Multicellular rosettes link mesenchymal-epithelial transition to radial intercalation in the mouse axial mesoderm

Abstract

Mesenchymal-epithelial transitions are fundamental drivers of development and disease, but how these behaviors generate epithelial structure is not well understood. Here, we show that mesenchymal-epithelial transitions promote epithelial organization in the mouse node and notochordal plate through the assembly and radial intercalation of three-dimensional rosettes. Axial mesoderm rosettes acquire junctional and apical polarity, develop a central lumen, and dynamically expand, coalesce, and radially intercalate into the surface epithelium, converting mesenchymal-epithelial transitions into higher-order tissue structure. In mouse Par3 mutants, axial mesoderm rosettes establish central tight junction polarity but fail to form an expanded apical domain and lumen. These defects are associated with altered rosette dynamics, delayed radial intercalation, and formation of a small, fragmented surface epithelial structure. These results demonstrate that three-dimensional rosette behaviors translate mesenchymal-epithelial transitions into collective radial intercalation and epithelial formation, providing a strategy for building epithelial sheets from individual self-organizing units in the mammalian embryo.

Keywords: Par3; Pard3; axial mesoderm; embryo; mesenchymal-epithelial transition; morphogenesis; mouse; node; notochord; radial intercalation; rosette.

Figure 1.. Emergence and coalescence of an epithelial sheet in the mouse axial mesoderm

(A) Axial mesoderm cells in wild-type mouse embryos visualized with Brachyury (top row) and F-actin (phalloidin) (middle and bottom rows). Emerged axial mesoderm cells (cyan), unemerged axial mesoderm cells (magenta). Maximum-intensity projections (top and bottom rows). Surface projections (middle row). (B) Schematics of embryo orientation (left) and surface extraction method (right). Anterior (A), posterior (P). (C and D) Emerged apical area (C) and number of emerged clusters (D) in the axial mesoderm epithelium. Mean±SEM between embryos, each dot indicates one embryo (5-15 embryos/stage). Ventral views, anterior up. Bar, 25 μm. See also Figure S1 and Table S1.

Figure 2.. Axial mesoderm cells generate epithelial structures prior to emergence

(A) ZO-1-GFP localization at different distances from the surface of an E7.25 embryo. Total signal (white), surface signal (cyan), and subsurface signal (magenta) (5 μm projections). (B) Maximum-intensity projection (white), surface projection (cyan), and subsurface projection (magenta) for the embryo in A. (C) Fraction of the axial mesoderm epithelium located on the embryo surface at the indicated stages. Mean±SEM between embryos, each dot indicates one embryo (5-15 embryos/stage). (D) ZO-1-GFP localization at different stages. Emerged cells (cyan), unemerged cells (magenta) (maximum-intensity projections). Ventral views, anterior up. Bars, 25 μm. See also Table S1.

Figure 3.. Axial mesoderm cells assemble into cyst-like rosettes that radially intercalate into the surface endoderm

(A-D) Localization of membrane-GFP, Brachyury, and nuclei (Hoechst) (top panels). Axial mesoderm rosettes (yellow), surface endoderm cells (purple), membrane-GFP (black) (bottom panels). (E-H) Localization of ZO-1-GFP, Brachyury, and nuclei (Hoechst) (top panels). Axial mesoderm rosettes (yellow), surface endoderm cells (purple), β-catenin (black) (bottom panels). (I) Three-dimensional rendering of rosettes with internal lumens. Sagittal views, anterior up (left and middle panels), transverse views (right panels). Arrows indicate regions shown in right panels from top to bottom. (J) Number of basal, endoderm-contacting, and partially emerged rosettes/embryo at the indicated stages. Mean±SEM between embryos. (K) Percentage of rosettes with lumens at the indicated stages. (L and M) Time series of Ttr-Cre; Rosa26^mTmG/+ embryos expressing membrane-GFP (green) in visceral endoderm cells and membrane-Tomato (magenta) in all other cells. Arrowheads indicate the rosette center; dotted lines indicate position of transverse views. Optically reconstructed transverse views in A-H, L, and M (bottom panels), Maximum-intensity projections, anterior up in M (top panels). 165 rosettes in 29 embryos in J and K. Bars, 25 μm. See also Figure S2, Video S1, and Table S1.

