Barone V et al. (2024), Local and global changes in cell density induce...

ECB-ART-53078

Development 2024 Apr 15; doi: 10.1242/dev.202362.

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Local and global changes in cell density induce reorganisation of 3D packing in a proliferating epithelium.

Barone V , Tagua A , Román JÁA , Hamdoun A , Garrido-García J , Lyons DC , Escudero LM .

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Tissue morphogenesis is intimately linked to the changes in shape and organisation of individual cells. In curved epithelia, cells can intercalate along their own apicobasal axes adopting a shape named "scutoid" that allows energy minimization in the tissue. Although several geometric and biophysical factors have been associated with this 3D reorganisation, the dynamic changes underlying scutoid formation in 3D epithelial packing remain poorly understood. Here we use live-imaging of the sea star embryo coupled with deep learning-based segmentation, to dissect the relative contributions of cell density, tissue compaction, and cell proliferation on epithelial architecture. We find that tissue compaction, which naturally occurs in the embryo, is necessary for the appearance of scutoids. Physical compression experiments identify cell density as the factor promoting scutoid formation at a global level. Finally, the comparison of the developing embryo with computational models indicates that the increase in the proportion of scutoids is directly associated with cell divisions. Our results suggest that apico-basal intercalations appearing just after mitosis may help accommodate the new cells within the tissue. We propose that proliferation in a compact epithelium induces 3D cell rearrangements during development.

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	Fig. 1. 3D segmentation of sea star embryos over time: cell packing and morphological analysis at the cellular level. (A) Schematic representation of wild-type (top) and WT-comp (bottom) sea star embryos. (B) Maximum projections of a representative wild-type sea star embryo (top) or WT-comp embryo (bottom) expressing the membrane marker mYFP at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (C) Computer rendering of the segmented sea star embryo at the 512-cell stage from a frontal (left) and lateral (right) perspective. (D) 3D representation of a four-cell motif with scutoid (top) or frusta (bottom) conformations. The apical and basal z-slices of the motives are shown. Coloured overlays show the section area for each cell in the corresponding 3D representation. (E-I) Quantifications of average scutoid frequency (E), surface ratio anisotropy (F), cell density (G), cell volume (H) and cell convexity (I). Wild type, n=150 timepoints, six embryos, four experiments; WT-comp, n=150 timepoints, six embryos, five experiments for all panels except F (where n=125 timepoints, five embryos, four experiments). Data are mean±s.d. Mann–Whitney tests with Bonferroni multiple comparisons correction (black) and Kruskal–Wallis tests with Dunn multiple comparisons correction (blue), except in F where one-way ANOVA test with Tukey multiple comparison correction was used (light blue); ns, non-significant; P<0.05; P<0.01; **P<0.001.
	Fig. 2. Asynchronous compaction and scutoid formation in the sea star embryo. (A) (Top) Schematic representation of wild-type embryos highlighting animal and vegetal poles. (Bottom) Maximum projections of a representative animal pole and vegetal pole at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (B-E) Quantification of opening areas (B), cell density (C), cell convexity (D) and proportion of scutoids (E). Wild-type animal, n=75, three embryos, three experiments; wild-type vegetal, n=75, three embryos, two experiments. (F) (Top) Schematic representation of WT-comp embryos highlighting animal and vegetal poles. (Bottom) Maximum projections of a representative animal pole and vegetal pole at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (G-J) Quantifications of opening areas (G), cell density (H), cell convexity (I) and proportion of scutoids (J). WT-comp animal, n=75, three embryos, two experiments; WT-comp vegetal, n= 75, three embryos, three experiments. In B and I, relative time 0 corresponds to the first cell division occurring at the 64-cell stage and relative time 1 corresponds to the first cell division occurring at the 512-cell stage. The pink areas indicate the average duration of mitotic waves for each stage. Data are mean±s.d. Mann–Whitney tests with Bonferroni multiple comparisons correction; ns, non-significant; P<0.05; **P<0.001.
	Fig. 3. Single-cell tracking of scutoids relative to cell division. (A) Computer rendering of 3D Voronoi models generated from wild-type and WT-comp embryos. (B) Quantification of the proportion of scutoids. Wild-type embryo: n=150 timepoints, six embryos, four experiments. WT 3D Voronoi, n=150 timepoints; WT-comp embryo, n=150 timepoints, six embryos, five experiments; WT-comp 3D Voronoi, n=150 timepoints. Data are mean±s.d. Mann–Whitney tests (black) and Kruskal–Wallis tests (blue) with Bonferroni multiple comparisons correction; ns, non-significant; P<0.05; P<0.01; *P<0.001. (C,D) Representative scutoids whose onset is classified as independent of (C) or after (D) mitosis. Coloured overlays show the section area for each cell in the corresponding 3D representation. Scale bars: 10 μm. Quantifications of the proportion of scutoids with onset after mitosis in the whole dataset (E) or per embryo (F) are shown. Wild type, 97 scutoids, six embryos, four experiments; WT-comp, 207 scutoids, six embryos, five experiments. Data ate mean±s.d. (red and black dotted lines, respectively). Two-tailed Student's t-test. P<0.05.
	Fig. 4. Relationship between compaction, cell density, convexity and the formation of scutoids. Schematic representation of the process of compaction in sea star embryos. Cell proliferation drives the lateral expansion of the epithelium via oriented cell divisions. Initially, cell divisions cause the reduction of interstitial space, until the epithelium is sealed. In this phase, when cells still have space to expand laterally, cell divisions do not drive the formation of AB-T1s. Once the epithelium is sealed, and cells are now confined, oriented cell divisions create lateral compression forces that result in lowered convexity and in cells adopting the scutoidal shape.

