Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A-B) 2D-projected confocal images of the near-blastopore region (orange arrowhead) at stages 18 and 24. Schematics indicates approximate imaging locations and plane (Blue shaded box). (A) En face views of F-actin, keratin 8, and fibronectin and fibronectin in parasagittal section. The sagittal section is rotated 90° to align the blastopore with en face images. The regions of tissue rotation are located approximately 100 to 150 µm from the center of the blastopore. (B) En face views of keratin 8 and fibronectin, with a zoomed inset (yellow box). Aligned fibronectin fibrils are most prominent within 80 to 120 µm of the blastopore. (C) Polar histograms (rose plots) of fiber orientations at stage 18 and 24 (n, number of embryos used; Square goodness-of-fit test, ∗∗∗∗p<0.0001) (D) Workflow for image processing and analysis of immunostained samples. (E) Fibronectin morphological features from stage 18 and stage 24. (C and E) measured within a 100 by 100 µm region ventral to the blastopore. Each symbol represents the mean value per embryo (Mann-Whitney U, ∗p=0.02). Bars; mean ± 95% CI. (A-B) scale bars, 20 µm. (A-B) Xenopus illustrations © Natalya Zahn (2022).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A) Dorso-anterior index (DAI) of the severity of dorsalized and ventralized phenotypes. (B) Representative phenotypes of each treatment at stage 20, showing characteristic morphological differences between dorsalized, ventralized, and control embryos. (C) Final frame of brightfield timelapse sequence, overlaid with a yellow deformation map (see also Video S6). (D) Vorticity overlaid randomized dot plots deformed by calculated displacements, and max-projected across all timepoints to visualize movement patterns. Representative images are shown for each treatment. (E) SWIRL predicted vortices highlight vortex structure and organization. (F-G) Quantitative comparison of predicted vortex characteristics in dorsalized (8 vortices from 11 embryos), control (32 vortices from 19 embryos), and ventralized (11 vortices from 11 embryos) embryos. Each symbol represents a single predicted vortex (Mann-Whitney U, ∗∗p=0.0012; ∗∗p=0.0061; ∗∗∗p=0.0002; ∗∗∗∗p<0.0001). (F) Vortex compactness (mean ± 95% CI). (G) Vortex swirling strength (mean ± 95% CI). (H) Fibronectin networks in posterior tissues from stage 24 embryos within a 100 by 100 µm region ventral to the blastopore. (I) Fibronectin orientation frequency in each treatment (n, number of embryos used; Square goodness-of-fit test, ∗∗∗∗p<0.0001) (J) Morphological features of fibronectin network in dorsalized, control, and ventralized embryos. Each symbol represents the mean value per embryo (Mann-Whitney U, ∗p=0.0426; ∗∗∗∗ p < 0.0001). Bars indicate mean ± 95% CI. (B-C) scale bars, 100 µm; 20 µm in (H). (A) DAI diagram with permission of the publisher. Xenopus illustrations © Natalya Zahn (2022).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Control, Sequencing, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Supracellular Mechanics and Counter-Rotational Bilateral Flows Orchestrate Posterior Morphogenesis

doi: 10.1101/2025.11.18.689090

Figure Lengend Snippet: (A) Methods used for targeted disruption of fibronectin organization and integrin-fibronectin interactions. (B) Representative phenotypes showing morphological changes following fibronectin disruption. (C) 2D max-projected confocal image of fibronectin within a 100 by 100 µm region ventral to the blastopore. (D) Morphological features of fibronectin matrix for control (mAb 4H2) and function-blocking (mAb P8D4) treatments. Each symbol represents the per-embryo mean (Mann-Whitney U, ∗p=0.0350; ∗∗p=0.0023). Bars indicate mean ± 95% CI. (E) Final frame of brightfield timelapse sequence overlaid with yellow deformation map (see also Video S7). (F) Time-projected displacement of random dot plot overlaid with vorticity. Disruptions to fibronectin result in less distinct or absent bi-directional vortices compared to controls. (G) SWIRL predicted vortex structure and spatial distribution across treatments. (H-J) Vortex characteristics across treatments. Each symbol in (H) and (I) represents a single predicted vortex: 4H2 (19 vortices from 10 embryos), P8D4 (8 from 11), COMO (21 from 13), and FNMO (18 from 13). In (J), each symbol represents an embryo with a predicted vortex pair. Vortex formation was significantly disrupted in P8D4- and FNMO-treated embryos, with a reduction in detected vortices and alterations in vortex compactness (H) and swirling strength (I). (J) Vortex asymmetry index (Mann-Whitney U, ∗p<0.0290; ∗∗p=0.0054; ∗∗p=0.0024; ∗∗∗p=0.0002). Bars indicate mean ± 95% CI, except in (H). Scale bars, 100 µm in (B,E); 20 µm in (C).

Article Snippet: Immunofluorescence staining was carried out with primary antibodies against fibrillin-2 (JB3, Developmental Studies Hybridoma Bank; 1:200), collagen-2 (II-II6B3, Developmental Studies Hybridoma Bank; 1:100), laminin-a1 (L9393, Sigma-Aldrich; 1:500), mouse anti-fibronectin monoclonal antibody (4H2, courtesy of Douglas DeSimone, University of Virginia, Charlottesville, VA, USA; 1:500), rabbit anti-fibronectin polyclonal antibody (F3648, Sigma; 1:200), acetylated tubulin (T6793; Sigma; 1:125), keratin (1h5; Developmental Studies Hybridoma Bank; 1:500), and GFP (N86/8; Developmental Studies Hybridoma Bank; 1:50) and incubated overnight on a nutator at 4 °C.

Techniques: Disruption, Control, Blocking Assay, MANN-WHITNEY, Sequencing