Figure 4.. Par3 is required for epithelial organization in the mouse axial mesoderm

(A) Localization of Par3 (red) and ZO-1 (green) in a control embryo at E7.25. (B) Localization of Brachyury (top panels) and ZO-1-GFP in control and Par3^EpiΔ embryos. Emerged axial mesoderm cells (cyan) and unemerged axial mesoderm cells (magenta). (C-E) Emerged epithelial area (C), emerged cluster number (D), and emerged cluster area (E) in control and Par3^EpiΔ embryos. Each dot indicates one embryo (C and D) or cluster (E). (F) Emerged cell number in control and Par3^EpiΔ embryos at E7.5. Each dot indicates one embryo. (G) Emerged cell area in control and Par3^EpiΔ embryos at E7.5. Each dot indicates the average cell area in one cluster. (H) Localization of ZO-1-GFP in control and Par3^EpiΔ embryos at E7.5. Ventral views, anterior up (maximum-intensity projections). Mean±SEM between embryos (C, D, and F) or clusters (E and G), *p<0.03, **p≤0.0003 compared to controls (unpaired t-test), 3-15 embryos/genotype at each stage. Controls in C-G show combined data for Par3^EpiΔ and Par3^−/− littermate controls. Bars, 25 μm. See also Figures S3–S5 and Table S1.

Figure 5.. Live imaging reveals defects in epithelial expansion, coalescence, and emergence in Par3 mutants

(A and B) Stills from time-lapse movies of control embryos expressing ZO-1-GFP (white). Emerged axial mesoderm cells (cyan) and unemerged axial mesoderm cells (magenta). (C and D) Pre-cluster (C) and cluster (D) behaviors in control and Par3^EpiΔ embryos. (E) Time series of pre-cluster behaviors. (F) Time series of cluster and pre-cluster coalescence. Clusters and pre-clusters are highlighted in unique colors and adopt a shared color when two or more coalesce. (G) Number of coalescence events. (H) Time series and quantification of emerged apical cell area in control and Par3^EpiΔ embryos. The time point before the emerged apical surface was first visible was defined as t=0. Colors indicate three examples of cells that were first visible at t=12 min. Each line indicates one cell. Embryos were imaged starting at E7.25. Ventral views, anterior up (maximum-intensity projections). Mean±SEM between embryos, *p=0.03, **p≤0.0002 compared with controls (unpaired t-test), 3-5 embryos/genotype. Bars, 10 μm. See also Figure S6, Videos S2–S5, and Table S1.

Figure 6.. Par3 is required for apical aPKC localization and lumen formation in axial mesoderm rosettes

(A) Localization of β-catenin, ZO-1-GFP, and aPKC during rosette formation. Pseudocolors (top panels) and dotted lines (middle and bottom panels) indicate cells in rosettes or cells flanking spots and edges. (B) Number of basal, endoderm-contacting, and partially emerged rosettes/embryo in control and Par3^EpiΔ embryos at the indicated stages. (C) Percentage of partially emerged rosettes with lumens in control and Par3^EpiΔ embryos. (D) Schematic of ZO-1 and aPKC localization in rosettes. (E) aPKC localization in the indicated ZO-1-GFP regions in control and Par3^EpiΔ embryos. (F) Localization of ZO-1-GFP, aPKC, and β-catenin in control and Par3^EpiΔ embryos. Dotted lines, rosette outlines. Ventral views, anterior up. Mean±SEM between embryos, *p=0.03 (unpaired t-test), **p≤0.0008 (Fisher’s exact test), 92-117 rosettes in 14-23 embryos/genotype in B, 34-52 rosettes in 14-23 embryos/genotype in C, and 58-92 ZO-1-GFP regions in 8 embryos/genotype in E. Control data in B are reproduced from Figure 3J. Bars, 10 μm. See also Figure S7 and Table S1.

Figure 7.. Par3 is required to relocalize Pals1 from a cytoplasmic compartment to the apical membrane

(A) Localization of ZO-1-GFP, Pals1, and aPKC during rosette formation. Boxes and arrowheads indicate examples of Pals1 granules shown in D. (B) Localization of Pals1 and β-catenin in late rosettes. (C) Schematic of Pals1 localization in rosettes. (D) Close-ups of Pals1 granules in A. (E) Pals1 localization in the vicinity of the indicated ZO-1-GFP regions in control and Par3^EpiΔ embryos. No Pals1 detected (no Pals1), Pals1 in cytoplasmic granules (granular), Pals1 in granules and at the apical membrane (transitional), Pals1 primarily at the apical membrane (apical). (F) Localization of Pals1, β-catenin, and aPKC in control and Par3^EpiΔ embryos. Dotted lines, rosette outlines. (G) Localization of ZO-1-GFP, Pals1, and aPKC in control and Par3^EpiΔ embryos. White triangles, aPKC-positive Pals1 granules. Arrowheads, aPKC-negative Pals1 granules. Arrow, Pals1-positive apical membrane. (H) Model. Axial mesoderm cells form multicellular rosettes with central junctional and apical polarity that radially intercalate into the surface endoderm. Ventral views, anterior up (5 μm projections in A, B, and G, 5 μm projections for aPKC and Pals1 and single z-planes for β-catenin in F). **p=0.003 (Fisher’s exact test), 60-84 ZO-1-GFP regions in 6-7 embryos/genotype in E. Bars, 10 μm. See also Figure S7 and Table S1.