Figures and data
Live-imaging of Drosophila epithelial tissue dynamics in vivo.
A) Translucent Drosophila pupa with the pupal wing highlighted in magenta. B) The pupal wing (with magnified inset, B’, on the centre zone of the wing where we consistently image) with cell boundaries labelled using E-cadherin-GFP (green) and nuclei with Histone2-RFP (magenta). C) Magnified view of the pupal wing epithelium after wounding, with the white dashed line indicating the wound edge. D) Schematic showing a cross section through the upper layer of epithelium of the pupal wing, with hemolymph (insect blood containing hemocytes and adipocytes) beneath and rigid cuticle above E) Multiple cell divisions (arrows) occur in the unwounded pupal wing epithelial tissue over the course of 8 minutes. F) A cell division (arrow) occurs in a wounded epithelial tissue with the white dashed line indicating the wound edge.
Deep Learning Detection of Cell Divisions, Division Orientation and Cell Boundaries.
Four deep learning models were developed to analyse epithelial cell behaviours. A) The first version of the division detection model receives 3 frames from the Histone2-RFP channel, which can be combined into a single RGB image, as is standard for a U-Net model. B) The second version of the model input has 10 frames, 5 each from the Histone2-RFP and E-cadherin-GFP channels. The model produces a white circle (white spot) where it detects a division. C) The cell division locations are then passed through the U-NetOrientation model to determine the division orientation. This model takes 10 frames of a division as the input. D) Segmentation of the focused cell boundaries without using a deep learning model. The focused stack image is inputted to Tissue Analyzer for segmentation and the result is compared to a hand-labelled ground truth. Green cells are correctly segmented and red cells are incorrectly segmented. E) The 3-focal plane image is inputted into the U-NetBoundary model and then segmented using Tissue Analyzer; this result is then compared to the hand-labelled ground truth.
Dice scores for the deep learning models
Analysis of cell division density in living epithelial tissue in vivo.
A) The density of cell divisions in the unwounded tissue, with faded blue region showing the standard deviation. The red line is the line of ‘best fit’ of the unwounded data. B) A heatmap of the division density correlation over distance and time in unwounded epithelial tissue. Red indicates positive, and blue negative correlation. C) The density of cell divisions in the wounded tissue, with either small or large wounds, with faded regions showing associated standard deviation. The red line is the line of best fit of the unwounded data. The micrographs show representative divisions identified at two different time-points post-wounding. D) Diagram of the annular bands around a wound, each 10μm wide (white dashed line); white circles indicate cell divisions. E-F) Heatmaps of the change in division density for small and large wounds compared with a best fit linear model of unwounded data. Red areas have more divisions, and blue less, than equivalent regions in the unwounded data. The dashed lines highlight areas in which cell divisions decrease and the dotted lines highlight areas in which divisions increase compared to unwounded data. Schematics below the heatmaps in E and F show the radial division densities 100min and 110min after wounding, respectively.
Analysis of division orientation in living epithelial tissue in vivo.
A) Distribution of the division orientations with respect to the proximal-distal axis of the pupal wing in unwounded tissue. Cell division orientations of 0□ and 90□ are illustrated in the micrographs. B) Distribution of the division orientations with respect to the wing in unwounded tissue (green) and the daughter cell orientations 20 mins after dividing (magenta), with examples of the orientation of division before and after cell shuffling (B’). C) Heatmap of the space-time correlation of division orientation. Red indicates positive correlation, blue negative and white no correlation. D) Diagram of cell division orientation with respect to a wound; lower values are dividing towards the wound and higher values away. E) Mean division orientation towards the wound as a function of distance from wound for small and large wounds. For unwounded tissues an arbitrary point is chosen as a “virtual wound”. F-G) Distribution of the division orientations with respect to small and large wounds. The spectrum of colours (same as in D) indicates the bias in orientation towards the wound. H-I) Distribution of the division orientations with respect to the wound in small and large wounds (green), and the daughter cell orientation 20 mins after dividing (magenta).