	Fig. 1. 3D segmentation of sea star embryos over time: cell packing and morphological analysis at the cellular level. (A) Schematic representation of wild-type (top) and WT-comp (bottom) sea star embryos. (B) Maximum projections of a representative wild-type sea star embryo (top) or WT-comp embryo (bottom) expressing the membrane marker mYFP at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (C) Computer rendering of the segmented sea star embryo at the 512-cell stage from a frontal (left) and lateral (right) perspective. (D) 3D representation of a four-cell motif with scutoid (top) or frusta (bottom) conformations. The apical and basal z-slices of the motives are shown. Coloured overlays show the section area for each cell in the corresponding 3D representation. (E-I) Quantifications of average scutoid frequency (E), surface ratio anisotropy (F), cell density (G), cell volume (H) and cell convexity (I). Wild type, n=150 timepoints, six embryos, four experiments; WT-comp, n=150 timepoints, six embryos, five experiments for all panels except F (where n=125 timepoints, five embryos, four experiments). Data are mean±s.d. Mann–Whitney tests with Bonferroni multiple comparisons correction (black) and Kruskal–Wallis tests with Dunn multiple comparisons correction (blue), except in F where one-way ANOVA test with Tukey multiple comparison correction was used (light blue); ns, non-significant; P<0.05; P<0.01; **P<0.001.
	Fig. 2. Asynchronous compaction and scutoid formation in the sea star embryo. (A) (Top) Schematic representation of wild-type embryos highlighting animal and vegetal poles. (Bottom) Maximum projections of a representative animal pole and vegetal pole at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (B-E) Quantification of opening areas (B), cell density (C), cell convexity (D) and proportion of scutoids (E). Wild-type animal, n=75, three embryos, three experiments; wild-type vegetal, n=75, three embryos, two experiments. (F) (Top) Schematic representation of WT-comp embryos highlighting animal and vegetal poles. (Bottom) Maximum projections of a representative animal pole and vegetal pole at 128-, 256- and 512-cell stages. Scale bars: 50 μm. (G-J) Quantifications of opening areas (G), cell density (H), cell convexity (I) and proportion of scutoids (J). WT-comp animal, n=75, three embryos, two experiments; WT-comp vegetal, n= 75, three embryos, three experiments. In B and I, relative time 0 corresponds to the first cell division occurring at the 64-cell stage and relative time 1 corresponds to the first cell division occurring at the 512-cell stage. The pink areas indicate the average duration of mitotic waves for each stage. Data are mean±s.d. Mann–Whitney tests with Bonferroni multiple comparisons correction; ns, non-significant; P<0.05; **P<0.001.
	Fig. 3. Single-cell tracking of scutoids relative to cell division. (A) Computer rendering of 3D Voronoi models generated from wild-type and WT-comp embryos. (B) Quantification of the proportion of scutoids. Wild-type embryo: n=150 timepoints, six embryos, four experiments. WT 3D Voronoi, n=150 timepoints; WT-comp embryo, n=150 timepoints, six embryos, five experiments; WT-comp 3D Voronoi, n=150 timepoints. Data are mean±s.d. Mann–Whitney tests (black) and Kruskal–Wallis tests (blue) with Bonferroni multiple comparisons correction; ns, non-significant; P<0.05; P<0.01; *P<0.001. (C,D) Representative scutoids whose onset is classified as independent of (C) or after (D) mitosis. Coloured overlays show the section area for each cell in the corresponding 3D representation. Scale bars: 10 μm. Quantifications of the proportion of scutoids with onset after mitosis in the whole dataset (E) or per embryo (F) are shown. Wild type, 97 scutoids, six embryos, four experiments; WT-comp, 207 scutoids, six embryos, five experiments. Data ate mean±s.d. (red and black dotted lines, respectively). Two-tailed Student's t-test. P<0.05.
	Fig. 4. Relationship between compaction, cell density, convexity and the formation of scutoids. Schematic representation of the process of compaction in sea star embryos. Cell proliferation drives the lateral expansion of the epithelium via oriented cell divisions. Initially, cell divisions cause the reduction of interstitial space, until the epithelium is sealed. In this phase, when cells still have space to expand laterally, cell divisions do not drive the formation of AB-T1s. Once the epithelium is sealed, and cells are now confined, oriented cell divisions create lateral compression forces that result in lowered convexity and in cells adopting the scutoidal shape.