Figure 5.
PS Multiview imaging of mTurq2-Col4a1 mouse model. (A) Schematic of established live imaging method. The skin explant is mounted on an agarose pad and imaged using spinning disc microscopy. To visualize the tissue in Z, the image is software-reconstructed. Scale bar, 50 µm. (B) Schematic of PS Multiview live imaging. The skin explant is mounted as previous, but over an agarose ridge. Both planar (XY) and sagittal (“XZ”) focal planes are then imaged. Scale bar, 50 µm. (C) Snapshots taken from a time-lapse live imaging movie of hair follicle invagination in E14.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. (D) Representative image showing mean fluorescence intensity calculation methodology. Orange brackets highlight the hair follicle base region where signal intensity was measured. Magenta brackets highlight the IFE region where the signal intensity was measured. Scale bar, 50 µm. (E) Quantification of mTurq2-COL4A1 signal in IFE, placode, and hair germ stage. Each dot represents an individual fluorescence intensity measurement as shown in D. Larger dots show means of independent experiments. Error bars = SEM. ***P = <0.001, Mann–Whitney U test. ns = not significant. (F) Snapshots taken from a time-lapse live imaging movie of basal cell division in E15.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. Interphase, metaphase, and post-cytokinesis stages are shown (left to right). The bottom panel shows the same images with membrane curvature measurements. Scale bar, 10 µm. (G) Quantification of the average angle of BM deformation as shown in F. Each dot represents an average of two angles. n = 23 dividing cells. Error bars = mean + SD. *P = <0.05 (0.0366), **P = <0.01, ***P = <0.001, one-way ANOVA with Dunn’s multiple comparisons test. (H) Snapshots taken from time-lapse live imaging movies of placode, late-placode, and hair germ stages as labeled, in E14.5 and E15.5 Col4a1/+;mTmG/mTmG backskins. The bottom panel shows separate channels, note clear mTurq2-COL4A1 “baskets” surrounding the developing dermal condensate. Scale bar, 50 µm.

PS Multiview imaging of mTurq2-Col4a1 mouse model. (A) Schematic of established live imaging method. The skin explant is mounted on an agarose pad and imaged using spinning disc microscopy. To visualize the tissue in Z, the image is software-reconstructed. Scale bar, 50 µm. (B) Schematic of PS Multiview live imaging. The skin explant is mounted as previous, but over an agarose ridge. Both planar (XY) and sagittal (“XZ”) focal planes are then imaged. Scale bar, 50 µm. (C) Snapshots taken from a time-lapse live imaging movie of hair follicle invagination in E14.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. (D) Representative image showing mean fluorescence intensity calculation methodology. Orange brackets highlight the hair follicle base region where signal intensity was measured. Magenta brackets highlight the IFE region where the signal intensity was measured. Scale bar, 50 µm. (E) Quantification of mTurq2-COL4A1 signal in IFE, placode, and hair germ stage. Each dot represents an individual fluorescence intensity measurement as shown in D. Larger dots show means of independent experiments. Error bars = SEM. ***P = <0.001, Mann–Whitney U test. ns = not significant. (F) Snapshots taken from a time-lapse live imaging movie of basal cell division in E15.5 mTurq2-Col4a1/+;mTmG/mTmG backskin. Interphase, metaphase, and post-cytokinesis stages are shown (left to right). The bottom panel shows the same images with membrane curvature measurements. Scale bar, 10 µm. (G) Quantification of the average angle of BM deformation as shown in F. Each dot represents an average of two angles. n = 23 dividing cells. Error bars = mean + SD. *P = <0.05 (0.0366), **P = <0.01, ***P = <0.001, one-way ANOVA with Dunn’s multiple comparisons test. (H) Snapshots taken from time-lapse live imaging movies of placode, late-placode, and hair germ stages as labeled, in E14.5 and E15.5 Col4a1/+;mTmG/mTmG backskins. The bottom panel shows separate channels, note clear mTurq2-COL4A1 “baskets” surrounding the developing dermal condensate. Scale bar, 50 µm.

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