using an unpaired, two-sided Student's t-test (**P<0.01, ****P<0.001), and used Tukey's Honestly Significant Difference (HSD) test with Holm–Bonferroni adjustments for multiple comparisons. We employed letter-based groupings for a compact representation of the significant differences amongst treatment groups. Groups not statistically different from one another share the same letter, while significantly different groups are designated distinct letters.