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Fig. 4 Spatial heterogeneity in Her6:Venus expression is altered in embryos with destabilised protein. (A) Representative examples of 3D-reconstructed confocal images of the transverse view of the telencephalon in Her6:Venus (HV; top panels) and Her6:Venus-PEST (HVP; bottom panels) embryos at 20-26 hpf showing HV and HVP expression (yellow), nuclei (H2B-mKeima, blue) and membranes (CAAX-RFP, red). Arrows indicate highly expressing HVP cells. OP, olfactory placode. (B) Percentage of Venus-expressing cells in HV versus HVP from a single experiment. (C) Distribution of nuclear HV and HVP at 20-26 hpf (temporal breakdown by repeat in Fig. S5C). Markers represent individual nuclei per time point, boxes indicate median and interquartile range. (D) Comparison of population CV obtained from Venus (HV and HVP) and H2B-mKeima expression from HV and HVP embryos (temporal breakdown by repeat in Fig. S5D). (E,F) Local intensity mapping of Venus in neighbouring cells observed in HV (E) and HVP (F) embryonic telencephalon imaged simultaneously at 24 hpf. Dots indicate intensities in individual nuclei (Cell 1) that were paired with their nearest neighbour (Cell 2) based on 3D Euclidean distance. (G) Intensity ratios observed in neighbouring cells (see E,F) showing Venus and H2B-mKeima quantified in HV versus HVP embryos at 20-26 hpf. Bars indicate mean±s.d., dots represent mean per embryo. ns (1): P=0.0901; ns (2): P=0.9989. (H,I) Local intensity mapping of Venus in neighbouring cells observed in DAPT- (H) and DMSO- (I) treated HVP embryos at 24 hpf. Dots indicate intensities in individual nuclei (Cell 1) that were paired with their nearest neighbour (Cell 2) based on 3D Euclidean distance. (J) Average intensity ratios observed in neighbouring cells (see H,I) showing Venus and H2B-mKeima quantified in HVP embryos at 24 hpf treated with DAPT versus DMSO. Bars indicate mean±s.d., dots represent mean per embryo. ns: P>0.9999. *P≤0.05; ***P≤0.001; ****P≤0.0001 (statistical test details found in Table S6). ns, not significant. Scale bars: 30 µm.